## INSIGHTS INTO GPCR DRUG DISCOVERY & DEVELOPMENT
Exploring GPCR–Ligand Interactions and Signaling Pathways with Binding and Functional Assays

“… Examples of β-arrestin and G protein-biased ligands demonstrate how our new understanding of these two types of signaling pathways, gained initially at a biochemical level, can potentially be harnessed for therapeutic benefit.”

—Dr. Robert J. Lefkowitz, awardee 2012 Nobel Prize in Chemistry for groundbreaking discoveries on GPCR receptors

eurofins
DiscoverX
The Eurofins Discovery PRODUCTS COMPANY

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## TABLE OF CONTENTS

**Introduction..............................................................................................................................................................................................1-3**
GPCR Biology and the Drug Discovery & Development of New Therapeutics.................................................................................................. 1-3
**Expert Insight.......................................................................................................................................................................................... 4-7**
Q&A GPCR Drug Discovery & Development Insights with Dr. Terry Kenakin.................................................................................................... 4-7
**Articles................................................................................................................................................................................................... 8-30**
GPCR Innovations from Eurofins DiscoverX®............................................................................................................................................................ 8-12
Why Study GPCR Arrestin Recruitment?.................................................................................................................................................................. 13-14
Four Reasons Why You Should Measure Signaling Bias in Your Molecules...................................................................................................15-17 
Ligand Bias: Increasing Specificity and Reducing Side Effects of Drug Compounds..................................................................................18-19
Can We Use Assay “Volume Control” to Unveil Efficacies in Allosteric Lead Molecules for GPCRs?.................................................... 20-22
Unlocking Orphan GPCRs: Application of GPCR Pharmacotrafficking and Cell-Based Assays............................................................... 23-26
Uses and Advantages of Membrane Preparations for GPCRs............................................................................................................................27-28 
Ideal Methods to Study Cellular Transport of Membrane Proteins.................................................................................................................. 29-30
**Knowledge-Based Videos......................................................................................................................................................................... 31**
GPCR Assays: Choosing the Best Assay for Your Programs..................................................................................................................................... 31
Internalization Assays: Identify Safer Therapeutics with Cell-Based Assays...................................................................................................... 31
Pharmacotrafficking Assays: Discover Pharmacological Chaperones that Rescue Disease-Relevant Proteins......................................... 31
**Application Notes............................................................................................................................................................................... 32-55**
Uncover Novel G-Protein or Arrestin-Biased Ligands Using GPCR Assays: Study of Biased Agonism on Opioid Receptors ........ 32-36
Quantify GPCR Endocytosis and Recycling with PathHunter® GPCR Internalization Assays: Analyzing Therapeutics for 
Opioid and Cholecystokinin Receptors ................................................................................................................................................................... 37-43
Ligand Characterization of Mas-Related GPCR-X2 via Cell-Based Assays: Assessing Regulation of Mast Cell Function & 
Implications in Safety Pharmacology.......................................................................................................................................................................44-49
Automating Cell-Based Bioassays on Tecan’s Fluent® Automation Workstation: Measuring β-Arrestin Recruitment of 
Cannabinoid Receptor 1...............................................................................................................................................................................................50-55
**White Paper.........................................................................................................................................................................................56-62**
The New World of GPCR Allosteric Modulation: Another Shot on Goal........................................................................................................56-62
**Resources............................................................................................................................................................................................. 63-96**
The Human GPCRome Dendrogram and Assays for GPCR Targets.........................................................................................................................63
GPCR Families, Targets, and Couplings....................................................................................................................................................................64-66
Product Types......................................................................................................................................................................................................................... 67
GPCR Product Topics............................................................................................................................................................................................................ 68
Tools and References............................................................................................................................................................................................................ 69

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# INTRODUCTION

# GPCR Biology and the Drug Discovery & Development of New Therapeutics

## GPCRs IMPORTANCE

G protein-coupled receptors (GPCRs) represent the largest family of validated therapeutic targets with over 800 known human GPCRs. 
Their physiological and pathological involvements are vast, ranging from regulation of hunger to the development of fetal structures, and 
from roles in cardiovascular disorders to rare diseases (see Table 1.). Therapeutics targeting GPCRs represent over 40% of all currently 
marketed drug therapeutics acting on GPCRs either directly or indirectly.

The importance of GPCRs was elevated in 2012 with the awarding of the Nobel Prize in Chemistry to American Drs. Robert Lefkowitz 
and Brian Kobilka for their efforts on the inner workings of GPCRs, and revealing some of the mystery about how cells could sense their 
environment. Tremendous interest in the discovery and development of drugs targeting GPCRs continues, spurred on by advances in 
information, tools, technologies, additional GPCR crystal structures, and the identification of numerous orphan GPCRs. GPCRs are the 
largest family of validated therapeutic targets, and are of continued interest for drug and treatment development for diseases, including 
cancer, Alzheimer’s disease, and diabetes.

| Physiological |  | Pathological |  |
| --- | --- | --- | --- |
| Addiction | Pheromone response | Cancers | Fertility disorders |
| Behavioral/mood regulation | Regulation of heart rate | Cardiovascular disorders(e.g. high blood pressure) | Neurological diseases(e.g., Alzheimer's, schizophrenia, depression,Parkinson's disease) |
| Embryonic development | Regulation of cellular machinery | Endocrine and metabolic disorders(e.g. diabetes, obesity,hypo- and hyperthyroidism,and nephrogenic diabetes insipidus) | Ocular disorders |
| Homeostasis | Smell(olfactory) | Endocrine and metabolic disorders(e.g. diabetes, obesity,hypo- and hyperthyroidism,and nephrogenic diabetes insipidus) | Rare diseases |
| Hunger regulation | Taste | Endocrine and metabolic disorders(e.g. diabetes, obesity,hypo- and hyperthyroidism,and nephrogenic diabetes insipidus) | Respiratory disorders(e.g. asthma) |
| Learning and memory | Vision | Endocrine and metabolic disorders(e.g. diabetes, obesity,hypo- and hyperthyroidism,and nephrogenic diabetes insipidus) | Retinitis pigmentosa |
| Metabolism |  | Endocrine and metabolic disorders(e.g. diabetes, obesity,hypo- and hyperthyroidism,and nephrogenic diabetes insipidus) |  |
| Pain tolerance |  | Endocrine and metabolic disorders(e.g. diabetes, obesity,hypo- and hyperthyroidism,and nephrogenic diabetes insipidus) |  |

Table 1. GPCR physiological and pathological involvements

## GPCR SIGNALING PATHWAYS

GPCRs are 7-transmembrane domain receptors with a broad array of ligands, including growth factors, ions, lipids, nucleotides, 
hormones, and neurotransmitters. Upon activation by ligands, GPCRs undergo a conformational change that translates the extracellular 
signal to an intracellular response through G protein-dependent and -independent pathways (β-arrestin recruitment) 
(see Figure 1.).

These ligands are classified by their mechanism-of-action (MOA) and function as agonists, antagonists, inverse agonists, or allosteric 
modulators (positive or negative). When the ligand behaves as an agonist, several downstream effects are produced. These effects include 
up- or down-regulation of G protein-dependent second messenger signaling (via cAMP or calcium), and G protein-independent β-arrestin 
recruitment with subsequent receptor internalization.

In the G protein-dependent pathway (Figure 1. A.), heterotrimeric (α/β /γ) G-proteins contain a GDP molecule bound to the Gα subunit, 
which holds the trimer together. Upon activation, GDP is exchanged for GTP, leading to the dissociation of the Gβ /Gγ dimer from Gα. 
Both parts remain anchored to the membrane and become free to act upon their downstream effectors and initiate unique intracellular 
signaling responses. The activated Gα subunit interacts with and regulates many effector molecules such adenylyl cyclase that can 
ultimately lead to the accumulation of cAMP (a second messenger) seen in this example.

---

**Figure 1. GPCR signaling pathways.** A. G protein-dependent and B. G protein-independent pathways.

In the G protein-independent independent pathway (Figure 1. B.), the ligand-activated GPCR is phosphorylated by a specific GPCR kinase 
(GKRs, not shown here), leading to β-arrestin recruitment. This β-arrestin binding blocks G protein-mediated signaling and results in the 
internalization (or endocytosis) of the GPCR and ending of the attenuation of GPCR signaling (known as desensitization). Subsequently, 
the GPCR is recycled back to the plasma membrane or degraded in the lysosome.

A key difference between both pathways is the GPCR/G-protein interaction is catalytic (i.e., one receptor can sequentially activate 
multiple G proteins) leading to an amplified signal. In contrast, the GPCR β-arrestin interaction “arrests” the signaling leading to a nonamplified, one-to-one signal. Overall, GPCRs can signal through one or both pathways as well as internalize/recycle without the presence 
of a ligand.

## GPCRS IN DRUG DISCOVERY AND DEVELOPMENT

What needs to be considered are both the GPCR drug MOA and which downstream transduction pathway is triggered: These can have key 
consequences in the development of drugs targeting GPCRs. Depending on the objective of the drug developer, there may be a need to 
study a ligand or drug effect on more than one of these signaling pathways. For example, a drug developer may need compound-profiling 
data from β-arrestin recruitment and second messenger assays to measure possible ligand bias. Additionally, there may be a need to test 
effects on receptor internalization to examine drug tolerance or unwanted side effects.

Investigating how small molecule or biologic therapeutics bind to GPCRs and modulate their downstream cellular signal pathways 
provides insights into the therapeutic MOA and resulting phenotypic responses. Drug discovery programs for  developing therapeutics 
targeting GPCRs go through several steps, from the identification and characterization of the GPCR through pre-clinical testing (see 
Figure 2. for a detailed breakdown of these steps).

---

Pre-Clinical & 
Quality Control

| GPCR target hunting &t characterization | High-throughput screening | Lead characterization &t optimization | Quality Control lot release studies |
| --- | --- | --- | --- |
| MOA determination | Secondary &t orthogonal screening | Ligand bias studies | Stability testing of biologics |
| Deorphanization | Hit-to-lead | Structure activity relationship for potency &t efficacy |  |
| Multiple GPCR pathways analysis | Rank order hits | Multiple species (orthologs) analysis |  |
| Mutation / disease analysis | Pharmacochaperone discovery | Internalization studies |  |
| Assay development |  |  |  |

**Figure 2. GPCR drug discovery and development applications**

As drug development progresses, hundreds of potential therapeutic agents targeting GPCRs continue to enter into clinical trials. These 
drug candidates include small molecule agonists and antagonists that target known GPCRs. Over the past decade, there has been an 
increased number of biological drugs, allosteric modulators, and biased agonists as well as candidates targeting orphan GPCRs without 
a known ligand or approved drug. Additionally, there has been a shift towards a focus on metabolic and neurological disorders as well as 
cancer. Future developments of basic tools to study GPCRs and greater understanding of GPCRs, as well as in the number and quality of 
therapeutics, will go hand-in-hand to advance the GPCR space.

---

# EXPERT INSIGHT

# Q&A GPCR Drug Discovery & Development Insights with Dr. Terry Kenakin

Q&A with Terry Kenakin, Ph.D., Professor of Pharmacology at University of North Carolina School of Medicine, key 
opinion leader in GPCRs and drug discovery, author of several publications on pharmacology in drug discovery, and 
collaborator and consultant for many global biotechnology and pharmaceutical companies.

## Q: WHAT IS THE IMPORTANCE OF GPCRS IN DRUG DISCOVERY AND DEVELOPMENT?

**A:** GPCRs have long been regarded as a low-hanging fruit drug target as they are conveniently located on the cell

surface and control a myriad of cell functions, but at various times over the decades, they have been the most utilized target for new 
therapies. An important theme in drug discovery is the realization that GPCRs are Nature’s prototypical allosteric protein linking the 
internal signaling machinery in the cell cytosol with the outside world (receptor compartment extracellular space). What makes GPCRs 
especially attractive as drug targets is their versatility. Specifically, GPCRs are pleiotropically coupled to many signaling systems in 
 the cell and with the advent of methods to discern biased signaling, we can customize the cellular signal emerging from the activated 
receptor through different molecular scaffolds. In addition to drugs having efficacies of varying strength, they also yield ligands with 
different qualities of efficacy presented to the cell.  In this regard, GPCRs, historically thought to be switches, are now known to be 
micro-circuits taking different signals in and yielding different signals out. Through the application of quantitative pharmacology, 
biologists and medicinal chemists are learning to control these signaling patterns to provide more focused drugs with fewer unwanted 
effects.

## Q: HOW HAS THIS IMPORTANCE EVOLVED IN THE LAST DECADE?

**A:** Arguably, there has been a perfect storm in pharmacology and drug discovery in the past 15 years brought on from two sources: 
molecular dynamics and the increasing availability of new functional assays to detect the outcome of drug-receptor interactions. The 
appreciation of receptor proteins as dynamic systems of varying micro-conformations (ensembles) has enabled the understanding of 
GPCR signaling bias (the ability to dissect multiple signals from the receptor to emphasize some at the expense of others). The increased 
availability of functional assays has allowed us to quantify these varying signals; in early years when the response was considered a 
monotonic signal from the receptor to the cell, there was little opportunity to visualize the texture of signaling produced by receptor 
activation. Now with “more eyes to see,” we can discern subtleties of receptor signaling and observe differences in drugs that previously 
could not be detected.

In addition, the understanding of receptor allostery has advanced with the advent of the discovery of increasing numbers of allosteric 
ligands. Historically, new drug screening had been conducted in an orthosteric mode (binding) thereby understandably providing 
orthosteric lead molecules and few allosteric ones. With increased screening in functional systems (more suited to detect allosteric 
molecules) has come a concomitant increase in allosteric lead molecules. As we study these new allosteric ligands, we are seeing 
the versatility of allosteric drug ligands through the properties of saturation of effect and practice of probe dependence, and these 
ideas have opened new therapeutic vistas. So, the emphasis for small molecule drug development has shifted from copies of natural 
neurotransmitters and hormones and molecules that sterically block them to a flexible array of allosteric molecules capable of blocking, 
enhancing or otherwise modifying natural signals in drug therapy. In addition, biologic molecules are emerging onto the therapeutic 
horizon in the form of replacement proteins, peptides, vaccines, antibodies, and nucleotide-based therapies (gene therapy, RNAi therapy). 
The exciting aspect of this new class is that many biologics can do things small molecules cannot do due to an interactive conversation 
with the human immune system.

## Q: WHAT ARE THE COMMONLY STUDIED GPCR FAMILIES BEING RESEARCHED WITHIN THE LAST DECADE?

**A:** Historically family A GPCRs (rhodopsin family - monoamines, neurotransmitters) were the bread and butter of receptor therapies. 
From there, family B (secretin family - peptide) receptors emerged, but this led to an appreciation of the difficulties encountered trying 
to find small drug-like molecules for them. Theoretical ideas around the requirement of multiple binding domains on these receptors (so called “affinity traps”) appear to predict that a single small molecule would be insufficient to stabilize a peptide receptor active state. A 
paradigm shift in the approach to peptide receptors came with the realization that small molecule allosteric ligands could yield nonpeptide therapies for these targets. In addition, advances in the formulation and delivery of peptides has greatly improved the therapeutic 
utilization of family B receptors. Finally, there are active research programs pursuing the other 3 families of GPCRs (glutamate, adhesion 
and frizzled/taste) as well, an exciting prospect as the physiological function of many of these is still to be determined. In fact, proteins 
classified as GPCRs through their seven transmembrane domain structure with no known function (so called “orphan receptors”) form 
a large uncharted area in GPCR receptor pharmacology. Studies of these utilize “reverse pharmacology” in which a ligand found for 
an orphan receptor is then used as a probe to uncover the physiological role of the receptor and reveal new mechanisms for future 
therapies.

## Q: WHAT INSIGHTS DO YOU KNOW ABOUT FOR FUTURE THERAPEUTIC GPCR TARGETS?

**A:** The most obvious change in the field has come about 
with the discovery of biased signaling for GPCRs. At first 
thought to be a new phenomenon, it is now known simply 
to be standard probe dependent allostery practiced by all 
receptor proteins. It was detected when new functional 
assays became available to dissect the signaling emerging 
from activated receptors. The fact that biased signaling 
is a natural behavior of standard proteins predicts that 
synthetic ligands will more often than not produce 
signaling of a different quality than natural hormones 
and neurotransmitters. Gone is the belief that a synthetic

"What makes GPCRs especially attractive as drug targets is their versatility. Through the application of quantitative pharmacology, biologists and medicinal chemists are learning to control these signaling patterns to provide more focused drugs with fewer unwanted effects."

- Terry Kenakin, Ph.D., UNC School of Medicine

analog of a natural agonist will produce the same quality of signal to a cell, but only of greater or lesser magnitude. This both opens new 
avenues for better therapy, but also places a burden on researchers to fully characterize the effects of synthetic molecules aimed for 
therapy. In addition, with the great advances in screening technologies over the past years (high throughput screening, DNA-encoded 
libraries, virtual screening, structure-assisted screening, fraction-based screening), the present general notion indicates it is possible to 
“drug” (find a ligand for) any defined target. This puts the onus on the choice of protein target as being the critical and weak link in the 
discovery process and target validation, and thus having the target taking the center stage in discovery programs.

## Q: WHAT ROLES DO GPCRS PLAY IN VIRAL THERAPEUTICS?

**A:** There are basically three mechanisms GPCRs could be involved in viral function:

1. The first mechanism is typified by HIV-1 and AIDs. Specifically, the HIV-1 virus utilizes a GPCR (the CCR5 chemokine receptor) directly as 
the portal allowing cell infection. Allosteric blockers of CCR5-HIV interaction such as maraviroc are used as a therapy in this setting.

2. A second viral GPCR association is virally encoded GPCRs whereby the viral genome encodes for GPCRs that are then set free in the host 
to support viral replication and growth. Examples of these are various herpesvirus-encoded GPCRs and US28 of human cytomegalovirus 
(HCMV). Many of these GPCRs are constitutively active (spontaneously form active states that signal with no need of an agonist) and thus 
become pathological entities in the host. Interestingly, these pose unique therapeutic problems as only an inverse agonists can be used to 
quell the viral GPCR signal.

3.  A third association is a heightened pathology resulting from the virus targeted to GPCRs in the host; in these cases, the GPCRs are 
targeted for symptomatic treatment. With regard to COVID-19, the pro-inflammatory GPR4 mediating leukocyte infiltration in vascular endothelium and the C5a-C5a receptor mediating platelet hyperactivity have been implicated as targets for GPCR-based therapy in 
COVID-19 treatment.

## Q: WHAT ARE SOME OF THE LARGEST CHALLENGES RESEARCHERS ENCOUNTER WHEN STUDYING GPCRS?

**A:** There are two areas of heightened importance with the changing landscape of GPCR therapeutic research:

The first is accepting the premise that screening technologies may be not be such a limiting step in the discovery chain (i.e., we can find 
ligands for nearly all GPCR targets). The problem area becomes which target to choose? Considering that of the 30,000 genes in the 
human genome, there are approximations that about 3,000 are “druggable” (small molecules will change their behavior) and another 
3,000 are associated with disease. This leaves an intersection of between 800- to -1,500 viable drug targets and a great many of these 
are GPCRs. Currently, industry and academic programs target ~300-350 targets leaving a great many still to be explored. Incorrect

choice of target minimally costs programs years of precious discovery time and resources.

A second hurdle is translation of *in vitro* observed activity to complex systems (sometimes under the control of pathophysiological 
processes). The advent of biased signaling which predicts complex signaling patterns from GPCRs that may be altered by cell type and the 
relative stoichiometry of receptors to signaling components of the cell, means that predictions of *in vivo* GPCR-mediated effects are not 
straightforward. In this regard, the rise of phenotypic screening is leading to identification of new mechanisms of GPCR control of cell 
function, which may be exploited by discovery.

## Q: HOW FAR ALONG HAS GPCR RESEARCH EVOLVED FROM THE TRADITIONAL IN VIVO ANIMAL MODELS TO IN VITRO CELL-BASED ASSAY?

**A:** The history of drug discovery has been rooted in the phenomenological observation of drug effect in whole systems (animal models, 
isolated organs). A major stumbling block in this process has been dissimulation between human and animal material, i.e. correspondence 
of GPCR activity in animal test systems to humans. Biochemical binding technology followed by cellular signaling studies bridged this 
gap as the genome provided human drug targets and test systems devoid of species variance. This led to a “biochemical age” of discovery 
where simple system pharmacology (so called “synoptic pharmacology”) was de-emphasized and recombinant systems took the lead. 
However, with the realization that the complete system is greater than the sum of its parts, there is now a return to whole system 
pharmacology. Cellular systems and label-free technology enable observation of whole system pharmacology in human tissue. Ironically, 
this describes nearly a full circle from whole system to whole system, but with unique differences in the systems.

## Q: WHY IS IT NECESSARY TO CONSIDER MULTIPLE ASSAY TYPES TO CHARACTERIZE GPCRS?

**A:** GPCRs are interactive systems whose behavior is intimately associated with other proteins and molecules in the cell and extracellular 
space. The allosteric nature of GPCRs dictates a bidirectional flow of allosteric energy between the extracellular ligand binding site and 
the binding sites in the cytoplasm for signaling molecules. The absence of these partners leads to differences in observed activity. Thus, 
the binding of efficacious agonists to bare GPCR proteins devoid of G protein partners is hugely different from systems where those G 
proteins are present. This extends to many other signaling components present in the cell (i.e. β-arrestin) thereby dictating the need for 
the complete system to reflect pharmacological activity, i.e., functional cell systems. In addition, this question can be applied to new drug 
screening. Specifically, when a single probe of the receptor is used to detect ligand interaction with that receptor (i.e., radioligand), then 
any ligands that interact with the receptor but do not report that interaction to the screening probe, will be missed (i.e., many allosteric 
ligands). In contrast, a functional assay utilizes the full cadre of signaling proteins in the cell interrogating the receptor and reporting 
alteration of receptor conformation and function when ligands bind, i.e., this is a much broader *brush to paint* for detection of compound 
detection. It can be likened to a music box with a full complement of tuning forks vs a music box with only one tuning fork.

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## Q: WHAT IS THE INDUSTRY TREND TO MOVE FORWARD WITH IN VITRO ASSAYS VERSUS IN VIVO EXPERIMENTATION?

**A:** Clearly, *in vivo* drug effects are preferable since this is a view of what drugs do therapeutically. However, drug concentration is 
variable *in vivo* and real time is an issue. So it is critical to link drug concentration at the target with the observed effect. *In vitro*
experiments obviate this dissimulation in that the concentration of drug is defined and constant and the resulting drug effect can be 
observed. Therefore, to determine true drug value, *in vitro* experiments can be invaluable, i.e., if the drug is presented to the target then 
this effect will occur. Any dissimulation between *in vitro* and *in vivo* results therefore may be a function of variation in pharmacokinetics; 
this may or may not affect evaluations of the candidate value as a drug candidate, but it is an important variable in the progression 
process. Thus, lack of *in vivo* effect can be a result of failure of pharmacodynamic or pharmacokinetic effects and this must be 
differentiated in drug progression studies. Determination of whether pharmacokinetics limits *in vivo* utility is critical to the progression of 
new chemical scaffolds.

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## ARTICLE

## GPCR Innovations from Eurofins DiscoverX

To help researchers fully characterize their specific GPCR and 
GPCR-binding small molecule or biologic of interest, Eurofins 
DiscoverX® encourages exploring all possible scenarios with a 
variety of available biologically relevant cell-based functional 
and binding assays and membrane preparations. These products 
are target specific, sensitive, and robust for detecting receptormediated second messenger signals, β-arrestin recruitment, receptor 
internalization, and ligand binding.

## GPCR INNOVATIONS FROM Eurofins DiscoverX

| Highlights of GPCR Product Solutions |  |  |  |
| --- | --- | --- | --- |
| Applications | Benefits | Product Portfolio | Credibility |
| Biologically relevant functional assays | Homogeneous assays with easy-to-follow, rapid, no-wash protocols | Cell lines and ready-to-use assay kits | Accepted by industry and the scientific community(e.g.,100s of peer-reviewed publications, billions of data points screened) |
| Broad coverage of human GPCRome, including both liganded and orphan GPCRs | Luminescent and fluorescent readouts | Flexible toolbox(do-it-yourself) cell-based assay development products | Thorough technical documentation |
| Ortholog receptor assays | Sensitive detection | Membrane preps suitable for radio- and fluorescent ligand binding assays | Helpful and experienced customer support |
| Multiple modalities (second messenger,β-arrestin,and internalization)for ligand bias studies | Robust dynamic range | Qualified,optimized,and highly reproducible bioassays for development through QC lot-release applications |  |
| Pharmacological profiling(e.g.,agonist,antagonist,allosteric modulation) | Low false positive rate |  |  |
| Ability to multiplex various readouts(in one cell line) | ReproducibleSuitable for miniaturization and high-throughput screening | Custom product development services |  |

---

## CHOOSE THE SOLUTION THAT BEST MEETS YOUR GPCR PROGRAM NEEDS

Whether you are developing small molecule or biologic therapeutic drugs, Eurofins DiscoverX® provides you with a 
variety of drug discovery and development products and services with multiple assay types to meet your specific 
GPCR research needs.

• Broad Coverage – >90% targets covered across the human GPCRome to always find an assay for your target of interest

• Highly Applicable – Ideal for the development of small molecule and biologic therapeutics, from target ID through pre-clinical drug 
discovery programs

• Various Assay Options – Multiple assay types (e.g., β-arrestin, cAMP, calcium, internalization, ligand binding, trafficking) offering a diverse 
array of GPCR assay solutions for maximum flexibility

• Largest Collection – >1,600 qualified functional and binding assays through products and services seen in >800 peer-reviewed 
publications, giving you the most comprehensive GPCR assay solutions available for the confidence you need

---

## GPCR PRODUCT TYPES OVERVIEW

Eurofins DiscoverX® offers a variety of product types for quickly testing through thorough characterization of your small molecule or 
biologic GPCR ligand or therapeutics for multiple applications.

