Discovery of an intrasubunit nicotinic acetylcholine receptor-binding site for the positive allosteric modulator Br-PBTC

Abstract

Nicotinic acetylcholine receptor (nAChR) ligands that lack agonist activity but enhance activation in the presence of an agonist are called positive allosteric modulators (PAMs). nAChR PAMs have therapeutic potential for the treatment of nicotine addiction and several neuropsychiatric disorders. PAMs need to be selectively targeted toward certain nAChR subtypes to tap this potential. We previously discovered a novel PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which selectively potentiates the opening of α4β2*, α2β2*, α2β4*, and (α4β4)₂α4 nAChRs and reactivates some of these subtypes when desensitized (* indicates the presence of other subunits). We located the Br-PBTC-binding site through mutagenesis and docking in α4. The amino acids Glu-282 and Phe-286 near the extracellular domain on the third transmembrane helix were found to be crucial for Br-PBTC's PAM effect. E282Q abolishes Br-PBTC potentiation. Using (α4^E282Qβ2)₂α5 nAChRs, we discovered that the trifluoromethylated derivatives of Br-PBTC can potentiate channel opening of α5-containing nAChRs. Mutating Tyr-430 in the α5 M4 domain changed α5-selectivity among Br-PBTC derivatives. There are two kinds of α4 subunits in α4β2 nAChRs. Primary α4 forms an agonist-binding site with another β2 subunit. Accessory α4 forms an agonist-binding site with another α4 subunit. The pharmacological effect of Br-PBTC depends both on its own and agonists' occupancy of primary and accessory α4 subunits. Br-PBTC reactivates desensitized (α4β2)₂α4 nAChRs. Its full efficacy requires intact Br-PBTC sites in at least one accessory and one primary α4 subunit. PAM potency increases with higher occupancy of the agonist sites. Br-PBTC and its derivatives should prove useful as α subunit-selective nAChR PAMs.

Keywords: allosteric regulation; drug design; molecular docking; nicotinic acetylcholine receptors (nAChR); positive allosteric modulator; receptor desensitization.

Figure 1.

Structures of dFBr, Br-PBTC, and its analogs. Charged nitrogens at physiological pH are indicated.

Figure 2.

Comparing effects of Br-PBTC and dFBr on activation and desensitization of (α4β2)₂α4 and (α4β2)₂β2 nAChRs. A, concatameric nAChR of defined stoichiometries were expressed in HEK cell lines to investigate PAM effects of Br-PBTC and dFBr. Primary subunits are shown as open circles. Accessory subunits are shown as gray-shaded circles. Black AChs show agonist sites at the primary subunit interface. Gray ACh represents agonist sites at the accessory subunit interface. B, concentration–response curves for PAM potentiation of activation of nAChRs by EC_40–50 nicotine. Data for Br-PBTC are from a previous publication (15). Br-PBTC is more potent and efficacious than dFBr at potentiating activation of (α4β2)₂α4 nAChRs. As reported, Br-PBTC and dFBr showed decreased potentiation or even inhibition at 3 μm and higher concentrations (15, 60). These concentrations are not shown for comparison of the potentiation effect of the two compounds. C, reactivation effects on desensitized nAChRs by various concentrations of PAMs. Nicotine (0.5 μm) was preincubated with cultured cells for 6 h before addition of PAMs. Br-PBTC greatly reactivated (α4β2)₂α4 but had a very small effect on (α4β2)₂β2 nAChRs. dFBr is less potent than Br-PBTC, but it partially reactivates both (α4β2)₂β2 and (α4β2)₂α4. Results are presented as mean ± S.E., sample size n = 3.

Figure 3.

Location of α4 point mutations on the amino acid sequence and an α4 homology model. Mutated residues in the α4 subunit are shown as spheres with appropriate van der Waal radii. The α4 model shown here is derived from a revised Torpedo nAChR structure (25). Pink spheres represent carbon of side chains. The AGMI C-tail sequence is highlighted in red.

Figure 4.

Molecular surface cross-sections of an open-state α4 homology model and an (α4β2)₂β2-desensitized structure. A, cross-section of an open-state α4 homology model built from the corrected T. marmorata α1 subunit structure. Br-PBTC can be docked into an intrasubunit cavity. B, cross-section of an α4 subunit in a published X-ray crystal structure of desensitized (α4β2)₂β2 (22). In the desensitized structure, the intrasubunit cavity is not large enough to accommodate a Br-PBTC molecule near the triad of Glu-282, Phe-286, and the C-tail, which have been found to be important for Br-PBTC activity. C, side view of the open-state α4 homology model reveals a large intrasubunit cavity containing Glu-282, Phe-286, and the C-tail. D, side view of the desensitized α4 shows no such cavity. Cross-sectional planes, denoted by green lines, in both C and D are equidistant from the cell membrane's extracellular surface. The additional length of α4 at the bottom of D is there because a short length of amphipathic helices between M3 and M4 was resolved in the (α4β2)₂β2 crystal structure, whereas all residues between M3 and M4 in the α1 structure used to build the α4 model in C were deleted.

Figure 5.

