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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)
Agonist-induced increase in [cAMP]i in CHO cellsstably expressing MC3 receptors
(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
Agonist-induced increase in [cAMP]i in CHO cellsstably expressing MC3 receptors
(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
log [Lys-γ3-MSH] (M)
% 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)
Agonist-induced increase in [cAMP]i in CHO cellsstably expressing MC3 receptors
(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
log [Lys-γ3-MSH] (M)
% 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
log [Lys-γ3-MSH] (M)
% A
ctiv
atio
n
-12 -10 -8 -6 -4
0
20
40
60
80
100
120
Lys-γ3-MSH Alone+ 30uM PAM1
log [Lys-γ3-MSH] (M)
% A
ctiv
atio
n
-12 -10 -8 -6 -4
0
20
40
60
80
100
120
Lys-γ3-MSH Alone+ 30µM PAM2
log [Lys-γ3-MSH] (M)
% A
ctiv
atio
n
-12 -10 -8 -6 -4
0
20
40
60
80
100
120
Lys-γ3-MSH Alone+ 30µM PAM3
log [Lys-γ3-MSH] (M)
% A
ctiv
atio
n
-13 -11 -9 -7
0
20
40
60
80
100
120Lys-γ3-MSH Alone
+ 30µM PAM1
log [Lys-γ3-MSH] (M)
% A
ctiv
atio
n
-13 -11 -9 -7
0
20
40
60
80
100
120Lys-γ3-MSH Alone+ 30µM PAM2
log [Lys-γ3-MSH] (M)
% A
ctiv
atio
n
-13 -11 -9 -7
0
20
40
60
80
100
120Lys-γ3-MSH Alone+ 30µM PAM3
log [Lys-γ3-MSH] (M)
% A
ctiv
atio
n
-12 -10 -8 -6 -4
0
20
40
60
80
100
120
Lys-γ3-MSH Alone+ 30uM PAM4
log [Lys-γ3-MSH] (M)
% 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
MCR3 AG cAMP HTRF - Plate 4, Cpd 11
Log [M]
% A
ctiv
atio
n
-9 -8 -7 -6 -5 -4-30-20-10
0102030405060708090
100MRT00067246MRT00067246
ADRB2 cAMP HTRF - Plate 4, Cpd 11
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
MCR3 AG cAMP HTRF - Plate 1, Cpd 13
Log [M]%
Act
ivat
ion
-9 -8 -7 -6 -5 -4-30-20-10
0102030405060708090
100MRT00181059MRT00181059
ADRB2 cAMP HTRF - Plate 1, Cpd 13
Log [M]
% A
ctiv
atio
n
-9 -8 -7 -6 -5 -4-30-20-10
0102030405060708090
100MRT00181059MRT00181059