## Stable Cell Lines

• Qualified cryopreserved, target-specific cell lines

• Unlimited culture ready for all drug discovery and development programs

• High functional expression

• Optimal performance with Eurofins DiscoverX detection kits

## Membrane Preparations

• Applicable for competitive radioligand binding and GTPγS functional studies

• Simple assay, single-use preps

• Robust performance with reproducible results

• Provides high total binding

## Assay Ready Cells and Kits

• Complete kits with ready-to-use cryopreserved cells, plates, required reagents, and simple homogeneous 
protocols

• Applicable for target identification, screening, proof-of-concept, rank-order potencies, and more

• Amenable for miniaturization and high-throughput screening

• Bioassays developed for potency testing in QC lot release to meet robustness, precision, linearity, and 
reproducibility

## Toolbox “Do-it-Yourself” Products

• Create your own cell-based assays to evaluate mutants, orthologs, unique targets, and more

• Cost saving assay development kits, retroparticles (retrovirus), and expression vectors

• Time saving engineered parental cells

• Enables development of nearly any target in most cell types

## Custom Assays & Bulk Lot Production

• Custom cell pools, clonally-derived lines, membrane preps, or cell-based assays

• Leverage internal expertise (with decades of experience) and flexible milestone structure

• Assay protocol development and 100% method transfer

---

## DECIDING ON THE BEST GPCR SOLUTION

Achieving full understanding of your GPCR and/or GPCR-ligand/therapeutics is possible through using multiple cell-based assays 
and evaluating GPCR families, targets, variants (e.g., orthologs, isotypes), biological function, or signaling pathway as well as ligand/
therapeutic binding, functional status, MOAs, and pharmacological profiles. By knowing the applications and benefits for each GPCR 
cell-based assay type and detection kit, you can chose the appropriate assay for your GPCR research goals and drug discovery and 
development program.

| GPCR Assay Types | Cell-Based Assay Applications & Benefits | Eurofins DiscoverX® Solutions |
| --- | --- | --- |
| β-Arrestin Functional Assays | G protein-independent assays engineered to detect GPCR activation via recruitment of β-arrestin by ligand activated GPCRs | PathHunter® arrestin cell lines and assay ready kits (eXpress™ & bioassay) |
| β-Arrestin Functional Assays | Study virtually any Class A or B GPCR, including orphan receptors | Toolbox products |
| β-Arrestin Functional Assays | Less likely to miss partial agonists or get false agonist response with positive allosteric modulators (PAMs) | PathHunter detection kit |
| β-Arrestin Functional Assays | Superior for screening antagonists (IC50more reflective of ligand binding affinity) and partial agonists |  |
| β-Arrestin Functional Assays | Use with any GPCR regardless of coupling status |  |
| cAMP Functional Assays | Monitor the functional status of GPCRs directly by quantifying cellular cAMP accumulation levels in a dose-dependent manner | cAMP Hunter® cell lines and assay ready kits (eXpress™ & bioassay) |
| cAMP Functional Assays | Characterize ligand pharmacology with precision (superior agonist detection) | HitHunter® cAMP assays (detection kits) |
| cAMP Functional Assays | Evaluate ligand bias with complementary assays (e.g. β-arrestin assay) |  |
| cAMP Functional Assays | Reproducible performance with large assay windows and broad sensitivity ranges |  |
| Calcium Functional Assays | Use with cell lines or ready-to-assay cells expressing naturally G1-或G2-coupled GPCRs | Native calcium cell lines
ChemisCREEN™和ChemibRITE cell lines and assay ready cells |
| Calcium Functional Assays | Measure intracellular calcium mobilization (flux) in cells based on the GPCR activation status | HitHunter Calcium assay (detection kit) |
| Calcium Functional Assays | Qualified for use with FLIPR®和 fluorescent dye-based assays
Robust and flexible assays with broad sensitivity ranges and large assay windows
Use with cell lines or ready-to-assay cells expressing naturally G1-coupled or force-coupled (non-G1-) GPCRs |  |
| Internalization Assays | Measure GPCR internalization in live cells | PathHunter Internalization cell lines and assay ready kits (eXpress) |
| Internalization Assays | Secondary and orthogonal screening tool to help in identifying safer drugs (e.g., evaluating drug tolerance, unwanted side effects, and diseases) | Toolbox products |
| Internalization Assays | Quantify effect of ligand on internalization of GPCRs localized to intracellular endosomes | PathHunter detection kit |
| Internalization Assays | Monitor your activated GPCR without expensive microscopy or antibodies
Use with most GPCRs regardless of coupling status |  |
| Internalization Assays |  |  |
| Pharmacotrafficking Assays | Identify small molecule compounds called pharmacological chaperones or pharmacochaperones | PathHunter Pharmacotrafficking cell lines |
| Pharmacotrafficking Assays | Characterize pharmacochaperones that enter the cell and function by promoting proper folding of misfolded mutant receptors and enable their trafficking to the cell membrane and further to the endosome | Toolbox products |
| Pharmacotrafficking Assays | Interrogate your compounds' function in disease processes associated with protein trafficking and internalization due to protein misfolding | PathHunter detection kit |
| Pharmacotrafficking Assays | Quantitative chemiluminescent results without the need for antibodies or imaging |  |
| Pharmacotrafficking Assays |  |  |
| Radioligand Binding and GTPγS Functional Assays | Utilize high-quality membrane preparations to identify ligands that bind your GPCR | ChemisCREEN membrane preparations |
| Radioligand Binding and GTPγS Functional Assays | For saturation and competitive radioligand binding and GTPγS functional studies |  |
| Radioligand Binding and GTPγS Functional Assays | Obtain consistent, reproducible results and high total binding values with large signal-to-background ratio |  |

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| GPCR Detection Kits | Applications & Benefits |
| --- | --- |
| HitHunter® cAMP Assays | Detect cAMP accumulation with a luminescent read-outRobust, highly sensitive, no-wash,and easy-to-use high-throughput assay for monitoring GPCR activation based on cAMPproduction in cellsEnzyme Fragment Complementation (EFC)-based detection system for small molecule and biologics testing applicationsReduce optimization steps by using the detection kit qualified for use with cAMP Hunter™ GPCR cell linesCompatible for use with most GPCR cell linesAvailable in multiple sizes; based on the number of data-points |
| HitHunter Calcium Assays | Easily measure intracellular calcium to the determine activation status of GPCRDye-based assay with a fluorescent read-out and single-step,no-wash(homogeneous) protocol |
| PathHunter® Detection Kit | Luminescent detection kit for EFC-based cell lines engineered to study protein-protein interactions,protein translocation,receptor trafficking,and receptor internalizationAvailable in multiple sizes |

Author Dana Haley-Vicente, Ph.D., is a 
Senior Marketing Manager of Eurofins 
®
DiscoverX

Co-author Daniel Bassoni is a Technical 
Support Manager of Eurofins DiscoverX

---

# ARTICLE

# Why Study GPCR Arrestin Recruitment?

## GPCR β β-ARRESTIN SIGNALING

Arrestins are ubiquitously expressed and function in the activation 
of GPCRs, desensitization of most 7-transmembrane receptors (7-
TMs), and regulation of other signaling molecules such as protein 
kinases.

There are currently four known arrestins:

• Visual and cone arrestins play a major role in photoreceptor cell 
regulation

•	 β-arrestin1 and 2 interact with the majority of commonly targeted 
7-TMs receptors

For GPCRs, ligand-induced β-arrestin (1 or 2) recruitment activates 
signaling cascades independently of G-protein signaling to provide 
a stoichiometric, non-amplified signal. β-arrestin recruitment

assays offer an easy-to-use alternative to second messenger cAMP and calcium G-protein dependent pathways to enable enhanced 
profiles of compound pharmacology –  a universal assay that expands opportunities for development of novel drugs.

## BENEFITS OF A NON-AMPLIFIED SYSTEM

The β-arrestin signaling pathway is an ideal system for discovering antagonists, studying GPCR deorphanization, and dissecting ligand 
pharmacology differences.

## cAMP SECOND MESSENGER G-PROTEIN DEPENDENT SIGNALING PATHWAY

An amplified system that can lead to rapid saturation using low levels of GPCR activating ligands.  This is a more sensitive system 
based on partial receptor occupancy giving a full signal due to the amplified signaling event (receptor reserve phenomena) after ligand 
activation.  Agonist can easily be detected since it does not take much agonist to obtain a full signal, but differentiating partial from full 
agonists can be difficult, and cAMP assays tend to have lower sensitivities to antagonists than do arrestin assays.

## β β-ARRESTIN SIGNALING PATHWAY

A stoichiometric (one receptor: one ligand), non-amplified system that requires full ligand occupancy of the ligand bound to the receptor 
to give a full signal. This leads to a lower sensitivity to agonists, but improved ability to detect differences of efficacy between agonists, 
and superior sensitivity for antagonists (compared to second messenger systems). This makes β-arrestin an ideal system for fine-tuning 
GPCR biology when screening antagonists as well as for deorphanizing GPCRs and distinguishing between full, super, and partial agonists.

---

## MULTIPLE APPLICATIONS OF β β-ARRESTIN ASSAYS

• Perform multiple pathway analysis in the same cell line

• Uncover unique pharmacology

• Correctly rank order ligands

• Deorphanize GPCRs

• Evaluate difficult GPCRs

## REFERENCES

1. Lundstrom K. New winds in GPCR-based drug discovery. *Future Medicinal* 
*Chemistry*. 2016; 8(6): 605-608.
2. Bassoni D., *et. al*. Measurements of β-arrestin recruitment to activated seven 
transmembrane receptors using enzyme complementation. *Methods Mol. Biol*. 
2012; 897: 181-203

• Compare ligand responses in different species receptors 
(orthologs)

• Study mutant or isoform differences

• Investigate tissue-specific variations using different cell types

• Enable ligand bias studies

Author Dana Haley-Vicente, Ph.D., is a 
Senior Marketing Manager of Eurofins 
®
DiscoverX

Learn more at discoverX.com/arrestin.

---

# Four Reasons Why You Should Measure Signaling Bias in Your Molecules

Most GPCRs are pleiotropically coupled to multiple signaling pathways in cells; the availability of functional assays to separately measure 
agonist-induced activation of these pathways has revealed that agonists do not activate all pathways in a uniform manner, i.e., some 
activate selected pathways at the expense of others and thus are “biased” toward some signals in the cell. The way in which cells mix 
these signals for an overall cellular response also reveals that efficacy has quality as well as quantity for different agonists. This has led 
to a revolution in GPCR candidate selection since an important further selection criterion for agonism, in the form of bias determination, 
now offers new ways to advance molecules.

## THERE ARE FOUR DISTINCT REASONS WHY BIAS MEASUREMENTS ARE IMPORTANT

1. Bias can make better drugs: The identification of biased ligands can 
lead to molecules that (a) emphasize beneficial signaling pathways, 
(b) de-emphasize harmful pathways, and (c) de-emphasize harmful 
pathways and preclude the natural signaling system from activating 
those same harmful pathways. In addition, bias can expand the 
pursuit of targets otherwise thought excluded from consideration 
due to activation of harmful signaling (i.e., dysphoria for k-opioid 
agonists).

2. Bias identifies molecules for progression from high-throughput 
screens: Counter-screens of hits found in high throughput screens 
in biased assays can identify hits that are truly different on a 
molecular level, thereby optimizing the subsequent identification of 
useful therapeutic phenotypes in animal models and more complex 
therapeutic assays.

3. Bias reduces efficacy fingerprints to measurable scales that may 
then be optimized through medicinal chemistry: It is now known 
that efficacy has quality as well as quantity, which arises from the 
mixture of elemental signaling pathways in the cytosol. Radar plots 
or clustering have been used to represent these efficacy types, but

bias measurements further allow the reduction of these complex phenotypes to graded activation of signaling pathways. Therefore, an 
identified favorable efficacy in therapeutic cells could be deconstructed to a combination of these scales to allow medicinal chemists 
to optimize the pattern and amplify the favorable phenotype.

4. Bias can more effectively quantify selectivity: Selective agonism on more than one signaling pathway can identify possibly false levels 
of selectivity or at least offer a rational scheme for determining selectivity based on all known signaling for a given molecule.

In general, and at little extra cost, the measurement of signaling bias increases value in already known lead compounds and can assist in 
the prediction of the activity of these compounds in other, including therapeutic, assays.

## BIAS DETERMINATION

Eurofins DiscoverX® provides the largest portfolio of GPCR assays and cell lines and a unique service that utilizes state-of-the-art tools 
developed by Professor Terry Kenakin at the UNC School of Medicine for the characterization of ligand bias.  With the appropriate 
β-arrestin, internalization, and second messenger assays to quantify selective response and statistical tools to scale these effects, 
harnessing bias to produce selective ligands is now made simple.

---

## EMPHASIS OF BENEFICIAL SIGNALING PATHWAYS

Parathyroid hormone (PTH) alternately builds and degrades bone physiologically; it has been shown that PTH does not build bone in 
β-arrestin knockout mice (Ferrari *et al.* 2005; Gesty-Palmer *et al.* 2006; 2009) suggesting that PTH agonists biased toward β-arrestin 
signaling would be optimally beneficial in the treatment of osteoporosis.

## DE-EMPHASIS OF HARMFUL SIGNALING PATHWAYS

Opioid agonists such as morphine provide useful analgesia but debilitating respirator depression. It has been shown in β-arrestin 
knockout mice that morphine produces less respiratory depression thereby suggesting that an opioid agonist biased toward G protein 
signaling and away from β-arrestin may be a superior analgesic (Raehal *et al.* 2005; DeWire *et al.* 2013; Charfi *et al.* 2015).

## DE-EMPHASIS OF HARMFUL PATHWAYS WITH INHIBITION OF NATURAL ACTIVATION OF THOSE SAME HARMFUL PATHWAYS

The increase of angiotensin signaling in congestive heart failure, resulting in elevation of arterial pressure, is a normal response to 
decreased organ perfusion; this leads to short-term improvement in organ perfusion. However, long-term elevation in arterial pressure 
is deleterious to a failing myocardium; therefore, decreased angiotensin responsiveness, in the form of angiotensin receptor blockade 
with drugs such as losartan, is the indicated therapy. However, angiotensin also provides beneficial effects through β-arrestin signals 
and these are lost during standard losartan therapy. New angiotensin antagonists such as TRV120027 block the deleterious effects of 
angiotensin, but also provide beneficial β-arrestin signals through biased signaling leading to an improved profile for congestive heart 
failure therapy (Violin *et al.* 2006; 2010).

## MAKING k-AGONISM A VIABLE THERAPEUTIC OPTION

κ-Opioid agonist activity has been implicated in the modulation of reward, mood, cognition, and perception making these molecules 
of possible benefit as antidepressants and anxiolytics in affective disorders, drug addiction, and psychotic disorders. However, κ-opioid 
agonists also produce serious dysphoria thereby precluding their application to these therapies. Biased κ-opioid agonists may circumvent 
these limitations by reducing dysphoric effects and emphasizing beneficial effects (White *et al.* 2014).

## LIGANDS WITH DIFFERENT BIAS IN SIGNALING ARE DIFFERENT ON A MOLECULAR LEVEL

Biased signaling is the result of the stabilization of unique ensembles of receptor states by agonists (Kenakin and Morgan, 1989; Kenakin, 
1995; 2002). Therefore, if two agonists are found to produce signaling with different bias, it is likely they are producing different receptor 
active states, which may behave very differently *in vivo*. Under these circumstances, these two agonists would also likely produce 
different signaling patterns in complex assays such as *in vivo* animal models.

## RADAR PLOTS DISPLAY UNIQUE PATTERNS OF EFFICACY

Multiple agonist activities can be displayed on multiple ordinate axes in the form of radar plots to show two dimensional arrays of “webs 
of efficacy” to distinguish agonists, i.e., see such webs for β-adrenoceptor agonists (Evans *et al.* 2010) and k-opioid receptors (Zhou *et* 
*al.* 2013). Interestingly, each agonist tested reveals a different web, thereby indicating the uniqueness of agonist efficacy quality profiles; 
these may relate to the unique therapeutic phenotypic responses to these agonists.

---

## CLUSTERING DISPLAYS AGONIST EFFICACY PHENOTYPES

Through testing agonists in multiple functional assays, measures of efficacy for each signaling pathway (in the form of transducer 
coefficients, (Kenakin *et al.* 2012)) can be used in clustering programs to group agonists in terms of their efficacy (Kenakin, 2015). Such 
clusters may enable simple manipulation of these phenotypes through medicinal chemistry.

## THERAPEUTICALLY RELEVANT RECEPTOR SELECTIVITY

β 2-adrenoceptor agonists for agonist therapy require high activity at β 2-adrenoceptors and correspondingly low activity at cardiac 
β 1-adrenoceptors. The β 2-adrenoceptor bronchodilator clenbuterol has a 500-fold selectivity of β 2– over β 1– activating capability for 
cyclic AMP, indicating a favorable profile (Casella *et al.* 2011). However, this selectivity reduces to 5.7-fold when the receptor-mediated 
β-arrestin effects for these same receptors are measured. The measurement of selectivity for different signaling pathways may be critical 
to the assessment of true selectivity for new agonists for therapy.

## REFERENCES

1. Casella I, Ambrosio C, Grò MC, Molinari P, Costa T. Divergent agonist selectivity 
in activating β 1- and β 2-adrenoceptors for G protein and arrestin coupling. 
*Biochem J*. 2011;438: 191-202.
2. Charfi I, Audet N, Bagheri Tudashki H, Pineyro G. Identifying ligand specific 
signaling within biased responses: focus on δ opioid receptor ligands. *Br J* 
*Pharmacol*. 2015;172: 435–448.
3. DeWire SM, Yamashita DS, Rominger DH, Liu G, Cowan CL, Graczyk TM, *et al.*
A G protein-biased ligand at the μ-opioid receptor is potently analgesic with 
reduced gastrointestinal and respiratory dysfunction compared with morphine. 
*J Pharmacol Exp Ther.* 2013;344: 708–717.
4. Evans BA, Sato M, Sarwar M, Hutchinson DS, Summers RJ. Ligand directed 
signaling at β-adrenoceptors. *Br J Pharmacol*. 2010;159: 1022–1038.
5. Ferrari SL, Pierroz DD, Glatt V, Goddard DS, Bianchi EN, *et al.* Bone response 
to intermittent parathyroid hormone is altered in mice null for β-arrestin2. 
*Endocrinology*. 2005;146: 1854–1862.
6. Gesty-Palmer D, Chen M, Reiter E, Ahn S, Nelson CD, *et al.* Distinct betaarrestin and G protein-dependent pathways for parathyroid hormone receptorstimulated ERK1/2 activation. *J Biol Chem.* 2006;281: 10856–10864.
7. Gesty-Palmer D, Flannery P, Yuan L, Corsino L, Spurney R, *et al.* A β-arrestinbiased agonist of the parathyroid hormone receptor (PTH1R) promotes bone 
formation independent of G protein activation. *Sci Transl Med.* 2006; 1:1ra1.
8. Kenakin T. Agonist-receptor efficacy. II. Agonist trafficking of receptor signals. 
*Trends Pharmacol Sci.* 1995;16: 232–238.
9. Kenakin T. Drug efficacy at G protein-coupled receptors. *Annu Rev Pharmacol* 
*Toxicol.* 2002;42: 349–379.

10. Kenakin T. Forum: New Lives for Seven Transmembrane Receptors. *Trends* 
*Pharmacol. Sci.*, 2015;36: 705-706.
11. Kenakin T, Morgan PH. Theoretical effects of single and multiple transducer 
receptor coupling proteins on estimates of the relative potency of agonists. 
*Mol Pharmacol.* 1989;35: 214–222.
12. Kenakin T, Watson C, Muniz-Medina V, Christopoulos A, Novick S. A simple 
method for quantifying functional selectivity and agonist bias. *ACS Chem* 
*Neurosci.* 2012;3(3): 193-203.
13. Raehal KM, Walker JK, Bohn LM. Morphine side effects in beta-arrestin 2 
knockout mice. *J Pharmacol Exp Ther.* 2005;314: 1195–1201.
14. Violin JD, Dewire SM, Barnes WG, Lefkowitz RJ. G protein-coupled receptor 
kinase and beta-arrestin-mediated desensitization of the angiotensin II type 
1A receptor elucidated by diacylglycerol dynamics. *J Biol Chem.* 2005;,281: 
36411–36419.
15. Violin JD, DeWire SM, Yamashita D, Rominger DH, Nguyen L, *et. al.* Selectively 
engaging β-arrestins at the angiotensin II type 1 receptor reduces blood 
pressure and increases cardiac performance. *J Pharmacol Exp Ther.* 2010;335: 
572–579.
16. White KL, Scopton AP, Rives ML, Bikbulatov RV, Polepally PR, *et al.*
Identification of novel functionally selective k-opioid receptor scaffolds. 
*Mol Pharmacol.* 2014;85: 83–90.
17. Zhou L, Lovell KM, Frankowski KJ, Slauson SR, Phillips AM, *et al.* Development 
of functionally selective, small molecule agonists at kappa opioid receptors. 
*J Biol Chem.* 2014288: 36703–36716.

Article author Terry Kenakin, Ph.D., is a 
pharmacology professor at UNC School 
of Medicine

---

# ARTICLE

# Ligand Bias: Increasing Specificity and Reducing Side Effects of Drug Compounds

Ligand bias is the ability of compounds to exert differential signal 
transduction responses on activation of a GPCR. This ligand bias 
effect is a relatively new area for drug discovery and offers novel 
approaches to develop pharmaceuticals that have enhanced specificity 
and reduced side effects.

When a ligand binds to a GPCR, it alters the conformation of the 
protein, which is transduced through the plasma membrane to induce 
cell signaling.  There are multiple ways signal transduction is mediated 
through GPCRs:

• G-proteins to modulate second messengers such as cAMP or calcium

•	 β-arrestins

β-arrestins are involved in regulating the duration of receptor signaling by associating with activated receptors and blocking further 
G-protein binding and signaling. β-arrestins subsequently form complexes with kinases such as MAPK and Erk to provide alternate 
signaling events.

## APPLICATIONS FOR LIGAND BIAS

Potential applications for ligand bias are highlighted by observations that these distinct signaling pathways can have significantly diverse 
effects either as a result of a medical condition, such as hypertension, or due to unforeseen consequences of drug action, such as opiates.

## ANGIOTENSIN II LIGAND BIAS

Drug candidate TRV027, currently in Phase IIb trials for acute heart failure from Trevena Pharmaceuticals, is one such drug that uses 
ligand bias in GPCR signaling to tease out the positive effects from the negative effects of receptor activation.

Angiotensin II plays a central role in heart failure by regulating blood pressure, cardiac cell function, and renal function. In cases of acute 
heart failure, angiotensin II has been shown to make the symptoms worse via second messenger signaling that results in vasoconstriction, 
reducing blood flow to the heart. Therefore, a drug that blocks this deleterious effect of angiotensin II has proven to show positive results 
for patients with acute heart failure.

In contrast, angiotensin II also promotes cardiac health and protects cardiac cells from dying through signaling via β-arrestin. Blocking 
this positive effect of angiotensin II activity through the β-arrestin response can make the heart condition worse.  The development of 
drugs, such as TRV027, that specifically activate the β-arrestin pathway while simultaneously inhibiting second messenger signaling from 
angiotensin II type 1 receptor (AT1R) can block the negative effects of angiotensin II while promoting its positive effects.

---

## OPIOID LIGAND BIAS

G proteins are not always the bad guys in signaling. For pain sensation, the body has the ability to naturally suppress pain sensation by 
downregulating the transmission of pain signals to the brain. Naturally occurring endorphins released by inhibitory neurons activate 
opioid receptors on synaptic terminals. This dampens neuronal signal transmission by reducing cAMP levels in the terminal.

Opiate drugs, such as morphine, produce pain relief by mirroring the actions of endorphins. Unfortunately, since opiate drugs are 
systemically applied, they also act on opioid receptors present in other parts of the body, such as the gut and lungs.  The effects are 
significant with constipation and cramping in the gut and respiratory suppression in the lungs. These perceived negative effects are 
largely driven via β-arrestin signaling. Therefore, while opiates provide effective pain control, the side effects limit dosage and produce 
significant complications.

The next generation of opiate drugs in development are biased ligands that target the second messenger pathway and minimize 
β-arrestin engagement, thus reducing the negative side effects of opioid pain treatment. Oliceridine (TRV130), a Phase 3 drug for 
intravenous treatment for the management of moderate-to-severe acute pain, is one such drug that activates the second messenger 
response, and not the β-arrestin response, using biased GPCR signaling.

## ASSAYS TO MEASURE LIGAND BIAS

The development of biased ligands requires tools to measure signaling via different pathways plus methods for analyzing results in order 
to quantify bias.

• Assays for β-arrestin recruitment and signaling via cAMP modulation or calcium mobilization are needed to define compound activity in 
both potency and efficacy terms since bias can be presented in either pathway.

• Quantifying bias between pathways that accounts for natural system bias is important. The method proposed by Dr. Terry Kenakin at the 
University of North Carolina achieves this by utilizing the Black and Leff operational model and normalizing compound activity to reference 
ligand responses.

Clearly, biased ligand development is opening a new chapter in drug discovery by taking compound specificity a step further to address 
not only receptor specificity but also downstream signaling selectivity to ensure the most positive outcome for the patient with minimum 
side effects.

Article author Neil Charter, Ph.D., is 
Senior Director of Operations Support 
®
for Eurofins DiscoverX

---

# Can We Use Assay "Volume Control" to Unveil Efficacies in Allosteric Lead Molecules for GPCRs?

Drugs interact with living physiology; an *in vitro* assay of drug activity can be considered a snapshot of the full movie seen *in vivo* of the 
drug exposed to a range of tissues of varying sensitivities. An assay measures signaling at one sensitivity, namely that of the particular 
*in vitro* assay being utilized, but the more of these snapshots we have, the better will be our understanding of what drugs will do *in vivo*.

## SIGNALING AND TISSUE VOLUME CONTROL

It is well known that physiological tissues control signal magnitude through adjustment of receptor expression levels and the relative 
stoichiometry of receptors to signaling components. This natural control of signaling is operative for all tissues *in vivo*. Such adjustment 
of tissue sensitivity can have a dominating control on the effects of agonists. For example, a low efficacy agonist such as prenalterol 
can produce nearly full agonism in thyroxine-treated guinea pig atria and no agonism (instead become an antagonist) in skeletal muscle 
such as the digitorum longus (Kenakin, 1985). An *in vitro* assay of drug activity measures signaling at one defined sensitivity, but if the 
sensitivity of the *in vitro* assay differs from that of the relevant therapeutic tissues *in vivo*, then a possibly costly dissimulation can occur. 
leading to an incorrect choice of drug candidate moving forward. This is why testing drugs in an array of assays of differing sensitivity 
can be a valuable tool to correctly characterizing hidden efficacies of new molecules.

