Learning Is Power

Using Multispan’s Human and Model Species GPCR Ortholog Panel to Guide Lead Compound Development

Use of ortholog screens helps to minimize a potential source of failure for preclinical candidates optimized against human GPCR targets. Incorporation of ortholog screens early in hit series evaluation can provide valuable insights for lead optimization. Multispan’s panel of GPCR orthologs can enable critical assessment of species-specific variations in target binding activity and modulation of GPCR signaling that could impact in vivo efficacy in small and large animal models.

Introduction

Demonstration of compound efficacy in small and large animal models of GPCR modulation remains a significant hurdle in the progression of preclinical candidates across a wide spectrum of indications of pharmaceutical interest. In addition to the challenges of optimizing pharmacology and toxicity, developers must translate results based on the screening of human GPCR targets into rodents and other model animals. In these model species, the structure of the receptor, its response to allosteric modulators and biases to signaling pathways and downstream effectors may not be fully conserved. Lead generation campaigns will obviously seek to prioritize hit series that impact therapeutically relevant pathways in both humans and model species while understanding the effects of ortholog sequence variations on activity.
Figure 1 Major therapeutic mechanisms of GPCR-signal modulation. Modulators may be orthosteric (at the natural ligand binding site, panels a and b) or allosteric (at a distal site, panels c-f) Changes in downstream signaling may be positive (green) or negative (red), Biased signals may be limited to changes in one or more pathways (panels e and f).
To meet this challenge, Multispan has assembled a panel of human GPCRs and their small and large animal orthologs. In this article, we discuss how integrating ortholog screens into lead generation campaigns can both provide invaluable insights into compound profiles to aid in prioritization and help avoid failures in animal efficacy models.

The importance of ortholog screening in the development of next generation GPCR modulators

GPCR-targeted therapies currently account for 34% of approved drugs and as GPCRs continue to be studied, de-orphanized and clinically validated, the number of potential druggable targets and range of clinical indications will continue to expand1. As a result, there is optimism that improved understanding of GPCR biology and downstream signaling can be exploited to create a new generation of targeted therapeutics with improved selectivity, specificity, and side effect profiles.
There are multiple ways in which therapeutics may modify GPCR function (Figure 1), and their successful design therefore relies heavily on conservation of receptor structure and function between humans and model animals. In addition to classical competitive orthosteric agonists or antagonists (Figure 1 Panel a and b), GPCR signaling activity may also be modified agonistically or antagonistically by allosteric modulators (AM) that bind at sites distinct from the natural (orthosteric) ligand (Figure 1 Panel c and d). Furthermore, downstream signaling can be biased to one pathway or another by a given ligand. Both positive (PAM) and negative allosteric modulators (NAM) can also bias signaling through downstream pathways2,3 (Figure 1 Panel e and f). As a class, PAM and NAM currently comprise 7% of GPCR-targeted therapeutics in development (PMID) but as they should offer improved selectivity, specificity, and side effect profiles (PMID) over orthosteric drugs, this fraction is likely to grow.
Successful exploitation of the therapeutic possibilities of GPCRs depends on the ability of lead generation teams to screen and profile candidates in therapeutically relevant in vitro screens4. Translation of in vitro results into efficacy studies in model species obviously introduces additional challenges even when pharmacology and toxicology are set aside. The corresponding GPCR ortholog may be poorly conserved, either at the orthosteric site, in regions involved in allosteric modulation in the human ortholog or even differentially biased to signal through different downstream pathways. As a result, the biological activity of potential modulators may not be well conserved between species, and this will impact their viability in preclinical models. It follows that understanding of GPCR ortholog signaling in the test species and compound evaluation across orthologs during lead generation are essential steps in preclinical candidate profiling.
To address this challenge, Multispan has created a panel of cell-based assays (Table 1) for clinically validated GPCR orthologs of human and small and large animals. Figure 2 suggests a typical screen-to-lead approach and emphasizes how ortholog screens fully embedded in lead generation programs can reduce translational uncertainty.

