Identification and characterization of allosteric modulators of GPCRs: The utility of HTRF and incorporation into generalised screening … · MRC Technology Centre for Therapeutics

MRC Technology Centre for Therapeutics Discovery

Identification and characterization of allosteric

modulators of GPCRs: The utility of HTRF and

incorporation into generalised screening strategies

Jeff Jerman

ELRIG – Cisbio Workshop

Sep 2012

Presentation Outline

Melanocortin receptors

Allosteric modulation of 7TM

HTS and compound profiling considerations

� Major challenges

� Suggested PAM screening strategies

Comparative Pharmacology

Summary

Melanocortin receptors

MelanocyteEndothelial Cells

FibroblastMonocytes

ACTH

Adrenal CortexAdipocytes

MacrophagesBrainGut

Placenta

γ-MSH α-MSH = ACTH

Brain

γ -MSH ACTH

α -MSH

BrainPeripheral Tissues

MC1 MC2 MC3 MC4 MC5

γ -MSH ACTH

α -MSH

γ -MSH ACTH

α -MSH

Pigmentation Inflammation

SteroidogenesisCardiovascular

Function, Inflammation

Feeding Control (obesity)

Control of the Sebaceous Gland

MC3 receptor stimulators are predicted to drive resolution of inflammation

Targeting novel compound mechanism of action

Orthosteric vs Allosteric binding/functional modality

Advantages of Positive Allosteric Modulation (PAM)

1. Improved selectivity

2. Saturability (self-limiting) biological effect

3. Temporal and spatial resolution

Agonist vs PAM Assay configurations

7TM HTS can be configured to detected both

agonism and Positive Allosteric modulation

simultaneously

� The ‘simple’ inclusion of a submaximal

(EC20) of agonist facilitates this

Test Compound

Cells Detect ActivationSensitive to Agonism

(orthosteric and allosteric)

Cells Detect ActivationSensitive to Agonism

AND Potentiation

EC20 of native agonist

Agonists

PAMS

Test Compound

7TM PAM HTS/CP – Major Challenges (1)

Prediction and control of an EC20 stimulus

� Endogenous melanocortin agonists are ‘sticky’ peptides (loss and/or carry over)

� Changes in receptor expression/coupling can dramatically affect pEC50

� The predictability and stability of the EC20 determines the sensitivity to PAMS

� HTRF affords both sensitivity and stability in response

0

20

40

60

80

100

120

-11 -10 -9 -8 -7 -6 -5 -4

log [Agonist] (M)

% M

ax R

espo

nse

Agonist Alone

Agonist + Pos mod

Small potentiation

Use of agonist EC20 maximises

potentiation widow

0

20

40

60

80

100

120

-11 -10 -9 -8 -7 -6 -5 -4

log [Agonist] (M)

% M

ax R

espo

nse

Agonist Alone

Agonist + Pos mod

Small potentiation

Use of agonist EC20 maximises

potentiation widow

Automated Assay Protocol

Compound pre-exposure time can be optimised to sensitise the

assay to PAMs

Relatively lengthy incubation enables study of ‘slow-on’ binders (e.g. PAM)

Agonist-induced cAMP accumulation (generic HTRF automation protocol)

Library Plate20ul, 1mM, 100%DMSO

Dilution Plate50ul, 40uM, 4%DMSO

Assay Plate10ul, 10uM, 1%DMSO

Agonist addition(EC100,EC0,EC20)

1st Addition Detection Mix (cAMP-d2)

2ulPBS Diluent Plate

48ul

Cell Plate10-15K cells/well

Stop reaction

2.5ul

2.5ul

5ul

5ul

Incubation, 30min, RT

Read

Incubation, 60min, RT2nd Addition Detection Mix

(anti-cAMP-cryptate)

5ul

HTS Performance

Raw Data

Plate #

HT

RF

Rat

io

0 50 100 150 200 250 300 3500

5000

10000

15000

20000EC100 EC20 EC0

% Control (max)

Plate #

% A

ctiv

atio

n

0 50 100 150 200 250 300 3500

20

40

60

80

100

120EC100 EC0

Z Prime

Plate #

Z'

0 50 100 150 200 250 300 3500.0

0.2

0.4

0.6

0.8

1.0

EC20 %Control (max)