## POSITIVE ALLOSTERIC MODULATION

A case in point are positive allosteric modulators (PAMs) for GPCRs. These unique molecules are becoming important therapeutic entities 
as they have many beneficial features for agonist signal modification. Specifically, they can augment natural responses and preserve 
the way in which physiological systems are wired (not alter the complex patterns as they function in Nature). In addition, they produce 
saturable effects that allow them to change the operational sensitivity of systems without signal overload. PAMs may have quite different 
patterns of activity in tissues of varying sensitivity and these patterns can be used to quantify their efficacies and predict effects *in vivo*. 
One pattern is sensitization of tissues to agonist response as shown in Figure 1. A. for a tissue of moderate sensitivity. This single assay 
yields important information such as, in this case, the fact that the PAM sensitizes the tissue to the agonist. However, there are two 
important questions left unanswered; (1) in a more sensitive tissue, will direct agonism to the PAM be seen? And (2) does the potentiation 
of agonist effect occur because of effects on agonist affinity, efficacy, or both? Answers to these questions can be obtained by testing the 
PAM in two more assays of varying sensitivity.

**A**

**B**

**C**

**Figure 1. The effects of a PAM possessing direct efficacy and potentiating activity for efficacy.** A. In a tissue of moderate sensitivity, sinistral displacement of the 
agonist dose response curve is observed. B. In a sensitive tissue, the direct agonism to the PAM is shown by the elevated baseline. C. In a tissue of low sensitivity where 
the agonist is a partial agonist, the efficacy increase produced by the PAM is shown by the increased maximal response to the agonist.

---

## DIRECT PAM AGONISM

Figure 1. B. shows the same PAM in an assay of higher sensitivity. There are many chemical series whereby PAM activity transfers to 
direct agonism in highly sensitive tissues; presumably, this is related to the fact that PAMs stabilize the formation of receptor active 
states. Therefore, the data this figure predicts that if this PAM encounters a sensitive tissue, it would produce direct agonism. In many 
cases PAMs are used to avoid direct agonism as these may lead to side effects. Thus, a PAM without agonism would produce effects only 
when the system is activated naturally and otherwise will produce no effect (thus reducing side effects). The detection of direct efficacy 
may be a harbinger of the side effects of a therapeutic PAM.

## DO PAMs INCREASE AGONIST AFFINITY, EFFICACY, OR BOTH?

PAMs can potentiate responses through elevation of agonist affinity, efficacy, or both of those; it can be very important which of these 
activities is operative for a therapeutic PAM. If the PAM is aimed to rejuvenate a failing system, i.e. neurotransmission in Alzheimer’s 
disease, then an increase only in agonist affinity may not produce increased signaling as the original signal is weak and after PAM 
treatment would still be weak. Instead, what is needed is an increase in efficacy whereby a low level signal is magnified. Under these 
3.32
circumstances, an inoperative receptor system could be revitalized by the efficacy-based PAM. For example, a D112 mutation of 
the muscarinic M4 receptor makes it completely refractory to the natural agonist acetylcholine yet application of efficacy-active PAM 
LY2033298 can completely restore the receptor sensitivity to the agonist (Leach *et al.* 2011). In general, efficacy-based PAMs would be 
predicted to produce more robust sensitization of signaling systems.

## PAMs CAN INCREASE EFFICACY

It is not possible to differentiate whether sensitization to a full agonist is due to increased affinity or efficacy as both of those will 
produce a leftward shift of the agonist dose response curve. However, if the agonist is a partial agonist, then increases in efficacy will be 
shown as an increased maximal response, i.e. increases in affinity will not change the maximal response. Therefore, if the PAM could be 
tested in an assay where the agonist is a partial agonist, then increased efficacy effects can be unveiled, i.e. see Figure 1. C. Once efficacy 
involvement has been identified, then any possible role for changes in affinity can be detected through a comparison of dose-response 
curves to the functional allosteric model (Bdioui *et al.* 2018). Thus, testing of the PAM in the three systems would fully characterize the 
activity as PAM agonism with increased efficacy; an example of such a molecule can be found in the muscarinic M2 receptor PAM BQCA 
(benzylquinolone carboxylic acid ) (Bdioui *et al.* 2019).

## WHAT IS TISSUE "VOLUME CONTROL"?

Assay volume control can be attained through various means such as differences in receptor expression levels, alkylation of surface 
receptors, choice of host cell, and augmentation of signal disposition (i.e., GRK co-expression for β-arrestin). Exploration of these 
approaches could yield valuable information for the full characterization of candidate efficacies.  It has been estimated that 50% of 
new drug candidate failures occur because of therapeutic efficacy (Arrowsmith, 2011); this is in addition to issues of safety that add 
to this high rate of attrition. Demonstration of such failure usually occurs in the most complex systems showing pathology-controlled 
pharmacology at the end stage of discovery and development, a costly point in the process. While complete examination of all of the 
efficacies of a candidate molecule may not prevent such failures at the outset, it may prevent follow-up failures, i.e. why follow up a 
failed molecule with one that has the same profile of efficacies?  Full candidate characterization also can help identify which collection 
of efficacies are important therapeutically as an effective treatment may require multiple activities, not a single one. For example, the 
blockade of β-adrenoceptors has been identified as an important treatment for heart failure. However, the testing of 16 β-blockers in 
clinical trials shows an unequal spectrum of benefit with the multiple efficacy molecule carvedilol emerging as the best (Metra *et al.*
2004).  In general, investment into determining a full characterization of the efficacies of new candidate molecules may yield dividends in 
late-stage development; thus, an array of *in vitro* assays can be a valuable asset in this regard.

$$
\beta
$$

---

## REFERENCES

1. Arrowsmith J. Trial watch: phase II failures: 2008–2010. *Nat Rev Drug Discov.*
2011;10: 328–329.
2. Bdioui S, Verdi J, Pierre N, Trinquet E, Roux T, Kenakin T. Equilibrium Assays Are 
Required to Accurately Characterize the Activity Profiles of Drugs Modulating 
G<sub>q</sub>-Protein-Coupled Receptors. *Mol. Pharmacol.* 2018;94: 992-1006.
3. Bdioui S, Verdi J, Pierre N, Trinquet E, Roux T, Kenakin T. The pharmacologic 
characterization of allosteric molecules: G<sub>q</sub> protein activation. *J. Recept., Signal* 
*Transd.* 2019;39 (2): 106-113.
4. Kenakin TP. Prenalterol as a selective cardiostimulant: differences between organ 
and receptor selectivity. *J Cardiovasc Pharmacol*. 1985;7 (1): 208-10.
5. Leach K, Davey AE, Felder CC, Sexton PM, Christopoulos A. The Role of 
Transmembrane Domain 3 in the Actions of Orthosteric, Allosteric, and Atypical 
Agonists of the M4 Muscarinic Acetylcholine Receptor. *Mol. Pharmacol*. 2011;79: 
855-865. 
6. Marco Metra M, Livio Dei Cas L, Andrea di Lenarda A, Philip Poole-Wilson P. 
Beta-Blockers in Heart Failure: Are Pharmacological Differences Clinically 
Important? *Heart Failure Reviews.* 2004;9: 123–130.

Article author Terry Kenakin, Ph.D., is a 
pharmacology professor at UNC School 
of Medicine

---

# ARTICLE

# Unlocking Orphan GPCRs: Application of GPCR Pharmacotrafficking and Cell-Based Assays

Orphan GPCRs comprise one-third of the druggable GPCR space¹. Despite their pharmacotherapeutic potential, they remain 
underexploited owing to difficulties in interrogating their physiological roles and signaling pathways. Further, the lack of chemical probes 
or antibodies needed for elucidating their roles makes oGPCRs largely untapped for pharmaceutical interventions in a variety of diseases.

To address this gap, a systematic approach has been developed to identifiy chemical probes of oGPCRs using a recently developed 
novel platform called the Ligand-Induced Forward Trafficking (LIFT) assay. Established to circumvent impediments to the application of 
automated high-throughput technologies to pursue oGPCR screening, the innovative LIFT assay design features:

• A universal assay system suitable for high throughput screening (HTS)

• No requirement of a *priori* knowledge of receptor pharmacology

• Universal positive control

• Reasonably cost-effective and integrative platform for lead ID

• Rapid ability to validate utility

Scientists at the Conrad Prebys Center for Chemical Genomics at Sanford Burnham Prebys (SBP) Medical Discovery Institute in San Diego, 
Calif., have conducted a full HTS campaign to identify chemical probes of the orphan GPCR, GPR20. They used a highly curated library of 
compounds in the LIFT approach, thereby providing a new approach to determine compound:target engagement.

Development and overall validation of the LIFT assay platform, as 
well as the utility of hits emerging from this HTS campaign was 
conducted in collaboration with Eurofins DiscoverX® using the GPCR 
PathHunter® Pharmacotrafficking assay, PathHunter b-arrestin 
recruitment assay, and HitHunter® cAMP assay platforms, as well as 
the *gpcr*MAX panel services.

## IDENTIFICATION OF CHEMICAL PROBES FOR oGPCRS

## Overview of the LIFT Assay

The LIFT assay platform was developed to enable the 
identification of chemical probes for oGPCRs using the concept 
of pharmacochaperone-induced trafficking of mutant receptors. 
Pharmacochaperones are cell-permeable small molecules that bind 
to mutant receptors, stabilizes them, and enables their trafficking 
to the membrane². Assays that report on receptor trafficking, such 
as the cell-based pharmacotrafficking assays from DiscoverX, have 
been valuable in the identification of target-specific ligands, and the 
interrogation of their mechanisms-of-action (MOA). When applied 
to oGPCRS, the assay consists of a target orphan receptor harboring 
a synthetic point mutation that causes retention of the target in 
the endoplasmic reticulum (ER) due to improper folding. (Note that 
the cellular protein synthesis machinery can accommodate point 
mutations usually found in conserved Cholesterol Consensus Motif 
(CCM) residues among many class A oGPCRs³.) Upon binding of

**LIFT Platform** 
with Eurofins DiscoverX (products) 
and Eurofins Discovery (services) Assays

**Figure 1. Flow schematic of the LIFT assay platform for ligand identification.** 
The LIFT platform uses DiscoverX’s pharmacochaperone, cAMP, and β-arrestin 
assays along with Eurofins Discovery services *gpcr*MAX panel to validate the 
forward trafficking approach to identify chemical probes against oGPCRs.

---

a chemical ligand (pharmacochaperone), the mutated receptor adopts a stabilized conformation in the ER that is forward-trafficked 
through the Golgi, to the plasma membrane, and ultimately to the early endosome. The Eurofins DiscoverX® ’s Enzyme Fragment 
Complementation (EFC) platform, which is the base technology of these assays, reports the forward trafficking of the receptor yielding a 
robust luminescent signal that can be measured. The overview of the general oGPCR LIFT lead discovery strategy is shown in Figure 1.

As proof of concept, LIFT was applied to the human orphan GPR20, and a large compound collection (~450,000 compounds) was 
screened with the goal of identifying small molecule probes of GPR20. Since the approach involves mutating the target receptor, it is 
to be noted that the induced mutation is not related to human disease, but only as a means to retain the receptor in the ER. Hence, 
assays for wild-type receptor including cAMP and β-arrestin recruitment assays are required for hit validation. Given the orphan 
nature of GPR20, compound selectivity to GPR20 was also assessed using *gpcr*MAX panel assays. Overall, applying a wide array of 
technologies, such as second messenger, receptor internalization, and cellular impedance assays formed a comprehensive strategy for 
probe discovery, delineating functional cell-based assays to serve as critical tools for interrogating the MOA in drug discovery therapeutic 
characterization.

## HOW PHARMACOCHAPERONE ASSAY CAN MONITOR RECEPTOR TRANSLOCATION?

Eurofins DiscoverX’s PathHunter® cell-based pharmacotrafficking assays provide a simple and quantitative tool for broad pharmacological 
characterization and interrogation of compound function in disease processes associated with receptor protein trafficking and 
internalization due to protein misfolding. Featured as part of the repertoire of cell-based assays surrounding translocation and trafficking 
of membrane proteins, the benefits of its use over other methods are described here.

These pharmacotrafficking assays provide a validated, cell-based chemiluminescent assay platform that enables identification and 
optimization of compounds that function as pharmacochaperones by promoting proper folding of misfolded receptor proteins and 
assisting in trafficking to their intended location.

Figure 2. illustrates the EFC-based PathHunter® pharmacotrafficking assay in two formats wherein the binding of a chemical ligand 
(pharmacochaperone) to a misfolded, tagged receptor protein stabilizes the protein in a conformation that enables its trafficking from 
the ER through the golgi, then onward to the cell membrane, and further internalized to the endosome. Both formats involve two inactive 
β-galactosidase (β-gal) enzyme fragments (a small enzyme donor and a large enzyme acceptor (EA)) to come together to form an active 
β-gal enzyme that hydrolyzes a substrate to generate a chemiluminescent signal that can be measured on any standard luminometer. 
In these assays, the membrane protein (GPCR in this case) is tagged with the small enzyme donor called ProLink™ (PK) and the EA is 
localized to either the cell membrane or endosome. In the Membrane-EA format (Figure 2. (A)), complementation of the two β-gal 
enzyme fragments (EA & PK) occurs at the membrane. In contrast, in the Endosome-EA format (Figure 2. (B)), the complementation of 
the fragments occur in endosomes after receptor internalization. In the LIFT platform strategy, the Endosome-EA assay format has been 
employed for monitoring mutant receptor translocation.

Using the fully optimized LIFT assay on β 2-adrenergic receptor (ADRB2), a screening of a Library of Pharmacologically Active Compounds 
(LOPAC) typically used for demonstrating HTS feasibility of an assay was performed as LOPAC contains several compounds with known 
activity at ADRB2, as well as many compounds with no effect for this receptor. (www.sigmaaldrich.com/life-science/cell-biology/
4
bioactive-small-molecules/lopac1280-navigator.html). The ADRB2 LIFT assay performed well with a Z’ factor = 0.5 and having identified 
all ADRB2 ligands in the LOPAC library. The complete results of this pilot study are reported in (Morfa CJ *et al.* 2018).

---

**Figure 2. Pharmacochaperone assay principle using the EFC technology.** Membrane-EA format: Cell lines co-express the PKtagged receptor protein that is retained in the ER (due to protein misfolding) and EA localized to the cell membrane. Endosome-EA 
format: Cell lines co-express the PK-tagged receptor protein and the EA localized to the early endosomes. In both formats, the 
complementation of the two β-gal enzyme fragments (EA & PK) at either the transmembrane or endomes results in an active 
β-gal enzyme that hydrolyzes the substrate to generate a chemiluminescent signal.

## COMPOUND SCREENING & HIT CONFIRMATION

HTS screening and hit confirmation using the LIFT assay platform was employed to identify novel ligands of GPR20. The assay performed 
(4)
well exhibiting a Z factor = 0.5′. Among the hits (> 300 compounds), compound SBI-6630 emerged as a confirmed hit showing a robust 
concentration response in the GPR20 LIFT assay (EC₅₀ = 0.067 µM) and a high degree of selectivity for GPR20 over ADRB2 (Figure 3. A.). 
SBI-6630 was further validated as a potent and selective GPR20 agonist using wild-type human GPR20 (hGPR20) expressed in HEK-293 
cells. The ability of SBI-6630 to reduce forskolin-stimulated intracellular cAMP was measured using the HitHunter® cAMP assay. 
The compound exhibited robust activity with a similar potency to that observed in the primary LIFT assay (EC₅₀ = 0.150 µM). As before, 
SBI-6630 showed a high degree of selectivity for hGPR20 with no activity observed in cells lacking GPR20 (parental HEK-293).

$$
\ Z\,f a c t o r=0.5^{\prime\ (4)}
$$

$$
\left(\mathrm {E C} _ {5 0} = 0. 0 6 7 \mu \mathrm {M}\right)
$$

$$
\left(\mathrm {E C} _ {5 0} = 0. 1 5 0 \mu \mathrm {M}\right)
$$

---

**B Compound Hit Assay with** 
**Wild-Type Human GPR20**

## Wild-Type Human GPR20 120

**Figure 3. SBI-6630 shown as a validated hit agonist of GPR20.** A. Dose-response activity of SBI-6630 against mutated GPR20 and ADBR2.
B. Dose-response activity of SBI-6630 against wild-type GPR20 and parental cells lacking GPR20.

## CONCLUSIONS

A novel approach that addresses critical limitations 
surrounding deorphanization of GPCRs is presented as a 
solution. Further, employing functional downstream assays 
to validate hits, and confirm probe compound selectivity 
and receptor signaling specificity, underscores the overall 
value of the strategy encompassing the forward trafficking 
approach. Representative data shown here, in addition to 
data from previous work⁴, verifies the capability of the 
pharmacotrafficking assay platform to identify chemical 
ligands of oGPCRs using existing high-throughput

"While GPCRs have been historically one of the most successful sources of drug targets, there is still a number of unexploited therapeutic opportunities in the GPCRome ... Opportunities to deorphanize GPCRs with no known ligand by taking advantage of the many cell-based assays available."

- Layton Smith, Ph.D., VP at Fate Therapeutics & Executive Director, Medical Discovery Institute

screening automation and compound libraries. The implementation of Eurofins DiscoverX®’s EFC technology in the adoption of a forward 
trafficking approach not only ensures specificity, but also exerts suitability for automated screening platforms at an economical and 
time-sensitive standpoint, especially with the availability of target- and MOA-selective ready-to-run assays.

## REFERENCES

1. Hopkins AL, Groom CR: The druggable genome. *Nat Rev Drug Discov.* 2002; 
1: 727–730.
2. Leidenheimer NJ, Ryder KG: Pharmacological chaperoning: a primer on 
mechanism and pharmacology. *Pharmacol Res.* 2014; 83: 10–19.
3. Hanson MA, Cherezov V, Griffith MT, *et al.* A specific cholesterol binding site 
is established by the 2.8 A structure of the human beta2-adrenergic receptor. 
*Structure.* 2008;16: 897–905.
4. Morfa CJ, Bassoni D, Szabo A, McAnally D, Sharir H, Hood BL, *et al.*
Pharmacochaperone-based high-throughput screening assay for the 
discovery of chemical probes of orphan receptors. *Assay Drug Dev Technol.* 
2018 Oct;16 (7): 384-396.

Article author Arvind Kannan, Ph.D., is a 
Marketing Manager for Eurofins DiscoverX®

Co-author Layton Harris Smith, Ph.D., is Vice 
President of Preclinical Pharmacology and 
Toxicology, Fate Therapeutics and Executive 
Director, Head of Biology & Pharmacology 
for The Conrad Prebys Center for Chemical 
Genomics at Sanford Burnham Medical 
Discovery Institute

---

# ARTICLE

# Uses and Advantages of Membrane Preparations for GPCRs

Cellular membrane proteins play important functions in many biological processes. Understanding their roles often requires that these 
proteins be isolated from their cellular context. This can be achieved by the disruption of the cell membrane using various mechanical 
methods and differential centrifugation (Roy, A. 2015). Membrane preps generated through such methods result in small fragments of cell 
membranes expressing large quantities of receptor proteins.

Eurofins DiscoverX® membrane preparations (aka membrane preps) are purified membranes derived from stable cell lines optimized 
for high expression levels of the targeted GPCR protein. They are excellent for studying binding affinities of GPCR targets, without the 
requirement for cell culture.

## MEMBRANE PREPARATIONS’ USAGE

Eurofins DiscoverX membrane preps are intended for two main 
purposes: (1) to screen for ligand binding affinities using a 
radiolabeled ligand approach; and (2) to screen for GPCR activity 
using a radiolabeled GTPγS approach. For binding affinities and 
GPCR activation levels, these can be calculated based on changes in 
the resulting amount of radioactive signal in the assay. An increase 
in signal is seen for GTPγS and saturation binding assays, while a 
decrease in signal is seen for competition binding assays.

$$
\mathrm {G T P} \gamma \mathrm {S}
$$

$$
\mathsf{G T P Y}
$$

## RADIOLABELED BINDING ASSAYS

Radiolabeled ligand studies can be performed using DiscoverX’s 
membrane preps in two formats – saturation binding and 
competition binding. The differences between these formats are 
described below.

## SATURATION BINDING

The saturation binding format is achieved by adding increasing doses 
of radiolabeled ligand in the presence (non-specific binding curve) 
or absence (total binding curve) of extremely excessive amounts of 
non-labeled ligand and subtracting non-specific from total binding 
to get a saturation binding curve. Saturation binding assays can be 
used in combination with information on IC₅₀ or EC₅₀ from other 
sources to determine the K (<sub>d</sub>issociation equilibrium constant or 
d
binding affinity) of the ligand.

$$
\ _{d}
$$

## COMPETITION BINDING

**Figure 1. Saturation binding experiment for the ghrelin receptor (Cat. No.** 
**HTS187M).** Ghrelin receptor membrane prep (5.0 µg/well) was incubated with 
increasing amount of [¹²⁵I]-ghrelin in the absence (total binding, TB) or presence 
(non-specific binding, NSB) of 200-fold excess unlabeled ghrelin. Specific binding 
(SB) was determined by subtracting NSB from TB. The K<sub>d</sub> of ghrelin and B<sub>max</sub> (total 
number of receptors) of the system can be calculated from the specific binding 
curve.

$$
[{^{125}}]].
$$

$$
\mathsf{B}_{\mathsf{m a x}}
$$

$$
K_{d}
$$

The competition binding format involves using one low dose of radiolabeled ligand, and increasing concentrations of non-labeled ligand. 
This will produce a binding-characteristic-dependent competition curve showing how the amount of radiolabeled ligand-bound decreases 
as the concentration of non-labeled ligand increases. Competition binding assays can be used to determine the K (<sub>i</sub>nhibitory constant) of 
i
the test ligand, as well as the IC₅₀ of the unlabeled ligand with respect to the radiolabeled ligand.

$$
\ \mathrm{I}\mathrm{C}_{50}
$$

$$
K\mathrm{.}
$$

---

## GTPγγS ACTIVITY ASSAYS

GTPγS assays work by using a radiolabeled non-hydrolyzable GTP 
analog. This analog is added into the buffer with the membrane prep. 
Then the membrane is provided with the known or experimental 
ligand for the GPCR expressed on the membrane. If the GPCR is 
activated, the now-available Gα protein will exchange its GDP 
for GTP. Due to the non-hydrolyzable nature of GTPγS, the Gα
protein will then remain permanently bound to the GTPγS. After the 
removal of excess unbound GTPγS, radioactive detection methods 
can then be used to determine how much GTPγS remains bound 
by Gα, and therefore how much of the target GPCR was activated 
by the applied ligand. This assay is a useful alternative approach to 
checking agonism vs. antagonism and determining potency or rank 
order of experimental ligands in an easy-to-use format that doesn’t 
require cell culture steps.

$$
\mathsf{G P v y}
$$

$$
\mathsf{G T P y S},
$$

$$
\mathsf{G T P Y}
$$

$$
\mathsf{G T P Y},
$$

$$
\mathrm{G a}
$$

$$
\mathsf{G T P y}
$$

## MEMBRANE PREPS VALIDATION

The Eurofins DiscoverX® membrane preps have been tested for and 
provide reference data in either GTPγS or binding assay applications, 
not both. They have also been validated with radioligands and not 
been specifically tested using fluorescent or other ligand labeling 
methods. These membrane preps are intended for filtration-based 
ligand labeling strategies and were not validated using scintillating 
plates or bead-bound methods. Attempting to use these other 
strategies will require some optimization. Overall, if you are 
interested in studying binding affinities of GPCR targets, without a 
requirement for cell culture, pre-prepared membrane preps are an 
excellent assay tool to have in your toolbox.

$$
\mathsf\{G{P Y y}
$$

## REFERENCE

1. Roy A. Membrane preparation and solubilization. *Methods Enzymol.* 2015;557: 
45-56.

## Log [CP-55940], M 45-56.

Article author Natalie Gath, Ph.D., is 
a Technical Support Scientist at 
Eurofins DiscoverX.

**Figure 2. Competition binding experiment for glucagon-like peptide-1 GLP-1** 
**receptor (Cat. No. HTS020M).** GLP-1 membrane preparation or wild-type (WT) 
Chem- 1 membrane prep (Cat. No. HTS000MC1) was incubated with 0.5 nM 
[<sup>125</sup>I]-GLP-1 (7-36) and increasing concentrations of unlabeled GLP-1 (7-36), 
producing a competition curve. Using this curve, Ki and IC₅₀ of the unlabeled 
GLP-1 (7-36) can be calculated. More than 7-fold signal to background was 
obtained.

$$
\mathrm{I C}_{50}
$$

**Figure 3. GTP**γγ**S assay for cannabinoid-2 (CB2) receptor (Cat. No. HTS020M).**
5 µg/well CB2 membrane prep or WT Chem-1 membrane prep (Cat. No. 
HTS000MC1) were incubated with 0.3 nM [<sup>35</sup>S]-GTPg S, 10 µM GDP, and 
increasing amounts of unlabeled CP-55940. Bound radioactivity was determined 
by filtration and scintillation counting. An EC₅₀ of 3.7 pmol/mg and signal to 
background of > 3.4 fold were obtained from this curve.

$$
[ ^ {3 5} S ] - G T P \gamma S,
$$

$$
\mathrm{E C}_{50}
$$

---

# ARTICLE

# Ideal Methods to Study Cellular Transport of Membrane Proteins

## TRANSLOCATION AND TRAFFICKING OF MEMBRANE PROTEINS

Membrane proteins (such as receptors like GPCRs and ion channels) are translated and undergo proper folding in the endoplasmic 
reticulum (ER) and then transported to the cell surface (plasma membrane). Additionally, as part of the normal recycling and receptor 
activation processes, membrane proteins internalize from the plasma membrane into endosomes. The appropriate localization of 
membrane proteins is essential to maintain their biological function, but in some cases, these protein transport events are altered leading 
to a non-functional membrane protein and undesirable effects that are often associated with serious human diseases (e.g. cystic fibrosis, 
Alzheimer’s disease, and Huntington’s disease).

**Figure 1. PathHunter® internalization and pharmacotrafficking assays.**

Explore Eurofins DiscoverX®  products and solutions related to these methods: Translocation assays, GPCR Internalization assays, and 
Pharmacotrafficking assays.

## PREVENTING UNDESIRABLE TRANSLOCATION EVENTS

Membrane protein defects that are caused by mutations, deletions, and truncations can lead to protein misfolding in the ER and prevent 
their trafficking to the plasma membrane. This mislocalization renders the membrane protein non-functional and leads to diseases like 
cystic fibrosis. Many endogenous and synthetic receptor ligands (like morphine) can change the rate of membrane protein internalization 
and recycling. Drug-induced receptor internalization and recycling are important pharmacological properties since they can limit their 
clinical effectiveness and result in unwanted side effects, and tachyphylaxis drug tolerance problems.

