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Structure-based discovery of selective positive allosteric modulators of antagonists for the M2 muscarinic acetylcholine receptor.

ABSTRACT: Subtype-selective antagonists for muscarinic acetylcholine receptors (mAChRs) have long been elusive, owing to the highly conserved orthosteric binding site. However, allosteric sites of these receptors are less conserved, motivating the search for allosteric ligands that modulate agonists or antagonists to confer subtype selectivity. Accordingly, a 4.6 million-molecule library was docked against the structure of the prototypical M2 mAChR, seeking molecules that specifically stabilized antagonist binding. This led us to identify a positive allosteric modulator (PAM) that potentiated the antagonist N-methyl scopolamine (NMS). Structure-based optimization led to compound '628, which enhanced binding of NMS, and the drug scopolamine itself, with a cooperativity factor (?) of 5.5 and a KB of 1.1 ?M, while sparing the endogenous agonist acetylcholine. NMR spectral changes determined for methionine residues reflected changes in the allosteric network. Moreover, '628 slowed the dissociation rate of NMS from the M2 mAChR by 50-fold, an effect not observed at the other four mAChR subtypes. The specific PAM effect of '628 on NMS antagonism was conserved in functional assays, including agonist stimulation of [35S]GTP?S binding and ERK 1/2 phosphorylation. Importantly, the selective allostery between '628 and NMS was retained in membranes from adult rat hypothalamus and in neonatal rat cardiomyocytes, supporting the physiological relevance of this PAM/antagonist approach. This study supports the feasibility of discovering PAMs that confer subtype selectivity to antagonists; molecules like '628 can convert an armamentarium of potent but nonselective GPCR antagonist drugs into subtype-selective reagents, thus reducing their off-target effects.

SUBMITTER: Korczynska M

PROVIDER: S-EPMC5877965 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Structure-based discovery of selective positive allosteric modulators of antagonists for the M<sub>2</sub> muscarinic acetylcholine receptor.

Korczynska Magdalena M Clark Mary J MJ Valant Celine C Xu Jun J Moo Ee Von EV Albold Sabine S Weiss Dahlia R DR Torosyan Hayarpi H Huang Weijiao W Kruse Andrew C AC Lyda Brent R BR May Lauren T LT Baltos Jo-Anne JA Sexton Patrick M PM Kobilka Brian K BK Christopoulos Arthur A Shoichet Brian K BK Sunahara Roger K RK

Proceedings of the National Academy of Sciences of the United States of America 20180216 10

Subtype-selective antagonists for muscarinic acetylcholine receptors (mAChRs) have long been elusive, owing to the highly conserved orthosteric binding site. However, allosteric sites of these receptors are less conserved, motivating the search for allosteric ligands that modulate agonists or antagonists to confer subtype selectivity. Accordingly, a 4.6 million-molecule library was docked against the structure of the prototypical M<sub>2</sub> mAChR, seeking molecules that specifically stabilize ...[more]

PMID: 29453275

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Project description:Accumulating evidence suggests that selective M4 muscarinic acetylcholine receptor (mAChR) activators may offer a novel strategy for the treatment of psychosis. However, previous efforts to develop selective M4 activators were unsuccessful because of the lack of M4 mAChR subtype specificity and off-target muscarinic adverse effects. We recently developed VU0152100, a highly selective M4 positive allosteric modulator (PAM) that exerts central effects after systemic administration. We now report that VU0152100 dose-dependently reverses amphetamine-induced hyperlocomotion in rats and wild-type mice, but not in M4 KO mice. VU0152100 also blocks amphetamine-induced disruption of the acquisition of contextual fear conditioning and prepulse inhibition of the acoustic startle reflex. These effects were observed at doses that do not produce catalepsy or peripheral adverse effects associated with non-selective mAChR agonists. To further understand the effects of selective potentiation of M4 on region-specific brain activation, VU0152100 alone and in combination with amphetamine were evaluated using pharmacologic magnetic resonance imaging (phMRI). Key neural substrates of M4-mediated modulation of the amphetamine response included the nucleus accumbens (NAS), caudate-putamen (CP), hippocampus, and medial thalamus. Functional connectivity analysis of phMRI data, specifically assessing correlations in activation between regions, revealed several brain networks involved in the M4 modulation of amphetamine-induced brain activation, including the NAS and retrosplenial cortex with motor cortex, hippocampus, and medial thalamus. Using in vivo microdialysis, we found that VU0152100 reversed amphetamine-induced increases in extracellular dopamine levels in NAS and CP. The present data are consistent with an antipsychotic drug-like profile of activity for VU0152100. Taken together, these data support the development of selective M4 PAMs as a new approach to the treatment of psychosis and cognitive impairments associated with psychiatric disorders such as schizophrenia.

Project description:Homomeric ?7 nicotinic acetylcholine receptors (nAChR) have an intrinsically low probability of opening that can be overcome by ?7-selective positive allosteric modulators (PAMs), which bind at a site involving the second transmembrane domain (TM2). Mutation of a methionine that is unique to ?7 at the 15' position of TM2 to leucine, the residue in most other nAChR subunits, largely eliminates the activity of such PAMs. We tested the effect of the reverse mutation (L15'M) in heteromeric nAChR receptors containing ?4 and ?2, which are the nAChR subunits that are most abundant in the brain. Receptors containing these mutations were found to be strongly potentiated by the ?7 PAM 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS) but insensitive to the alternative PAM 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea. The presence of the mutation in the ?2 subunit was necessary and sufficient for TQS sensitivity. The primary effect of the mutation in the ?4 subunit was to reduce responses to acetylcholine applied alone. Sensitivity to TQS required only a single mutant ? subunit, regardless of the position of the mutant ? subunit within the pentameric complex. Similar results were obtained when ?2L15'M was coexpressed with ?2 or ?3 and when the L15'M mutation was placed in ?4 and coexpressed with ?2, ?3, or ?4. Functional receptors were not observed when ?1L15'M subunits were coexpressed with other muscle nAChR subunits. The unique structure-activity relationship of PAMs and the ?4?2L15'M receptor compared with ?7 and the availability of high-resolution ?4?2 structures may provide new insights into the fundamental mechanisms of nAChR allosteric potentiation. SIGNIFICANCE STATEMENT: Heteromeric neuronal nAChRs have a relatively high initial probability of channel activation compared to receptors that are homomers of ?7 subunits but are insensitive to PAMs, which greatly increase the open probability of ?7 receptors. These features of heteromeric nAChR can be reversed by mutation of a single residue present in all neuronal heteromeric nAChR subunits to the sequence found in ?7. Specifically, the mutation of the TM2 15' leucine to methionine in ? subunits reduces heteromeric receptor channel activation, while the same mutation in neuronal ? subunits allows heteromeric receptors to respond to select ?7 PAMs. The results indicate a key role for this residue in the functional differences in the two main classes of neuronal nAChRs.

Dataset Information

Structure-based discovery of selective positive allosteric modulators of antagonists for the M2 muscarinic acetylcholine receptor.

Publications

Structure-based discovery of selective positive allosteric modulators of antagonists for the M<sub>2</sub> muscarinic acetylcholine receptor.

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