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GPCR Ligand Bias Assays

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DiscoveRx GPCR cell based assays for identifying novel ligands, studying receptor pharmacology and uncovering ligand bias.

DiscoveRx GPCR cell based assays for identifying novel ligands, studying receptor pharmacology and uncovering ligand bias.

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  • 1. Revealing Ligand & GPCR Bias Using A Combination of 2nd Messenger, β-Arrestin & Internalization EFC Assay Formats
  • 2. DiscoveRx – Your Drug Discovery Partner Cell-Based Assays 246 GPCR cell lines Catalog 15 Cell Based Kinases Products 26 NHR cell lines 6 Pathway assays Custom Profiling and Assay Screening Development
  • 3. DiscoveRx Core Technology Active Enzyme Inactive Fragments Active Enzyme Enzyme PK PKComplementation + Biochemical Cell Biology HitHunterTM Product Line PathHunterTM Product Line • Cellular GPCR Assays • GPCR Signaling Assays • Cellular NHR Assays • Generic Kinase Assays • Cellular Kinase Assays • Protease Technology • Cellular Protease Assays • Unactive Kinase Technology • Pathway / Signaling Assays
  • 4. Evolution of Understanding GPCR Function1. Binding - On/ Off Radioligand binding studies2. Receptor Activation Through Ga Second messenger assays3. Receptor De-sensitization via b-Arrestin Internalization & recruitment studies
  • 5. Evolution of Understanding GPCR Function4. b-Arrestin involved in desensitization Balanced Signal but also has signaling properties Kinases (MAPK, P3K, AKT) Originally thought that all downstreampathways equally activated for a given agonist5. Novel conformations of the same Biased Signals receptor can initiate different signaling cascades Now understood that agonists can activate only a subset of signals
  • 6. Evolution of GPCRs
  • 7. Functional GPCR Activities & Ligand BiasEvents following ligand binding to GPCRs Second Messenger Internalization Calcium Signaling Arrestin cAMP Differential activation of these pathways occurs using the same ligand (Ligand Bias)
  • 8. GPCR Activation: Multiple Biological Processes G-protein Activation Arrestin Recruitment InternalizationOpportunity for GRK 1 2 3 ligand bias Calcium cAMP •2nd Messenger Signaling •Alternate pathway •Long term desensitization •Short term desensitization •Altered Trafficking •Contributes to Internalization •Degradation / Recycling Long Acting Agonists Functional Antagonists, Avoid Unwanted Signaling Receptor removal Monitoring each function can aid in compound characterization
  • 9. PathHunter™ Protein Interaction Technology Active Enzyme Inactive Fragments Active Enzyme PK PK EnzymeComplementation + Weak/No activity High activityModified EnzymeComplementation for Protein interactions + Features: • Generic, homogeneous approach applicable to a wide range of drug target classes • Flexible, small peptide tag with options for high and low affinity • Only technology successfully transitioned from biochemical to cell based format
  • 10. GPCR Activation & b-Arrestin Binding PathHunter™ b-Arrestin Assays 50000 ADRB2  Small protein tag (42 aa) 40000  Dynamic protein interaction assay 30000 RLU  Target-specific signal 20000  Chemiluminescent or fluorescent detection 10000  Transfers easily from benchtop to full HTS 0 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 campaigns Isoproterenol [M]
  • 11. Arrestin-Based GPCR Signaling Thaw & Plate Cells SSTR2 (Gi) CHRM5 (Gq) ACHR5 3500 EC50=2.0 nM EC50=3.1 μM; 3000 S/B=42 14000 S/B=5.9 2500 12000 12-48 hrs 2000 10000 RLU RLU 1500 8000 6000 1000 4000 500 2000 0 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 0 Somatostatin 28 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 1 hr 11hr hour Oxotremorine M ADRB2 (Gs) Type B Glucagon (GCGR) 40000 50000 EC50=11.2 nM EC50=15.3nM; 1 hour 40000 S/B=6.6 30000 S/B=11 30000 RLU RLU 20000 20000 10000 10000 0 0 10 -14 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 Isoproterenol [M] Glucagon [M] Read Luminescence