The detrimental cellular events caused by protein mislocalization and excessive protein internalization can be reversed by the 
development of compounds called pharmacochaperones (short for pharmacological chaperones) and better drugs (such as partial 
agonists). Pharmacochaperones, correct, or stabilize the folding of the abnormally misfolded proteins allowing them to translocate 
properly from the ER to the plasma membrane. Once at the cell surface, a partial agonist can bind to the membrane protein initiating a 
partially efficacious response compared to a full agonist, while also reducing unwanted side effects and preventing other internalization 
issues.

---

## METHODS OF MEASURING SUBCELLULAR TRANSLOCATION OF MEMBRANE PROTEINS

Researchers currently monitor membrane protein translocation events using high content imaging methods.

• Immunohistochemistry (IHC) or immunocytochemistry (ICC) – Provides visualization of membrane protein localization

• Fluorescence-activated cell sorting (FACS) – Provides quantitative detection of membrane protein localization

## PROBLEMS WITH HIGH CONTENT IMAGING METHODS:

• Low throughput

• Requires antibodies or fluorescent tags

• Uses specialized equipment that can often be expensive

In contrast, cell-based assays for studying protein function, trafficking, and internalization are becoming more mainstream for screening 
and profiling potential drugs to prevent translocation issues.

## BENEFITS OF USING CELL-BASED ASSAYS FOR STUDYING TRANSLOCATION ISSUES:

• High throughput

• Quantitative

• Affordable with no need for specialized equipment; assays can be read using standard luminometer

These methods are complementary to each other and should all be considered when studying cellular membrane protein 
translocation events.

Author Dana Haley-Vicente, Ph.D., is a 
Senior Marketing Manager of Eurofins 
®
DiscoverX

---

# KNOWLEDGE-BASED VIDEOS

## GPCR Assays: Choosing the Best Assay for Your Programs

Learn more about multiple GPCR assays, and choose the best ones to use and why for your research and drug discovery and development 
program.

Find out what GPCR product types are available and discover assays including cAMP and calcium accumulation, β-arrestin recruitment, 
internalization, pharmacotrafficking, radioligand binding, and GTPyS assays to expedite your GPCR research and therapeutic development. 
Video covers GPCR families, targets, assay technology and protocol, and applications for target ID through QC lot release. (30 min.) 
Short Recording (5 min.)

View Related Products at discoverx.com/gpcrs.

## Internalization Assays: Identify Safer Therapeutics with Cell-Based Assays

Learn about PathHunter® Internalization assays developed to measure translocation of both single-pass and multi-pass receptors 
(like receptor tyrosine kinases, checkpoint receptors, and GPCRs) to intracellular endosomes.

Identify safer therapeutics with simple, quantitative cell-based assays for measuring receptor internalization. (13 min.)

View Related Products at discoverx.com/translocation.

## Pharmacotrafficking Assays: Discover Pharmacological Chaperones that Rescue Disease-Relevant Proteins

Learn more about PathHunter pharmacotrafficking assays to identify small molecule compounds that rescue disease relevant mutant 
membrane proteins like GPCRs, ion channels, and transporters.

Discover how these quantitative, antibody- and image-free cell-based assays can be used to screen compounds (called pharmacological 
chaperones) that stabilize mutated membrane proteins allowing for their proper trafficking to the cell membrane. Applications discussed 
include rescuing conditions like retinitis pigmentosa, obesity, diabetes, cystic fibrosis, and cardiac arrhythmias. (30 min.)

View related products at discoverx.com/pharmacotrafficking.

---

Uncover Novel G-Protein or Arrestin-Biased Ligands Using a Suite of GPCR Signaling Cell-Based Assays: 
A Study of Biased Agonism on Opioid Receptors

Daniel Bassoni, Elizabeth Quinn, Ph.D., Manisha Pratap, and Tom Wehrman, Ph.D.
®
Eurofins DiscoverX, Fremont, CA, USA

## INTRODUCTION

It is well understood that GPCR activation results in G-protein 
dependent, as well as G-protein independent signaling events 
such as β-arrestin recruitment and receptor internalization. For 
the G-protein dependent pathway, agonist binding promotes 
a conformational change of the receptor that results in the 
activation of receptor-associated heterotrimeric G-proteins. In 
turn, these G-proteins activate downstream second messenger 
molecules such as cyclic AMP (cAMP), inositol triphosphate, 
diacylglycerol, and calcium. For the G-protein independent 
pathway, agonist binding promotes G-protein receptor kinases 
(GRKs) to phosphorylate the C-terminal tail of most GPCRs, 
resulting in the recruitment of β-arrestin. Bound arrestin then 
serves to sterically block further G-protein activation, limiting 
the length of G-protein signaling and results in receptor 
desensitization followed by internalization into clathrin coated 
<sup>1</sup>
pits. In addition to regulating G-protein signaling, β-arrestins can 
also initiate a distinct second set of G-protein independent signals 
such as the activation of several downstream protein kinases. 
Overall, the complex relationship that exists between G-proteins 
and β-arrestin signaling determines both the efficacy and potential 
2,3
side effects of GPCR targeted drugs. Research from the last two 
decades has shown that different agonists of the same GPCR can 
induce different signaling responses, a phenomenon termed as 
biased agonism. For example, one agonist may trigger G-protein 
dependent signaling events such as cyclic AMP production. In 
contrast, another agonist may show a preference for G-protein 
independent events such as arrestin recruitment. Therefore, in the 
context of therapeutics, quantitatively examining these signaling 
pathways can aid in defining the compound function, and can lead

$$
\beta\ r a r e s t i n
$$

to the discovery of novel biased ligands with unique efficacy and 
safety profiles.

Several therapeutically relevant examples of biased agonism 
3, 4, 5
have been described with the opioid system that is involved 
in the body’s response to pain. Activation of the δ human opioid 
receptor (OPRD1, hDOR) alleviates persistent pain leading to the 
desired analgesic effect. However, sustained or repeated receptor 
activation results in receptor desensitization that is thought to 
be the main cause of opioid tolerance *in vivo⁶*. Despite having 
similar potencies and efficacies *in vitro*, δ opioid receptor agonists 
can differ dramatically in their desensitization and receptor 
internalization profiles. Importantly, in the context of pain, it has 
now been shown that δ agonist-induced analgesic effects are 
retained when receptors remain at the cell surface and are lost 
6, 7
following receptor activation and internalization. Similar to 
hDOR, there are many disease-associated GPCRs that cannot be 
maximally utilized for effective drug development due to adverse 
side effects of downstream receptor signaling. Therefore, there 
is a need to characterize and develop novel ligands that target 
only the pathways that lead to beneficial therapeutic effects, thus 
increasing efficacy while decreasing unwanted side effects.

To address this need, Eurofins DiscoverX offers a portfolio of 
cell-based assays for GPCR drug discovery research. Whether the 
goal of an experiment is to quantify 2nd messenger accumulation, 
measure arrestin recruitment, or understand receptor 
internalization, an assay is available for drug characterization. 
While these assays are designed to capture different signaling 
events, all of the assays are based on the Enzyme Fragment

---

Complementation (EFC, discoverx.com/efc) technology for 
generating a chemiluminescent signal that can be read on any 
standard luminometer. EFC involves tagging the receptor of interest 
with an inactive β-galactosidase (β-gal) small enzyme fragment 
called ProLink or PK (also called Enzyme Donor or ED). 
Its complementary inactive larger fragment, Enzyme Acceptor (EA), 
is either tagged with a relevant signaling protein (e.g. β-arrestin) or 
added in with the detection reagents. Ligand-mediated signaling 
events bring the PK/ED and EA fragments together, resulting in the 
formation of an active β-gal enzyme. This active enzyme hyrdrolyzes 
an added substrate to generate a chemiluminescent signal. Figure 1 
illustrates the working principles of these cell-based assays.

In this paper, we demonstrate that the PathHunter® β-arrestin, 
PathHunter GPCR Internalization, and HitHunter® cAMP second 
messenger assays can be used in parallel to uncover novel, biased 
ligands with specific receptor activation and internalization profiles. 
We systematically characterized the activation and internalization 
profiles of OPRD1 in response to multiple hDOR agonists using 
three functional readouts. Our results demonstrate that compound 
potency and efficacy differences that exist between endogenous and 
synthetic ligands can be easily uncovered using a combination of 
arrestin recruitment, internalization, and second messenger assays. 
®
Thus, Eurofins DiscoverX ’s suite of functional, cell-based assays is 
an ideal platform for ligand bias discovery and the characterization 
of compounds using empirically derived guidelines.

**Figure 1. Functional cell-based assays by Eurofins DiscoverX.** A. cAMP accumulation, B. β-arrestin recruitment, and C. GPCR internalization. 
For a complete list of assays, visit discoverx.com/gpcrs.

---

## METHODS

## Clonal Cell Lines

Cells stably overexpressing hDOR in the cAMP (cAMP Hunter™, Cat. 
No. 95-0108C2), arrestin recruitment (PathHunter® Arrestin, Cat. 
No. 93-0241C2), and GPCR internalization (PathHunter Activated 
GPCR Internalization, Cat. No. 93-0673C3) formats were plated at 
5,000 cells per well of a 384-well plate and incubated overnight at 
37°C and 5% CO₂.

$$
37\,^{\circ}\mathrm{C}
$$

$$
\mathrm{C0}_{2}
$$

## HitHunter cAMP assay

hDOR cAMP Hunter cells were treated with increasing 
concentrations of known compounds for 30 minutes at 37°C and 
5% CO₂. cAMP signal was detected using the HitHunter® cAMP 
Detection Kit (Cat. No. 90-0075) according to the recommended 
protocol. HitHunter cAMP assays are competitive immunoassays, 
where free cAMP from cell lysates competes with ED-tagged 
cAMP for binding to the anti-cAMP antibody.  In the presence of 
ample cellular cAMP, fewer ED-labeled cAMP molecules are able to 
bind to the antibody. As a result, there is a high level of ED-labeled 
cAMP that is free to complement with EA from the detection 
reagent. The resulting active full β-gal enzyme hydrolyzes a 
substrate to produce a chemiluminescent signal. A positive signal is 
directly proportional to the amount of cellular cAMP. (Figure 1. A.).

$$
\mathrm {C O} _ {2}.
$$

$$
37\,^{\circ}\mathrm{C}
$$

$$
\beta-g a l
$$

## PathHunter β-Arrestin Assay

hDOR PathHunter β-Arrestin cells were treated with increasing 
concentrations of the compounds for 90 minutes at 37°C and 5% 
CO₂. In this system, the PK fragment is appended to the C-terminus 
of the GPCR. β-arrestin is fused to the EA fragment. Activation 
of a single GPCR stimulates the binding of arrestin, forcing 
complementation of the two enzyme fragments. The resultant 
increase in β-gal enzyme activity is measured by addition of 
chemiluminescent PathHunter Detection Reagents (Cat. No. 93-
0001). (Figure 1. B.)

$$
\beta-A r r e t i n
$$

$$
\mathrm{C0}_{2}.
$$

$$
37\,^{\circ}\mathbb{C}
$$

$$
\beta
$$

$$
\beta -
$$

## PathHunter Activated GPCR Internalization Assay

hDOR PathHunter GPCR Internalization cells were treated with 
increasing concentrations of the compounds for 180 minutes at 
37°C and 5% CO₂. PathHunter Internalization assays employ the PK

$$
37\,^{\circ}\mathrm{C}
$$

$$
\mathrm{C0}_{2}.
$$

tag that is localized to the surface of intracellular endosomes and 
the EA fused to β-arrestin. Stimulation of the untagged receptor 
results in β-arrestin binding to the activated GPCR, internalization 
of the receptor, and trafficking to cellular endosomes. The resultant 
enzyme complementation leads to β-gal enzymatic activity that is 
measured by addition of chemiluminescent PathHunter Detection 
Reagents (Cat. No. 93-0001). (Figure 1. C.)

$$
\beta\ {r a r s e t n}
$$

$$
\beta-a r r e t i n
$$

$$
\mathsf{G P C R},
$$

## Agonists

Four hDOR agonists (DADLE, [Met⁵]-enkephalin, [Leu]-enkephalin, 
and SNC80, a strongly internalizing compound and functional 
antagonist), were analyzed by the three types of GPCR functional 
assays. For comparison, the data was normalized to [Met⁵]-
enkephalin in potency (set equal to 1) and efficacy (set equal to 
100%).

All data was read on a multi-mode plate reader and analyzed using 
GraphPad Prism®.

## RESULTS AND DISCUSSION

Our experiments demonstrate that potency and efficacy 
differences exist between endogenous enkephalin peptides and 
synthetic analogs for the human δ opioid receptor. Furthermore, 
these differences can be easily uncovered using a combination of 
second messenger, arrestin recruitment, and internalization assays, 
as shown in Figure 2. According to the internalization assay, 
SNC80 was clearly defined as a super agonist. Although the hDOR 
receptor undergoes rapid internalization, distinct ligand-specific 
differences were observed during re-sensitization when the GPCR 
became available. These results correlate with published literature 
that indicates that receptor internalization, as determined by a 
large reduction in cell surface receptors, influences the efficacy 
6, 7
of an agonist. Therefore, using a single pathway approach 
to GPCR analysis *in vitro* could lead to incorrect prediction of 
compound activity *in vivo*. From a drug development standpoint, 
using multiple GPCR signaling readouts during the screening and 
lead optimization process can facilitate the identification of novel 
biased ligands with unique activation and internalization profiles.

---

**Figure 2. Comparison of human** δ **opioid receptor cell-based assays.** A. c A MP, B. β-arrestin recruitment, and C. GPCR internalization assays of the human δ opioid 
receptor (OPRD1, hDOR) receptor.

## CONCLUSIONS

Understanding G-protein and arrestin biased signaling has 
important implications in the discovery and development of novel 
®
therapeutics. Using Eurofins DiscoverX ’s suite of functional cellbased assays, our experiments indicate that novel biased ligands 
can be identified and characterized for multiple activities *in vitro*
using the same robust, reproducible, and high throughput-friendly 
chemiluminescent format. We have completed a systematic 
analysis of many GPCRs and their related ligands to study the 
relationship between different readouts of GPCR activation. 
More than half of the receptors tested showed significant 
pharmacological differences depending on the pathway studied,

indicating that functional selectivity is not an isolated event, but 
is prevalent across multiple receptor classes and ligand types. 
We have embarked on an era of GPCR drug discovery that will 
enable researchers to develop smarter, safer, and more effective 
drugs based on a deeper understanding of receptor activity and 
compound mechanism. With the largest and most comprehensive 
menu of over 1600 functional and binding GPCR assays for 
screening and profiling, Eurofins DiscoverX delivers simple, 
functional, quantitative, and high throughput cell-based assays for 
small molecule and biologics drug discovery analysis.

---

## REFERENCES

1. DeWire SM, Ahn S, Lefkowitz RJ and Shenoy SK. β-Arrestins and cell signaling. 
*Annu Rev Physiol* 69: 483-510, 2007.
2. Lefkowitz RJ. Part 1 and Part 2: Seven Transmembrane Receptors ibioseminars. 
Online video. March, 2010.
3. Whalen EJ, Rajagopal S, and Lefkowitz RJ. Therapeutic potential of β-Arrestin 
and G protein-biased agonists. *Trends Mol Med.*17(3): 126-39, 2011.
4. Kyle D. Functionally biased agonism of mu and kappa opioid receptors. In Res 
Dev Opioid-Related Ligands (pp. 177-197). Washington DC: ACS Symposium 
Series, American Chemical Society, 2013.
5. DeWire SM, Yamashita DS, Rominger DH, *et al*. A G protein-biased ligand at 
the mu-opioid receptor is potently analgesic with reduced gastrointestinal and 
respiratory dysfunction compared with morphine. *J Pharm Exp Ther* 344: 708-
717, 2013.
6. Lecoq I, Marie N, Jauzac Ph and Allouche S. Different Regulation of Human 
d-Opioid Receptors by SNC-80 [(+)-4-[(a R)-a ((2S,5R)-4-Allyl-2,5-dimethyl-
1-piperazinyl)-3-methoxybenzyl]-N,Ndiethylbenzamide] and Endogenous 
Enkephalins. *J Pharm Exp Ther* 310(2): 666-677, 2004.
7. Pradhan AAA, Becker JAJ, Scherrer G, Tryoen-Toth P, Filliol D, *et al*. (2009) 
*In Vivo* Delta Opioid Receptor Internalization Controls Behavioral Effects of 
Agonists. *PLoS ONE* 4(5): 1-11, 2009.

## RESOURCE REFERENCES

1. Gilchrist A, Gauntner TD, Fazzini A, *et al*. Identifying bias in CCR1 antagonists 
using radiolabelled binding, receptor internalization, β-arrestin translocation 
and chemotaxis assays. *Br J Pharmacol*. 2014; 171(22): 5127-5138. doi:10.1111/
bph.12835 
2. Winpenny D, Clark M, Cawkill D. Biased ligand quantification in drug discovery: 
from theory to high throughput screening to identify new biased μ opioid 
receptor agonists. *Br J Pharmacol*. 2016; 173(8): 1393-1403. doi:10.1111/
bph.13441
3. Dhopeshwarkar A, Mackie K. Functional Selectivity of CB2 Cannabinoid 
Receptor Ligands at a Canonical and Noncanonical Pathway. *J Pharmacol Exp* 
*Ther.* 2016; 358(2): 342-351. doi:10.1124/jpet.116.232561 
4. Wang T, Li Z, Cvijic ME, *et al*. Measurement of β-Arrestin Recruitment for GPCR 
Targets. 2017 Nov 20. In: Sittampalam GS, Grossman A, Brimacombe K, *et al*., 
editors. *Assay Guidance Manual* [Internet]. Bethesda (MD): Eli Lilly & Company 
and the National Center for Advancing Translational Sciences; 2004. Available 
from: https://www.ncbi.nlm.nih.gov/books/NBK464634/ 
5. Mores KL, Cummins BR, Cassell RJ, van Rijn RM. A Review of the Therapeutic 
Potential of Recently Developed G Protein-Biased Kappa Agonists. *Front* 
*Pharmacol.* 2019; 10:407. Published 2019 Apr 17. doi:10.3389/fphar.2019.00407
6. Jaeger K, Bruenle S, Weinert T, *et al*. Structural Basis for Allosteric Ligand 
Recognition in the Human CC Chemokine Receptor 7. *Cell.* 2019; 178(5): 1222-
1230.e10. doi:10.1016/j.cell.2019.07.028
7. Peters, A., Rabe, P., Krumbholz, P. *et al*. Natural biased signaling of 
hydroxycarboxylic acid receptor 3 and G protein-coupled receptor 84. *Cell* 
*Commun Signal* 18, 31 (2020). https://doi.org/10.1186/s12964-020-0516-2

---

Quantify GPCR Endocytosis and Recycling with PathHunter® GPCR Internalization Assays – Analyzing 
Therapeutics for Opioid and Cholecystokinin Receptors

Daniel Bassoni, Elizabeth Quinn, Ph.D., Manisha Pratap, and Tom Wehrman, Ph.D.
®
Eurofins DiscoverX, Fremont, CA, USA

## INTRODUCTION

G-protein coupled receptors (GPCRs) are the largest and most 
druggable class of cell surface receptors¹. Following agonistmediated receptor activation and subsequent G-protein signaling, 
GPCRs are phosphorylated by G-protein receptor kinases (GRKs), 
which results in the binding of β-arrestin to the activated receptor.

**Figure 1. The life cycle of a GPCR includes activation, endocytosis, and** 
**recycling back to the plasma membrane.** Repeated or prolonged exposure 
to a ligand can re-route receptors to the lysosome, thus contributing to the 
downregulation of receptors.

Bound arrestin sterically blocks further G protein activation, 
thus limiting the length of G-protein signaling and leading to 
receptor desensitization and internalization into clathrin coated 
<sup>2</sup>
pits. Once internalized, GPCRs are either targeted to lysosomes 
for degradation, retained within the endosomal compartment, or 
recycled back to the cell membrane³, as seen in Figure 1.

The rate and extent of receptor internalization can dramatically 
impact the biological efficacy of a ligand or therapeutic. Studies 
have shown that agonist treatment can lead to tachyphylaxis, 
a decrease in responsiveness to the drug with repeated dosing⁴. 
Moreover, GPCR agonists can induce tolerance, whereby a higher 
amount of drug is required with repeated doses to obtain the 
5,6
same therapeutic effect. Both tachyphylaxis and drug tolerance, 
which can limit the clinical effectiveness of a drug, have been 
shown to correlate with the tendency of the ligand to induce 
receptor internalization following prolonged or repeated receptor 
4-6
activation. Using a mouse model of inflammatory pain, Pradhan 
*et al.* showed that despite having similar *in vitro* potencies and 
efficacies, δ-opioid receptor (DOR) agonists were only able to 
retain full analgesic efficacy when receptors remained at the cell 
surface⁶.

Although various approaches for monitoring receptor 
internalization have been developed, these techniques are often 
difficult to quantify, rely on the availability of highly specific 
antibodies and expensive imaging instruments, and are not 
amenable to a high-throughput format⁷. As a result, internalization 
profiles of candidate drugs may not be investigated until very late 
in the discovery process following expensive and labor-intensive 
animal testing. Therefore, there is a need for a simple, scalable, and 
quantitative internalization assay that can be used to characterize 
therapeutic activity earlier in the drug discovery process in order 
to elucidate the mechanism by which a GPCR-targeted drug exerts 
its effect *in vivo*. As a solution, PathHunter GPCR Internalization 
Assays by Eurofins DiscoverX are specifically designed to quantitatively measure internalization and recycling patterns of 
GPCR drug targets to enable the discovery and characterization 
of novel agonists. These assays assist in differentiating between 
strongly and weakly internalizing agonist ligands or therapeutics, 
and provide a simple, non-imaging, non-antibody based 
chemiluminescent technique that is amenable to high throughput 
screening.

## PathHunter GPCR Internalization Assays

®
Eurofins DiscoverX has developed two different formats of 
®
the PathHunter GPCR Internalization Assays – the activated 
internalization assay and the total internalization assay. While 
both formats measure receptor internalization in whole cells, the 
activated internalization assay measures receptor internalization 
mediated by β-arrestin recruitment. On the other hand, the total 
internalization assay measures GPCR endocytosis brought about 
by all relevant mechanisms including the natural recycling of 
GPCRs. Both of these formats are based on the established Enzyme 
Fragment Complementation (EFC; discoverx.com/efc) technology. 
EFC is based on two recombinant β-galactosidase (β-gal) enzyme 
fragments – a large enzyme acceptor (EA) fragment and a smaller 
enzyme donor (ED, also called ProLink™ (PK) fragment. Separately, 
these fragments are inactive, but when combined, they form an 
active β-gal enzyme that hydrolyzes its substrate to produce a 
chemiluminescent signal.

As illustrated in Figure 2, the PathHunter Activated GPCR 
Internalization Assays combine engineered clonal cell lines stably 
expressing the wild-type (untagged) GPCR, PK-tagged endosomes, 
and a β-arrestin/EA fusion protein. Activation of the untagged 
GPCR induces β-arrestin recruitment and internalization of 
the GPCR/arrestin-EA complex to the PK-tagged endosomes, 
resulting in the complementation of the PK-EA fragments to form 
the active β-gal enzyme. In comparison (not shown here), the 
PathHunter Total GPCR Internalization Assays combine engineered 
clonal cell lines stably expressing a PK-tagged GPCR and EAtagged endosomes. Activation of the PK-tagged GPCR induces 
internalization to the EA-tagged endosomes, bringing about the 
formation of the active β-gal enzyme. Overall, both assays result

$$
\beta
$$

in a positive, chemiluminescence gain-of-signal with large signalto-noise ratios using a simple, mix-and-read detection system 
for optimal assay performance. Additionally, these internalization 
assays, like most EFC assays, can be used to screen small molecules 
or biologics in a fast, scalable, and highly quantitative manner.

**Figure 2. PathHunter Activated GPCR Internalization Assays provide a novel,**

**non-imaging approach for measuring GPCR activation and internalization in** 
**live cells.** In this system, enzyme fragment complementation occurs following 
β-arrestin recruitment and receptor internalization and localization at the early 
endosome.

## To demonstrate that the PathHunter Activated GPCR

Internalization Assays can be used as a powerful tool to measure 
receptor internalization, we characterized the receptor trafficking 
properties of agonists of two different classes of GPCRs—DOR and 
Cholecystokinin-A receptor (CCKAR) agonists using various GPCR 
cell lines. DORs are involved in pain modulation as mentioned 
above, while CCKAR plays an active role in various parts of the 
digestive system including the pancreas, gallbladder, and stomach. 
Using a combination of activation and internalization assays for 
the same GPCR target, we also demonstrate that these assays 
can be used to uncover potency and efficacy differences between 
ligands.

---

## METHODS

## Clonal Cell Lines

PathHunter® Activated GPCR Internalization Cell Lines expressing 
the receptors CCR5 (Cat. No. 93-0653C3), CHRM5 (Cat. No. 
93-0657C3), SSTR2 (Cat. No. 93-0676C2), FPRL1 (Cat. No. 93-
0666C3), DOR (Cat. No. 93-0673C3) and CCKAR (Cat. No. 93-
0650C3) and PathHunter β-Arrestin Cell Line expressing CCKAR 
(93-0190C2) and cAMP Hunter™ Cell Line expressing DOR (Cat. No. 
95-0108C2) were cultured as recommended in the cell line-specific 
datasheets. Prior to running the assays, cells were plated at 5,000 
cells per well of a 384-well plate and incubated overnight at 37ºC 
and 5% CO₂.

$$
\mathrm{C O}_{2}
$$

$$
37^circ\mathrm{C}
$$

## Ligands

Reference ligands CCL3 (Cat. No. 92-1002), Oxotremorine-M (Cat. 
No. 92-1106), Somatostatin-28 (Cat. No. 92-1068), WKYMVm-NH2 
(Cat. No. 92-1075), DADLE (Cat. No. 92-1055), [Met⁵]-Enkephalin 
(Cat. No. 92-1086) were obtained from Eurofins DiscoverX®. SNC-
80 (Cat. No. 0764) and A-71623 (Cat. No. 2411) were obtained 
from Tocris Bioscience (Minneapolis, MN). Cholecystokinin (CCK) 
Fragment 26-33 Amide, Non-sulfated, sCCK8 (Cat. No. C2901) 
and [Leu]-Enkephalin (Cat. No. L9133) were obtained from Sigma 
Aldrich (St. Louis, MO).

