Importance of calcium assay parameters in drug discovery Sabine - - PowerPoint PPT Presentation

SLIDE 1

Sabine Rouanet

• Dr. Isabelle Bertrand
• Dr. Stéphane Krief
• Dr. Thierry Calmels

Importance of calcium assay parameters in drug discovery

Success is the ability to go from one failure to another with no loss of enthusiasm (Sir Winston Churchill)

Actelion Pharmaceutical Allschwill

Hamamatsu 10th FDSS

User Meeting June 2014

Directeur:

• Pr. Jean-Charles Schwartz

SLIDE 2

GPCRs signaling

Gq Gs Gi Gq Gs Gi

Functional selectivity Several ligand-specific receptor conformations can be associated to biased functionnal signaling

SLIDE 3

Precise affinity required for GPCR antagonism

• Identification of biased signaling
• Studying drug specificity
• Accurate affinity values for pre-development compounds
• Advance SAR analysis

Agonist A Agonist B

20 40 60 80 100 120 20 40 60 80 100

Bias plot for histamine H2 agonists

Histamine Amthamine % of 10 µM HA response on GTPγ

35S assay

% of 10 µM HA response on calcium assay

Correlation calcium & CRE-MRE reporter assays

% of Max reference agonist response

• n GTPγ35S binding assay

% of Max reference agonist response on calcium assay

Agonism Antagonism

β β β β-arrestin Gs / cAMP

Agonism Inverse Agonism Inverse Agonism Agonism Cardioprotective effects

• blockercarvedilol

β β β β-arrestin Gs / cAMP

Agonism Inverse Agonism Inverse Agonism Agonism Cardioprotective effects

• blockercarvedilol

Thanawala VJ et al, Curr Opin Pharmacol. 2014 Mar 26;16C:50-57

SLIDE 4

Calcium mobilization assays at Bioprojet:

HTS Evaluate agonism efficacy and affinity Evaluate type of antagonism Identification of biased ligands

(Identify and classify hits) (Schild regression analysis)

SLIDE 5

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)

GPCR antagonism and Calcium assay in drug discovery

SLIDE 6

pA2 = pKb + log ( 1+ 2 [ A ] / Ka )

At low [agonist] occupancy [ A ] < < < Ka

pA2 tend towards the pKb Use of the pA2 as a universal determinant of antagonist potency

• Arthur Christopoulos et al, 1999, Euro J Pharmacol, 382:217–227
• 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

Calculation of pA2 at low agonist responses

Overcome the potential bias associated with non equilibrium conditions Estimate insurmountable antagonists affinity

pA2 ~ pKb + log (1)

Concentration response curve dextral displacement Max response reduction

Non equilibrium

SLIDE 7

pA2 = log ( DR – 1 ) – log B

pA2 = - log [M] of antagonist producing a 2 fold shift

• f the agonist concentration response curve

Use of Dose Ratio (DR) values as surrogate parameter for calculation of pA2

Competitive surmountable Antagonism at equilibrium Non competitive (Insurmountable) Antagonism at Hemi-equilibrium DR at EC50 DR at low agonist response

At DR = 2 pA2 = – log B

SLIDE 8

• 1. Adherent vs suspension cells
• 3. Ligand diffusion
• 2. Receptor functionality at the cell membrane

Calcium assay parameters and GPCRs-ligand accessibility

SLIDE 9

• 1. Adherent vs suspension cells
• 3. Ligand diffusion

GPCRs and ligand accessibility

• 2. Receptor functionality at the cell membrane

SLIDE 10

Settings: 10µl/sec, height 9.6 mm, sensitivity 200ms, gain 1

1E-9 1E-8 1E-7 1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 100 100010000 100000 2000 4000 6000 8000 10000 12000 14000

Calcium Flux on HEK293 cell suspension

Ki= 230 nM

EC50=330nM

Agonist Agonist 1µM + BP1 antagonist Agonist 1µM + BP2 antagonist

Max-Min (Fluorescence Arbitrary Units) Concentrations (µM)

