Importance of calcium assay parameters in drug discovery · Importance of calcium assay parameters...

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

GPCRs signaling

Gαq Gαs Gαi Gαq Gαs Gαi

Functional selectivity

Several ligand-specific receptor conformations

can be associated to biased functionnal signaling

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

0 20 40 60 80 100 120

0

20

40

60

80

100

Bias plot for histamine H2 agonists

Histamine

Amthamine

% o

f 10

µM

HA

response o

n G

TP

γ35S

assay

% of 10 µM HA response on calcium assay

Correlation calcium & CRE-MRE reporter assays

% o

f M

ax

refe

rence

ago

nis

tre

sponse

on G

TP

γ35S

bin

din

gassay

% of Max reference agonist response on calcium assay

Agonism Antagonism

ββ ββ -arr

esti

n

Gαs / cAMP

Agonism

Inverse

Agonism

Inverse

Agonism

Agonism

Cardioprotective effectsβ-blockercarvedilol

ββ ββ -arr

esti

n

Gαs / cAMP

Agonism

Inverse

Agonism

Inverse

Agonism

Agonism

Cardioprotective effectsβ-blockercarvedilol

Thanawala VJ et al,

Curr Opin Pharmacol.

2014 Mar 26;16C:50-57

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)

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

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 responsecurve dextral displacement

Max response reduction

Non equilibrium

pA2 = log ( DR – 1 ) – log B

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

of 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

1. Adherent vs suspension cells

3. Ligand diffusion

2. Receptor functionality at the cell membrane

Calcium assay parameters and

GPCRs-ligand accessibility


3. Ligand diffusion

GPCRs and ligand accessibility


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 100010000100000

0

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

Ma

x-M

in (

Flu

ore

sce

nce

Arb

itra

ry U

nits)

Concentrations (µM)

1E-9 1E-8 1E-7 1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 100 100010000100000

0

1000

2000

3000

4000

5000

6000

Agonist

Agonist 1µM + BP1

Agonist 1µM + BP2

Ki= 57.4 nM

Ki> 5 µM

EC50= 500 nM

Ma

x-M

in (

Flu

ore

sce

nce

Arb

itra

ry U

nits)

Concentrations (µM)

Calcium Flux on MSR1-HEK293 adherent cells

MSR1: macrophage scavenger receptor 1

AdherentKi (nM)

SuspensionKi (nM)

BP1 antagonist

BP2 antagonist 230 nM57 nM

> 5 µM Inactive


3. Ligand diffusion



Calcium assay on recombinant-GPCR1 expressing HEK293 cells :

Agonist EC50 = 300 nM

Arb

itra

ryF

luore

scence u

nits

(A.F

.U)

Time

Calcium assay on native-GPCR1 in HUVEC cells :A

rbitra

ryF

luore

scence u

nits

(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

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

(D

R-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)

AU

C (

Inte

gra

le)

Agonist, EC50= 1.2 µM

Agonist + 1 nM Antagonist BPx





1E-30.01 10 100 1000 10000 100000

0

2000

4000

6000

8000

10000

12000

14000

Agonist, EC50= 1.2 µM






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

(D

R-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-M

in (

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


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

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

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 well96 well plate (full or ½ size wells)

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




(small molecule ligand)

For antagonism charaterization

Agonist injection: 10 µl / sec at 9.6 mm height

10 100 1000 10000

0

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.


------------------------------------------------------------A 2,45896 0,98345

B 0,53107 0,1622

------------------------------------------------------------

R SD N P

------------------------------------------------------------

0,91802 0,25649 4 0,08198

EC50= 5 µM

RF

U (

Ma

x-M

in)

Concentration (nM)

10 100 1000 10000

0

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

(D

R-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

RF

U (

Ma

x-M

in)

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

0

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.