  • 12. Highlights of PathHunter™ Arrestin Format CHO A2 ADRB2 Clone Ligand Rank Order Salbutamol 1750 Isoproterenol Stochiometric Signal Generation 1500 1250 (-) Epinephrine Deoxyepinephrine Clenbuterol Compound Efficacy 1000 RLU Salmeterol 750 Dobutamine 500 Formoterol Antagonist Mode 250 0 Isoetharine Metaproterenol 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 Ritodrine Agonist [M] Terbutaline CHO A2 GLP2R Predictable Pharmacology 10000 7500 RLU 5000 Specificity 2500 0 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5  Serum samples GLP 2 (1-33) human [M] • Chemokines 8000 7000 • Bioactive lipids 6000 20% 40% FBS FBS 5000 60% FBS • Antibody Supernatants RLU 4000 80% FBS 3000 2000 1000 0 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 Anti-CCL20 mAb [g/mL] + 10nM CCL20
  • 13. PathHunter Performance ADRB2 >50% are 9X or better S:B Ratio Clenbuterol Formoterol Isoproterenol PathHunter clones (Receptors tested with reference agonist)  Real-time analysis  Signal to noise
  • 14. Arrestin is G-protein IndependentBasis for Arrestin Ligand Bias cAMP Assay Arrestin Assay CHO Gi FPRL1 CHO Arrestin FPRL1 1250 60000 (+) Pertussis tox  G-protein inhibitor 1000 50000 40000 (-) Pertussis tox 750 independent (+) Pertussis tox RLU RLU (-) Pertussis tox 30000 500 20000 250 10000 0 0 10 -14 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 WKYMVm-NH2 [M] + 20 M Forskolin WKYMVm-NH2 [M] CXCR7 30000 C5L2 40000  Arrestin in the 30000 20000 RLUs absence of G-protein RLU 20000 10000 activation 10000 0 0 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 SDF1a [M] Complement C5a [M] Lambertus et al. J. Biomol Screening (2009) Arrestin binding and internalization can occur in the absence of G-protein activity
  • 15. Second Messenger Native Gi, Gs, and Gq coupled cell lines HitHunter® biochemical reagents for cAMP detection Complex pharmacology- Agonists, antagonists, partial agonists & allosterism Differentiating compounds - Large assay windows, ideal for difficult Gi targets Miniaturizable Fluorescent and Aequorin-based calcium detection
  • 16. Arrestin Biased AgonismAGTR1 Ca++ Reference agonist Reference agonist 1500 4000 1250 1000 3000 RLU RLU 750 2000 500 1000 250 0 0 10 -15 10 -14 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 Angiotensin II [M] Angiotensin II [M] 4000 Biased agonist SII Biased agonist SII 1500 3000 1250 1000 RLU RLU 2000 750 500 1000 250 0 0 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 [Sar1, Ile4,8]-Angiotensin II [M] [Sar1, Ile4,8]-Angiotensin II [M]
  • 17. PathHunter is Ideal for Investigating Ligand Bias GCGR cAMP Glucagon 2000 Glucagon 1250 DHG+ 300nM Glucagon DHG 1000 1500 RLU 750 1000 RLU 500 500 250 0 0 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 Ligand [M] Ligand [M] DHG is a ligand for the GCGR receptor-- antagonist in Arrestin but a partial agonist in cAMP
  • 18. Lessons Learned 7TMR Signaling Both G-protein dependent & independent pathways Linked but not dependent pathways Can be modulated independently by compounds PathHunter™ Arrestin Technology Broadly applicable measure of arrestin signaling Ideal for difficult targets and orphans HTS friendly Opportunity for multiplexed measurements
  • 19. GPCR InternalizationApplicable to the majority of GPCRsReduces the number of cell surface receptors acutely Recycled Targeted for degradation Functional antagonismCompartmentalized signaling has been observed
  • 20. PathHunter™ GPCR Internalization Assays:2 Convenient Platforms. Same Answer. Does the receptor internalize? Activated Internalization EA EA Arrestin Recruitment EA EA EA EA Endo EA Complemented Enzyme Complemented Enzyme - Gain-of-signal, large S:B - Arrestin dependent - Gain-of-signal, large S:B -Short-term desensitization at - Arrestin dependent membrane - Long-term desensitization at endosome