Plate #

% A

ctiv

atio

n

0 50 100 150 200 250 300 3500

10

20

30

40Mean

HTS Performance

Frequency Distribution

% Activity

Fre

quen

cy

-60 -40 -20 0 20 40 600

1000

2000

3000

4000

5000

6000

7000

8000

Frequency Distribution

% Activity

Cum

ulat

ive

Fre

quen

cy

-20 0 20 40 60 80 100 1200

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

8469

3372

1658912

520 269 147 62 12 2

-60 -40 -20 0 20 40 600

1000

2000

3000

4000

5000

6000

7000

8000

30.22%

66.71%

2.24%

0.59%

0.00%

0.05%0.19%

>=10080 -10060 - 8040 - 6040 - 2020 - 0<=0

% Activity Distribution

% Activity Bin

-60 -40 -20 0 20 40 600

1000

2000

3000

4000

5000

6000

7000

8000

109760 compounds @ 10mM (1% DMSO)

Cutoff(%) # Hits % HR40 912 0.8350 520 0.4760 269 0.2570 147 0.1380 62 0.0690 12 0.01100 2 0.00

Mean Z’ = 0.84 (±0.06)

Low Control %CV = 5.3 (±2.7)

High Control %CV= 3.6 (±1.5)

GalR2 IP1 HTS – Agonist incubation time

Agonist pEC50 is not dependant on incubation time

(within these limits)

This suggests an EC20 should be stable throughout

an HTS run/day

Galanin-induced IP1 accumulation in CHO (Gα16) cells stably expressing hGalR2

log [hGalanin] (M)

665/

620n

m H

TRF

rat

io

-10 -9 -8 -7 -6 -50

5000

10000

15000

20000

25000

buffer

30mins45mins60mins75mins90mins

Agonist Incubation

GalR2 IP1 HTS – Detection reagent stability

Signal window is stable up to 24hrs

Long read times (e.g. high plate numbers

plates/reader delays) will not adversely affect

assay performance

Galanin-induced IP1 accumulation in CHO (Gα16) cells stably expressing hGalR2

log [hGalanin] (M)

665/

620n

m H

TRF

rat

io

-10 -9 -8 -7 -6 -50

5000

10000

15000

20000

25000

0.5hr read1hr read1.5hr read2hr read2.5hr read>24hr read

GalR2 IP1 HTS – Stability and variance of EC20

Day 1 Day 2

5nM Galanin produces a robust and stable response (~EC20)

CVs <10%, window 2.5 fold, Z’ 0.6 – 0.7

Agonist-induced increase in [IP1] in CHO cellsstably expressing GalR2 receptors

% M

ax R

espo

nse

Hig

h C

on

tro

l

Lo

w C

on

tro

l 1 5 10 500

20

40

60

80

100

120

0

27

48

80

[Galanin] (nM)

Agonist-induced increase in [IP1] in CHO cellsstably expressing GalR2 receptors

% M

ax R

esp

on

se

Hig

h C

on

tro

l

Lo

w C

on

tro

l 1 5 10 50

0

20

40

60

80

100

120

[Galanin] (nM)

-1

22

43

78

PAM HTS/CP – Major Challenges (2)

Deconvolution of PAM/Agonist hits

Removing false positives and/or non-preferred mechanism(s)

Ago

nist

s (o

rtho

& a

llo)

Test Compound aloneTest Compound alone

(Host cells)

Test Compound +

Competitive Antagonist

Test Compound +

EC20 of screen agonist

Test Compound +

EC20 of screen agonist+

Competitive Antagonist

Test Compound +

EC20 of alternative agonist

Test Compound +

EC20 of alternative agonist +

Competitive Antagonist (of alternative agonist)

EC20 and/or MoAAssay in cells with

different expression level

Leftward shift MoA assays to determine

efficacy and potency (of modulation)

Orthologue Assay(s)

PA

Ms

(& N

AM

s)S

peci

ficity

&

sel

ectiv

ity

Binding Assay(s)

Deconvolution of PAM/Agonist modalities (Single Shot Triage)

PAM_%ActvityPAM Activity (% Lys-γ3-MSH Max)

Ago

nist

Act

ivity

(%

Lys

-γ3-

MS

H M

ax)

AgonistsActive in the absence of native ligand

PAMSActive only in the presence of native ligand

Native agonist present

Nat

ive

agon

ist a

bsen

t

absent

Control 1Control 2Control 3Test

Control 1Control 2Control 3Test

Agonist-induced increase in [cAMP]i in CHO cellsstably expressing MC3 receptors

-10 -9 -8 -7 -6 -5 -4

0

20

40

60

80

100

120Cmpd 1

Cmpd 2

Cmpd 3

Cmpd 4

Cmpd 5Cmpd 6

Cmpd 7

log [Compound] (M)