Table 1: MULTISCREEN™ Ortholog GPCRs

Receptor FamilyReceptorSpeciesParentalStable Cell Lines Division-Arrested Cells Membranes
AdenosineA1ratHEK293TCr1427DCr1427MCr1427
A2AmouseHEK293TCm1428ADCm1428AMCm1428A
A2AmouseHEK293TCm1428BDCm1428BMCm1428B
A2AmouseCHO-K1Cm1428-1DCm1428-1MCm1428-1
A2AratHEK293TCr1428DCr1428MCr1428
A2BratHEK293TCr1429DCr1429MCr1429
A3ratHEK293TCr1430DCr1430MCr1430
AngiotensinAT1mouseCHO-K1Cm1417-1DCm1417-1MCm1417-1
AT1mouseCHO-K1Cm1417-1aDCm1417-1aMCm1417-1a
Bile AcidGPBAratCHO-K1Cr1361-1DCr1361-1MCr1361-1
GPBAmouseCHO-K1Cm1361-1DCm1361-1MCm1361-1
BombesinBB1ratC6C1211-1DC1211-1MC1211-1
BradykininB1mouseHEK293TCm1198DCm1198MCm1198
B2mouseHEK293TCm1199DCm1199MCm1199
B2ratHEK293TCr1199DCr1199MCr1199
B2dogHEK293T cd1199 DCd1199MCd1199
B2rhesus monkeyHEK293TCpr1199DCpr1199MCpr1199
B2guinea pigHEK293TCc1199DCc1199MCc1199
B2rabbitHEK293TCb1199DCb1199MCb1199
Calcium-SensingCaSRratHEK293THr1233DHr1233MHr1233
CannabinoidCB1mouseCHO-K1Cm1229-1ADCm1229-1AMCm1229-1A
CB1mouseCHO-K1Cm1229-1BDCm1229-1BMCm1229-1B
CB1dogHEK293TCd1229DCd1229MCd1229
CB1dogHEK293TCd1229aDCd1229aMCd1229a
CB1dogHEK293TCd1229bDCd1229bMCd1229b
CB2ratCHO-K1Cr1230-1DCr1230-1MCr1230-1
CB2ratCHO-K1Cr1230-1aDCr1230-1aMCr1230-1a
CB2mouseCHO-K1 β-Arrestin2CAm1230BA2-1DCAm1230BA2-1MCAm1230BA2-1
CB2mouseCHO-K1Cm1230-1DCm1230-1MCm1230-1
CB2mouseCHO-K1Cm1230-1aDCm1230-1aMCm1230-1a
CB2ratCHO-K1 β-Arrestin2CAr1230BA2-1DCAr1230BA2-1MCAr1230BA2-1
CB2ratCHO-K1 β-Arrestin2CAr1230BA2-1aDCAr1230BA2-1aMCAr1230BA2-1a
ChemokineCCR6mouseHEK293TCm1014DCm1014MCm1014
CCR9dogHEK293T Gαqi5CGd1017DCGd1017MCGd1017
CCR9mouseHEK293TCm1017DCm1017MCm1017
CCR9mouseHEK293T Gαqi5CGm1017DCGm1017MCGm1017
CCR9ratHEK293T Gαqi5CGr1017DCGr1017MCGr1017
Citric Acid Cycle IntermediatesGPR91mouseCHO-K1Cm1144-1DCm1144-1MCm1144-1
GPR91ratCHO-K1Cr1144-1DCr1144-1MCr1144-1
EndothelinETAratCHO-K1Cr1216-1DCr1216-1MCr1216-1
Free Fatty AcidGPR119mouseCHO-K1Cm1298-1DCm1298-1MCm1298-1
GPR120mouseHEK293T Gαqi5CGm1294DCGm1294MCGm1294
GPR120ratHEK293TCr1294DCr1294MCr1294
GPR40cynomolgus monkeyCHO-K1Cpc1101-1DCpc1101-1MCpc1101-1
GPR40cynomolgus monkeyCHO-K1CApr1101-1DCApr1101-1MCApr1101-1
GPR40cynomolgus monkeyCHO-K1 β-Arrestin2CApc1101-1DCApc1101-1MCApc1101-1
GPR40dogCHO-K1Cd1101-1DCd1101-1MCd1101-1
GPR40mouseHEK293TCm1101DCm1101MCm1101
GPR40ratHEK293TCr1101DCr1101MCr1101
GPR40ratCHO-K1Cr1101-1DCr1101-1MCr1101-1
GPR40rhesus monkeyCHO-K1Cpr1101-1DCpr1101-1MCpr1101-1
GPR40ratCHO-K1 β-Arrestin2CAr1101-1DCAr1101-1MCAr1101-1
GPR41mouseCHO-K1 Gα16CGm1102-1DCGm1102-1MCGm1102-1
GPR41ratHEK293TCr1102DCr1102MCr1102
GPR43mouseHEK293TCm1104DCm1104MCm1104
GPR43ratHEK293TCr1104DCr1104MCr1104
GPR43dogCHO-K1Cd1104-1ADCd1104-1AMCd1104-1A
GPR43dogCHO-K1Cd1104-1BDCd1104-1BMCd1104-1B
GPR43cynomolgus monkeyCHO-K1Cpc1104-1ADCpc1104-1AMCpc1104-1A
GPR43cynomolgus monkeyCHO-K1Cpc1104-1BDCpc1104-1BMCpc1104-1B
GPR43rhesus monkeyCHO-K1Cpr1104-1ADCpr1104-1AMCpr1104-1A
GPR43rhesus monkeyCHO-K1Cpr1104-1BDCpr1104-1BMCpr1104-1B
GhrelinGhrelinmouseHEK293TCm1197DCm1197MCm1197
GlucagonGIPmouseHEK293TCm1290DCm1290MCm1290
GIPratHEK293TCr1290DCr1290MCr1290
GIPdogHEK293TCd1290DCd1290MCd1290
GIPdogHEK293TCd1290aDCd1290aMCd1290a
GIPrabbitHEK293TCb1290DCb1290MCb1290
GIPpigHEK293TCp1290DCp1290MCp1290
