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Identification by virtual screening and functional characterisation of novel positive and negative allosteric modulators of the ?7 nicotinic acetylcholine receptor.

ABSTRACT: Several previous studies have demonstrated that the activity of neurotransmitters acting on ligand-gated ion channels such as the nicotinic acetylcholine receptor (nAChR) can be altered by compounds binding to allosteric modulatory sites. In the case of ?7 nAChRs, both positive and negative allosteric modulators (PAMs and NAMs) have been identified and have attracted considerable interest. A recent study, employing revised structural models of the transmembrane domain of the ?7 nAChR in closed and open conformations, has provided support for an inter-subunit transmembrane allosteric binding site (Newcombe et al 2017). In the present study, we have performed virtual screening of the DrugBank database using pharmacophore queries that were based on the predicted binding mode of PAMs to ?7 nAChR structural models. A total of 81 compounds were identified in the DrugBank database, of which the 25 highest-ranked hits corresponded to one of four previously-identified therapeutic compound groups (carbonic anhydrase inhibitors, cyclin-dependent kinase inhibitors, diuretics targeting the Na⁺-K⁺-Cl^- cotransporter, and fluoroquinolone antibiotics targeting DNA gyrase). The top-ranked compound from each of these four groups (DB04763, DB08122, furosemide and pefloxacin, respectively) was tested for its effects on human ?7 nAChR expressed in Xenopus oocytes using two-electrode voltage-clamp electrophysiology. These studies, conducted with wild-type, mutant and chimeric receptors, resulted in all four compounds exerting allosteric modulatory effects. While DB04763, DB08122 and pefloxacin were antagonists, furosemide potentiated ACh responses. Our findings, supported by docking studies, are consistent with these compounds acting as PAMs and NAMs of the ?7 nAChR via interaction with a transmembrane site.

SUBMITTER: Smelt CLC

PROVIDER: S-EPMC6078708 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Identification by virtual screening and functional characterisation of novel positive and negative allosteric modulators of the α7 nicotinic acetylcholine receptor.

Smelt Charles L C CLC Sanders Victoria R VR Newcombe Joseph J Burt Richard P RP Sheppard Tom D TD Topf Maya M Millar Neil S NS

Neuropharmacology 20180720

Several previous studies have demonstrated that the activity of neurotransmitters acting on ligand-gated ion channels such as the nicotinic acetylcholine receptor (nAChR) can be altered by compounds binding to allosteric modulatory sites. In the case of α7 nAChRs, both positive and negative allosteric modulators (PAMs and NAMs) have been identified and have attracted considerable interest. A recent study, employing revised structural models of the transmembrane domain of the α7 nAChR in closed a ...[more]

PMID: 30009834

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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

Identification by virtual screening and functional characterisation of novel positive and negative allosteric modulators of the ?7 nicotinic acetylcholine receptor.

Publications

Identification by virtual screening and functional characterisation of novel positive and negative allosteric modulators of the α7 nicotinic acetylcholine receptor.

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