% A

ctiv

atio

n

PAM-induced increase in agonist-mediated [cAMP]iin CHO cells stably expressing MC3 receptors

-10 -9 -8 -7 -6 -5 -4

0

20

40

60

80

100

120

Cmpd 2

Cmpd 3Cmpd 7

log [Compound] (M)

% A

ctiv

atio

n

Increase in [cAMP]i in CHO cellsstably expressing MC3 receptors

-10 -9 -8 -7 -6 -5 -4

0

20

40

60

80

100

120Cmpd 1

Cmpd 2

Cmpd 3

Cmpd 4Cmpd 5

Cmpd 6

Cmpd 7

log [Compound] (M)

% A

ctiv

atio

n

Agonists PAMS

-EC20 +EC20

Deconvolution of PAM/Agonist modalities (Full Curve)


(leftward shift)

-12 -11 -10 -9 -8 -7 -6 -5 -4 -3

0

20

40

60

80

100

120

Lys-γ3-MSH Alone

PAM Alone

log [Lys-γ3-MSH] (M)

% A

ctiv

atio

n


(leftward shift)

-12 -11 -10 -9 -8 -7 -6 -5

0

20

40

60

80

100

120 Lys-γ3-MSH Alone

+ 100µM PAM

+ 31.6µM PAM

+ 10µM PAM

+ 3.16µM PAM

+ 1µM PAM

+ 316nM PAM

+ 100nM PAM


% A

ctiv

atio

n

Agonist-induced increase in [cAMP]i in CHO cellsstably expressing β2 -Adrenoceptors

(leftward shift)

-12 -11 -10 -9 -8 -7 -6 -5

0

20

40

60

80

100

120

Metaproterenol Alone

+ PAM [25µM]

log [Metaproterenol] (M)

% A

ctiv

atio

n

Shared robotic HTRF protocols facilitate;

� Assay in both single-point and full-

curve mode (& transitions between)

� Flexibility in assay design (pEC50mod

vs leftward shift)

� Simultaneous ‘counter’ assay (Ag vs

PAM, off-target selectivity)


(leftward shift)

-11 -10 -9 -8 -7 -6

0

20

40

60

80

100

120

Lys-y3-MSH CRC Alone

+ 1µM Cmpd 7+ 3µM Cmpd 7+ 10µM Cmpd 7


% A

ctiv

atio

n

Potentiation of affinity

No overt agonism

Moderate pEC50 of modulation (EC20 mode)translates to very effective leftward shift

PAM-induced increase in agonist-mediated [cAMP]iin CHO cells stably expressing MC3 receptors

-10 -9 -8 -7 -6 -5 -4

0

20

40

60

80

100

120

Cmpd 2

Cmpd 3Cmpd 7

log [Compound] (M)

% A

ctiv

atio

nApparent Potency in different assay formats

PAM HTS/CP – Major Challenges (3)

Differences in pharmacology between cAMP detection systems

� PAM ‘activity’ may not necessarily align between detection formats


% A

ctiv

atio

n

-12 -10 -8 -6 -4

0

20

40

60

80

100

120

Lys-γ3-MSH Alone+ 30uM PAM1


% A

ctiv

atio

n

-12 -10 -8 -6 -4

0

20

40

60

80

100

120

Lys-γ3-MSH Alone+ 30µM PAM2


% A

ctiv

atio

n

-12 -10 -8 -6 -4

0

20

40

60

80

100

120

Lys-γ3-MSH Alone+ 30µM PAM3


% A

ctiv

atio

n

-13 -11 -9 -7

0

20

40

60

80

100

120Lys-γ3-MSH Alone

+ 30µM PAM1


% A

ctiv

atio

n

-13 -11 -9 -7

0

20

40

60

80

100

120Lys-γ3-MSH Alone+ 30µM PAM2


% A

ctiv

atio

n

-13 -11 -9 -7

0

20

40

60

80

100

120Lys-γ3-MSH Alone+ 30µM PAM3


% A

ctiv

atio

n

-12 -10 -8 -6 -4

0

20

40

60

80

100

120

Lys-γ3-MSH Alone+ 30uM PAM4


% A

ctiv

atio

n

-12 -10 -8

0

20

40

60

80

100

120Lys-γ3-MSH Alone+ 30uM PAM4

HTRF HTRF*CRE

*GeneBLAzer; β-lactamase coupled to a cyclic AMP response element (CRE)