  • 21. PathHunter™ Activated GPCR Internalization Assays:Broadly Applicable Neurotensin Chemokine Muscarinic 1000 750 750 500 RLU RLU 7500 500 250 250 5000 RLU 0 0 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 2500 CCL3 [M] Oxotremorine-M [M] NTSR1 0 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 CCR5 M5 Neurotensin [M]  Tested across 35 different families BOTTOM NTSR1 177.4 TOP 6785  Over 65 targets available (over 80 by 3/1) LOGEC50 HILLSLOPE -8.850 1.651 EC50 1.4128e-009  Building out specific panels S/B = 47.0  Compound selectivity  Timing, magnitude, cell type specificity
  • 22. GPCR “Trio Project” For Ligand Bias 24 targets tested in all 3 formats, average of 5 agonists per target Pharmacology differences observed in 11 of 24 Expected overlap between cAMP/Calcium and Arrestin Only 1% of ligands are completely biased, 5-10% show some bias More differences with internalization Currently expanding studies and following up on “biased” ligands 22
  • 23. DiscoveRx Trio Initiative CXCR2 CCR1 BDK2 No Bias detected Efficacy Bias Potency Bias Calcium 2nd Messenger cAMP cAMP 125 125 150 CXCL1 CCL3 100 Lys-Bradykinin 125 % cAMP inhibition CCL23 % cAMP Inhibition % Activation 100 CXCL2 Bradykinin CXCL3 100 75 [Phe8"Psi"BDK 75 CXCL5 75 50 50 CXCL6 Interleukin-8 50 25 25 25 0 0 0 0. 1 01 1 01 1 1 10 0 10 0 10 00 00 1 10 01 1 1 0 00 01 1 10 01 1 1 0 00 0 01 00 00 10 0 0. 10 00 0. 10 00 00 0. 00 10 0 00 0. 10 00 0. 10 00 0. 00 0. 0. 10 0. 0. 0. 0. Agonist + 15 M Forskolin Agonist + 15 M Forskolin Agonist Arrestin Arrestin Arrestin 150 150 150 CXCL1 CCL3 125 125Arrestin 125 CXCL2 CCL23 Lys-Bradykinin % Activation % Activation % Activation 100 CXCL3 100 100 Bradykinin CXCL5 [Phe8"Psi"-BDK 75 75 75 CXCL6 50 Interleukin-8 50 50 25 25 25 0 0 0 0 0 0 0 00 00 00 00 01 1 0 1 01 1 10 00 0 .0 .0 .0 .0 1 10 1 0 1 01 00 10 00 01 00 0. 1 10 00 00 0. 0. 1. 2. 3. -4 -3 -2 -1 10 00 00 0. 00 10 0. 10 00 0 00 0. 10 0. 10 00 0. 0. Agonist 0. Agonist AgonistInternalization Internalization Internalization Internalization 150 125 CXCL1 600 125 CXCL2 CCL3 500 100 Lys-Bradykinin % Activation 100 CXCL3 CCL23 % Activation Bradykinin % Activation CXCL5 400 75 75 [Phe8"Psi"-BDK CXCL6 50 300 50 Interleukin-8 25 200 25 0 100 0 0 01 1 01 00 1 0 1 10 00 10 0. 00 10 0. 0. 0. 1 10 0 0. 1 01 1 01 1 00 10 00 00 1 1 01 10 0 01 1 00 0 10 00 00 10 0. 00 00 0. 00 10 10 0. 00 0 00 0. 10 0. 00 10 0. Agonist 0. 0. Agonist Agonist
  • 24. Uncover Biased Ligands Using the CompleteDiscoveRx GPCR Portfolio 2nd Messenger Arrestin InternalizationProlonged ProlongedG-protein Desensitization Biased Signalingactivation Functional Antagonism Delta Opioid Receptor Case Study• Opioid system controls responses to pain - OPRD1 (hDOR) activation alleviates persistent pain - Desensitization leads to persistent pain• Receptor responds to endogenous & synthetic ligands• hDOR undergoes rapid internalization• Compound-specific differences in re-sensitization have been identified
  • 25. SNC-80 Results in OPRD1 Desensitization• SNC-80 showed 95% inhibition slower sustained for 30 minutes• Faster & stronger desensitization vs endogenous enkephalins• Fresh ligand could not restore signal
  • 26. SNC-80 Results in Prolonged OPRD1Internalization•SNC-80 identified as a strongly internalizing compound•Agonist recycling is compound-specific
  • 27. In vivo Effects of a Non-Internalizing Ligand First 2ndARMS390 and SNC80 are full agonists Challenge Challengeby GTPgSSNC80 –EC50: 122nMARMS390-EC50: 170nM Summary•SNC80 is a stronglyinternalizing ligand•SNC80 behaves as afunctional antagonistin vivo
  • 28. Differential GPCR Biology: OPRD1 2nd Messenger Arrestin Internalization 200000 2000 25000 20000 1500 150000 15000 RLU RLURLU 100000 1000 10000 50000 500 5000 0 0 0 10 -14 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 Agonist [M] + 20 M Forskolin Agonist [M] Agonist [M]  DAMGO is a weak agonist  DAMGO weakly recruits Arrestin  SNC-80 strong Arrestin- compared to SNC-80  SNC-80 strong Arrestin response mediated internalization vs DAMGO
  • 29. More Information - CCKAR •Full agonist in calcium •Similar in vivo half lives •Why is a compound that is less potent, more active in vivo?