GIPrhesus monkeyHEK293TCpr1290DCpr1290MCpr1290
GIPrhesus monkeyHEK293TCpr1290aDCpr1290aMCpr1290a
GIPferretHEK293TCf1290DCf1290MCf1290
GIPferretHEK293TCf1290aDCf1290aMCf1290a
GLP-1cynomolgus monkeyCHO-K1Cpc1267-1DCpc1267-1MCpc1267-1
GLP-1cynomolgus monkeyCHO-K1Cpc1267-1aDCpc1267-1aMCpc1267-1a
GLP-1ratCHO-K1Cr1267-1DCr1267-1MCr1267-1
GLP-1dogCHO-K1Cd1267-1DCd1267-1MCd1267-1
GLP-1dogHEK293TCd1267-1aDCd1267-1aMCd1267-1a
GLP-1dogHEK293TCd1267-1bDCd1267-1bMCd1267-1b
GLP-1mouse CHO-K1Cm1267-1DCm1267-1MCm1267-1
GLP-1mouse CHO-K1Cm1267-1aDCm1267-1aMCm1267-1a
GLP-1pigCHO-K1Cp1267-1DCp1267-1MCp1267-1
GLP-1rabbitCHO-K1Cb1267-1DCb1267-1MCb1267-1
GLP-1rabbitCHO-K1Cb1267-1aDCb1267-1aMCb1267-1a
GLP-2ratHEK293TCr1268DCr1268MCr1268
GlucagondogHEK293TCd1266DCd1266MCd1266
GlucagonpigHEK293TCp1266DCp1266MCp1266
GlucagonratHEK293TCr1266DCr1266MCr1266
Glucagoncynomolgus monkeyCHO-K1Cpc1266-1DCpc1266-1MCpc1266-1
Glucagoncynomolgus monkeyCHO-K1Cpc1266-1aDCpc1266-1aMCpc1266-1a
Glucagoncynomolgus monkeyHEK293TCpc1266DCpc1266MCpc1266
Glucagoncynomolgus monkeyHEK293TCpc1266aDCpc1266aMCpc1266a
Glycoprotein HormoneTSHratHEK293TC1177-1DC1177-1MC1177-1
Gonadotrophin-Releasing HormoneGnRHdogHEK293TCd1283DCd1283MCd1283
HistamineH1mouseHEK293TCm1027DCm1027MCm1027
H3mouseHEK293TCm1029DCm1029MCm1029
H3mouseCHO-K1Cm1029-1DCm1029-1MCm1029-1
H4mouseHEK293TCm1030DCm1030MCm1030
H4mouseHEK293T Gαqi5CGm1030DCGm1030MCGm1030
KiSS1-Derived PeptideKiSS1ratCHO dhfr-C1036-1DC1036-1MC1036-1
Leukotriene/LipoxinBLT2monkeyHEK293TCp1272aDCp1272aMCp1272a
BLT2monkeyHEK293TCp1272DCp1272MCp1272
BLT2mouseHEK293TCm1272aDCm1272aMCm1272a
BLT2mouseHEK293TCm1272DCm1272MCm1272
LysophospholipidLPA1mouseRH7777Cm1048-6ADCm1048-6AMCm1048-6A
LPA1mouseRH7777Cm1048-6BDCm1048-6BMCm1048-6B
LPA1ratRH7777Cr1048-6BDCr1048-6BMCr1048-6B
LPA5mouseRH7777Cm1145-6DCm1145-6MCm1145-6
S1P2mouseCHO-K1Cm1051-1DCm1051-1MCm1051-1
S1P2ratCHO-K1Cr1051-1DCr1051-1MCr1051-1
S1P3mouseCHO-K1 Gαqi5CGm1049-1DCGm1049-1MCGm1049-1
S1P3ratCHO-K1 Gαqi5CGr1049-1DCGr1049-1MCGr1049-1
S1P4mouseCHO-K1 Gαqi5CGm1052-1DCGm1052-1MCGm1052-1
S1P4ratCHO-K1 Gαqi5CGr1052-1DCGr1052-1MCGr1052-1
Melanin-Concentrating HormoneMCH1ratHEK293T Gαqi5C1031-4DC1031-4MC1031-4
MelatoninMT2mouseHEK293TCm1224DCm1224MCm1224
MT2ratHEK293TCr1224DCr1224MCr1224
Metabotropic GlutamatemGlu2ratHEK293T Gαqi5HGr1189bDHGr1189bMHGr1189b
mGlu4dogCHO-K1 Gαqi5HGd1191-1DHGd1191-1MHGd1191-1
mGlu4mouseCHO-K1 Gαqi5HGm1191-1aDHGm1191-1aMHGm1191-1a
mGlu4mouseCHO-K1 Gαqi5HGm1191-1bDHGm1191-1bMHGm1191-1b
mGlu4mouseCHO-K1 Gαqi5HGm1191-1cDHGm1191-1cMHGm1191-1c
mGlu4mouseCHO-K1 Gαqi5HGm1191-1dDHGm1191-1dMHGm1191-1d
mGlu4ratHEK293TCr1191DCr1191MCr1191
MuscarinicM3mouseCHO-K1Cm1024-1DCm1024-1MCm1024-1
M4mouseCHO-K1Cm1025-1DCm1025-1MCm1025-1
Neuropeptide SNPSmouseHEK293TC1355-1DC1355-1MC1355-1
Neuropeptide W/BNPBW1ratHEK293T Gαqi5C1124-1DC1124-1MC1124-1
Neuropeptide YY2ratCHO-K1Cr1274-1DCr1274-1MCr1274-1
OpioiddeltaratCHO dhfr-C1351DC1351MC1351
kapparatCHO dhfr-C1352-1DC1352-1MC1352-1
kapparatCHO dhfr-Cr1352-8DCr1352-8MCr1352-8
muratCHO dhfr-Cr1350-1aDCr1350-1aMCr1350-1a
NOPratCHO dhfr-Cr1354DCr1354MCr1354
OrphanGPR35dogHEK293T Gα16CGd1096DCGd1096MCGd1096
GPR35dogCHO-K1 Gα16CGd1096-1DCGd1096-1MCGd1096-1
GPR35mouseCHO-K1Cm1096-1DCm1096-1MCm1096-1