1E-9 1E-8 1E-7 1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 100 100010000 100000 1000 2000 3000 4000 5000 6000

Agonist Agonist 1µM + BP1 Agonist 1µM + BP2

Ki= 57.4 nM

Ki> 5 µM EC50= 500 nM

Max-Min (Fluorescence Arbitrary Units) Concentrations (µM) Calcium Flux on MSR1-HEK293 adherent cells

MSR1: macrophage scavenger receptor 1

Adherent Ki (nM) Suspension Ki (nM) BP1 antagonist BP2 antagonist

230 nM 57 nM > 5 µM Inactive

SLIDE 11

• 1. Adherent vs suspension cells
• 3. Ligand diffusion

GPCRs and ligand accessibility

• 2. Receptor functionality at the cell membrane

SLIDE 12

Calcium assay on recombinant-GPCR1 expressing HEK293 cells :

Agonist EC50 = 300 nM

Arbitrary Fluorescence units (A.F.U) Time

SLIDE 13

Calcium assay on native-GPCR1 in HUVEC cells :

Arbitrary Fluorescence units (A.F.U) Time

Agonist EC50 = 1.1 µM

Involvement of receptor reserve, agonist-induced structural modifications …. ?

Importance of GPCR expressing cells when looking at the calcium response

SLIDE 14

1 10 100 1000 10000 100000 1800000 2000000 2200000 2400000 2600000 2800000 3000000 3200000

0.00E+000 5.00E-009 1.00E-008

• 1.0
• 0.5

0.0 0.5 1.0 1.5 2.0 2.5 pA2= 8.46 log (DR-1) log [agonist] Parameter Value Error

• A
• 0.72622

0.37705 B 2.09302E8 6.22684E7

• R

SD N P

• 0.95848

0.41616 3 0.18409

Agonist, EC50 = 1.2 µM

pA2 = 8.46

Agonist (nM) AUC (Integrale)

Agonist, EC50= 1.2 µM Agonist + 1 nM Antagonist BPx Agonist + 3 nM Antagonist BPx Agonist + 10 nM Antagonist BPx Agonist + 30 nM Antagonist BPx Agonist + 100 nM Antagonist BPx 1E-30.01 10 100 1000 10000 100000 2000 4000 6000 8000 10000 12000 14000 Agonist, EC50= 1.2 µM Agonist + 1 nM Antagonist BPx Agonist + 3 nM Antagonist BPx Agonist + 10 nM Antagonist BPx Agonist + 30 nM Antagonist BPx Agonist + 100 nM Antagonist BPx

pA2 = 8.82

0,00E+000 2,00E-009 4,00E-009 6,00E-009 8,00E-009 1,00E-008

• 0,4
• 0,2

0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 pA2=1.5 nM log (DR-1) log [BP1.7577] Y = A + B * X Parameter Value Error

• A
• 0,23066

0,29386 B 1,57048E8 4,85289E7

• R

SD N P

• 0,95543

0,32433 3 0,19079

• Agonist, EC50 = 1.6 µM

Max-Min (F.A.U) Agonist (nM) Log[agonist]

Calcium assay on native-GPCR1 expressing cells :

Evaluation of BPx antagonist (from 1nM to 100 nM) against 3µM reference agonist

No major difference observed when calculating pA2 using Max-Min or A.U.C data

SLIDE 15

• 1. Adherent vs suspension cells
• 3. Ligand diffusion

GPCRs and ligand accessibility

• 2. Receptor functionality at the cell membrane

SLIDE 16

Calcium assay:

• Rapid and transient signaling system under non equilibrium condition
• Influenced by the diffusion characteristics of the injected agonist

SLIDE 17

Calcium assay:

• Rapid and transient signaling system under non equilibrium condition
• Influenced by the diffusion characteristics of the injected agonist