## PathHunter Activated GPCR Internalization Assays

Cells were treated with increasing concentrations of ligands for 
180 minutes at 37°C and 5% CO₂. The resultant EFC after agonist 
treatment leads to an increase in enzyme activity that is measured 
by addition of chemiluminescent PathHunter Detection Reagents 
(Cat. No. 93-0001). All data was read on a multi-mode plate reader 
and analyzed using GraphPad Prism®.

$$
37\,^{\circ}\mathrm{C}
$$

$$
\mathrm{C0}_{2}.
$$

## Calcium Flux Assays

Naturally Gq-coupled PathHunter β-Arrestin CCKAR cells were 
loaded for intracellular calcium flux measurement with a Calcium 
PLUS
No Wash Kit (Cat. No. 90-0091L) assay solution supplemented 
with 2.5 mM probenecid to avoid calcium dye leakage from the 
cells. The cells were then incubated for 1 hour at 37°C and 5% CO₂. 
The agonist was added after 10 seconds of baseline acquisition.

$$
37\,^{\circ}\mathrm{C}
$$

$$
\mathrm{C O}_{2}.
$$

Increase of intracellular calcium concentration was monitored 
using the FLIPR® Tetra detection system from Molecular Devices 
(Sunnyvale, CA). Visit discoverx.com/calcium for more details.

## HitHunter cAMP Assays

Human DOR cAMP Hunter cells were treated with increasing 
concentrations of known ligands for 30 minutes at 37°C and 
5% CO₂. The cAMP signal was detected using the HitHunter® 
cAMP XS+ Detection Kit (Cat. No. 90-0075) according to the 
recommended protocol. HitHunter cAMP assays are competitive 
immunoassays, where free cAMP from cell lysates competes for 
antibody binding against labeled cAMP (ED-cAMP conjugate). 
Unbound ED-cAMP is free to complement EA to form an active 
enzyme by EFC which hydrolyzes substrate to produce signal. A 
positive signal is directly proportional to the amount of cellular 
cAMP. Visit discoverx.com/cAMP for more details.

$$
37\,^{\circ}\mathrm{C}
$$

$$
\mathrm{C O}_{2}.
$$

## Data Normalization

Where indicated, data was normalized to the reference agonist in 
potency (set equal to 1) and efficacy (set equal to 100%) to enable 
a graphic representation of the agonist comparisons.

## RESULTS AND DISCUSSION

At first, a series of experiments were run using the GPCR 
internalization assay across a range of different GPCR receptors. 
Figure 3 shows representative dose-response curves for the human 
CCR5, M5, SSTR2, and FPRL1 receptors, demonstrating that these 
assays are broadly applicable to a large number of GPCR families and 
receptor types to provide quantitative comparisons of ligand activity.

To determine whether PathHunter GPCR Internalization assays can 
be used to differentiate between strongly and weakly internalizing 
agonist ligands, we characterized the receptor trafficking properties 
5
of four DOR agonists (DADLE, [Met ]-enkephalin, [Leu]-enkephalin, 
and SNC-80, a known strongly internalizing compound and 
functional antagonist). For comparison, the data was normalized 
5
to [Met ]-enkephalin in potency (set equal to 1) and efficacy (set 
equal to 100%). Our results demonstrate that treatment with 
SNC-80 leads to high levels of DOR receptor internalization (Figure 
4. A.). Moreover, SNC-80 could be defined as a superagonist in the assay based on a maximal response greater than that of the 
endogenous agonist, [Met⁵]-enkephalin. We also examined the 
inhibitory effects of the four agonists on forskolin-stimulated cAMP 
accumulation using the DOR cAMP Hunter™ Cell Line. In contrast to

the internalization profiles, we observed similar ligand potencies and 
efficacies for both SNC-80 and enkephalins using the cAMP assay 
(Figure 4. B.).

**Figure 3. The PathHunter® GPCR Activated Internalization Assay is broadly applicable to a large number of GPCR families.** Cells expressing the wild type untagged 
A. Chemokine CCR5, B. Cholinergic Muscarinic CHRM5, C. Somatostatin SSTR2, and D. Formylpeptide FPRL1 were treated with increasing concentrations of control 
agonist and assayed using PathHunter detection reagents. In all cases, the results show a positive, gain-of-signal assays with large signal-to-noise ratios for the agonists 
tested.

---

1000 Agonist Agonist + 20 μM Forskolin

**Figure 4. Comparison of DOR internalization versus cAMP accumulation by known receptor agonists.** Cells overexpressing the DOR receptor in the PathHunter® 
Activated GPCR Internalization A. and cAMP Hunter™ B. assay formats were treated with agonists and assayed using PathHunter and HitHunter detection reagents, 
respectively, as described in Methods. Despite similar ligand potencies and efficacies based on the inhibition of cAMP accumulation, SNC-80 which is a known strongly 
internalizing compound and functional antagonist, is clearly defined as a superagonist in the internalization assay only.

To help explain these results, we associated these finding with 
other studies. Lecoq *et al.* first studied the desensitization and 
internalization patterns of the DOR receptor following exposure 
to endogenous enkephalin ligands or the synthetic opioid agonist, 
SNC-80. Their results showed that δ opioid agonists differed 
dramatically in their desensitization and receptor internalization 
profiles despite having similar potencies and efficacies *in vitro*. 
Moreover, DORs were more rapidly desensitized by SNC-80 
resulting in high levels of internalization via clathrin-coated 
pits, whereas exposure to the natural enkephalin ligands led to 
internalization followed by sorting to recycling endosomes⁵. Using 
a mouse model of inflammatory pain, Pradhan *et al.* established 
a direct link between receptor localization and function by 
demonstrating that δ opioid agonist-induced analgesic effects 
are retained when receptors remain at the cell surface, but lost 
following receptor activation and internalization⁶. Overall, the 
PathHunter internalization assays results correlated well with the 
aforementioned experiments.

The PathHunter Activated GPCR Internalization assays could 
be broadly applied to other therapeutically relevant targets, as 
demonstrated through our experiments on the receptor trafficking 
properties of CCKAR and related agonists. Asin *et al.* described 
the *in vivo* behavior of a highly selective CCKAR agonist, A-71623. 
Despite being less potent compared to the endogenous ligand 
cholecystokinin (CCK), A-71623 showed improved potency and 
prolonged duration by suppressing food intake and locomotor 
activity in food deprived rats¹⁰. Using a combination of activation 
(calcium flux) and receptor internalization assays, our results 
demonstrate that agonist treatment with the sulfated version 
of CCK (sCCK8) leads to high levels of receptor internalization, 
whereas treatment with A-71623 does not (Figure 5. A.). When 
we compared the potency of the ligands using a calcium flux 
assay, our results confirmed that A-71623 is less potent than the 
endogenous sCCK8 ligand. In the case of CCKAR, one hypothesis 
is that the lack of receptor internalization following A-71623 
treatment in the food deprived rats could explain the greater 
therapeutic benefit of the less potent synthetic analog compared 
to the endogenous ligand by increasing the length of time the drug 
can interact with its therapeutic target.

---

Our results demonstrate that these assays can be used to 
directly measure internalized GPCRs localized to intracellular 
endosomes in a dose-response dependent manner, and identify 
strongly internalizing ligands that appear as superagonists. 
Furthermore, we saw that both DOR and CCKAR receptors undergo 
rapid internalization. We observed dramatic differences in the 
internalization profiles between endogenous agonists and synthetic 
analogs that were easily uncovered using our simple, mix-andread chemiluminescent readout. Additionally, we completed a 
systematic pharmacologic analysis of a set of chemokine receptors 
and their related endogenous ligands using assays for G-protein 
signaling, β-arrestin recruitment, and receptor internalization. 
For chemokines targeting the same receptor, differences in 
the efficacies for G-protein- or arrestin-mediated signaling or 
receptor internalization were observed. From a drug development 
standpoint, using PathHunter® GPCR Internalization assays 
as orthogonal screening tools during the hit-to-lead and lead 
optimization stages can provide novel information about a ligand’s 
functionality early in the drug discovery process that cannot be 
inferred using a simple receptor activation readout alone (2nd 
messenger signaling or β-arrestin recruitment). Understanding 
differences in receptor trafficking and how localization of the GPCR 
impacts the biological efficacy of a ligand could have important 
implications for drug discovery in many therapeutic areas.

$$
\beta\mathsf{a r r e s t i n}
$$

**Figure 5. Comparison of CCKAR internalization versus calcium flux by known** 
**receptor agonists.** Cells overexpressing the CCK-A receptor in the PathHunter 
Activated GPCR Internalization A. and PathHunter β-Arrestin B. formats were 
treated with agonists and assayed using PathHunter and Calcium No Wash<sup>PLUS</sup> 
reagents, respectively, as described in Methods. A-71623 showed a lower potency 
in calcium flux as well as ~25% efficacy relative to the sulfated version of the 
endogenous CCK ligand (sCCK8) in the GPCR internalization assay.

---

## CONCLUSIONS

GPCR activation and internalization patterns in response to ligand 
activation have important implications in the discovery and 
development of novel therapeutics. Although integral to GPCR 
pharmacology, quantification of receptor internalization has proven 
cumbersome and difficult. Frequently applied tools to study GPCR 
trafficking include a combination of low-throughtput fluorescence 
microscopy with complex automated image analysis software and 
expensive antibodies⁷. To overcome these challenges in monitoring 
cell surface receptor biology, we developed a generic, quantitative, 
8, 9
cellular assay based on the EFC technology.

## REFERENCES

Eurofins DiscoverX®’s comprehensive menu of more than 100 
PathHunter® GPCR Internalization assays offers a simple, nonimaging, non-antibody based chemiluminescent assay that is 
amenable to high-throughput screening. By providing routine 
analysis of receptor internalization at an early point in the drug 
discovery process, Eurofins DiscoverX delivers on the promise of 
a deeper understanding of ligand activity and the development of 
novel small molecule or biologic drug candidates with better safety 
and efficacy profiles.

1. Ma P and Zemmel R. Value of novelty? *Nat Rev Drug Discovery* 1: 571-572, 
2002.
2. Whalen E, Rajagopal S and Lefkowitz, R. Therapeutic potential of β-Arrestin 
and G protein-biased agonists. *Trends in Mol Med* 17(3): 126-139, 2011.
3. Hanyaloglu A and von Zastrow M. Regulation of GPCRs by endocytic 
membrane trafficking and its potential implications. *Annu Rev Pharmacol* 
*Toxicol* 48(3): 537-568, 2008.
4. Lamian V, Rich A, Ma Z, Li J, Seethala R, Gordon D and Dubaquie Y. 
Characterization of agonist-induced motilin receptor trafficking and its 
implications for tachyphylaxis. *Mol Pharm* 69: 109-118, 2006.
5. Lecoq I, Marie N, Jauzac Ph and Allouche S. Different regulation of 
human δ-Opioid receptors by SNC-80 [(+)-4-[(αR)-α-((2S,5R)-4- Allyl- 
2,5-dimethyl-1-piperazinyl)- 3- methoxybenzyl]-N, Ndiethylbenzamide] and 
endogenous enkephalins. *J Pharm Exp Ther* 310(2): 666-677, 2004.
6. Pradhan A, Becker J, Scherrer G, Tryoen-Toth P, Filliol D, Matifas A, Massotte D 
Gaveriaux-Ruff C and Kieffer BL. *In Vivo* delta opioid receptor internalization 
controls behavioral effects of agonists. *PLoS ONE* 4(5): 1-11, 2009.
7. Haasen D, Schnapp A, Valler MJ and Heilker R. G protein-coupled receptor 
internalization assays in the high-content screening format. *Methods in* 
*Enzymology* 414: 121-39, 2006.
8. Eglen RM. Enzyme fragment complementation: a flexible high throughput 
screening assay technology. *Assay Drug Dev Technol* 1: 97-104, 2002.
9. Bassoni D, Raab WJ, Achacoso P, Loh CY and Wehrman TS. Measurements of 
β-Arrestin recruitment to activated seven transmembrane receptors using 
enzyme complementation. Receptor Binding Techniques: *Methods in Molecular* 
*Biology*, Vol 897, 3rd edition, XXIV, 327, 70, 2012.
10. Asin K, Bednarz L, Nikkel A, Gore P, Montana W, Cullen M, Shiosaki K, Craig R 
and Nadzan A. Behavioral Effects of A71623, a highly selective CCK-A agonist 
tetrapeptide. *Am. J. Physiol*. 263 (Regulatory Integrative Comp. Physiol. 32: 
Rl25-R135, 1992.

## RESOURCES REFERENCES

1. Loh H, Tao P and Smith A. Role of receptor regulation in opioid tolerance 
mechanisms. *Synapse* 2: 457-462, 2002.
2. Ahn S, Kahsai AW, Pani B, *et al.* Allosteric "beta-blocker" isolated from a 
DNA-encoded small molecule library. *Proc Natl Acad Sci U S A.* 2017; 114(7): 
1708-1713. doi:10.1073/pnas.1620645114
3. Cahill TJ 3rd, Thomsen AR, Tarrasch JT, *et al*. Distinct conformations of GPCR-
β-arrestin complexes mediate desensitization, signaling, and endocytosis. *Proc* 
*Natl Acad Sci U S A.* 2017; 114(10): 2562-2567. doi:10.1073/pnas.1701529114
4. Wingler LM, Elgeti M, Hilger D, *et al*. Angiotensin Analogs with Divergent Bias 
Stabilize Distinct Receptor Conformations. *Cell*. 2019;176(3): 468-478.e11. 
doi:10.1016/j.cell.2018.12.005

---

# Ligand Characterization of Mas-Related GPCR-X2 via Cell-Based Assays:

# Assessing the Regulation of Mast Cell Function and Implications in Safety Pharmacology

Andrew Green, Gordon Leung, Lakshmi Anantharaman, Arvind Kannan, and Neil Charter
Eurofins DiscoverX®, Fremont, CA 94538

## INTRODUCTION

Effectors of mast cell degranulation (MCD) such as histamine, 
serotonin, heparin, and certain cytokines principally drive IgEmediated allergic reactions. On the other hand, non-immune 
mediated activation of mast cells has been identified to cause 
pseudo-allergic Adverse Drug Reactions (ADR), due in part 
to multiple receptor expression profiles on their surfaces. 
Furthermore, the recognition of a wide range of activating 
ligands, including certain classes of therapeutics targeting mast 
cell receptors warrants a closer examination into their safety 
pharmacological roles. Among other receptor classes, orphan 
GPCRs have emerged as active proponents in non-immunogenic 
mast cell activation profiles with possible roles in pseudo-allergic 
or anaphylactoid reactions. As a result, efforts in drug discovery 
and development have focused on developing safer therapeutics 
aimed at reducing anaphylactoid reactions. For instance, a study by 
McNeil *et al*. (2015) has formed the basis of exploring the role of 
orphan receptors in non-IgE mediated occurrence of histamine-like 
symptoms associated with secretagogues and therapeutic drugs.

A relatively new subfamily of GPCRs associated with the MAS1 
oncogene was identified based on transcriptome analysis by Dong 
*et al*. (2001). The encoding genes were termed as Mas-Related 
genes (MRGs) and the receptors as MAS-related GPCRs (MRGPR). 
One of its members, MrgprX2 encoded by the gene MRGPRX2, 
has been identified to be promiscuous and activated by several 
synthetic small molecules and peptides including U.S. Food and 
Drug Administration (FDA) approved drugs. While a role for the

former “orphan” MrgprX2 was established in 2015, subsequent 
studies have identified MrgprX2 receptor-mediated agonism to be 
implicated in causing adverse reactions associated with clinical 
candidate molecules during their development (Grimes *et al*. 2019).

Overall, these studies have since necessitated more complex risk 
assessments and targeted safety testing for drugs in development 
to minimize ADRs, including anaphylaxis and allergic reactions.

## RECEPTOR INTERACTIONS AND CLINICAL RELEVANCE

Mast cells are principal players in IgE-, and more recently, 
MrgprX2-mediated anaphylactoid reactions. They can be activated 
by immunogenic effectors and by several types of receptors 
including Toll-like receptors (TLR), protease-activated receptors 
(PARs), opioid receptors, c5a complement, and T-cells depending 
on localization and type of mast cell (Hennino *et al.* 2006, Spoerl 
*et al*. 2017) opening up routes along non-immunogenic activation 
related pathways (Figure 1.).

---

**Figure 1. Mast cell degranulation including non-immunological activation route (adapted from Spoerl et al. 2017).**

Historically and despite significant efforts, the exact function 
and role of the MrgprX2 orphan receptor remained elusive given 
its extensive mast cell expression profile. Previously, studies 
with the human MrgprX2 receptor had revealed surrogate ligand 
pairings, but were unsuccessful in linking the receptor to its 
biological function; primarily due to the lack of understanding of 
orthologue receptor genetics. The catalyst for the recent focus on 
this receptor was a study from Johns Hopkins University (McNeil 
*et al*. 2015) that elucidated a physiological role for the receptor 
for the first time. The group focused on the mouse orthologue 
gene, recapitulating the same pharmacology observed with the 
human MrgprX2 receptor and their knockout studies confirmed 
a link between receptor expression to mast cell function and 
anaphylaxis.

A few other studies emerged to better understand the receptor’s 
link to drug-induced anaphylaxis. Of particular mention is a 
study led by Dr. Andrew Brown’s team at GlaxoSmithKline (GSK), 
which identified a series of orthologue receptors, typically 
of pharmacological interest during drug development, with a

bioinformatics approach (Grimes *et al*. 2019). Subsequently, 
recombinant expression studies and compound profiling, including 
Eurofins DiscoverX®’s β-arrestin recruitment and calcium signaling 
assays, provided a rigorous characterization platform of compound 
pharmacology.

In retrospect, a series of drug discovery programs, where 
anaphylaxis in animals had impeded development, the (drug) 
candidates were shown to exhibit MrgprX2 pharmacology at the 
corresponding orthologue gene.  Notably, these programs studied 
diverse therapeutic types.  Further analysis and insight correlated 
diverse pharmacology to a ligand molecule’s net charge or pH 
rather than any specific peptide sequence or structural features.  
Indeed, the promiscuity of the MrgprX2 receptor supported Dr. 
Brown’s hypothesis that it functions as a sensor for exogenous 
and potentially harmful (viral) peptides, invoking a mast cell host 
response (Grimes *et al*. 2019).  This is analogous to the function of 
the formyl peptide receptor (FPR1), a promiscuous receptor without 
an established cognate ligand, sensing patterns of bacterial 
peptides.

---

## NEED FOR SAFETY LIABILITY SCREENS

Receptor activation towards regulating mast cell degranulation has 
a purported key safety liability, as observed in studies such as that 
of Lafleur *et al*. 2020. Since assays with mast cells are challenging 
(principally due to access to insufficient cells and difficulty creating 
viable cultures), their study characterized an extended panel of 
established and novel ligands with an emphasis on investigating 
the pharmacology of compounds (peptide) charge.

Several targets can represent pharmacological panels in safety 
liability screens. With appropriate testing methodologies, improved 
understanding of their pharmacological relevance can be met, 
which in turn can show the impact of a particular target (and its 
mechanism-of-action) on the safety liability of the drug at a much 
earlier stage in the discovery process. In general, it was observed 
that the outcomes of MrgprX2 activation bear a strong correlation 
with histamine release in CD34+ stem cell-derived mature human 
mast cells (La Fleur *et al*. 2020). For that study, the Eurofins 
DiscoverX® ‘s PathHunter® platform was employed to analyze 
MrgprX2 for both calcium release and β-arrestin recruitment. 
The study endorsed the use of the cell-based functional assays 
in safety liability screens as a predictive and phenotypically 
representative model of mast cell activation.

MRGPRX2 is part of Mas-related gene (MRG) family of GPCRs 
comprising almost 50 members sub-divided into subfamilies (A-H 
and X), of which the X-species is specific to primates including 
humans, macaques, and rhesus monkeys. Transcriptomic analyses 
have shown that MRGPRX2 to be one of the highest expressed 
receptors in human mast cells (Motakis *et al*. 2014). Studies 
related to ligand activation of the MrgprX2 receptor have brought 
forth its promiscuous nature of exerting unexpected/un-intended 
histamine-like allergenic effects, necessitating the investigation of 
interaction of potential ligands with MrgprX2.

In this application note, we independently generated a new set 
of data with selected agonists, previously identified as MrgprX2 
agonists, using calcium mobilization and β-arrestin recruitment 
assays feasible with the application of the PathHunter MrgprX2

cell line. The goal of this study was to show the high-throughput 
and time-savings value of using engineered, cell-based assays to 
study its activation through these screening assays.

## MATERIALS

Compound 48/80 (Sigma Aldrich, UK), mastoparan (Tocris 
Biosciences, Bristol, UK), cortistatin-14 (Genscript, USA), 
octreotide, indolicidin, and vancomycin (Sigma Aldrich, UK); U2OS 
(ATCC Cat. No. HTB-96, RRID:CVCL_0042) or HEK 293-MSRII cells; 
and Eurofins DiscoverX PathHunter CHO-K1 MRGPRX2 β-Arrestin 
Cell Line (Cat. No. 93-0309C2), AssayComplete™ Cell Plating 0 
Reagent (CP0, Cat. No. 93-0563R0A), and PathHunter Detection 
Kit (Cat. No. 93-0001).

## METHODS

## Calcium Mobilization Assay

Assay comprised of cell line expansion and propagation, dye loading, 
and agonist activity assay to determine the mobilization of calcium 
in the cell.

## Cell Handling

U2OS or HEK 293-MSRII cell lines were expanded from freezer 
stocks according to standard procedures. Cells were seeded in a 
total volume of 20 µL into black-walled, clear-bottom, Poly-D-lysine 
coated 384-well microplates, and then incubated at 37°C and 5% 
CO  for the appropriate time prior to testing.
2

## Dye Loading

Assays were performed in 1X Dye Loading Buffer consisting of 1X 
dye, 1X Additive A, and 2.5 mM Probenecid in HBSS / 20 mM HEPES.  
Probenicid was prepared fresh. Cells were loaded with dye prior to 
testing. Media was aspirated from cells and replaced with 20 µL Dye 
Loading Buffer. Cells were incubated for 45 minutes at 37°C and 5% 
CO₂.

## Agonist Format

For agonist determination, cells were incubated with ligand samples 
(compound 48/80, mastoparan, cortistatin-14, octreotide, indolicidin, 
and vancomycin) to induce a response. After dye loading, cells were removed from the incubator, and 10 µL HBSS / 20 mM HEPES was 
added.  Cells were incubated for 15 minutes at room temperature in 
the dark to equilibrate plate temperature. Intermediate dilution of 
sample stocks was performed to generate 4X sample in assay buffer. 
Compound agonist activity was measured on a FLIPR Tetra (MDS).  
Calcium mobilization was monitored for 2 minutes and 10 µL 4X 
sample in HBSS / 20 mM HEPES was added to the cells 5 seconds 
into the assay. Refer to Figure 2. for the assay principle.

$$
1 0 \mu \mathrm {L} 4 \mathrm {X}
$$

## bb-ARRESTIN RECRUITMENT ASSAY

5000 Cells/well of the PathHunter® CHO-K1 MRGPRX2 β-arrestin 
cells were seeded with 20 μL AssayComplete CP0 reagent into 
white 384-well plates (Corning) and incubated overnight (37°C 
and 5% CO₂). Test agents were serially diluted in DMSO, added to 
OptiMEM/0.1% BSA (to 5x final), and incubated with 5 μL of cells 
for 90 minutes at 37°C and 5% CO₂ followed by incubation for 30 
minutes at room temperature. Luminescence was detected using 
an Envision plate reader (Perkin Elmer) after the addition of 12.5 
μL PathHunter detection reagent at room temperature for 2 hours. 
Refer to Figure 3. for the assay principle.

$$
\beta
$$

$$
2 0 \mu \mathrm {L}
$$

$$
(37^{\circ}\mathbb{C}
$$

$$
\mathrm {C O} _ {2})
$$

$$
5 \mu L
$$

$$
37\ ^{\circ}\mathbb{C}
$$

$$
\mathrm{C0}_{2}
$$

## RESULTS

Several agonists (cortistatin-14, indolicidin, compound 48/80, 
mastoparan, octreotide, and vancomycin) were assessed in 
PathHunter MRGPRX2 cell lines for β-arrestin recruitment and 
calcium release using Eurofins DiscoverX® assays.

$$
\beta
$$

Measuring intracellular calcium mobilization based on the 
activation status of specific GPCRs can provide information 
that uncovers pharmacological rank-orders of lead agonists and 
calcium signaling flux. The calcium mobilization assay (Figure 
2.) uses an inactive dye probe that, when it penetrates the cell 
membrane, switches to an active form, whose intensity can be 
measured via fluorescence.

For measuring β-arrestin recruitment, the PathHunter β-arrestin 
assay (Figure 3.) was used. This assay is based on DiscoverX’s 
proprietary Enzyme Fragment Complementation (EFC) technology, 
which involves two recombinant β-galactosidase (β-gal) enzyme

$$
\beta
$$

$$
\beta
$$

$$
\beta
$$

fragments that act as an enzyme acceptor (EA) and an enzyme 
donor (ED). Separately, the fragments are inactive, but when 
combined, they form an active enzyme. In this assay, MrgprX2 
cell lines were engineered to co-express the ED, called ProLink™ 
(PK), tagged to the receptor, MrgprX2, and the EA tagged to 
β-arrestin. Activation of the MrgprX2-PK by agonist treatment 
(cortistatin-14) induces β-arrestin-EA recruitment, forcing 
complementation of the two β-gal enzyme fragments. The 
resulting functional enzyme hydrolyzes substrate to generate a 
chemiluminescent signal.

$$
\mathrm{E D},
$$

$$
\beta\mathsf{-a r r e s t i n\ –E A}
$$

$$
\beta-g a l
$$

**Figure 2. Calcium mobilization assay principle.** The calcium mobilization assay 
(see DiscoverX.com/calcium) is a Calcium No Wash<sup>PLUS</sup> Assay wherein an esterified 
(inactive) calcium dye (probe) penetrates the cell membrane and becomes active 
once inside the cell.  The active form of the dye becomes fluorescent after 
binding to intracellular calcium. Additive A is added to prevent the dye from 
being released out from the cell. Ligand binding stimulates GPCR activation, 
resulting in the release of intracellular calcium stores from the endoplasmic 
reticulum (ER), which leads to an increase in fluorescence in the presence of the 
activated calcium dye.

$$
W a h^{P L U S}
$$

---

Published studies using a wide range of chemotypes were 
previously characterized using calcium release and β-arrestin 
recruitment assays for MrgprX2. The studies adopted the same 
clone used in this application note and have been useful in 
ascribing the role of receptor activation to its purported effect 
of non-immune mediated mast cell activation (Grimes *et al*. 2019 
and Lafleur *et al*. 2020). Results herein are in good agreement 
with those previously published with respect to rank orders of 
potency agonists (Grimes *et al*. 2019) maintained relative to 
cortistatin-14 used as reference agonist for both assays (Figure 
4.). Vancomycin activity is negligible with the concentration 
range used in these assays, but consistent with its expected 
low potency. These studies also highlight the potential for the 
β-arrestin assay to report higher levels of efficacy relative to 
cortistatin-14 that was not observed in the calcium mobilization 
assay. Our recent data set is limited, but these apparent 
differences in efficacy may warrant further consideration.