GPR35mouseCHO-K1 Gα16CGm1096-1DCGm1096-1MCGm1096-1
GPR88ratCHO-K1Cr1141-1DCr1141-1MCr1141-1
MRGX2rhesus monkeyCHO-K1Cpr1257-1DCpr1257-1MCpr1257-1
OxysterolEBI2mouseCHO-K1 Gαqi5CGm1242-1DCGm1242-1MCGm1242-1
EBI2mouseCHO-K1Cm1242-1DCm1242-1MCm1242-1
ProstanoidEP2mouseHEK293TCm1202DCm1202MCm1202
EP2mouseHEK293TCm1202-3DCm1202-3MCm1202-3
EP2ratHEK293TCr1202DCr1202MCr1202
EP2ratHEK293TCr1202-134DCr1202-134MCr1202-134
PurinergicGPR17mouse1321N1Cm1526-3DCm1526-3MCm1526-3
GPR17rat1321N1Cr1526-3DCr1526-3MCr1526-3
GPR17rat1321N1Cr1525-3DCr1525-3MCr1525-3
P2Y2rat1321N1Cr1161-3DCr1161-3MCr1161-3
P2Y12ratCHO dhfr-C1170-1DC1170-1MC1170-1
P2Y14ratHEK293T Gαqi5CGr1057DCGr1057MCGr1057
P2Y14monkeyHEK293T Gαqi5CGp1057DCGp1057MCGp1057
Serotonin5-HT2AmouseHEK293TCm1324DCm1324MCm1324
5-HT2AmouseCHO-K1Cm1324-1DCm1324-1MCm1324-1
5-HT2AdogHEK293TCd1324DCd1324MCd1324
5-HT2BmouseHEK293TCm1325DCm1325MCm1325
5-HT4AmouseCHO-K1Cm1518-1DCm1518-1MCm1518-1
TachykininNK3ratHEK293TCr1305DCr1305MCr1305
NK3mouseHEK293TCm1305DCm1305MCm1305
Trace AmineTA1ratHEK293TC1357DC1357MC1357
TA1ratHEK293T Gαqi5Cr1357DCr1357MCr1357
UrotensinUTratCHO dhfr-C1035-1DC1035-1MC1035-1
Vasopressin/OxytocinOTrabbitHEK293TCb1299DCb1299MCb1299
OTrabbitHEK293TCb1299LDCb1299LMCb1299L
V1ArabbitHEK293TCb1042DCb1042MCb1042
V1BrabbitHEK293TCb1043DCb1043MCb1043
V1BrabbitHEK293TCb1043LDCb1043LMCb1043L
V2rabbitHEK293TCb1044DCb1044MCb1044
V2rabbitHEK293TCb1044LDCb1044LMCb1044L
V2dogCHO-K1Cd1044-1DCd1044-1MCd1044-1
In addition to providing preliminary data on potential efficacy across species, ortholog screens included at or immediately downstream of primary screening may be useful in prioritizing human hit series and inform structure-based lead design if this is available. During lead generation, ortholog assays add an additional dimension in the analysis of structure-activity relationships and the selection of leads. Meanwhile, human counterscreens may use a subset of receptors structurally or functionally related to the target or a multiclass panel such as the MULTISCREEN-231™.
Efforts to develop agonists of GPR43 provide a clear example of the translational challenges outlined above. GPR43 (also known as Free Fatty Acid Receptor-2 (FFAR2) is stimulated by short-chain fatty acid (SCFA) ligands and is involved in multiple roles in inflammation, metabolism and the immune system5. In the intestinal epithelium, it is implicated in mediating the relationship between the gut microbiota and innate immunity.
A substantial body of evidence6 suggests that SCFA-stimulated GPR43 signaling plays a complex role in dysregulation of gut inflammation in ulcerative colitis and Crohn’s disease (collectively referred to as inflammatory bowel disease, (IBD). Both agonists and antagonists of GPR43 have been investigated as selective antiflammatories in these indications7. Rat and murine orthologs of GPR43 are ~80% conserved relative to human. Commercial and academic discovery campaigns6,8. have identified both orthosteric and allosteric agonists and antagonists which are selective for the human ortholog and with reduced potency against the murine and rat receptors. In one study, only one of two potent allosteric agonists developed against the human ortholog showed any activity in a murine efficacy model9.