This phenomenon of particles distribution is governed by the first and second laws of Fick Diffusion Movement of a fluid from higher concentration to lower concentration The particles will mix until they are evenly distributed

SLIDE 18

The diffusion phenomenom for the agonist may be of importance regarding :

Depth and rate of agonist injection 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) Nature and size of considered agonists (aminergic, lipidic, peptidic … ligands)

SLIDE 19

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

SLIDE 20

10 100 1000 10000 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000

Ago Ago + X 100nM Ago + X 300nM Ago + X 1 µM Ago + X 3 µM Ago + X 10 µM

• 7,0
• 6,5
• 6,0
• 5,5
• 5,0
• 1,4
• 1,2
• 1,0
• 0,8
• 0,6
• 0,4
• 0,2

0,0 0,2 pA2 = 4.63 (23 µM) log(DR-1) log [BS6.890] yscale(Y) = A + B * xscale(X) where scale() is the current axis scale function. Parameter Value Error

• A

2,45896 0,98345 B 0,53107 0,1622

• R

SD N P

• 0,91802

0,25649 4 0,08198

EC50= 5 µM

RFU (Max-Min)

Concentration (nM) 10 100 1000 10000 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000

Expected pA2 = 7

• 7,0
• 6,5
• 6,0
• 5,5
• 5,0
• 0,2

0,0 0,2 0,4 0,6 0,8 1,0 pA2= 7.05 (89.1 nM) log (DR-1) log [BS6-890] yscale(Y) = A + B * xscale(X) where scale() is the current axis scale function. Parameter Value Error

• A

2,93449 0,20493 B 0,41618 0,03387

• R

SD N P

• 0,99021

0,05356 5 0,00116

EC50=2µM

RFU (Max-Min)

Concentration (nM)

Ago Ago + X 100nM Ago + X 300nM Ago + X 1 µM Ago + X 3 µM Ago + X 10 µM 10 100 1000 10000 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000

• 7,0
• 6,5
• 6,0
• 5,5
• 5,0
• 0,8
• 0,6
• 0,4
• 0,2

0,0 0,2 0,4 0,6 0,8 1,0 1,2 pA2 = 6.23 (589 nM) log (DR-1) log [BS6.890] yscale(Y) = A + B * xscale(X) where scale() is the current axis scale function. Parameter Value Error

• A

4,63522 0,85702 B 0,74365 0,14135

• R

SD N P

• 0,96572

0,22351 4 0,03428

Ago Ago + X 100nM Ago + X 300nM Ago + X 1 µM Ago + X 3 µM Ago + X 10 µM

pA2 = 6.23

EC50=835nM

RFU (Max-Min)

Concentration (nM)

pA2 = 7.05

Inactive

180 µl height

240 µl height

100 µl height

Compound BPx antagonism using CHO expressing recombinant hu-GPCR

FDSS µCell Determined pA2

Agonist injection height (related volume)

7.05

9.6 mm (240 µl)

6.23

7.2 mm (100 µl

Inactive

4 mm (100 µl)

Expected pA2 = 7 (FlexStation)

SLIDE 21

The diffusion phenomenom for the agonist may be of importance regarding :

Depth and rate of agonist injection 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)

Nature and size of considered agonists (aminergic, lipidic, peptidic ligands)

SLIDE 22

0.01 0.1 1 10 100 1000 10000 100000 5000 10000 15000 20000

EC50=310nM

Max-Min (F.A.U) Concentration (nM)

Agonist Agonist 100nM + antagonist BPx, Ki = 17 nM

CHO-GPCR cells in 96 well plate

Antagonism study using

large peptidic endogenous agonist

Settings: 10µl/sec, height 3 mm sensitivity 200ms, gain 2 Settings: 80µl/sec, height 3 mm sensitivity 200ms, gain 2 Determined Ki for antagonist and EC50 for agonist far from expected

• ~ 1 nM and 30 nM, respectively

No signal

SLIDE 23

Settings: 200µl/sec, height 3 mm, sensitivity 200ms, gain 2 Schild regression analysis

