Actelion Pharmaceutical Allschwill Hamamatsu 10th FDSS User Meeting June 2014 Importance of calcium assay parameters in drug discovery Sabine Rouanet Dr. Isabelle Bertrand Dr. Stéphane Krief Directeur: Dr. Thierry Calmels Pr. Jean-Charles Schwartz Success is the ability to go from one failure to another with no loss of enthusiasm (Sir Winston Churchill)
GPCRs signaling G � q G � s G � i G � q G � s G � i Functional selectivity Several ligand-specific receptor conformations can be associated to biased functionnal signaling
Precise affinity required for GPCR antagonism • Advance SAR analysis • Studying drug specificity • Accurate affinity values for pre-development compounds • Identification of biased signaling Correlation calcium & G � s / cAMP G � s / cAMP CRE-MRE reporter assays Bias plot for histamine H2 agonists Agonism Agonism % of Max reference agonist response 35 S assay 100 on GTP γ 35 S binding assay Histamine 80 Amthamine % of 10 µM HA response on GTP γ β -arrestin β -arrestin 60 Inverse Inverse Agonism Agonism Agonism Agonism 40 β β β β β β Agonist A 20 � -blockercarvedilol � -blockercarvedilol Agonist B Cardioprotective effects Cardioprotective effects 0 Thanawala VJ et al, 0 20 40 60 80 100 120 Inverse Inverse % of 10 µM HA response on calcium assay Agonism Agonism Curr Opin Pharmacol. % of Max reference agonist response on calcium assay 2014 Mar 26;16C:50-57 Agonism Antagonism
Calcium mobilization assays at Bioprojet: HTS (Identify and classify hits) Evaluate agonism efficacy and affinity Evaluate type of antagonism (Schild regression analysis) Identification of biased ligands
GPCR antagonism and Calcium assay in drug discovery Need to obtain precise affinity by Kb determination in Calcium assays Kb is applicable at equilibrium conditions that are not encountered with functional calcium assays (incubation exceeds 4 times the dissociation t 1/2 of ligand/receptor)
Use of the pA2 as a universal determinant of antagonist potency pA2 = pKb + log ( 1+ 2 [ A ] / Ka ) At low [agonist] occupancy [ A ] < < < Ka pA2 ~ pKb + log (1) pA2 tend towards the pKb Concentration response Calculation of pA2 at low agonist responses curve dextral displacement Max response reduction Overcome the potential bias associated with non equilibrium conditions Estimate insurmountable antagonists affinity • Steven J Charlton and Georges Vauquelin, 2010, British J Pharmacol 161:1250–1265 • Terry Kenakin, 2009, A pharmacology Primer: Theory, Application and Methods, Chapter 11, Academic Press • Terry Kenakin et al, 2006, JPET 319:710–723 • Arthur Christopoulos et al, 1999, Euro J Pharmacol, 382:217–227 Non equilibrium
pA2 = - log [M] of antagonist producing a 2 fold shift of the agonist concentration response curve Use of Dose Ratio (DR) pA2 = log ( DR – 1 ) – log B values as surrogate parameter for calculation of pA2 pA2 = – log B At DR = 2 Competitive surmountable Non competitive (Insurmountable) Antagonism at equilibrium Antagonism at Hemi-equilibrium DR at low agonist response DR at EC50
Calcium assay parameters and GPCRs-ligand accessibility 1. Adherent vs suspension cells 2. Receptor functionality at the cell membrane 3. Ligand diffusion
GPCRs and ligand accessibility 1. Adherent vs suspension cells 2. Receptor functionality at the cell membrane 3. Ligand diffusion
Settings: 10µl/sec, height 9.6 mm, sensitivity 200ms, gain 1 Calcium Flux on HEK293 cell suspension Calcium Flux on MSR1-HEK293 adherent cells 6000 MSR1: macrophage scavenger receptor 1 14000 Agonist Max-Min (Fluorescence Arbitrary Units) Agonist 1µM + BP1 antagonist Max-Min (Fluorescence Arbitrary Units) Agonist Agonist 1µM + BP2 antagonist EC50=330nM 5000 12000 Agonist 1µM + BP1 Agonist 1µM + BP2 EC50= 500 nM 10000 4000 8000 3000 6000 2000 4000 Ki> 5 µM 1000 2000 Ki= 230 nM Ki= 57.4 nM 0 0 1E-9 1E-8 1E-7 1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 100 100010000 100000 1E-9 1E-8 1E-7 1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 100 100010000 100000 Concentrations (µM) Concentrations (µM) Adherent Suspension Ki (nM) Ki (nM) BP2 antagonist 57 nM 230 nM > 5 µM Inactive BP1 antagonist
