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Identification by virtual screening and functional characterisation of novel positive and negative allosteric modulators of the α7 nicotinic acetylcholine receptor.
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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 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 α7 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 α7 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 α7 nAChR via interaction with a transmembrane site.
Fig. 1. Generation of pharmacophore queries used for virtual screening. The highest ranked clusters of binding mode solutions with previously characterised PAMs are shown within the α7 nAChR transmembrane domain (A). The Cα trace of TM1-3 helices of the principal subunit and TM2 helix of the complimentary subunit are shown for the open (cyan) and closed (pink) conformations. Also shown are binding mode clusters from which pharmacophore queries were generated for the open (green) and closed (orange) conformations. From the ligands in each cluster, pharmacophore queries were generated for the closed and open conformations (B and C, respectively). Note, only those selected for screening are shown. Features of the pharmacophore are represented as yellow spheres (hydrophobes), green spheres (rings), red hashed spheres (hydrogen bond acceptors) and blue hashed spheres (hydrogen bond donors). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2. Allosteric modulators of the α7 nAChR. The general structure of the ‘TQS-family’ of compounds that were used to generate the phamacophore query is shown on the left. Four compounds from the DrugBank database that were identified by virtual screening and selected for functional characterisation are shown on the right.
Fig. 3. Functional characterisation of furosemide on the α7 nAChR. A) Representative traces illustrating responses of α7 nAChRs to ACh (100 μM; left), together with ACh responses from the same oocyte after pre- and co-application of furosemide (1 mM; middle). Also shown (right) is a response to ACh (100 μM) following wash. The vertical scale bar corresponds to 200 nA and the horizontal scale bars to 2.5 s. B) Responses with varying concentrations of ACh in the absence (open circles) and presence (closed circles) of a fixed concentration (1 mM) of furosemide. Data are normalised to the maximum ACh response and are means ± SEM of three independent experiments. C) Concentration-response data illustrating potentiation of responses to ACh (50 μM) by varying concentrations of furosemide. Data are normalised to the EC20 concentration of ACh (50 μM) and are means ± SEM of three independent experiments.
Fig. 4. Functional characterisation of furosemide on α7 nAChR, 5-HT3AR and an α7/5-HT3AR chimera. The effect of furosemide was examined with either a maximal or an EC50 concentration of agonist (A and B, respectively). A) Furosemide (1 mM) was pre- and co-applied with a maximal concentration of agonist (3 mM ACh for α7 and α7/5-HT3AR chimera; 30 μM 5-HT for 5-HT3AR). B) Furosemide (1 mM) was pre and co-applied with an EC50 concentration of agonist (100 μM ACh for α7 and α7/5-HT3AR chimera; 1 μM 5-HT for 5-HT3AR). In both cases potentiation of the α7 nAChR was significantly greater than potentiation with the 5-HT3AR and with the α7/5-HT3A chimera. Data are means ± SEM (n = 4–8). Significant differences are indicated (**P < 0.01, ***P < 0.001).
Fig. 5. Influence of α7 nAChR mutations on the allosteric modulatory effect of furosemide. The docked position of furosemide is shown in the closed (A) and open (B) structural model of the α7 nAChR transmembrane region. The TM1-3 helices of the principal subunit (khaki) and TM2 and TM3 helices of the complimentary subunit (green) are shown. Amino acids examined by site-directed mutagenesis are indicated. C) ACh dose-response curves determined with wild-type α7 nAChR (dashed line) and with α7 nAChRs containing single point mutations S222M, L247A, S248A, M260L and T288A. Data are means of at least three independent experiments. D) Bar chart illustrating the influence of furosemide (1 mM) on responses to an EC50 concentration of ACh (10 μM for L247A and 100 μM for wild-type and all other mutated receptors). Data are normalised to the response observed in the same oocyte in the absence of furosemide. Data are means ± SEM of at least three independent experiments. Significant differences from wild-type are indicated (*P < 0.05, **P < 0.01). In addition, significant differences from agonist responses in the absence of furosemide are indicated (#P < 0.05, ##P < 0.01). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 6. Functional characterisation of DB04763, DB08122 and pefloxacin on the α7 nAChR. A-C) Representative traces, from oocytes expressing the α7 nAChR, in response to ACh (100 μM; left), together with an ACh response from the same oocyte after pre- and co-application of test compound (1 mM; middle). Also shown are responses to ACh (100 μM) after a 2 min wash (right). Data are shown for DB04763 (A), DB08122 (B) and pefloxacin (C), all at 1 mM. Vertical scale bars correspond to 500 nA and horizontal scale bars correspond to 5 s. D) Concentration-response data illustrating antagonism of responses to ACh (100 μM) by varying concentrations of DB04763 (filled circles), DB08122 (open circles) and pefloxacin (crossed circles). Data are normalised to the response to an EC50 concentration of ACh (100 μM) and are means ± SEM of three independent experiments. E) Responses to varying concentrations of ACh in the presence of a fixed concentration (100 μM) of DB04763 (filled circles), DB08122 (open circles) and pefloxacin (crossed circles). Data are normalised to the response to a maximum concentration of ACh (3 mM) and are means ± SEM of 3–4 independent experiments.
Fig. 7. Functional characterisation of DB04763, DB08122 and pefloxacin on the 5-HT3AR and α7/5-HT3AR chimera. The effects of DB04763 (A), DB08122 (B) and pefloxacin (C), were examined by pre- and co-application (100 μM) with a maximal concentration of agonist (3 mM ACh for α7 and α7/5-HT3AR chimera; 30 μM 5-HT for 5-HT3AR) on the α7 nAChR, 5-HT3AR and α7/5-HT3AR chimera. All three of the compounds inhibited the 5-HT3AR and α7/5-HT3AR chimera to a significantly lower extent than was observed with the α7 nAChR. Data are means ± SEM (n = 4–6). Significant differences are indicated (**P < 0.01, ***P < 0.001).
Fig. 8. Influence of α7 nAChR mutations on the allosteric modulatory effect of DB04763, DB08122 and pefloxacin. Data are shown for DB04763 (A), DB08122 (B) and pefloxacin (C). Bar charts illustrate the influence of the compounds (100 μM) on responses to an EC50 concentration of ACh (10 μM for L247A and 100 μM for wild-type and all other mutated receptors). Data are normalised to the response observed in the same oocyte in the absence of the compounds. Data are means ± SEM of at least three independent experiments. Significant differences from wild-type are indicated (*P < 0.05, **P < 0.01, ***P < 0.001). In addition, significant differences from agonist responses in the absence of the compounds are indicated (##P < 0.01, ###P < 0.001).
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