Agonist 0.03 nM to 3 µM Agonist + 1 nM Antagonist Agonist + 10 nM Antagonist Agonist + 100 nM Antagonist Agonist + 1 µM Antagonist Agonist + 10 µM Antagonist

[Antagonist]

Antagonism study on CHO-GPCR cells with large peptidic agonist

1E-4 1E-3 0.01 0.1 1 10 100 1000 10000 100000 2500 5000 7500 10000 12500 15000 17500 20000 22500 EC50 = 27 nM

pA2 = 8.4 (3.7 nM)

1E-9 1E-8 1E-7 1E-6 1E-5

• 1

1 2 3 4 Y Axis Title X Axis Title yscale(Y) = A + B * xscale(X) where scale() is the current axis scale function. Parameter Value Error

• A

9,21877 0,70212 B 1,09386 0,09832

• R

SD N P

• 0,9881 0,31091

5 0,00156

Agonist (nM) Max-Min (F.A.U)

Agonist, EC50 = 27 nM Agonist + Antagonist BPx 1nM Agonist + Antagonist BPx 10nM Agonist + Antagonist BPx 100nM Agonist + Antagonist BPx 1µM Agonist + Antagonist BPx 10µM

With large peptidic ligand, fast agonist injection is required to study antagonism

SLIDE 24

The diffusion phenomenom for the agonist may be of importance regarding :

Depth and rate of agonist injection

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) Nature and size of considered agonists (aminergic, lipidic, peptidic ligands)

SLIDE 25

HEK293 cell in 96 well plate

0.01 0.1 1 10 100 1000 1000 2000 3000 4000 5000 6000 7000 8000

HEK293 cell suspension in 96 well plate

EC50 = 3.8 nM EC50 = 2.5 nM

µCell settings (vit 10µl/sec, height 3 mm, sensitivity 200ms, gain 1)

Max-Min (Fluorescence Arbitrary Units) Agonist (nM)

Protocol with NO WASH BD assay buffer Regular protocol with Fluo4 in HBSS bufer

No Wash BD kit assay buffer Regular protocol Fluo4 in HBSS assay buffer Settings: 10µl/sec, height 3 mm, sensitivity 200ms, gain 1

Agonist

none 0.3 nM 1 nM 3 nM 10 nM 30 nM 100 nM 300 nM

Working window is too narrow to study antagonism at 10 µl/sec agonist injection

SLIDE 26

0.1 1 10 100 1000 1000 2000 3000 4000 5000 6000 7000 8000

µCell settings (vit 200µl/sec, height 3mm, sensitivity 200ms, gain 1)

EC50= 3.6 nM

Agonist (nM) Max-Min (Fluorescence Arbitrary Units)

Agonist + Antagonist 3nM Agonist + Antagonist 10nM Agonist + Antagonist 30nM Agonist + Antagonist 100nM Agonist + Antagonist 300nM Agonist

HEK293 cell in 96 well plate

Settings: 200µl/sec, height 3 mm, sensitivity 200ms, gain 1

Agonist

From 0.3 nM to 300 nM

Agonist + 3 nM Antagonist

Schild regression analysis

Agonist + 10 nM Antagonist Agonist + 30 nM Antagonist Agonist + 100 nM Antagonist Agonist + 300 nM Antagonist

Antagonism study using No Wash BD kit assay buffer

In No Wash buffer, At 200 µl/sec agonist injection, the Kb (pA2) can be determined for an antagonism

SLIDE 27

The diffusion phenomenom for the agonist may be of importance regarding :

Depth and rate of agonist injection 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)

Nature and size of considered agonists (aminergic, lipidic, peptidic ligands)

SLIDE 28

Agonist

none 0.01 nM 0.1 nM 1 nM 10 nM 100 nM 1 µM 10 µM

0.01 0.1 1 10 100 1000 10000 1000 2000 3000 4000 5000 6000 7000 8000

HEK-GPCR cell suspension, full size well (96 well plate)

