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; Sokhom.pin@cerevel.com
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+ | ||||||||
22-CVE-7155 SFN in vitro agonist_M2-1.pdf 1 | 11/8/22 8:34 AM |
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Cerevel Therapeutics Holdings Inc. published this content on 13 November 2022 and is solely responsible for the information contained therein. Distributed by Public, unedited and unaltered, on 13 November 2022 20:11:04 UTC.