------------------------------------------------------------

A 4,63522 0,85702B 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

RF

U (

Max-M

in)

Concentration (nM)

pA2 = 7.05

Inactive

180 µl height 240 µl height100 µl height

Compound BPx antagonism using CHO expressing recombinant hu-GPCR

FDSS µCell

Determined

pA2

Agonist injection height

(related volume)

7.059.6 mm

(240 µl)

6.237.2 mm

(100 µl

Inactive4 mm

(100 µl)Expected pA2 = 7

(FlexStation)






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

0.01 0.1 1 10 100 1000 10000 100000

0

5000

10000

15000

20000 EC50=310nM

Ma

x-M

in (

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

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

Schild regression analysis

Agonist0.03 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

0

2500

5000

7500

10000

12500

15000

17500

20000

22500EC50 = 27 nM

pA2 = 8.4

(3.7 nM)

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

-1

0

1

2

3

4

Y A

xis

Title

X Axis Title

yscale(Y) = A + B * xscale(X)

where scale() is the current axis scale function.


------------------------------------------------------------

A 9,21877 0,70212

B 1,09386 0,09832

------------------------------------------------------------

R SD N P

------------------------------------------------------------0,9881 0,31091 5 0,00156

Agonist (nM)

Ma

x-M

in (

F.A

.U)

Agonist, EC50 = 27 nM

Agonist + Antagonist BPx 1nM



Agonist + Antagonist BPx 1µM

Agonist + Antagonist BPx 10µM

With large peptidic ligand, fast agonist

injection is required to study antagonism

HEK293 cell in 96 well plate

0.01 0.1 1 10 100 1000

0

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-M

in (

Flu

ore

scen

ce

Arb

itra

ry U

nits)

Agonist (nM)

Protocol with NO WASH BD assay buffer

Regular protocol with Fluo4 in HBSS bufer

No Wash BD kit assay buffer

Regular protocolFluo4 in HBSS

assay buffer


Agonist

none

0.3 nM

1 nM

3 nM

10 nM

30 nM

100 nM

300 nM Working window is too narrow to studyantagonism at 10 µl/sec agonist injection

0.1 1 10 100 1000

0

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-M

in (

Flu

ore

scen

ce A

rbitra

ry U

nits)

Agonist + Antagonist 3nM





Agonist

HEK293 cell in 96 well plate


AgonistFrom 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

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

0

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

Ma

x-M

in (

F.A

.U)

Agonist (nM)

Agonist

Agonist 1µM + Antagonist BP1

Agonist 1µM + Antagonist BP2

HEK-GPCR cell suspension in 96 well plateSettings: 10µl/sec, height 9.6 mm, sensitivity 200ms, gain 1

Full size well1/2 size well

Agonist0.01 nMto 10 µM

Agonist1 µM

+ BP1

Agonist1 µM + BP2

Agonist0.01 nMto 10 µM

Agonist1 µM

+ BP1

Agonist1 µM

+ BP2

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

Settings: 200 µl/sec, height 3 mmsensitivity 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 suspension1/2 size well

HEK-GPCR, cell suspension full size well

1E-4 1E-3 0.01 0.1 1 10 100 1000 10000 1000001000000

0

2000

4000

6000

8000

10000

12000

EC50 = 94 nM

FA

U (

ma

x-m

in)

Concentration (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

0

2000

4000

6000

8000

10000

12000

FA

U (

ma

x-m

in)

Concentration (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 mmsensitivity 200ms, gain 1

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

1 10 100 1000 10000

1

10

100

1000

10000

KΒ w

ith w

hole

cell

CR

E-M

RE

-lu

cife

rase

from BP library chemical serie A

from BP library chemical serie B

reference antagonists

Correlation between KΒΒΒΒ 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 w

ith m

em

bra

ne G

TP

gS

KΒ with whole cell Calcium assay

Correlation between KΒΒΒΒ obtained on GTPγγγγ

35S & calcium assays




1 10 100 1000 10000

1

10

100

1000

10000




Correlation between KΒΒΒΒ obtained on GTPγγγγ

35S assays & MRE-CREluc whole cell assays

KΒ w

ith m

em

bra

ne

GT

PgS

KΒ with whole cell CRE-luciferase

Terry Kenekin, JPET 336:296–302, 2011

In fine, we have to keep in mindthat what really matters is

in vivo therapeutic efficacy …

Correlation calcium & CRE-MRE reporter assays

Correlation calcium &

GTPγγγγ35S binding assays

Correlation CRE-MRE reporter &

GTPγγγγ35S binding assays

Importance of calcium assay parameters in drug discovery · Importance of calcium assay parameters...

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Transcript of Importance of calcium assay parameters in drug discovery · Importance of calcium assay parameters...