  • 30. Relevant, Predictive, Informative High Content Compound Analysis Arrestin Endo 400000 sCCK8 7000 A-71623 6000 sCCK8 300000 A-71623 5000 RLU 200000 4000 RLU 3000 100000 2000 1000 0 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 0 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 [M] [M]Working hypothesis: A-71623 has prolonged duration of action due to poor internalization. Moving the 2nd Messenger Arrestin Internalization industry……. Potency Efficacy Potency Efficacy Potency Efficacy Compound X 10nM Full 5nM Full 5nM Partial Compound Y 50nM Full 35nM Partial 35nM Full Compound Z 20nM Partial 100nM Full 100nM Full
  • 31. Uncover Biased Ligands Using the DiscoveRx Complete GPCR Offering: CHRM2 CHRM2 cAMP CHRM2 Arrestin CHRM2 Internalization CHRM2 12500 12500 150000 10000 10000 7500 100000RLU 7500 RLU 5000 RLU 5000 50000 2500 2500 0 0 0 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 10 -10.0 10 -7.5 10 -5.0 10 -2.5 10 0.0 [M] [M] [M]  Oxo-M gives a functional response  Oxo-M shows weak  Oxo-M shows strong Arrestin-  Behaves as an agonist b-Arrestin recruitment mediated internalization
  • 32. Uncover Biased Ligands Using the DiscoveRxComplete GPCR Offering: NTSR1 NTSR1 Calcium NTSR1 Arrestin NTSR1 Internalization NTSR1 350 1250000 70000 300 1000000 60000 250 50000 200 750000RLU RLU 40000 RLU 150 500000 30000 100 20000 250000 50 10000 0 0 0 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 [M] [M] [M]  Kinetensin behaves as a weak  Kinetensin is a weak  Kinetensin results in weak agonist agonist, similar EC50s internalization *Currently building out data sets and algorithms (in collaboration) for Ligand Bias quantification*
  • 33. Why Are PathHunter Internalization Assays SuchAn Important Drug Discovery Tool?• Receptor Internalization Influences Agonist Efficacy -Fewer receptors at the cell surface, reduces potency -Selective and non-selective agonists can be compared• New classes of drugs can be developed - Functional Antagonists that remove receptors from cell surface can reduce unwanted side effects - Non-recycling receptors may be better targets for chronic diseases (multiple administrations)• Molecular Pharmacology - Determine a direct link between internalization/localization & function for a target
  • 34. DiscoveRx GPCR Portfolio Summary 2nd Messenger Arrestin InternalizationProlonged ProlongedG-protein Desensitization Biased Signalingactivation Functional Antagonism  Multi-Mode GPCR characterization provides:  Extensive ligand analysis – identification of biased ligands  Potential to select ligands with specific properties and durations  Correlate biological function with biochemical characterization  Similar Studies • OPRM1 - μ- Opioid receptors: correlation of agonist efficacy for signalling with ability to activate internalization: McPherson et al. Mol Pharm July 2010 • EP4 - Functional selectivity of natural and synthetic prostaglandin EP4 receptor ligands: Leduc et al. JPET July 2009
  • 35. Biased Ligand Discovery with DiscoveRxMonitor GPCR activity through multiple 7TMRsignaling pathways and uncover novel, biased ligands• Largest GPCR menu covering all 7TMR signaling pathways -- 2nd messenger, arrestin & internalization)• Preferred supplier offering all technology platforms in the same robust, easy-to-use, HTS-friendly format• Provide “Next Generation” GPCR platforms that can be used explore novel receptor biology including internalization and dimerization
  • 36. DiscoveRx Comprehensive GPCR Portfolio Thank You!GPCR Pathway DRX Products AdvantagesGi/Gs Native Gi/Gs cell lines, Native, non-force coupled cell lines with chemiluminescent HitHunter™ cAMP XS+ detectionGq Native Gq cell lines, Native, non-force coupled cell lines with fluorescent detection HitHunter™ Calcium No Wash PLUSGq HitHunter™ IP3 Secondary confirmation of Calcium responseG-protein PathHunter™ Arrestin De-orphanization, uncover novel pharmacology biased ligandIndependent discoverySignaling PathHunter™ Activated GPCR Monitor activated GPCR internalization without imaging, biased Internalization Assays ligand discovery PathHunter™ GPCR Dimerization Assays Specific and quantitative measure of GPCR heterodimers