In the case of MRGPRX2, a human GPCR involved in non-IgE mediated mast cell responses10, the murine (MrgprB2) and rat orthologs of have only 63% and 61% homology respectively with the human ortholog. Accordingly, MRGPRX2 and MrgprB2 respond differently to model agonists. The EC50 values (concentration required to give 50% response) of most ligands for MrgprB2 are significantly higher than those for MRGPRX211. This example of low sequence homology for functional orthologs of major clinical interest further underscores the importance of ortholog screens for compound profiling early in lead development.

Why you should use the MULTISCREEN™ ortholog panel in your next campaign

MULTISCREEN™ is the result of more than 20 years of commitment to the success of screens targeting GPCRs. The ability to make valid and meaningful decisions based on compound profiling assays across orthologs reflects Multispan’s stringent quality control in the development and use of its assays. Each GPCR target is expressed in a single, stable clonal cell line selected for responses to the receptor’s native ligand. Cell lines are constructed from the same parental cell lines using the same expression vectors. Ortholog clones are selected with similar pharmacological profiles using functional assays to validate receptor expression levels and their stability over multiple passages. Multiscreen™ cell lines therefore make it possible to establish, optimize and validate robust assay and quality control protocols for each GPCR target. This ensures that researchers can make ‘apples-to-apples’ comparisons across ortholog screens (Figure 3), thus improving decision making and reducing translational uncertainty during lead generation.
Questions? email Us info@multispaninc.com

References

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  3. Conn PJ, Christopoulos A, Lindsley CW. Allosteric modulators of GPCRs: a novel approach for the treatment of CNS disorders. Nat Rev Drug Discov. 2009;8(1):41-54.
  4. Sum CS, Murphy BJ, Li Z, et al. Pharmacological Characterization of GPCR Agonists, Antagonists, Allosteric Modulators and Biased Ligands from HTS Hits to Lead Optimization. 2019 Nov 1. In: Markossian S, 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-https://www.ncbi.nlm.nih.gov/books/NBK549462/
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  7. Park BO, Kang JS, Paudel S, et al. Novel gpr43 agonists exert an anti-inflammatory effect in a colitis model. Biomol Ther (Seoul). 2022;30(1):48-54.
  8. Lee T, Schwandner R, Swaminath G, et al. Identification and functional characterization of allosteric agonists for the G protein-coupled receptor FFA2. Mol Pharmacol. 2008;74(6):1599-1609.
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