Accelerating GPCR Drug Discovery and Development Products and Services for Target ID, Functional Screening, Lead Optimization, and QC Lot-Release Testing What’sWhat’s Inside: Inside: x >650 GPCR Assays x x Functional Readouts x cAMP Calcium β-Arrestin Internalization Pharmacotrafficking Complete Solutions Ready-to-Use Kits Cell Lines Screening Services Toolbox GPCR Pathways and Assays G-protein coupled receptors (GPCRs) play a crucial role in many physiological functions and in the pathology of multiple diseases including cancer, endocrine, and metabolic disorders. These receptors represent the largest class of validated therapeutic targets. Over 30% of currently marketed drugs target these receptors, and many GPCRs are still active targets for drug discovery. Largest portfolio of validated products and services to accelerate your GPCR drug discovery. >400 functional assays measuring β-arrestin recruitment to over 90% of class A and B GPCRs, including assays for over 75 orphan GPCR targets >150 naturally-coupled Gi/o, Gs and Gq GPCR cell lines for measurement of cAMP and calcium >100 functional cell lines for receptor internalization and trafficking The assays are available as stable, clonal cell lines or complete ready-to-use kits optimized for small molecules or biologics. With over 700 peer-reviewed customer publications, these assays are the most trusted pharmacologically- relevant functional GPCR assays in the industry. cAMP Assays Measure intracellular cAMP with easy-to-use protocols Characterize ligand pharmacology with precision Reproducible performance with large assay windows and broad sensitivity ranges Gαs cAMP Gαi Calcium Assays Measure intracellular calcium flux in adherent or suspension cells Fast, highly sensitive, and low-cost, HTS-friendly platform Homogeneous protocol with large assay windows and broad sensitivity ranges Ca2+ β-Arrestin Assays Universal G-protein independent method to quantify GPCR activation Easy, add-and-read protocol optimized for screening to QC lot-release testing Multiplex with calcium or cAMP readouts for cost-effective screening β-arrestin and ligand bias analysis Internalization Assays Quantitative measurement of GPCR internalization, desensitization, and resensitization Simple chemiluminescent readout, without imaging Easy protocol enabling rapid lead optimization and ligand bias studies Pharmacotrafficking Assays Detect trafficking in functionally-restored mutant GPCRs Identify pharmacochaperones that correct protein misfolding-related diseases Quantitative chemiluminescent results without imaging Learn more about all GPCR platforms at discoverx.com/gpcr One GPCR Target, Multiple Functional Readouts Identify Functionally Selective Ligands Biased GPCR ligands represent exciting opportunities for developing better and safer drugs, by selectively promoting the desired biological effect relevant for the disease, while simultaneously deselecting the deleterious ones1,2. DiscoverX provides the largest portfolio of GPCR assays and a unique service, “gpcrBIAS” for characterization of ligand bias. These assays offer wide assay windows to allow different functional readouts in single or multiplexed mode for the same GPCR target in a single cell line, to generate a unique efficacy profile of the ligand and capture the full repertoire of possible signaling responses. 20000 cAMP Acetylcholine Carbachol 15000 cAMP Oxotremorine-M 10000 RLU 5000 0 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 nist sin β-Arrestin 125000 100000 75000 RLU 50000 β-arrestin CHRM2 25000 0 10 -10 10 -8 10 -6 10 -4 10 -2 [M] Internalization 10000 7500 RLU 5000 2500 0 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 [M] The cholinergic receptor, muscarinic 2 (CHRM2), was expressed in CHO-K1 cells and three assay formats were created for cAMP, β-arrestin, and internalization readouts. Dose-response curves were generated for multiple ligands to determine their functional selectivity. Acteylcholine and Carbachol showed similar agonist responses and potencies in all three readouts. Oxotremorine-M showed similar cAMP response, but as a partial agonist in recruiting β-arrestin and a strong agonist for receptor internalization. Interrogating the three pathways for the same target revealed differences in the mechanism of action of each ligand. These assays provide the ability to quantify “bias” for each potential drug candidate to help determine the overall quality of efficacy possessed by each drug. “… 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.”