Cerevel Therapeutics : Binding and Signal Profiling of Full and Partial M4 Agonists

Binding and Signal Profiling of Full and Partial M4 Agonists

INTRODUCTION

●The M4 muscarinic acetylcholine receptor (mAChR) is

one of 5 mAChR subtypes (M1-M5) in the G-protein

coupled receptor (GPCR) superfamily

METHODS

• Radioligand binding was performed using a classical filtration method
• The G-protein activation assay (GTPgS) was performed with scintillation proximity assay (SPA) technology

Sokhom S. Pin,1 Angela Wonsey-Quinn,1 Deborah L. Smith,1 Leonard Te,1 May Fern Toh,1 Hanh Nho Nguyen,1 Philip Iredale1

1Cerevel Therapeutics, Cambridge, MA, USA

Presenting Author: Sokhom S. Pin; [e-mail]

SUMMARY

●The M4 mAChR is a 7-transmembraneGai-coupled

receptor that is expressed both presynaptically and

postsynaptically in neurons, within brain regions

associated with psychotic and cognitive functions,

including the striatum, the cortex, and

the hippocampus

OBJECTIVE

• The aim of this study was to understand the agonism and the binding profile of M4 ligands with different degrees of intrinsic activities by using multiple probes, evaluating different signaling events/pathways, and by performing experiments at steady state, as well as at multiple time points

RESULTS

●Full agonists are functionally balanced between the

• Activation of individual G-proteins,β-arrestin recruitment, and receptor trafficking were evaluated using bioluminescence resonance energy transfer (BRET) technology from Domain Therapeutics (Strasbourg, France)
• Second messenger activation was measured using homogenous time- resolved fluorescence ([HTRF]; for cyclic adenosine monophosphate [cAMP]) and with a fluorescent imaging plate reader ([FLIPR]; for Ca2+ release)

Radioligand binding

α

Giβ Gq

ATP	M2, M4	M1, M3, M5

HTRF

Scintillation proximity assay

(SPA)Bioluminescence resonance energy transfer (BRET) assay

[35S]GTPγS

[35S]GTPγS

FLIPR

Illustration under authorization of Domain Therapeutics.

A large number of selective M4 ligands were profiled for their binding and functional activity at M4, as well as for their functional selectivity at M1, M2, M3, and M5

Shown here is a set of representative compounds that include full agonists, partial agonists (<80% maximum effect [Emax]), antagonists, and agonist PAMs

Full agonists are functionally balanced between the G-protein and the β-arrestin pathway while partial agonists and agonist PAMs are biased toward the G-protein pathway

Binding profiles suggest that compounds with varying binding affinities have similar Koff with a broad range of Kon, as well as varying preferences for orthosteric versus allosteric sites

Presented at Society for Neuroscience (SfN)

November 12-16, 2022 • San Diego, CA, USA

G-protein and the β-arrestin pathway (representative

compound shown in Figure 1)

●Partial agonists and ago-positive allosteric modifiers

(PAMs) are biased toward the G-protein pathway

(representative compound shown in Figure 2)

●Using [3H] N-methyl scopolamine ([3H]NMS) as a

probe (Table 1), compounds with varying binding

affinities (Ki) appear to have similar dissociation rate

constants (Koff) with a broad range of association rate

constants (Kon)

●Compounds appear to have a varying degree of

preference for orthosteric versus allosteric sites (Ki of

CV-0000042 against [3H]MK-6884 is >100 nM; it is 16

nM for CV-0000294); see Figure 1A and Figure 2A

Table 1. M4 Binding Profile With [3H] N-Methyl Scopolamine

Compound ID

Binding

Equilbrium Ki (or Kd)

Kinetics Kd

([3H]NMS)

kon

koff

τ

(=koff/kon)

nM

M -1 min -1

min -1

min

nM

[3H]Scopolamine

0.2 ± 0.02 (Kd, n=30)

1.4 x 108 ± 1.4 x 107

0.02

± 0.002

48

± 4.0

1.5 ± 0.03

Scopolamine

0.33 ± 0.04 (n = 26)

CV-0000294-00-01

>1,000

3.2 x 103 ± 1.0 x 103

0.05

± 0.002

21

± 1.0

1,600 ± 4,400

CV-0000030-00-01

41 ± 8.0

5.3 x 105 ± 2.2 x 105

0.04

± 0.03

33

± 19

79

± 16

CV-0000042-00-01

3.1 x 105 ± 2.2 x 105

137 ± 23

0.07

± 0.04

20

±11

290 ± 78

CV-0000071-00-01

9.0 ± 0.13

1.2 x 106 ± 1.3 x 105

0.03

± 0.002

40

± 3.9

21

± 4.4

MK-6884

74 ± 12

1.8 x 105 ± 3.8 x 104

0.03

± 0.007

36

± 8.0

180 ± 73

Trospium Chloride

0.49 ± 0.07

4.3 x 108 ± 2.5 x 107

0.03

± 0.004

31

± 4.2

0.44 ± 0.18

Xanomeline

1.4 x 106 ± 7.5 x 105

6.0 ± 0.25

0.02

± 0.003

49

± 6.2

20

± 8.6

Clozapine

23 ± 11

5.5 x 105 ± 2.5 x 104

0.03

± 0.01

38

± 8.6

50

± 9.2

PCS1055 Dihydrochloride

1.0 ± 0.0

2.0 x 107 ± 5.2 x 106

0.01

± 0.001

98

± 11

0.5 ± 0.1

Acetylcholine Chloride

5.1 x 103 ± 1.3 x 103

>1,000

0.05

± 0.02

27

± 12

10,800 ± 6,700

TBPB

1.8 x 105 ± 5.1 x 104

60 ± 2.0

0.02

± 0.002

48

± 4.0

130 ± 46

CV-0000422-00-01

186 ± 1.0

4.0 x 104 ± 5.0 x 103

0.03

± 0.002

29

± 2.0

860 ± 50

Figure 1. Full agonist.				log (Emax/EC50)
A. Dissociation of [3H]NMS +/- CV-0000042	B. Receptor selectivity		C. Functional selectivity
	125					hM4 GTPgS				Gi1
						2.0		cAMP	4	Gi2
Binding	100					0.0
	Scopolamine (20 ∝ M)	t1/2	hM2 cAMP	hM4 cAMP		1
	60 min	-2.0
H]NMS	75		+500 nM CV-0000042	22 min		-4.0		GTPgS	-2	Gi3
[	50					-6.0			-5
3
Total%								Barr2+GRK recruitment		GoA
	25				hM5 ca2+		hM1 ca2+
								Barr2 recruitment		GoB
	0					hM3 ca2+		M4R internalization		Gz
	0	50	100	150	200
			Time, min

Figure 2. Partial agonist, ago-PAM.

A. Dissociation of [3H]NMS +/- CV-0000294	B. Receptor selectivity		C. Functional selectivity
	125					hM4 GTPgS			Gi1
					2.0			4
							cAMP
Binding						0.0		Gi2
100		Scopolamine (20 ∝ M)	60 min	hM2 cAMP	hM4 cAMP	GTPgS	Gi3
	-2.0
			t1/2					1
H]NMS	75		+10 ∝ M CV-0000294	18 min		-4.0			-2
3						-6.0			-5
[	50						Barr2+GRK recruitment	GoA
% Total
25				hM5 ca2+		hM1 ca2+
						Barr2 recruitment	GoB
	0
	0	50	100	150	200			M4R internalization	Gz
			Time, min			hM3 ca2+

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