*CRE

Summary

HTRF provides a sensitive and stable assay from which to configure PAM assay(s)

� In HTS mode the stability of EC20 is pivotal to PAM sensitivity

HTS and full curve hit profiling assays can be configured to share common (simple) robotised protocols

� pEC50 of modulation (underpinned by EC20)

� Partial or full leftward shifts provide texture to PAM activity

� Quantitative pharmacological analyses e.g. ETCM modelling to dissect potentiation of affinity vs efficacy

The technology lends itself to establishing appropriate and

necessarily extensive deconvolution assays

� Which can share a common detection platform

Summary

The accumulation nature of the signal affords greater flexibility

in compound pre-exposure

� Arguably improving the sensitivity to slow binders (PAMS)

� Circumventing confounding kinetic issues with more

transient detection systems (Ca2+)

The pharmacology of PAMs is complex and ‘perfect’ alignment

with other (cAMP) detection technologies is likely to be rare

� A plethora of biological factors can give rise to subtle

differences in apparent PAM pharmacology

Acknowledgments

University of Bristol

David Wynick

William Harvey Research Institute,

Queen Mary University

Mauro Perretti

Trinidad Montero-Melendez

Ahmad Kamal

Jenny Cook

Rachel Forfar

Debra Taylor

Hayley Jones

Paul Wright

Puneet Khurana

Alternative PAM assay configurations

0

20

40

60

80

100

-7 -6 -5 -4 -3 -2log [Agonist] (M)

% R

esp

ons

e

Ag Alone

Ag + PAM [1]

Ag + PAM [2]

Ag + PAM [3]

Ag + PAM [4]

0

20

40

60

80

100

-7 -6 -5 -4 -3 -2log [Agonist] (M)

% R

esp

on

se

Ag Alone

Ag + PAM CRC

Ag + PAM CRC

Ag + PAM CRC

Ag + PAM CRC

‘Leftward Shift’ ‘EC20’

Agonsit CRC Agonsit EC20

++++

++++

Exemplar curve signatures

‘Clean’ PAM

PAM with (allo) agonism

Non specific compound (receptor and or mechanism)

PAM MC3 PAM β2Ag MC3MCR3 PAM cAMP HTRF - Plate 4, Cpd 15

Log [M]

% A

ctiv

atio

n

-9 -8 -7 -6 -5 -4-30-20-10

0102030405060708090

100MRT00162442MRT00162442

MCR3 AG cAMP HTRF - Plate 4, Cpd 15

Log [M]

% A

ctiv

atio

n

-9 -8 -7 -6 -5 -4-30-20-10

0102030405060708090

100MRT00162442MRT00162442

ADRB2 cAMP HTRF - Plate 4, Cpd 15

Log [M]

% A

ctiv

atio

n

-9 -8 -7 -6 -5 -4-30-20-10

0102030405060708090

100MRT00162442MRT00162442

MCR3 PAM cAMP HTRF - Plate 4, Cpd 11

Log [M]

% A

ctiv

atio

n

-9 -8 -7 -6 -5 -4-30-20-10

0102030405060708090

100MRT00067246MRT00067246


Log [M]

% A

ctiv

atio

n

-9 -8 -7 -6 -5 -4-30-20-10

0102030405060708090

100MRT00067246MRT00067246


Log [M]

% A

ctiv

atio

n

-9 -8 -7 -6 -5 -4-30-20-10

0102030405060708090

100MRT00067246MRT00067246

MCR3 PAM cAMP HTRF - Plate 1, Cpd 13

Log [M]

% A

ctiv

atio

n

-9 -8 -7 -6 -5 -4-30-20-10

0102030405060708090

100MRT00181059MRT00181059


Log [M]%

Act

ivat

ion

-9 -8 -7 -6 -5 -4-30-20-10

0102030405060708090

100MRT00181059MRT00181059


Log [M]

% A

ctiv

atio

n

-9 -8 -7 -6 -5 -4-30-20-10

0102030405060708090

100MRT00181059MRT00181059

Documents

Identification and characterization of allosteric modulators of GPCRs: The utility of HTRF and incorporation into generalised screening … · MRC Technology Centre for Therapeutics