µCell settings: vit 10µl/sec, height 3mm, sensitivity 200ms, gain 1

inactive Ki=13nM EC50=35nM

Max-Min (F.A.U) Agonist (nM)

Agonist Agonist 1µM + Antagonist BP1 Agonist 1µM + Antagonist BP2

HEK-GPCR cell suspension in 96 well plate

Settings: 10µl/sec, height 9.6 mm, sensitivity 200ms, gain 1

Full size well 1/2 size well

Agonist 0.01 nM to 10 µM Agonist 1 µM + BP1 Agonist 1 µM + BP2 Agonist 0.01 nM to 10 µM Agonist 1 µM + BP1 Agonist 1 µM + BP2

Settings for full size well are not compatible with ½ size well

SLIDE 29

Settings: 200 µl/sec, height 3 mm sensitivity 200ms, gain 1

Antagonism study using ½ size well

There is no differences when Ki is calculated from experiments done in ½ or full size well, at 3mm height but at different rate agonist injection

HEK-GPCR, cell suspension 1/2 size well HEK-GPCR, cell suspension full size well

1E-4 1E-3 0.01 0.1 1 10 100 1000 10000 100000 1000000 2000 4000 6000 8000 10000 12000 EC50 = 94 nM

FAU (max-min) Concentration (nM)

Agonist, EC50 = 94 nM Agonist 1 µM + BP1, ki= 52.8 nM Agonist 1 µM + BP2, Ki= 258 nM 1E-3 0.01 0.1 1 10 100 1000 10000 100000 2000 4000 6000 8000 10000 12000

FAU (max-min) Concentration (nM)

Agonist, EC50 = 69 nM Agonist 1 µM + BP1, Ki= 52.3 nM Agonist 1 µM + BP2, Ki= ki= 207 nM EC50 = 69 nM

1/2 size Full size Agonist EC50 (nM) 69 94 Antagonist BP1 Ki (nM) 52.3 52.8 Antagonist BP2 Ki (nM) 207 258 96 Well plate

Settings: 10 µl/sec, height 3 mm sensitivity 200ms, gain 1

SLIDE 30

SUMMARY

• Use of the pA2 for antagonist potency to overcome the

potential bias associated with non equilibrium conditions

• Precise and defined agonist parameters needed for

any given GPCR when implementing calcium assay

• Receptor functionality at cell membrane
• GPCR expressing cells
• Ligand diffusion
• Receptor homo/hetero oligomerization
• Binding kinetics
• Receptor trafficking
• Other important parameters to consider

SLIDE 31

1 10 100 1000 10000 1 10 100 1000 10000

KΒ with whole cell CRE-MRE-luciferase

from BP library chemical serie A from BP library chemical serie B reference antagonists

Correlation between KΒ

Β Β Β obtained on calcium assay & MRE-CREluc whole cell assay

KΒ with whole cell Calcium assay

1 10 100 1000 10000 1 10 100 1000

KΒ with membrane GTPgS KΒ with whole cell Calcium assay Correlation between KΒ

Β Β Β obtained on GTPγ

γ γ γ

35S & calcium assays

from BP library chemical serie A from BP library chemical serie B reference antagonists 1 10 100 1000 10000 1 10 100 1000 10000 from BP library chemical serie A from BP library chemical serie B reference antagonists Correlation between KΒ

Β Β Β obtained on GTPγ

γ γ γ

35S assays & MRE-CREluc whole cell assays

KΒ with membrane GTPgS KΒ with whole cell CRE-luciferase

Terry Kenekin, JPET 336:296–302, 2011

In fine, we have to keep in mind that what really matters is in vivo therapeutic efficacy …

Correlation calcium & CRE-MRE reporter assays Correlation calcium & GTPγ γ γ γ35S binding assays Correlation CRE-MRE reporter & GTPγ γ γ γ35S binding assays