GPCRs and ligand accessibility 1. Adherent vs suspension cells 2. Receptor functionality at the cell membrane 3. Ligand diffusion
Calcium assay on recombinant-GPCR1 expressing HEK293 cells : Arbitrary Fluorescence units (A.F.U) Agonist EC50 = 300 nM Time
Calcium assay on native-GPCR1 in HUVEC cells : Agonist EC50 = 1.1 µM Arbitrary Fluorescence units (A.F.U) Time Involvement of receptor reserve, agonist-induced structural modifications …. ? Importance of GPCR expressing cells when looking at the calcium response
Calcium assay on native-GPCR1 expressing cells : Evaluation of BPx antagonist (from 1nM to 100 nM) against 3µM reference agonist Agonist, EC50= 1.2 µM Agonist, EC50= 1.2 µM Agonist + 1 nM Antagonist BPx Agonist + 1 nM Antagonist BPx 3200000 Agonist + 3 nM Antagonist BPx Agonist + 3 nM Antagonist BPx 14000 Agonist, EC50 = 1.6 µM Agonist + 10 nM Antagonist BPx Agonist + 10 nM Antagonist BPx Agonist, EC50 = 1.2 µM Agonist + 30 nM Antagonist BPx Agonist + 30 nM Antagonist BPx 12000 3000000 Agonist + 100 nM Antagonist BPx Agonist + 100 nM Antagonist BPx 10000 2800000 Y = A + B * X 2.5 Parameter Value Error Max-Min (F.A.U) ------------------------------------------------------------ Parameter Value Error 1,4 A -0,23066 0,29386 2.0 ------------------------------------------------------------ B 1,57048E8 4,85289E7 AUC (Integrale) A -0.72622 0.37705 ------------------------------------------------------------ 1,2 B 2.09302E8 6.22684E7 R SD N P ------------------------------------------------------------ ------------------------------------------------------------ 1.5 8000 1,0 0,95543 0,32433 3 0,19079 R SD N P ---------------------------------------------------- ------------------------------------------------------------ log (DR-1) pA2 = 8.82 0.95848 0.41616 3 0.18409 0,8 2600000 1.0 pA2 = 8.46 log (DR-1) 0,6 0.5 0,4 6000 pA2=1.5 nM 0.0 0,2 0,0 -0.5 2400000 pA2= 8.46 -0,2 -1.0 -0,4 4000 0,00E+000 2,00E-009 4,00E-009 6,00E-009 8,00E-009 1,00E-008 0.00E+000 5.00E-009 1.00E-008 log [BP1.7577] Log[agonist] log [agonist] 2200000 2000 2000000 0 1800000 1E-30.01 10 100 1000 10000 100000 1 10 100 1000 10000 100000 Agonist (nM) Agonist (nM) No major difference observed when calculating pA2 using Max-Min or A.U.C data
GPCRs and ligand accessibility 1. Adherent vs suspension cells 2. Receptor functionality at the cell membrane 3. Ligand diffusion
Calcium assay: • Rapid and transient signaling system under non equilibrium condition • Influenced by the diffusion characteristics of the injected agonist
Calcium assay: • Rapid and transient signaling system under non equilibrium condition • Influenced by the diffusion characteristics of the injected agonist Diffusion Movement of a fluid from higher concentration to lower concentration The particles will mix until they are evenly distributed This phenomenon of particles distribution is governed by the first and second laws of Fick
The diffusion phenomenom for the agonist may be of importance regarding : Depth and rate of agonist injection Nature and size of considered agonists (aminergic, lipidic, peptidic … ligands) Viscosity of the assay buffer (basic methodology vs NW kits) Volume and surface area of the assay well 96 well plate (full or ½ size wells)
The diffusion phenomenom for the agonist may be of importance regarding : Depth and rate of agonist injection (small molecule ligand) For antagonism charaterization Agonist injection: 10 µl / sec at 9.6 mm height
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