**Figure 3. Eurofins DiscoverX®’s EFC-based** β**-arrestin recruitment assay** 
**principle.** The PathHunter® MrgprX2 β-arrestin recruitment assay principle is 
based on the EFC platform. Binding of cortistatin-14 results in MrgprX2 activation 
thereby stimulating recruitment of β-arrestin-EA fusion protein to MrgprX2-PK. 
The resulting complementation of the two enzyme fragments results in an active 
β-gal enzyme whose activity can be measured using chemiluminescent PathHunter 
detection reagents.

**A Calcium Mobilization Assay**

**B** β**-Arrestin Recruitment Assay**

**C**

|  | Calcium Mobilization |  | β-Arrestin Recruitment |  |
| --- | --- | --- | --- | --- |
|  | EC50μM | % Max Cortistatin-14 | EC50μM | % Max Cortistatin-14 |
| Cortistatin-14 | 0.22911 | 100 | 0.11446 | 100 |
| Indolicidin | 0.2614 | 92 | 0.30684 | 139.1 |
| Compound 48/80 | 1.6901 | 100 | 3.8828 | 136 |
| Mastoparan | 4.3427 | 89 | 3.2843 | 60 |
| Octreotide | 4.8441 | 90 | 15.005 | 65 |
| Vancomycin | &gt;100 | 3 | &gt;100 | 12 |

$$
\mathrm {E C} _ {5 0} \mu \mathrm {M}
$$

$$
\underline{{\mathsf{E C}_{50}\,mu\mathsf{M}}}
$$

**Figure 4. Dose response and rank order potency for calcium mobilization and** β**-arrestin recruitment assays.** Readouts showing select agonists effect on (A.) calcium 
mobilization of intracellular calcium and on (B.) β-arrestin recruitment upon binding to MrgprX2 receptors. Both dose response curves are the mean of two replicates 
and show normalized data calculated from the maximum effect obtained with cortistatin-14. C. Table showing potency and efficacy of agonist relative to cortistatin-14.

---

## CONCLUSION

## Several targets are given consideration to represent

pharmacological panels in safety liability screens as it is crucial to 
identify any specific molecular interactions at profiling stages that 
may cause ADRs in humans. Hence, an improved understanding of 
the pharmacological relevance of targets, including establishment 
of in vitro testing methodologies that can show the impact of 
a particular target (and its mechanism-of-action) on the safety 
liability of the drug at a much earlier stage in the discovery 
process is needed.

In an effort to support the characterization and possible role 
of MrgprX2 in non-classical mast cell-mediated anaphylactoid 
reactions, Eurofins DiscoverX® collaborated with Andrew Brown 
(GSK) and helped identify compounds/peptides that activated 
MrgprX2 via calcium mobilization and β-arrestin recruitment

For more information, visit discoverx.com/gpcrs.

assays (Grimes *et al*. 2019). Further, assessing MrgprX2 activation 
in an engineered cell model can provide value as a rapid, highthroughput, economical mechanism-based screening tool for early 
mast cell degranulation or hazard identification during preclinical 
safety evaluation of therapeutics.

The importance and role of MrgprX2 as an emerging activator 
of non-IgE mediated MCD underscores its value in being a top 
panelist in safety pharmacology. Besides, its potential as a drug 
target for conditions/diseases related to mast cell function and 
regulation, including anaphylaxis, attests to its position as central 
to safety liability testing. Overall, MrgprX2 antagonism promotes 
a fresh interest as an emerging therapeutic target for treating 
pseudo-allergic reactions, and Eurofins DiscoverX qualified assays 
are ideal for supporting research programs centered on MrgprX2 
activation and mast cell regulation.

## REFERENCES

1. Dong X, Han SK, Zylka MJ, Simon MI, Anderson DJ. A diverse family of GPCRs 
expressed in specific subsets of nociceptive sensory neurons. *Cell.* 2001 Sep 7; 
106 (5): 619-32. 
2. McNeil BD, Pundir P, Meeker S, Han L, Undem BJ, Kulka M, Dong X. 
Identification of a mast-cell-specific receptor crucial for pseudo-allergic drug 
reactions. *Nature*. 2015 Mar; 519 (7542): 237-41.
3. Grimes J, Desai S, Charter NW, Lodge J, Moita Santos R, Isidro-Llobet A, Mason 
AM, Wu Z, Wolfe III LA, Anantharaman L, Green A, Bridges AM, DalmasWilk AD,  
Brown AJ. MrgX2 is a promiscuous receptor for basic peptides causing mast 
cell pseudo-allergic and anaphylactoid reactions. *Pharmacology research &* 
*perspectives*. 2019 Dec; 7 (6): e00547.
4. Hennino A., Bérard F., Guillot I., Saad N., Rozières A., Nicolas J.-F. 
Pathophysiology of urticaria. *Clin. Rev. Allergy Immunol*. 2006; 30: 3–11.

5. Spoerl D, Nigolian H, Czarnetzki C, Harr T. Reclassifying Anaphylaxis to 
Neuromuscular Blocking Agents Based on the Presumed Patho-Mechanism: 
IgE-Mediated, Pharmacological Adverse Reaction or "Innate Hypersensitivity"? 
International *Journal of Molecular Sciences*. 2017 Jun; 18 (6): 1223.
6. Lafleur MA, Werner J, Fort M, Lobenhofer EK, Balazs M, Goyos A. MRGPRX2 
activation as a rapid, high-throughput mechanistic-based approach for 
detecting peptide-mediated human mast cell degranulation liabilities. *Journal* 
*of Immunotoxicology.* 2020 Jan 1; 17 (1): 110-21.
7. Motakis E, Guhl S, Ishizu Y, Itoh M, Kawaji H, de Hoon M, Lassmann T, Carninci 
P, Hayashizaki Y, Zuberbier T, Forrest AR, Babina M, *et al*. Redefinition of the 
human mast cell transcriptome by deep-CAGE sequencing. *Blood*. 2014; 123 
(17): e58–e67.

---

# Automating Cell-Based Bioassays on Tecan®’s Fluent Automation Workstation: Measuring β-Arrestin Recruitment of Cannabinoid Receptor 1

Bringing Higher Throughput, Improved Assay Consistency, and Reproducibility for Easy Implementation in Quality Testing Programs

Lucia Bruzzone, Ph.D.<sup>1</sup>, Venkatesh Chari, Ph.D.<sup>2</sup>, Neil Charter, Ph.D.<sup>2</sup> , and Gaurav Agrawal, Ph.D.<sup>2</sup>
<sup>1</sup>Tecan, Zurich, Switzerland, <sup>2</sup>Eurofins DiscoverX®, Fremont, CA 94538

## INTRODUCTION

Through early discovery, screening, characterization, and 
particularly for market release and commercialization of biologics, 
drug candidates require physiologically relevant, functional 
cell-based assays as characterization tools that attest to their 
therapeutic activity. This biological activity is a critical attribute 
for drug candidates, and can be accurately measured using 
cell-based potency assays. Moreover, analyzing potency is a key 
measurement for the final market release of a therapeutic product 
such as a biologic or a biosimilar.

Cell-based assays implemented in quality controlled (QC) lotrelease testing programs of biologics are often indicators of any 
altered potency or the presence of impurities in the manufactured 
drug products intended for clinical use. Hence, such programs 
demand highly consistent and reproducible cell-based assays. 
There are specific requirements associated with implementing 
cell-based potency assays, including cell culture and handling 
expertise, skilled pipetting, and precise plate layout. These 
abilities directly contribute to an operator’s ability to handle live 
cells and routinely run sensitive assays with a high degree of 
precision and accuracy. The operator is expected to execute the 
assay consistently and uniformly across experiments performed 
on different plates and across different days. QC testing typically 
includes multiple operators adding another variable in attaining 
consistent results. Thus, it can be challenging for biopharma, 
contract research organizations (CROs), and contract development 
and manufacturing organizations (CDMOs) to achieve desirable 
assay reproducibility, particularly with borderline assays with 
less reproducible results. For these reasons, bringing automation 
to cell-based assays can provide better assay consistency and

increased throughput that can result in decreased operational 
costs. Introducing assay automation can also bring consistent 
method execution when assays are transferred between different 
testing sites with automation capabilities.

This Application Note presents collaborative work of Eurofins 
DiscoverX, the leader in cell-based assays for drug discovery and 
development, and Tecan, a leader in robotic lab automation. It 
compiles and discusses the results obtained during the automation 
of a Eurofins DiscoverX cell-based bioassay on Tecan’s Fluent**®**
Automation Workstation. In summary, the automation produced 
highly consistent and reproducible results with minor optimization. 
In addition, the results obtained from this automation were 
comparable to those obtained by an experienced QC scientist.

## PathHunterbb-ARRESTIN ASSAY

β-Arrestin recruitment is considered a hallmark mechanism for 
GPCR activation upon ligand binding. The recruitment of β-arrestin 
activates signaling cascades to provide a non-amplified signal ideal 
for antagonist mode screening, studying ligand pharmacologies, 
and de-orphanizing GPCRs. Using PathHunter**®** β-arrestin cellbased assays, researchers can run cAMP or calcium second 
messenger signaling assays in parallel using the same β-arrestin 
cell line. The benefit is this allows for easily distinguishing ligand 
pharmacological differences for multiple ligand mechanismof-actions.

$$
\beta\mathsf{a-a e s s i n}
$$

PathHunter β-arrestin assays utilize the well-established 
Enzyme Fragment Complementation (EFC) technology 
(Figure 1. A.) pioneered and patented by Eurofins DiscoverX.

$$
\beta\mathsf r r r s t i\mathsf n
$$

---

This robust technology is based on the complementation of two 
inactive β-galactosidase (β-gal) enzyme fragments – a large 
enzyme acceptor (EA) fragment and a small enzyme donor (ED) 
fragment. Separately, these fragments are inactive, but they 
form an active enzyme that hydrolyzes a substrate to produce 
a chemiluminescence signal when combined. For this study, 
®
we selected the PathHunter CHO-K1 CNR1 Bioassay that 
measures β-arrestin recruitment (Figure 1. B.) to demonstrate the 
automation of the Eurofins DiscoverX® bioassays on the Tecan 
<sup>®</sup>
Fluent instrument. CNR1, a type 1 cannabinoid receptor (CB1), 
encoded by the CNR1 gene, is expressed in the peripheral and 
central nervous systems. Endocannabinoids and phytocannabinoids 
such as THC, an active ingredient of cannabis, are known to 
activate CB1. In this bioassay, CNR1 is fused in frame with an 
ED fragment called ProLink™ (PK). The CNR1-PK fusion protein 
is then stably co-expressed in cells stably expressing a fusion 
protein of β-arrestin and EA. Binding of a ligand (CP 55,940, 
a synthetic cannabinoid) to CNR1 results in its activation

$$
\beta\ r a r s s t n
$$

$$
\beta\ r\ e r s s t i
$$

stimulates recruitment of β-arrestin-EA fusion protein to CNR1-
PK. This recruitment forces the complementation of the two 
enzyme fragments that results in the formation of an active 
β-galactosidase enzyme. This interaction leads to an increase in 
enzyme activity that can be measured using chemiluminescent 
PathHunter detection reagents.

$$
\beta\mathsf{-a r r e s t i n\ E A}
$$

$$
\beta-g g a a c o s
$$

For this study, the bioassay format of the assay was performed. 
Eurofins DiscoverX bioassays are derived from qualified stable cell 
lines and provide a thaw-and-use approach that alleviates any 
assay variability associated with cells derived from continuous 
culture (passage-to-passage variability). Bioassays are further 
optimized with a ready-to-use protocol where cells are thawed and 
plated directly into assay plates, thus not requiring cell culture. 
These bioassays are ideal for comparability studies, QC lot release 
testing, and are critical for accelerating the drug release into the 
market.

**A**

**B**

**Figure 1. Eurofins DiscoverX’s EFC assays.** A. The EFC assay principle is based on two recombinant β-galactosidase (β-gal) enzyme fragments - an enzyme acceptor 
(EA) and an enzyme donor (ED). Separately, the fragments are inactive, but when combined, they form an active enzyme that hydrolyzes its substrate to produce a 
chemiluminescence signal. B. The PathHunter CNR1 β-arrestin recruitment assay principle: Binding of CP 55,940 results in CNR1 activation thereby stimulating recruitment 
of β-arrestin-EA fusion protein to CNR1-PK (noting PK corresponds to the ED of β-gal). The resulting complementation of the two enzyme fragments results in an active 
β-gal enzyme whose activity can be measured using chemiluminescent PathHunter detection reagents.

$$
(\beta-g\boldsymbol{a})
$$

---

## MATERIALS

## Assay Components

®
PathHunter CHO-K1 CNR1 Bioassay Kit (Cat. No. 93-
0959Y2-00120), containing ready-to-use PathHunter CHO-K1 
™
CNR1bioassay cells, 96-well assay plate, AssayComplete Cell 
Plating 2 Reagent (CP2), protein dilution buffer, reference agonist, 
and detection reagent, was used from Eurofins DiscoverX®.

## INSTRUMENTATION

## Automation Workstation

®
Experiments were conducted on a Tecan Fluent 780 system that 
includes an eight-channel Air Flexible Channel Arm™ (Air FCA), a 
Multiple Channel Arm™ (MCA) with an extended volume adapter 
for pipetting up to 500 μl with 96 tips in parallel, and a long 
Robotic Gripper Arm™ (RGA) to reach below the Dynamic Deck™ 
(Figure 2. A.). A vertical laminar flow HEPA hood with UV light 
(Bigneat) was integrated with the Fluent system to ensure a clean 
environment. Liquid handling tasks were conducted with the Air 
FCA using 1000 μL and 200 μL filtered disposable tips, and with 
the MCA used150 μL filtered disposable tips. Sterile 15 mL Falcon™ 
tubes and 2 mL Eppendorf tubes were placed into tube runners for 
pipetting into sterile 96-well white, clear flat-bottom, TC-treated 
microplates with lid, and 96-well green V-bottom microplates 
(Eurofins DiscoverX, Cat. No. 92-0011). A Cytomat™ 2 C450-LiN

$$
\mathsf{A r m}^{\mathsf{T M}}
$$

$$
1 0 0 0 \mu \mathrm {L}
$$

$$
2 0 0 \mu \mathrm {L}
$$

$$
\ a|c n^{M}
$$

Automated Incubator with two stackers (for 21 and 10 microplates, 
respectively) was integrated with the Fluent system; enabling the 
storage and incubation of CHO-K1 cells in microplates during the 
48-hour incubation period with the agonist CP 55,940 (Figure 
2. B.). The automated incubator parameters were set at 37°C 
and 5% CO₂ to provide an ideal and stable environment for the 
cryopreserved ready-to-use cells.

$$
37\,^{\circ}\mathrm{C}
$$

$$
\mathrm{C O}_{2}
$$

Individual scripts were developed using the FluentControl™ 
software for cell seeding on Day 1 and sample preparation of 
the agonist’s serial dilutions with the subsequent addition of the 
detection reagents on Day 3. The chemiluminescence signal was 
®
measured with Tecan’s Spark multimode reader.

**Figure 2. Tecan’s Fluent Automation system.** A. The Fluent Automation Workstation used to automate the PathHunter CHO-K1 CNR1 bioassay, and B. the Cytomat 2 
C450-LiN Automated Incubator that was integrated with the Fluent system.

---

## METHODS

Automation and manual experiments were performed 
®
independently. The protocol for experiments run on the Fluent
automation system is outlined in the protocol schematic (Figure 
3.). For manually performed experiments, the protocol in the 
bioassay user manual was followed.

## Cell Culture

®
The PathHunter CHO-K1 CNR1 Bioassay cells were manually 
™
thawed and plated in AssayComplete Cell Plating 2 Reagent (CP2) 
at 5,000 cells-per-well in a 96-well plate and incubated overnight 
at 37°C and 5% CO₂.

$$
37\,^{\circ}\mathrm{C}
$$

$$
\mathrm{C0}_{2}.
$$

## Agonist

A 10 mM stock of CP 55,940 was prepared in DMSO as detailed in 
the user manual. An intermediate stock of 100 µM CP 55,940 was 
prepared in DMSO. Using this intermediate stock, an 11-point 1:3 
dilution series of CP 55,940 was prepared with a top dose of 5 µM 
at 5X using CP2 as the diluent. 20 µL of the above serial dilutions 
were subsequently added to cells plated in 80 µL CP2 to obtain a 
final dose of 1 µM of CP 55,940 at 1X.

$$
1 0 0 \mu \mathrm {M}
$$

$$
\mathrm{C P}
$$

$$
5 \mu M
$$

$$
2 0 \mu \mathrm {L}
$$

$$
8 0 \mu \mathrm {L} \mathrm {C P 2}
$$

$$
1 \mu M
$$

## PathHunter β-Arrestin Assay

PathHunter CHO-K1 CNR1 bioassay cells were treated with 
increasing concentrations of CP 55,940 for 120 minutes at 37°C 
and 5% CO₂. Activation of a single GPCR stimulates the binding of 
arrestin, forcing complementation of the two enzyme fragments 
(ED and EA) fused with the GPCR or β-arrestin, respectively. 
The resultant increase in β-gal enzyme activity is measured 
by the addition of the chemiluminescent PathHunter Bioassay 
Detection Kit (Cat. No. 93-0933). Increased complementation of 
the two β-gal fusion subunits is directly proportional to higher 
chemiluminescent signal.

$$
\mathrm {C O} _ {2}.
$$

$$
37\,^{\circ}\mathrm{C}
$$

$$
\beta-g a l
$$

$$
\beta-g a l
$$

## PROTOCOL SCHEMATIC

**Figure 3. Protocol schematic for PathHunter CHO-K1 CNR1 Bioassay performed** 
**on Tecan’s Fluent 780 System – steps highlighted in blue required minimal** 
**human intervention.**

---

## RESULTS

## Automated

Inter-day results show comparable EC₅₀ values across four 
independent experiments performed on four individual days with 
an average relative standard deviation (%RSD) of 13.5% (Figure 4.). 
Each experiment was performed in triplicate, and each replicate is 
plotted as a separate curve as shown below in Figure 4. A. All three 
curves show a high degree of overlap for every experiment, thereby 
demonstrating high intra-day intermediate precision.

$$
\mathrm{E C}_{50}
$$

In order to evaluate inter-day replicate consistency, the raw 
luminescence data from all four experiments were analyzed. 
As shown in Figure 4. C., the replicates for each data-point are 
visibly tight, with an average %RSD of less than 5%. These results 
demonstrate a high inter-day replicate consistency.

**Day 1**

**B**

| Day | Replicate | EC50 | Mean | %RSD | Mean EC50 | %RSD(4 days) |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | 1 | 1.10E-08 | 1.37E-08 | 16.90% | 1.30E-08 | 13.50% |
| 1 | 2 | 1.50E-08 | 1.37E-08 | 16.90% | 1.30E-08 | 13.50% |
| 1 | 3 | 1.50E-08 | 1.37E-08 | 16.90% | 1.30E-08 | 13.50% |
| 2 | 1 | 1.50E-08 | 1.43E-08 | 1.30% | 1.30E-08 | 13.50% |
| 2 | 2 | 1.20E-08 | 1.43E-08 | 1.30% | 1.30E-08 | 13.50% |
| 2 | 3 | 1.60E-08 | 1.43E-08 | 1.30% | 1.30E-08 | 13.50% |
| 3 | 1 | 1.10E-08 | 1.23E-08 | 9.40% | 1.30E-08 | 13.50% |
| 3 | 2 | 1.30E-08 | 1.23E-08 | 9.40% | 1.30E-08 | 13.50% |
| 3 | 3 | 1.30E-08 | 1.23E-08 | 9.40% | 1.30E-08 | 13.50% |
| 4 | 1 | 9.30E-09 | 1.04E-08 | 13.40% | 1.30E-08 | 13.50% |
| 4 | 2 | 1.20E-08 | 1.04E-08 | 13.40% | 1.30E-08 | 13.50% |
| 4 | 3 | 1.00E-08 | 1.04E-08 | 13.40% | 1.30E-08 | 13.50% |

**C**

| [CP55,940],M | Mean RLUs(n=3) | %RSD |
| --- | --- | --- |
| 0 | 3.00E+05 | 4.30% |
| 1.70E-11 | 3.00E+05 | 4.50% |
| 5.10E-11 | 3.00E+05 | 2.00% |
| 1.50E-10 | 3.20E+05 | 4.50% |
| 4.60E-10 | 3.90E+05 | 3.40% |
| 1.40E-09 | 6.30E+05 | 5.80% |
| 4.10E-09 | 1.40E+06 | 9.90% |
| 1.20E-08 | 2.60E+06 | 6.70% |
| 3.70E-08 | 4.20E+06 | 2.80% |
| 1.10E-07 | 5.00E+06 | 3.40% |
| 3.30E-07 | 5.30E+06 | 4.80% |
|  | Avg. %RSD= | 4.70% |

$$
\mathrm{E C}_{50}
$$

$$
\mathrm{M e a n\ E C}_{_{50}}
$$

**Day 2**

| Mean RLUs(n=3) | %RSD |
| --- | --- |
| 3.00E+05 | 5.80% |
| 3.00E+05 | 5.00% |
| 3.00E+05 | 3.30% |
| 3.30E+05 | 4.80% |
| 3.90E+05 | 4.00% |
| 7.00E+05 | 6.00% |
| 1.60E+06 | 8.40% |
| 2.70E+06 | 6.00% |
| 3.90E+06 | 3.70% |
| 5.00E+06 | 3.00% |
| 5.10E+06 | 4.40% |
| Avg. %RSD= | 3.30% |

**Day 3**

| Mean RLUs(n=3) | %RSD |
| --- | --- |
| 2.80E+05 | 4.40% |
| 2.70E+05 | 0.60% |
| 2.80E+05 | 4.30% |
| 3.10E+05 | 2.30% |
| 3.90E+05 | 2.20% |
| 7.10E+05 | 6.10% |
| 1.50E+06 | 4.10% |
| 3.00E+06 | 1.00% |
| 4.20E+06 | 2.20% |
| 5.00E+06 | 4.50% |
| 5.40E+06 | 3.40% |
| Avg. %RSD= | 3.20% |

**Day 4**

| Mean RLUs(n=3) | %RSD |
| --- | --- |
| 2.80E+05 | 4.30% |
| 2.60E+05 | 5.00% |
| 2.70E+05 | 2.20% |
| 3.10E+05 | 1.80% |
| 4.20E+05 | 4.10% |
| 8.30E+05 | 9.90% |
| 1.90E+06 | 11.00% |
| 3.80E+06 | 5.20% |
| 5.30E+06 | 5.80% |
| 6.60E+06 | 1.70% |
| 6.40E+06 | 1.00% |
| Avg. %RSD= | 4.90% |

**Figure 4. Results from the Tecan Fluent® 780 Automation Workstation.** A. Dose response curves for CP 55,940 demonstrating β-arrestin recruitment in a dosedependent manner for four days. B. Table summarizing EC₅₀ values and %RSD for each day; and mean EC₅₀ and %RSD across four days. C. Table illustrating %RSD of 
raw RLUs for each day. RLU = Relative Luminescence Units

$$
\mathrm{E C}_{50}
$$

$$
\ {mathsf E C}_{50}
$$

---

## MANUAL

To compare assay consistency and reproducibility, the same set 
of experiments were performed manually by a QC scientist with 
more than six years of experience running similar assays. These 
experiments were run across three separate days in triplicate. The 
team plotted data of each replicate per experiment as an individual 
curve (Figure 5.). Data analysis of the assays performed revealed

The assay design of the CNR1 bioassay used in this study was 
automation-friendly and performed optimally out-of-the-box 
with minimal optimization needed for running on Tecan’s Fluent 
system. Overall, analyzing the data obtained from automation and

## CONCLUSION

The work described in this Application Note demonstrates that 
automation of Eurofins DiscoverX®’s cell-based assays can 
consistently produce reproducible results, which is key for cellbased potency assays implemented in QC lot-release programs. 
The results from the assays run using automation were remarkably 
similar compared to the manually executed assays run by an 
experienced QC scientist with several years of experience. 
Automating cell-based potency assays in quality testing programs 
can play a significant role in streamlining assay workflows,

that the results obtained were very close to the automation results 
with the %RSD for each day being below 20%. Overall, the interday intermediate precision for the manually performed data was 
17.5% that is slightly lower than that obtained from the Fluent® 
system (13.5%).

| Day | Replicate | EC50 | Mean EC50 | %RSD | Mean EC50(3 days) | %RSD(3 days) |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | 1 | 1.80E-08 | 1.77E-08 | 3.30% | 1.47E-08 | 17.50% |
| 1 | 2 | 1.70E-08 | 1.77E-08 | 3.30% | 1.47E-08 | 17.50% |
| 1 | 3 | 1.80E-08 | 1.77E-08 | 3.30% | 1.47E-08 | 17.50% |
| 2 | 1 | 1.50E-08 | 1.35E-08 | 1.30% | 1.47E-08 | 17.50% |
| 2 | 2 | 1.20E-08 | 1.35E-08 | 1.30% | 1.47E-08 | 17.50% |
| 2 | 3 | 1.30E-08 | 1.35E-08 | 1.30% | 1.47E-08 | 17.50% |
| 3 | 1 | 1.40E-08 | 1.30E-08 | 7.70% | 1.47E-08 | 17.50% |
| 3 | 2 | 1.20E-08 | 1.30E-08 | 7.70% | 1.47E-08 | 17.50% |
| 3 | 3 | 1.30E-08 | 1.30E-08 | 7.70% | 1.47E-08 | 17.50% |

$$
\mathrm{E C}_{50}
$$

$$
\mathrm{E C}_{50}
$$

$$
\mathrm{E C}_{50}
$$

**Figure 5. Results from manual experiments.** A. Dose response curves for CP 55,940 
demonstrating β-arrestin recruitment in a dose-dependent manner for three days. 
B. Table summarizing EC₅₀ values and %RSD for each day; and mean EC₅₀ and %RSD 
across three days.

$$
\mathrm{E C}_{50}
$$

$$
\mathrm{E C}_{50}
$$

manually performed experiments show comparable results. Thus, 
assay automation can help ease congested QC testing pipelines 
that often result in delayed market release of critical drugs by 
sharing the workload without compromising the quality of results.

maintaining assay consistency, and ultimately saving considerable 
time and cost by dramatically reducing the handling of repetitive 
tasks and increasing assay throughput. This automation is 
particularly helpful when operators are running several assay 
plates with complex plate layouts. Overall, automating assay is 
key in circumventing challenges and minimizing day-to-day and 
operator-to-operator differences producing high-quality data.