— Robert Lefkowitz, Ph.D. 2012 Nobel Prize in Chemistry. Calcium and β-Arrestin Detection in the Same Well Several GPCR agonists activate multiple signaling pathways differentially. An increasing awareness of the subtleties of GPCR receptor signaling and the potential for ligand bias requires multiple endpoints to be monitored in any screening campaign. DiscoverX offers Gq-coupled arrestin targets as clonal cell lines or Ready-to-Use kits, to monitor intracellular calcium flux in real time, followed by the recruitment ofβ -arrestin in the same cell line or in a single well. This HTS-compatible duplexed assay increases your screening capabilities, removes challenges of running multiple assays, reduces data complexity, and provides significant cost savings and time efficiencies. Calcium β-Arrestin 200 100000 BDKRB1 EC50 = 12.2 native CHO-K1 75000 S:B = 12.7 X 100 50000 RFU RLU ∆ 25000 0 0 -12 -11 -10 -9 -8 -7 -6 -5 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 10 10 10 10 10 10 10 Bradykinin [M] Bradykinin [M] ® Naturally Gq-coupled PathHunter β-arrestin cell line expressing the Bradykinin B1 Receptor (BDKRB1) was first monitored for calcium mobilization using the Calcium No WashPLUS Kit as a fluorescent readout.β -arrestin recruitment was then analyzed in the same well using the PathHunter Detection Reagents. These duplexed assay provide a simple and efficient approach for measuring different signaling pathways for the same GPCR and define potency ranking of GPCR modulators. References: 1. Violin JD and Lefkowitz RJ. β-Arrestin-biased ligands at seven-transmembrane receptors. Trends in Pharmacological Sciences. 2007, 28 (8), 416–422. 2. Kingwell K. Pioneering biased ligand offers efficacy with reduced on-target toxicity. Nature Reviews Drug Discovery 2015, 14 (12), 809–810 Choose the Solution that Best Meets Your Program Needs Whether you are developing small molecules or biologics, DiscoverX provides you with a choice of different product formats to meet your specific research needs. Learn more at discoverx.com/gpcr. Human | Orphans | Orthologs >300 GPCR Targets | >650 Cell Lines Multiplexed Readouts Pharmacological Activity . cAMP . β-Arrestin . Agonism . Ligand Bias . Calcium . Internalization . Antagonism . Allosteric Modulation Test Assay Quickly Screen In-House Lead Optimization Build Your Own Assay Ready-to-Use Kits Cell Lines Screening Services Toolbox . Bioassay and eXpress . Stable cell lines for . Leading provider of . Rapidly develop assays kitsinclude cryopreserved continuous cell culture GPCR screening and for any GPCR with a ready-to-use cells and all and in-house banking profiling services β-arrestin, trafficking, or required reagents internalization readout . Save time and money, . Deliver consistent assay . Screen and profile . Create your own cell- no cell culture needed performance guaranteed against the broadest based assays in any for at least 10 passages menu of cell-based dividing mammalian functional assays with cell type the fastest turnaround time . Ease of assay develop- . Obtain reproducible . Select from a pre- . Analyze GPCR mutants, ment and method resultsusing optimized defined panel or create orthologs or novel cell transfer to global sites culture anddetection a personalized panel backgrounds reagents with specific assays Can’t Find Your Assay on Our Menu? Try Our Custom Assay Development Services For information on requesting a custom assay development, please visit discoverx.com/cad. w discoverx.com usa 1.866.448.4864 eu +44.121.260.6142 e [email protected] GPCRome The Largest GPCR Family Coverage MRGPRX3 P2RY11 FFAR3 FFAR2 MRGPRX1 HCAR3 FFAR1 F2RL2 F MRGPRX4 HCAR2 GPCRs HCAR1 OXER1 MRGPRX2 2 GPR31 UTS2R MRGPRD R MRGP MRGPRE L 2 4 MRGPRF 3 P2RY8 F2R F2RL1 GPR55 GPR18 GPR20 GPR92 GPR35 OXGR1 MAS1L GPR91 P2RY1 TBXA2R P2RY6 PTGER1 RG P2RY FR MAS1 P2RY GPR132 GPR65 PTGER3PTGFR GPR4 PTGER2 GPR68 PTA GPR34 PTGDR GPR171 PTGER4 GPR87 PTGIR P2RY14 P2RY12 P2RY13 LGR6 GPR183 CYSLTR1 LGR5 CYSLTR2 LGR4 GPR17 LHCGRTSHR GPR174 P2RY10 FSHR GPR23 RXFP2 LPAR6 RXFP1 GPR152 LTB4R OPN5 LTB4R2 GPR85OPN3 C5AR1 GPR173 C5AR2 GPR27 GPR1 GPBAR1 C3AR1 CMKLR1 GPR88 GPR135 GPR33 PTGDR2 GPR62 GPR32 GPR61 FPR1 GPR78 FPR2 GPR26 FPR3 GPR75 KISS1R GPR150 GALR1 GALR2 GALR3 ADRA2C MCHR1 ADRA2A MCHR2 ADRA2B OPRL1 ADRA1B OPRK1 OPRD1 ADRA1A OPRM1 ADRA1D NPBWR1 HTR4 NPBWR2TR1 HRH2 SS DRD3 SSTR4TR2 DRD2 SS SSTR3 DRD4 SSTR5 ADRB2 ER1 ADRB1 GP RXFP3 ADRB3 RXFP4 HTR6 AGTRL1 HTR1F GPR25 HTR1E GPR15 HTR1D AGTR1 HTR1B AGTR2 HTR1A Class A BDKRB1 DRD5 BDKRB2 DRD1 CXCR7 HTR7 GPR182 HTR5A CCBP2 TAAR8 XCR1 TAAR6 CCRL2 AR9 CX3CR1 TA CCR8 TAAR5 CCR4 TAAR3 CCR1 TAAR2 CCR3 TAAR1 CCR2 HRH1 CCR5 HTR2C CXCR1 HTR2A CXCR2 HTR2B CXCR5 CHRM5 CCR10 CHRM1 CXCR3 CHRM3 CXCR4 CX CHRM4 CCRL1CR6 CHRM2 HRH4 CCR9 HRH3 CCR6 CCR7 EDG3 GPR161 EDG1 GPR101 EDG5 GPR84 EDG8 Class B Class C GPR50 EDG6 MTNR1A