For research use only. Not for use in diagnostic procedures.

For more information on bioassays, visit DiscoverX.com/bioassays.

---

# WHITE PAPER The New World of GPCR Allosteric Modulation: Another Shot on Goal

## Authors

Terry Kenakin, Ph.D., Department of Pharmacology, University of 
North Carolina School of Medicine

Neil Charter, Ph.D., Eurofins DiscoverX®

Carsten Brock, Ph.D., Eurofins Cerep, Eurofins Discovery
Thierry Jolas, Ph.D., Eurofins Discovery

Although historically GPCRs have been a rich source of new drug 
molecules, the discovery of unique drug types for this target class 
has waned in the last 3 decades. In the same time frame, the 
emergence of functional screening and the appreciation of the 
allosteric nature of GPCRs has revitalized the field and led to an 
explosion of activity that has transformed GPCR discovery.

Thus allosteric screening and development are seen as another 
shot on goal for many mined out and/or intractable GPCR targets. 
It is useful to consider the particular mechanistic interactions of 
allosteric ligands with receptors, the unique molecules that emerge 
from these interactions, and finally the key role of pharmacological 
assays in their discovery and development.

## THE EMERGENCE OF RECEPTOR ALLOSTERY IN PHARMACOLOGY

**The emergence of allostery in pharmacology sup-**
**ports the notion that whether a discovery program** 
**seeks an allosteric ligand or not, it is increasingly** 
**likely that allosteric hits and leads will emerge** 
**from functional screening campaigns. Thus it is** 
**incumbent upon pharmacologists to at least be** 
**aware of how to study these unique molecules and** 
**develop their full potential in discovery programs.**

The study of allosterism was pioneered with enzymes, but the 
pursuit of this mechanism for receptors has lagged behind. 
One important reason for this is the historical reliance of highthroughput screening on basically orthosteric assays (binding). In 
all probability, many allosteric ligands were not detected leading 
to a paucity of these molecules for development. The advent of 
functional assay screening, where allosteric effects are readily 
seen, has led to a precipitous increase in the publication of papers 
on receptor allosterism beginning in 1992 and continuing through 
to the present day.  With this trend has come a resurgence of 
interest in allosteric receptor ligands (and its offshoot, biased 
receptor signaling) and an increase in the number of molecules 
available to study allosteric receptor mechanisms.

## ALLOSTERIC MECHANISMS AND NEW DRUG DISCOVERY

GPCRs are nature’s prototypical allosteric protein (everything 
the receptor does is allosteric) since these proteins simply act 
as energy conduits for extracellular molecules and intracellular 
signaling proteins. The molecular dynamics of the receptor 
suggests that they can form a multitude of active and inactive 
conformations through selective stabilization. These concepts also 
support the notion that efficacy and affinity are thermodynamically 
linked since binding is not a passive process.  Therefore, the pursuit 
of possible signaling effects (pluridimensional efficacy¹) for all 
molecules detected in a screening assay could lead to unique 
therapeutic profiles.  In this regard, the assay assumes a key role in 
GPCR drug discovery.

## WHAT MAKES ALLOSTERIC MOLECULES SO SPECIAL?

There are at least three unique features of allosteric ligandreceptor interactions that lead to potentially valuable therapeutic 
behaviors².

• Allosterism can change the very nature of receptors (efficacy, 
functional signaling)

• The fact that allosteric molecules bind to separate sites on the 
receptor allows them to modulate or potentiate receptor effects 
(re-set physiology)

• Allosteric effects are probe dependent, allowing precise 
discrimination between endogenous molecules and signaling 
proteins (effects can be molecule-specific)

---

## ALLOSTERIC MODULATION CAN CREATE A NEW RECEPTOR

Allosteric modulators have the unique capability of allowing the 
natural endogenous agonist to concomitantly bind to the receptor 
and, therefore, they can potentiate physiological response. An 
active research area in the pharmacology of drug discovery is 
the search for Positive Allosteric Modulators (PAMs) to revitalize 
failing physiological systems in disease. PAMs possess unique 
advantages over orthosteric agonists in that the systems affected 
respond only when activated. For example, a current therapy 
for diabetes employs GLP-1 agonists that potentiate the release 
of insulin. However, a limitation of this approach is that the 
constant activation of the GLP-1 receptors with an agonist 
causes intractable nausea in patients. In contrast, a GLP-1 PAM 
potentiates GLP-1 enhancement of insulin release in diabetes only 
when the system is activated, such as when the patient has a meal 
but otherwise produces no effect.  This intermittent activation 
will eliminate the current GLP-1 based nausea seen with agonist

therapies for diabetes. The potentiation of muscarinic response by 
the PAM BQCA is illustrated with the Eurofins Discovery services, 
M1 receptor β-arrestin recruitment assay shown in Figure 1. 
Patterns of concentration-response curves such as these can be fit 
3-5
to the functional allosteric model to yield universal parameters 
that characterize allosteric function in a system-independent 
manner.

Such analyses yield parameters for modification of endogenous 
agonist affinity (through a cooperativity term α) and efficacy 
(through a cooperativity term β) to guide medicinal chemistry 
structure-activity studies.  For the data shown in Figure 1, the 
cooperativity factor for BQCA effect is 24 indicating that BQCA 
will produce a 24-fold potentiation of acetylcholine β-arrestin 
response in all systems.

**CHRM1**
**BQCA**
 **Arrestin Recruitment**

**Black / Leff Operational Model**

**Figure 1. Concentration-response curves to acetylcholine for the receptor recruitment of** β**-arrestin in the absence and presence of a range of concentrations** 
**of the PAM BQCA.** Concentration-dependent shifts of the CR curves to the left indicate allosteric potentiation of acetylcholine response. Fitting these curves to the 
functional allosteric model, schematically shown on the right, yields universal parameters quantifying the allosteric effect in terms of the co-operative effects on 
acetylcholine binding and efficacy. Data from Eurofins Discovery.

---

## ALLOSTERIC MODULATORS RE-SET TARGET PHYSIOLOGY

Another feature of allosteric molecules is that they can produce a 
redefinition of target responsiveness including complete inhibition, 
reduced sensitivity, increased sensitivity, and full activation. 
Figure 2 shows the effect of the negative allosteric modulator

(NAM) UCB35625 on the binding of the chemokine CCL3. It can 
be seen that the receptor can still bind CCL3, but with a 2-fold 
reduced affinity; such fine-tuning of responsiveness can be useful 
therapeutically.

**Maximal Effect of UCB35625**

## Binding [ 125 I]-CCL3

**Figure 2. Displacement binding of [**<sup>125</sup>**I]-CCL3 by non-radioactive CCL3 (blue circles) and the allosteric NAM UCB35625 (green circles).** It can be seen that the maximal 
concentrations of UCB35625 do not displace bound [<sup>125</sup>I]-CCL3, but rather re-set the receptor affinity for the radioligand by a factor of 2. Data are redrawn from <sup>6</sup>.

---

## ALLOSTERIC MODULATORS PRACTICE PROBE-DEPENDENCE

Finally, allosterism is probe dependent, that is, an allosteric 
molecule can produce an effect for one probe of the receptor 
(i.e. agonist, radioligand), but have no effect on another 
(a different agonist, radioligand, etc.). This can be extremely 
valuable therapeutically as shown in Figure 3 for the allosteric 
HIV-1 entry inhibitors TAK779 and TAK652 in the prevention 
of HIV-1 infection and AIDS. Specifically, these molecules  
demonstrate probe dependence in the form of a 10-fold 
differential activity for blocking HIV-1 vs. the blockade of a 
beneficial effect for AIDS patients (CCL3-L1-induced CCR5 
receptor internalization found to be correlated with increased 
survival⁷).  It can be seen that while TAK779 is 10-fold more 
potent at blocking CCR5 internalization over HIV-1 infection, 
TAK652 reverses this profile to yield a beneficial 10-fold margin 
for blockade of HIV-1 infection while sparing the beneficial

CCL3L1-induced internalization effect⁸. In general, the judicious 
application of different target probes in assays can uncover such 
therapeutically relevant probe dependence.

**In general, allosteric modulators can re-format** 
**receptor sensitivity (including the revitalization** 
**of failing systems) and alter responses to** 
**agonists. They can also make targets “smart” in** 
**that they will discern different activators and** 
**respond to some but not others. These effects** 
**re-define the drug discovery playing field.**

Muniz-Medina *et. al.* (2009) *Mol. Pharmacol.* 75:490-501

**Figure 3. Inhibition of HIV-1 infection of HOS cells (green circles, dotted line curves) and blockade of CCL3L1-induced CCR5 internalization (blue circles, solid line** 
**curves).** Data are shown for two allosteric inhibitors of HIV-1 entry, TAK779 and TAK652-redrawn from <sup>8</sup>.

---

# NEW PLAYERS AS DRUG TARGET MOLECULES IN PHARMACOLOGICAL THERAPY

Allosteric mechanisms have spawned new types of molecules 
for therapeutic application. These new players can generally be 
classified as PAMs that increase an endogenous agonist response 
and thus can be used to augment failing physiology in disease. 
NAMs (Negative Allosteric Modulators) decrease endogenous 
agonism (and are basically antagonists, but with some very special 
qualities that differentiate them from standard competitive 
blockers. In addition, a special type of PAM that augments 
receptor-signaling protein interaction (and thus is an agonist) 
practices probe dependence for cytosolic signaling proteins; these 
are a new and important class of ligand referred to as the biased 
agonist. Considering two probes of the receptor as being two 
points of interaction with different cytosolic signaling proteins (i.e. 
G protein, and β-arrestin), biased agonists differentially activate 
one probe at the expense of another to produce a biased cell 
signal. These types of effects can be of enormous value in that 
beneficial signals can be emphasized (i.e. β-arrestin PTH response 
for osteoporosis⁹), deleterious signals can be de-emphasized 
(respiratory depression and addiction for opioids¹⁰) and deemphasized with blockade of natural activation of the same 
pathway (G protein-mediated vasoconstriction by angiotensin in 
q
heart failure¹¹). In addition, the editing of pleiotropic signaling may 
allow pursuit of previously forbidden drug targets for therapeutic

$$
\beta - a r r e s t i n)
$$

$$
\beta-r r e s t i n
$$

$$
\mathbb{G}_{\mathfrak{g}}
$$

advantage (i.e. κ-opioid receptors¹²).

Finally, the application of functional and binding assays may be 
used to identify a unique new class of antagonist, namely the 
PAM-Antagonist. These are a special subset of NAMs that actually 
become more potent upon activation of the functional system by 
the agonist allowing them to seek and destroy signaling agonistbound receptors¹³. PAM-Antagonists can reverse persistent 
pathological signaling (e.g. endothelin-based pre-eclampsia) 
and have extraordinarily high target coverage properties *in vivo*
(and long t for clearance) due to the cooperative binding with 
1/2
endogenous hormones and neurotransmitters as seen for the 
5-HT  receptor antiemetic palonosetron¹⁴). Figure 4 shows Eurofins 
3
Discovery services data used to characterize Org27569, a PAM 
Antagonist for the cannabinoid CB1 receptor⁵. The key to detecting 
these unique profiles is the orthogonal application of functional 
and binding assays.

$$
\ {{\sf t}}_{1/2}
$$

$$
\ \mathrm\ 5-\mathrm{H I}_{_3}
$$

Specifically, this behavior emanates from a positive a cooperativity 
to increase affinity and a fractional b activity to decrease efficacy. 
Thus, the presence of the agonist increases the affinity of the 
receptor for Org27659 that then becomes incapable of signaling 
once Org27659 is bound.

**Cannabinoid PAM-Antagonist ORG27569**

Binding of [ 3 H]CP55940 CP55940 Is Blocked Is Enhanced

**Figure 4. Opposing effects of the cannabinoid CB1 receptor PAM-Antagonist Org27659 on function (non-competitive blockade) and binding (increased agonist** 
**binding).** Data from Eurofins Discovery.

---

# PHARMACOLOGICAL ASSAYS AS THE WINDOW INTO PLURIDIMENSIONAL EFFICACY

The key to the discovery of ligand diversity is to have multiple 
views into pharmacological molecular activity. Classification of 
molecules into simple bins of agonist and antagonist has ceased 
to be relevant as molecules can be agonists for some signaling 
pathways and antagonists in others¹⁵. A more sophisticated 
classification system to characterize efficacy must be employed. 
For example, the multiple testing of ligand functional response 
yields textured patterns of ligand activity such as the display of 
µ-opioid signaling activity determined by clustering of data for 
16
sixteen opioid agonists in six functional assays shown in Figure 5. 
The hope is that such detailed profiling of new molecules will lead 
to more informed choices for compound progression.

$$
5^{16}.
$$

In general, the key to unlocking complex and potentially 
useful efficacy and affinity fingerprints in new molecules is 
the pharmacological assay. Data from multiple assays allows 
comparison to quantitative pharmacological models to yield 
universal and system-independent scales of activity for use in 
medicinal chemistry efforts to optimize activity.

**50% of new molecules in clinical testing fail due** 
**to lack of efficacy¹⁷. While some of this is due to** 
**lack of knowledge of what needs to be corrected** 
**in some diseases, some of this may also be due to** 
**the inadequate characterization of the efficacy of** 
**the candidate molecules put forward in the clinic.** 
**With more informed characterization of ligand** 
**efficacy fingerprints, perhaps better targeting** 
**for progression will result in a reduction in** 
**compound late-stage attrition.**

**Figure 5. Cluster analysis of sixteen** m**-opioid agonists in six different functional assays.** The gene cluster program GENE-E groups the agonists according to their 
Log(t/KA) values in each assay thereby grouping agonists according to their signaling profiles-redrawn from <sup>16</sup>.

---

## REFERENCES

1. Galandrin S, Bouvier M (2006) Distinct signaling profiles of beta1 and beta2 
adrenergic receptor ligands toward adenylyl cyclase and mitogen-activated 
protein kinase reveals the pluridimensionality of efficacy. *Mol Pharmacol*
70:1575–1584.
2. Kenakin, TP (2012) Biased signalling and allosteric machines: new vistas and 
challenges for drug discovery. Br. *J Pharmacol* 165: 1659-1669.
3. Kenakin T. (2005) New concepts in drug discovery: collateral efficacy and 
permissive antagonism. *Nat Rev Drug Discov* 4:919–927
4. Ehlert FJ (2005) Analysis of allosterism in functional assays. *J Pharmacol Exp* 
*Ther* 315:740–754
5. Price MR, Baillie GL, Thomas A, Stevenson LA, Easson M, Goodwin R, McLean 
A, McIntosh L, Goodwin G, Walker G, *et al.* (2005) Allosteric modulation of the 
cannabinoid CB1 receptor. *Mol Pharmacol* 68:1484–1495
6. Sabroe I,  Peck MJ, Van Keulen BJ, Jorritsma A, Simmons G, Clapham PR, 
(2000) A small molecule antagonist of chemokine receptors CCR1 and CCR3. *J* 
*Biol Chem.* 275:25985–25992.
7. Gonzalez E, Kulkarni H, Bolivar H, Mangano A, Sanchez R *et al.* (2005). The 
infl uence of CCL3L1 gene-containing segmental duplications on HIV-1/AIDS 
susceptibility. *Science* 307:14334–11440.
8. Muniz-Medina VM, Jones S, Maglich JM, Galardi C, Hollingsworth RE, 
Kazmierski WM *et al.* (2009). The relative activity of “function sparing” HIV-1 
entry inhibitors on viral entry and CCR5 internalization: is allosteric functional 
selectivity a valuable therapeutic property? *Mol Pharmacol* 75: 490–501.

receptor: a novel anabolic approach to increasing bone mass? Br *J Pharmacol*
164:59–67.
10. Raehal KM, Walker JK, and Bohn LM (2005) Morphine side effects in 
b-arrestin 2 knockout mice. *J Pharmacol* Exp Ther 314:1195–1201.
11. Violin JD, DeWire SM, Yamashita D, Rominger DH, Nguyen L, Schiller K, Whalen 
EJ, Gowen M, and Lark MW (2010) Selectively engaging b-arrestins at the 
angiotensin II type 1 receptor reduces blood pressure and increases cardiac 
performance. *J Pharmacol Exp Ther* 335:572–579.
12. White KL, Scopton AP, Rives ML, Bikbulatov RV, Polepally PR, Brown PJ, 
Kenakin T, Javitch JA, Zjawiony JK, and Roth BL (2014) Identification of novel 
functionally selective k-opioid receptor scaffolds. *Mol Pharmacol* 85:83–90.
13. Kenakin T, Strachan RT. (2018) PAM-Antagonists: A Better Way to 
Block Pathological Receptor Signaling? *Trends Pharmacol Sci.* S0165-
6147(18)30085-3.
14. Lummis, SCR, Thompson, AJ. (2013) Agonists and antagonists induce 
different palonosetron dissociation rates in 5-HT3A and 5-HT3AB receptors. 
*Neuropharmacol*. 73: 241-246
15. Kenakin T. (2008) Pharmacological Onomastics: What's in a name? Br *J* 
*Pharmacol* 153: 432-438.
16. Kenakin T (2015). New lives for seven transmembrane receptors as drug 
targets. *Trends Pharmacol Sci* 36:705–706.
17. Arrowsmith J. (2011) Trial watch: Phase II failures: 2008–2010. *Nat Rev Drug* 
*Discov* 10: 328–329.

9. Gesty-Palmer D and Luttrell LM (2011) “Biasing” the parathyroid hormone

## To learn more about Eurofins GPCR services visit here: eurofinsdiscoveryservices.com/gpcr

---

# The Human GPCRome Dendrogram and Assays for GPCR Targets

Eurofins DiscoverX® offers GPCR cell-based assays covering ~90% of the human GPCRome to meet your specific GPCR research and drug discovery & development needs. Using the dendrogram below for class A, B & C GPCRs, determine the availability of binding, second messenger activation, β-arrestin recruitment, and receptor internalization assays for your favorite GPCR target. Additional assays (or products) for GPCR targets not represented in the dendrogram are available at discoverx.com/gpcrs and through custom development requests via email at: customdevelopment@eurofins.com.

GPR109A GPR109B P2RY11 FFAR1 FFAR2 FFAR3 F2RL2 F2RL3 MRGPRX2MRGPRX4MRGPRX1MRGPRX3 MRGPRD UTR2 GPR31 OXER1 GPR81 MRGPRG MRGPRE MRGPRF P2RY8 F2R F2RL1 GPR55 GPR18 GPR20 GPR92 GPR35 OXGR1 GPR91 P2RY1 P2RY6 P2RY2 P2RY4 GPR132GPR65GPR4 GPR68PTAFR GPR34GPR171 GPR87P2RY14 P2RY12 P2RY13

Class A

Class B Class C

PTGER1 TBXA2RMAS1MAS1L PTGDR PTGER2 PTGER3PTGFR PTGER4PTGIR LGR4 LGR5 LGR6 LHCGRTSHR OPN5 RXFP1 RXFP2 FSHR

GPR27 GPR173 GPR85 OPN3

GPR62 GPR135 GPR88 GPBAR1

GPR78 GPR61 GPR150 GPR75 GPR26

ADRA2A ADRA2C

ADRA1A ADRA1B ADRA2B ADRA1D HRH2 HTR4 DRD2 DRD3

ADRB1 ADRB2 DRD4 ADRB3 HTR6 HTR1D HTR1E HTR1F

HTR1A HTR1B DRD1 DRD5 HTR5A HTR7 TAAR8 TAAR6 TAAR9 TAAR5 TAAR3 TAAR2 TAAR1 HTR2C HRH1 HTR2A HTR2B CHRM5 CHRM1 CHRM3 CHRM4 CHRM2 HRH4 HRH3 EDG3 EDG1 EDG5 EDG8 EDG6 EDG4 EDG2 EDG7 MC5R MC4R MC3R MC2R MC1R GPR6 GPR3 GPR12 CNR2 CNR1 GPR119 ADORA2B ADORA2A ADORA3 ADORA1 GPR52 GPR21 NTSR2 NTSR1 MLNR GHSR NMU2R NMU1R GPR39 TRHR GPR142 GPR139PPYR1NPY1R NPY5RPRLHRGPR83NPY2R NPFFR2 NPFFR1HCRTR2HCRTR1 TACR3 TACR1 TACR2 CCKBR CCKAR GPR45 GPR19 GPR120 GPR103 OXTR AVPR2 NPSR1 GPR63 AVPR1B AVPR1A GNRHR PROKR2 PROKR1

GLP2R CRHR2 GCGRGLP1R GHRHRPTHR2PTHR1GIPR GPR146 VIPR1SCTRVIPR2ADCYAP1R1 NMBR GRPR BRS3 EDNRB EDNRA GPR37L1 GPR37 GPR22 GPR176DARC

EBI2CYSLTR1 CYSLTR2 GPR17 GPR174 P2RY10 GPR23 LPAR6 GPR152 LTB4R C5AR1 C5L2 GPR1 LTB4R2 CMKLR1 GPR33 CRTH2 GPR32 C3AR1

FPR1 FPR2 FPR3 KISS1R GALR1 GALR2 GALR3 MCHR1 MCHR2 OPRL1 OPRK1 OPRD1 OPRM1 NPBWR1 NPBWR2 SSTR1 SSTR4 SSTR2 SSTR3 SSTR5 GPER RXFP3 AGTRL1 GPR25 RXFP4 GPR15 AGTR1 AGTR2 BDKRB1 BDKRB2 CXCR7 GPR182 XCR1 CCBP2 CX3CR1 CCRL2 CCR4 CCR8 CCR3 CCR1 CCR2 CXCR2 CXCR1 CCR5

CCR10 CXCR5 CXCR4 CXCR3 CCRL1 CXCR6 CCR6 CCR9 GPR161 CCR7 GPR84 GPR101 MTNR1A GPR50 GPR82 GPR151 MTNR1B GPR160 GPR141 GPR149 GPR148 GPR162 GPR153 CASR GABBR2 GRM5 GRM1 GPRC6A GRM3 GRM2 Functional & Binding GRM7 GRM6 GPCR Assays Available GABBR1 GRM8 GRM4 Binding CRHR1 CALCRL CALCR β-Arrestin Calcium cAMP Internalization GTPγS

---

## RESOURCES

## GPCR Families, Targets, and Couplings

**Class A GPCRs**

| Family | Target Gene ID(Common) | Coupling |  |
| --- | --- | --- | --- |
| 5-Hydroxytryptamine(Serotonin) | HTR1A(5HT1A) | G/Go |  |
| 5-Hydroxytryptamine(Serotonin) | HTR1B(5HT1B) | G/Go |  |
| 5-Hydroxytryptamine(Serotonin) | HTR1E(5HT1E) | G/Go |  |
| 5-Hydroxytryptamine(Serotonin) | HTR1F(5HT1F) | G/Go |  |
| 5-Hydroxytryptamine(Serotonin) | HTR2A(5HT2A) | Gq |  |
| 5-Hydroxytryptamine(Serotonin) | HTR2B(5HT2B) | Gq |  |
| 5-Hydroxytryptamine(Serotonin) | HTR2C(5HT2C) | Gq |  |
| 5-Hydroxytryptamine(Serotonin) | HTR5A(5HT5A) | G/Go |  |
| 5-Hydroxytryptamine(Serotonin) | HTR6(5HT6) | Gs |  |
| 5-Hydroxytryptamine(Serotonin) | HTR7A(5HT7A) | Gs |  |
| 5-Hydroxytryptamine(Serotonin) | HTR7B(5HT7B) | Gs |  |
| 5-Hydroxytryptamine(Serotonin) | HTR7D(5HT7D) | Gs |  |
| 5-Hydroxytryptamine(Serotonin) | Acetylcholine(Muscarinic) | CHRM1(M1) | Gq |
| 5-Hydroxytryptamine(Serotonin) | Acetylcholine(Muscarinic) | CHRM2(M2) | G/Go |
| CHRM3(M3) | Acetylcholine(Muscarinic) | Gq |  |
| CHRM4(M4) | Acetylcholine(Muscarinic) | Gs&amp;t G/Go |  |
| CHRM5(M5) | Acetylcholine(Muscarinic) | Gq |  |
| Adenosine | ADORA1(A1) | G/Go |  |
| Adenosine | ADORA2A(A2A) | Gs&amp;t Gq |  |
| Adenosine | ADORA2B(A2B) | Gs |  |
| Adenosine | ADORA3(A3) | G/Go |  |
| Adrenoceptor | ADRA1A(α1A) | Gq |  |
| Adrenoceptor | ADRA1B(α1B) | Gq |  |
| Adrenoceptor | ADRA1D(α1D) | Gq |  |
| Adrenoceptor | ADRA2A(α2A) | G/Go |  |
| Adrenoceptor | ADRA2B(α2B) | G/Go |  |
| Adrenoceptor | ADRA2C(α2C) | G/Go |  |
| Adrenoceptor | ADRB1(β1AR) | Gs |  |
| Adrenoceptor | ADRB2(β2AR) | Gs |  |
| ADRB3(β3AR) | Gs |  |  |
| Angiotensin | AGTR1(AT1) | Gq |  |
| Apelin | AGTRL1(APJ) | G/Go |  |
| Bile Acid(GPBA) | GPBAR1(GPR131) | Gs |  |
| Bombesin | BRS3(BB3) | Gq |  |
| Bombesin | GRPR(BB2) | Gq |  |
| Bombesin | NMBR(BB1) | Gq |  |
| Bradykinin | BDKRB1(B1) | Gq |  |
| Bradykinin | BDKRB2(B2) | Gq |  |
| Cannabinoid | CNR1(CB1) | G/Go |  |
| Cannabinoid | CNR2(CB2) | G/Go |  |
| Chemerin | CMKLR1(ChemR23) | G/Go |  |

$$
\mathrm{{G\ G_{i}/G_{o}}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathbf{\widehat{G}_{q}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathrm{G_{i}/G_{o}}
$$

$$
\mathfrak{G}_{s}
$$

$$
\mathtt{G}_{\mathrm{s}}
$$

$$
\mathtt{G}_{\mathrm{s}}
$$

$$
\mathtt{G}_{\mathrm{s}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathbf{\underline{{G}}_{\mathbf{q}}}
$$

$$
6{s\ }6{/}66{.}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathrm{{G6G_{o}}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathrm {G} _ {\mathrm {i}} / \mathrm {G} _ {\mathrm {o}}
$$

$$
\mathrm{G_{i}/G_{o}}
$$

$$
\mathrm{6_{{i}/6_{o}}}
$$

$$
\mathtt{G}_{\mathrm{s}}
$$

$$
\mathrm{{\_{S}}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
G _ {i} / G _ {o}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathrm {G} _ {\mathrm {i}} / \mathrm {G} _ {\mathrm {o}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathrm {G} _ {\mathrm {i}} / \mathrm {G} _ {\mathrm {o}}
$$

| Family | Target Gene ID(Common) | Coupling |
| --- | --- | --- |
| Chemokine | CCR1 | G1/G0 |
| Chemokine | CCR2 | G1/G0 |
| Chemokine | CCR3 | G1/G0 |
| Chemokine | CCR4 | G1/G0 |
| Chemokine | CCR5 | G1/G0 |
| Chemokine | CCR6 | G1/G0 |
| Chemokine | CCR7 | G1/G0 |
| Chemokine | CCR8 | G1/G0 |
| Chemokine | CCR9 | G1/G0 |
| Chemokine | CCR10 | G1/G0 |
| Chemokine | CCRL1 |  |
| Chemokine | CX3CR1 | G1/G0 |
| Chemokine | CXCR1(IL8RA) | G1/G0 |
| Chemokine | CXCR2(IL8RB) | G1/G0 |
| Chemokine | CXCR3 | G1/G0 |
| Chemokine | CXCR4 | G1/G0 |
| Chemokine | CXCR5 | G1/G0 |
| Chemokine | CXCR6 | G1/G0 |
| Chemokine | CXCR7(CMKOR1) |  |
| Chemokine | XCR1(XC1) | G1/G0 |
| Cholecystokinin | CCKAR(CCK1) | Gq |
| Cholecystokinin | CCKBR(CCK2) | Gq |
| Complement Peptide (Anaphylatoxin) | C3AR1(C3A) | G1/G0 |
| Complement Peptide (Anaphylatoxin) | C5AR1(C5A) | G1/G0 |
| Complement Peptide (Anaphylatoxin) | C5AR2(C5L2/GPR77) |  |
| Dopamine | DRD1(D1) | Gs |
| Dopamine | DRD2(D2) | G1/G0 |
| Dopamine | DRD3(D3) | G1/G0 |
| Dopamine | DRD4(D4) | G1/G0 |
| Dopamine | DRD5(D5) | Gs |
| Endothelin | EDNRA(ETA) | Gq |
| Endothelin | EDNRB(ETB) | Gs&amp;t G1/G0 |
| Formylpeptide | FPR1(FPR) | G1/G0 |
| Formylpeptide | FPR2(FPRL1/ALX) | G1/G0 |
| Formylpeptide | FPR3(FPRL2) | G1/G0 |
| Free Fatty Acid | FFAR1(FFA1/GPR40) | Gq |
| Free Fatty Acid | FFAR2(FFA2/GPR43) | Gq&amp;t G1/G0 |
| Free Fatty Acid | FFAR3(FFA3/GPR41) | G1/G0 |
| Free Fatty Acid | FFAR4(FFA4/GPR120) |  |
| Galanin | GALR1(GAL1) | G1/G0 |
| Galanin | GALR2(GAL2) | Gq&amp;t Gs |

**Class A GPCRs**

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathrm{{6}_{i/6_{o}}}
$$

$$
\ 6\!{\hat{G}}_{0}
$$

$$
\mathrm{G_{i}/G_{o}}
$$

$$
\ 66_{0}
$$

$$
\mathrm{G_{i}/G_{o}}
$$

$$
\mathrm{G_{i}/G_{o}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathrm {G} _ {\mathrm {i}} / \mathrm {G} _ {\mathrm {o}}
$$

$$
\mathrm{6i/6_{0}}
$$

$$
\mathrm{G_{i}/G_{o}}
$$

$$
\ 6\!{\hat{G}}_{0}
$$

$$
\mathrm {G} _ {\mathrm {i}} / \mathrm {G} _ {\mathrm {o}}
$$

$$
\mathrm {G} _ {\mathrm {i}} / \mathrm {G} _ {\mathrm {o}}
$$

---

**Class A GPCRs**

| Family | Target Gene ID(Common) | Coupling |  |
| --- | --- | --- | --- |
| Ghrelin | GHSR(GRLN) | $G_{q}$ |  |
| Glycoprotein Hormone | FSHR(FSH) | $G_{s}$ |  |
| Glycoprotein Hormone | LHCGR(LH) | $G_{s}$ |  |
| Glycoprotein Hormone | TSHR(TSH) | $G_{s} \text{ &amp; } G_{q}$ |  |
| Gonadotrophin-Releasing Hormone | GNRHR(GnRH) | $G_{q}$ |  |
| Histamine | HRH1(H1) | $G_{q}$ |  |
| Histamine | HRH2(H2) | $G_{q} \text{ &amp; } G_{s}$ |  |
| Histamine | HRH3(H3) | $G_{s}/G_{o}$ |  |
| Histamine | HRH4(H4) | $G_{s}/G_{o}$ |  |
| Hydroxycarboxylic Acid | HCAR1(HCA1/GPR81) | $G_{s}/G_{o}$ |  |
| Hydroxycarboxylic Acid | HCAR2(HCA2/GPR109A/HM74A/NIC1) | $G_{s}/G_{o}$ |  |
| Hydroxycarboxylic Acid | HCAR3(HCA3/GPR109B/NIC2) | $G_{s}/G_{o}$ |  |
| Kisspeptin | KISS1R(GPR54) | $G_{q}$ |  |
| Leukotriene | CYSLTR2(LTC4) | $G_{q}$ |  |
| Leukotriene | LTB4R(BLT1) | $G_{i}/G_{o} \text{ &amp; } G_{q}$ |  |
| Leukotriene | OXER1(GPR170) | $G_{i}/G_{o}$ |  |
| Lysophospholipid(LPA &amp; S1P) | LPAR1(LPA1/EDG2) | $G_{i}/G_{o}$ |  |
| Lysophospholipid(LPA &amp; S1P) | LPAR2(LPA2/EDG4) | $G_{q}$ |  |
| Lysophospholipid(LPA &amp; S1P) | LPAR3(LPA3/EDG7) | $G_{q}$ |  |
| Lysophospholipid(LPA &amp; S1P) | LPAR5(LPA5/GPR92) | $G_{q}$ |  |
| Lysophospholipid(LPA &amp; S1P) | S1PR1(S1P1/EDG1) | $G_{i}/G_{o}$ |  |
| Lysophospholipid(LPA &amp; S1P) | S1PR2(S1P2/EDG5) | $G_{s}$ |  |
| Lysophospholipid(LPA &amp; S1P) | S1PR3(S1P3/EDG3) | $G_{q}$ |  |
| Lysophospholipid(LPA &amp; S1P) | S1PR4(S1P4/EDG6) | $G_{q}$ |  |
| Lysophospholipid(LPA &amp; S1P) | S1PR5(S1P5/EDG8) | $G_{i}/G_{o}$ |  |
| Lysophospholipid(LPA &amp; S1P) | Melanin-Concentrating Hormone | MCHR1(MCH1) | $G_{i}/G_{o}$ |
| MCHR2(MCH2) | Melanin-Concentrating Hormone | $G_{q}$ |  |
| Melanocortin | MC1R(MC1) | $G_{s}$ |  |
| Melanocortin | MC3R(MC3) | $G_{s}$ |  |
| Melanocortin | MC4R(MC4) | $G_{s}$ |  |
| Melanocortin | MC5R(MC5) | $G_{s}$ |  |
| Melatonin | MTNR1A(MT1) | $G_{i}/G_{o}$ |  |
| Melatonin | MTNR1B(MT2) | $G_{i}/G_{o}$ |  |
| Motilin | MLNR(Motilin) | $G_{q}$ |  |
| Neuromedin U | NMU1R(NMU1) | $G_{q}$ |  |
| Neuropeptide B &amp; W | NPBWR1(GPR7) | $G_{i}/G_{o}$ |  |
| Neuropeptide B &amp; W | NPBWR2(GPR8) | $G_{i}/G_{o}$ |  |
| Neuropeptide FF | NPFFR1(GPR147) | $G_{i}/G_{o}$ |  |
| Neuropeptide S | NPSR1(NPS) | $G_{q}$ |  |

$$
\mathbf{\widehat{G}}_{\mathbf{q}}
$$

$$
\mathfrak{G}_{s}
$$

$$
\mathfrak{G}_{s}
$$

$$
6texts{{s s}\\ }tt G G{}ub{ubtextub}
$$

$$
\mathsf{G}_{\mathfrak{q}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
G _ {q} \& t G _ {s}
$$

$$
\mathrm {G} _ {\mathrm {i}} / \mathrm {G} _ {\mathrm {o}}
$$

$$
\mathrm{{G i{/6\partial}_{0}}}
$$

$$
\mathrm {G} _ {\mathrm {i}} / \mathrm {G} _ {\mathrm {o}}
$$

$$
\mathrm{G_{i}/G_{o}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
G _ {i} / G _ {o} \& t G _ {q}
$$

$$
\mathrm{G_{i}/G_{o}}
$$

$$
G _ {i} / G _ {0}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathrm{{G\ G_{i}/G_{o}}}
$$

$$
\mathfrak{G}_{\mathrm{s}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathbf{\hat{G}_{q}}
$$

$$
\mathrm{{G\ G_{i}/G_{o}}}
$$

$$
\mathrm{{\ 6\,G}_{o}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathtt{G}_{s}
$$

$$
\mathfrak{G}_{s}
$$

$$
\mathtt{G}_{s}
$$

$$
\mathrm{G_{i}/G_{o}}
$$

$$
\mathsf{G}_{\mathfrak{q}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathrm{{\ 6\,G}_{o}}
$$

$$
\mathrm{{G\ G_{i}/G_{o}}}
$$

$$
\mathrm{G_{i}/G_{o}}
$$

| Family | Target Gene ID(Common) | Coupling |  |
| --- | --- | --- | --- |
| Neuropeptide Y | NPY1R(Y1) | Gi/Go |  |
| Neuropeptide Y | NPY2R(Y2) | Gi/Go |  |
| Neuropeptide Y | NPY4R(Y4/PPYR1) | Gi/Go |  |
| Neurotensin | NTSR1(NTS1) | Gq |  |
| Opioid | OPRD1(δ) | Gi/Go |  |
| Opioid | OPRK1(κ) | Gi/Go |  |
| Opioid | OPRL1(NOP) | Gi/Go |  |
| Opioid | OPRM1(μ) | Gi/Go |  |
| Orexin | HCRTR1(OX1) | Gq |  |
| Orexin | HCRTR2(OX2) | Gq |  |
| Oxoglutarate | OXGR1(GPR9/GPR80) | Gq |  |
| P2Y | P2RY1(P2Y1) | Gq |  |
| P2Y | P2RY2(P2Y2) | Gq |  |
| P2Y | P2RY4(P2Y4) | Gq |  |
| P2Y | P2RY6(P2Y6) | Gq |  |
| P2Y | P2RY11(P2Y11) | Gs&lt;t Gq |  |
| P2Y | P2RY12(P2Y12) | Gi/Go |  |
| P2Y | Peptide P518 | QRFPR(QRFPGPR103) | Gq |
| Platelet-Activating Factor | PTAFR(PAF) | Gi/Go&lt;t Gq |  |
| Prokineticin | PROKR1(PKR1/GPR73) | Gs&lt;t Gq |  |
| Prokineticin | PROKR2(PKR2) | Gs |  |
| Prolactin-Releasing Peptide | PRLHR(PRRP) | Gq |  |
| Prostanoid | PTGDR(DP1) | Gs |  |
| Prostanoid | PTGDR2(DP2/CRTH2) | Gi/Go |  |
| Prostanoid | PTGER1(EP1) | Gq |  |
| Prostanoid | PTGER2(EP2) | Gs |  |
| Prostanoid | PTGER3(EP3) | Gi/Go&lt;t Gq |  |
| Prostanoid | PTGER4(EP4) | Gs |  |
| Prostanoid | PTGFR(FP) | Gq |  |
| Prostanoid | PTGIR(IP) | Gs |  |
| Prostanoid | TBXA2R(TP) | Gq |  |
| Prostanoid | Proteinase-Activated | F2R(PAR1) | Gi/Go |
| F2RL1(PAR2) | Proteinase-Activated | Gi/Go&lt;t Gq |  |
| F2RL3(PAR4) | Proteinase-Activated | Gi/Go&lt;t Gq |  |
| Relaxin Family Peptide | RXFP1(LGR7) | Gs |  |
| Relaxin Family Peptide | RXFP2(LGR8) | Gs |  |
| Relaxin Family Peptide | RXFP3(SALPR) | Gi/Go |  |
| Relaxin Family Peptide | RXFP4 | Gi/Go |  |

$$
\mathrm{{\ 6\,G}_{o}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

**Class A GPCRs**

$$
\mathrm{G_{i}/G_{o}}
$$

$$
\mathrm{6/66_{o}}
$$

$$
\mathrm{6/66_{0}}
$$

$$
\mathrm{G{}_{i}/{_{\ {mathsf o}}}}
$$

$$
\mathbf{G\}{_{mathfrak q}}}
$$

$$
\mathrm {G} _ {\mathrm {i}} / \mathrm {G} _ {\mathrm {o}}
$$

$$
G _ {i} / G _ {o}
$$

$$
\mathrm{6_{{i}/6_{o}}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathbf{\hat{G}}_{\mathbf{q}}
$$

$$
\mathbf{\widehat{G}}_{\mathbf{q}}
$$

$$
\mathtt{G}_{\mathtt{q}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
G _ {q}
$$

$$
6texts{s}\ 8\text{G}_{\text q}
$$

$$
G _ {s} \in t G _ {q}
$$

$$
\ 66_{0}
$$

$$
6_{!}/6_{0}\,6_{9}
$$

$$
\mathtt{G}_{\mathrm{s}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathfrak{G}_{\mathrm{s}}
$$

$$
\mathfrak{G}_{5}
$$

$$
\mathrm{{G6}_{i}/\mathrm{{G}_{o}}}
$$

$$
\mathfrak{G}_{5mathfrak}
$$

$$
\mathrm{G{}}_{\mathrm{s}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
66_{!}/6_{!}\,6\,6_{9}
$$

$$
G _ {i} / G _ {o}
$$

$$
66_{!}/6_{!}\,6\,6_{9}
$$

$$
\mathfrak{G}_{\mathrm{s}}
$$

$$
\mathfrak{G}_{\mathrm{s}}
$$

$$
\mathrm{G_{i}/G_{o}}
$$

$$
\mathrm {G} _ {\mathrm {i}} / \mathrm {G} _ {\mathrm {o}}
$$

---

**Class A GPCRs**

| Family | Target Gene ID(Common) | Coupling |
| --- | --- | --- |
| Somatostatin | SSTR1(SST1) | G1/G0 |
| Somatostatin | SSTR2(SST2) | G1/G0 |
| Somatostatin | SSTR3(SST3) | G1/G0 |
| Somatostatin | SSTR4(SST4) | G1/G0 |
| Somatostatin | SSTR5(SST5) | G1/G0 |
| Succinate | SUCNR1(GPR91) |  |
| Tachykinin | TACR1(NK1) | Gq |
| Tachykinin | TACR2(NK2) | Gq |
| Tachykinin | TACR3(NK3) | Gs&amp;tGq |
| Thyrotropin-Releasing Hormone | TRHR(TRH1) | Gq |
| Trace Amine | TAAR1(TA1) | Gs |
| Urotensin | UTS2R(UTR2) | Gq |
| Vasopressin&amp;t Oxytocin | AVPR1A(V1A) | Gq |
| Vasopressin&amp;t Oxytocin | AVPR1B(V1B) | Gq |
| Vasopressin&amp;t Oxytocin | AVPR2(V2) | Gs |
| Vasopressin&amp;t Oxytocin | OXTR(OT) | Gq |

$$
\mathrm{G_{i}/G_{o}}
$$

$$
\mathrm{{G G_{i}/G_{o}}}
$$

$$
\mathrm {G} _ {\mathrm {i}} / \mathrm {G} _ {\mathrm {o}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathrm{\mathfrak{G}_{\mathfrak{q}}}
$$

$$
\tt{G_{s}}\tt{S G_{q}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathbf{\hat{G}_{q}}
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathbf{\widehat{G}_{q}}
$$

$$
\mathfrak{G}_{\mathrm{s}}
$$

**Class B GPCRs**

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
\mathfrak{G}_{\mathfrak{S}}
$$

| Family | Target Gene ID(Common) | Coupling |
| --- | --- | --- |
| Calcitonin | CALCR(CT) | Gs |
| Calcitonin | CALCR-RAMP1(AMY1) | Gs |
| Calcitonin | CALCR-RAMP2(AMY2) | Gs |
| Calcitonin | CALCR-RAMP3(AMY3) | Gs |
| Calcitonin | CALCRL-RAMP1(CGRP1) | Gs |
| Calcitonin | CALCRL-RAMP2(AM1) | Gs |
| Calcitonin | CALCRL-RAMP3(AM2) | Gs |
| Corticotropin-Releasing Factor | CRHR1(CRF1) | Gs |
| Corticotropin-Releasing Factor | CRHR2(CRF2) | Gs |
| Glucagon | GCGR(Glucagon) | Gs |
| Glucagon | GHRHR(GHRH) | Gs |
| Glucagon | GIPR(GIP) | Gs |
| Glucagon | GLP1R(GLP-1) | Gs |
| Glucagon | GLP2R(GLP-2) | Gs |
| Glucagon | SCTR(Secretin) | Gs |
| Parathyroid Hormone | PTH1R(PTH1) | Gs&lt;t Gq |
| Parathyroid Hormone | PTH2R(PTH2) | Gs |
| VIP &amp; t PACAP | ADCYAP1R1(PAC1) | Gs&lt;t Gq |
| VIP &amp; t PACAP | VIPR1(VPAC1) | Gs&lt;t Gq |
| VIP &amp; t PACAP | VIPR2(VPAC2) | Gs |

$$
\mathtt{G}_{\mathtt{S}}
$$

$$
\mathtt{G}_{\mathrm{s}}
$$

$$
\mathtt{G}_{\mathrm{s}}
$$

$$
G _ {s} \& t G _ {q}
$$

$$
G _ {s} \in t G _ {q}
$$

$$
\mathsf{G}_{\mathsf{s}}\mathbin{\leqslant}\mathsf{G}_{\mathsf{q}}
$$

**Class C GPCRs**

| Family | Target Gene ID(Common) | Coupling |
| --- | --- | --- |
| Calcium-Sensing | CASR(CaS) | GqEtGi/Go |
| GABAB | GABBR1-GABBR2 | Gi/Go |
| Metabotropic Glutamate | GRM1(mGlu1) | Gq |
| Metabotropic Glutamate | GRM2(mGlu2) | Gi/Go |
| Metabotropic Glutamate | GRM3(mGlu3) | Gi/Go |
| Metabotropic Glutamate | GRM4(mGlu4) | Gi/Go |
| Metabotropic Glutamate | GRM5(mGlu5) | Gq |
| Metabotropic Glutamate | GRM6(mGlu6) | Gi/Go |
| Metabotropic Glutamate | GRM7(mGlu7) | Gi/Go |
| Liganded Orphans |  |  |
| Family | Target Gene ID(Common) | Coupling |
| Liganded Orphans | GPR1 |  |
| Liganded Orphans | GPR17 |  |
| Liganded Orphans | GPR35 | Gi/Go |
| Liganded Orphans | GPR39 | Gq |
| Liganded Orphans | GPR55 |  |
| Liganded Orphans | GPR84 | Gi/Go |
| Liganded Orphans | GPR119 |  |
| Liganded Orphans | GPR139(PGR3) |  |
| Liganded Orphans | GPR183(EBI2) | Gi/Go |
| Liganded Orphans | MRGPRD |  |
| Liganded Orphans | MRGPRX1(MRGX1) | Gq |
| Liganded Orphans | MRGPRX2(MRGX2) | Gq |

$$
G _ {q}
$$

$$
\mathsf{G A B A_{B}}
$$

$$
\mathrm{G_{i}/G_{o}}
$$

$$
\mathsf{G}_{\mathsf{q}}
$$

$$
\mathrm{G_{i}/G_{o}}
$$

$$
\ mathrm6_{\mathrm{i}}/6_{\mathrm{o}}
$$

$$
(mathfrak m G G3)
$$

$$
\mathfrak{G}_{\mathfrak{q}}
$$

$$
(mathsf m G G5)
$$

$$
\ 66_{0}
$$

$$
\mathrm{G_{i}/G_{o}}
$$

$$
\overline{{\ 6_{{mathrm{i}}}/{6_{0}}}}
$$

$$
G _ {q}
$$

$$
\mathrm{6i/6_{0}}
$$

$$
\mathrm{G_{i}/G_{o}}
$$

$$
\mathbf{G\}{}_{\mathbf{q}}}
$$

$$
G _ {q}
$$

Note: GPCR nomenclature and classification was determined using IUPHAR 
(guidetopharmacology.org), GPCR phylogenetic analysis was performed using 
ClustalW2 (ebi.ac.uk/Tools/msa/clustalw2), and dendrogram creation was done 
using iTOL (itol.embl.de). ~70 Non-liganded  orphan receptors (not shown in table) 
and over 100 GPCR orthologs (other species) assays (not shown in dendrogram) 
are available. Refer to our websites for the most recent list of available assays 
including additional GPCR products and services not shown in the dendrogram.

---

# RESOURCES

# Product Types

Eurofins DiscoverX® provides one of the most extensive drug discovery and development phase-appropriate solutions offerings of cellbased assays and reagents trusted by pharmaceutical and biotech companies. Accelerate your research with validated products and 
assays with the peace of mind that you are obtaining biologically-relevant MOA-reflective assays to take you through every step of 
discovery, through development, and into QC lot release testing, for the decades to come. These assays, cell lines, enzymes, and reagents 
have been validated in the screening of billions of data points in our internal CROs and pharma and biotech companies worldwide. Our 
expertise in your hands – Discover Confidently!

## Assay Kits

Complete off-the-shelf, cell-based functional assays with high reproducibility for rapid therapeutic assessment to QC 
lot release testing

## Cells

Stable cell lines, cell pools, frozen cells, & effector cells for a broad range of target classes & applications

## Membrane Preparations

Qualified GPCR & Ion Channel membrane preps for saturation and competitive radioligand binding & GTPγS 
functional studies

## Detection Kits & Reagents

Chemiluminescent & fluorescent readout kits & reagents to suit the diverse needs of your drug discovery & 
development programs

## Cell Culture Kits & Reagents

Optimized cell culture kits & reagents for consistent, reliable, & high quality results

## Control Ligands & Inhibitors

Highly purified, pre-validated reference ligands & inhibitors for use with Eurofins DiscoverX cell lines

## Toolbox Products

Complete set of vector & retroparticle-based tools & protocols to build your own stable cell lines & cell-based assays

Custom Capabilities
Custom cell lines, assays, & protein development capabilities optimized to your requirements

---

## RESOURCES

## GPCR Product Topics

Whether you are developing small molecule or biologic therapeutic drugs, Eurofins DiscoverX® provides you with a 
variety of drug discovery and development products with multiple assay types and applications to meet your specific 
GPCR research needs.

## GPCR Solutions

Discover the largest collection of GPCR assay solutions for developing small molecule or biologic therapeutics

HitHunter® cAMP Assays

Accurately detect cellular cAMP levels for a variety of biologics and small molecules ligands and applications

HitHunter Calcium Assay

Easily measure intracellular calcium within cells after activation status GPCRs and ion channels

PathHunter® β-Arrestin Assays

Universal screening & profiling platform that can be used for virtually any G-, G-, or G-coupled GPCR
i s q

$$
6_{i}-{6}_{s}
$$

$$
\mathsf{G}_{\textsf{q}}
$$

GPCR Internalization Assays

Simple orthogonal screening assays for monitoring GPCR internalization without microscopy to ultimately identify safer therapeutics

Pharmacotrafficking Assays

Identify pharmacochaperones that promote proper folding of misfolded protein & assist in trafficking to their intended location

---

# RESOURCES

# Tools and References

Eurofins DiscoverX® provides resources, tools, and references to help enable and accelerate your GPCR research and drug discovery 
and development programs. Access our industry-leading resources at discoverx.com/tools-resources, including downloading the white 
papers and application notes, along with original blog articles presented in this eBook. Other resources on this site include accessing a 
GPCR solution guides (product list), posters, presentations, a GPCR reference guide booklet, webinars, and peer-reviewed publications 
generated from experts in the field.

Access discoverx.com for the most recent list of available products and to learn more about the Enzyme Fragment Complementation 
(EFC) technology that is behind most of the Eurofins DiscoverX GPCR products.

## GPCR Solutions Guide

Assay guide and list of available GPCR assays, cell lines, membrane preps, & other products

## GPCR Reference Guide

Booklet highlighting the EFC technology & select validation data for Eurofins DiscoverX GPCR assays

## GPCR β-Arrestin Publications

Select list of over 150 peer-reviewed β-arrestin publications organized by GPCR families

## Publications

Peer-reviewed publications covering Eurofins DiscoverX products focused on GPCRs, kinases, ion channels, & more targets

## The Druggable GPCRome Poster

Downloadable poster showcasing the human GPCRome dendrogram, available assays & families, targets & coupling table

## EFC Platform

Robust, homogeneous detection assay system used for a wide variety of drug discovery & development applications

Visit discoverx.com/GPCRs to learn more about GPCRs in drug discovery & development or contact Technical Support for any questions.