Br-PBTC derivatives and dFBr docked to α4 using SwissDock. Docked Br-PBTC and dFBr with a view of the entire subunit is shown in A and B, and the C-tail is colored red. M2 and M3 domains are colored pink for clarity. SwissDock evaluates the whole α4 subunit and does not allow for side-chain rotation. Mutated side chains listed in Table 1 are colored olive green. All ligands dock with similar orientations, with the piperidine moiety placed next to Glu-282 and the aromatic ring buried further toward the cytoplasm. The lowest-energy conformational clusters of Br-PBTC (C) and dFBr (D) all fit in the same binding pocket within α4 between transmembrane domains M1, M3, and M4. SwissDock identifies hydrogen bonding (shown in green) between the carboxylate of Glu-282 and either the basic piperidine amine of Br-PBTC (C) or secondary amine of dFBr (D). Olive green denotes residues found to be critical for Br-PBTC potentiation: Glu-282 and Phe-286. The α-helix distortion near the intracellular end of M4 is due to an additional minimization step performed by SwissDock prior to docking. RMSD values between poses in Figs. 5 and 6 can be found in Table 2.

Figure 6.

Br-PBTC derivatives and dFBr docked to α4 using Autodock Vina. Docked Br-PBTC and dFBr with a view of the entire subunit is shown in A and B, and the C-tail is colored red. The search space and docked Br-PBTC and dFBr are shown in A and B. The search space outlined in red in α4 was chosen to encompass structural elements within α4 found to be critical for Br-PBTC function: the C-tail, Glu-282, and Phe-286. The search space focuses on an intrasubunit-binding pocket. Autodock Vina only docks within a defined search space on α4 and allows select side chains to rotate freely. The following α4 residues were allowed to rotate freely: Tyr-220, Leu-224, Ile-266, Leu-275, Leu-279, Glu-282, Tyr-283, Phe-286, Leu-593, and Leu-597. Rotatable residues are colored olive green in all panels. Br-PBTC (C) and dFBr (D) dock within a binding pocket between M1, M3, and M4 with piperidine moieties positioned next to the Glu-282 carboxylate. Among the rotatable residues (denoted as colored sticks), Leu-224, Glu-282, Tyr-283, and Phe-286 are in close contact with the ligand. RMSD values between poses in Figs. 5 and 6 can be found in Table 2.

Figure 7.

Br-PBTC derivatives can potentiate through α5 accessory subunits. A, (α4β2)₂β2, (α4^E282Qβ2)₂β2, (α4β2)₂α5, (α4^E282Qβ2)₂α5, and (α4^E282Qβ2)₂α5^Y430A were expressed in Xenopus oocytes. In (α4^E282Qβ2)₂α5, any observed potentiation can only come from PAM activity on α5 subunits because PAM sites on all α4 subunits are disabled. Varying concentrations of Br-PBTC and SR14273 were co-applied with a saturating 500 μm ACh. To test for potentiation, 1.56, 6.25, and 25 μm of SR13521, SR14270, SR14271, SR14273, and SR19678 were co-applied with 500 μm ACh, a saturating agonist concentration. All five compounds are known to potentiate α4β2* and (α4β2)₂α5 subtypes. Only SR14271 demonstrated agonist activity against (α4β2)₂α5 at 25 μm. Potentiation was observed in (α4β2)₂α5 at lower concentrations of SR14271. Results are presented as mean ± S.E. Sample size: B, n = 3; C, n = 3–4; D, n = 5; E, n = 5.

Figure 8.

Br-PBTC acts with higher potency on (α4β2)₂α4 but not (α4β2)₂β2 nAChRs as agonist concentration increases. Thus, the accessory ACh-binding site is critical for agonist-induced increase in sensitivity to potentiation by Br-PBTC. HEK cells expressing β2-α4 + α4 or β2-α4 + β2 were co-applied with varying concentrations of Br-PBTC in the presence of nicotine, ACh, or A85380. Responses were measured as fluorescence from a membrane potential-sensitive dye using a FlexStation benchtop fluorimeter. Results are presented as mean ± S.E. Sample size is n = 4.

Figure 9.

Br-PBTC acts more than 10-fold more potently against (α4β2)₂α4 and (α4β2)₂α4^E282Q when co-applied with ACh rather than sazetidine-A (Saz-A). Activation by ACh increased sensitivity to potentiation by Br-PBTC, but sazetidine did not. ACh binds to primary and accessory ACh sites, but sazetidine binds only to primary ACh sites. Thus, activation of the accessory site is critical for agonist-induced sensitivity to Br-PBTC. Activation of the accessory ACh site increases sensitivity to potentiation by Br-PBTC even when the PAM-binding site on the accessory α4 subunit is blocked by the E282Q mutation. Xenopus oocytes were injected with 1:2 mass ratio of β2-α4 + α4 or β2-α4 + α4^E282Q mRNA, which yields the nAChR constructs A (α4β2)₂α4 and B (α4β2)₂α4^E282Q. Br-PBTC was co-applied with either saturating ACh (500 μm) or saturating sazetidine-A (0.01 μm). The percent increase in peak current was used to assess Br-PBTC potentiation. Pentamer diagrams are presented on the right of the Br-PBTC concentration response curve for each nAChR construct. ACh-, sazetidine-A-, and Br-PBTC–binding sites are noted in the graph. Results are presented as mean ± S.E. Sample size is n = 4.

Figure 10.

Br-PBTC acts synergistically at α4α4 and α4β2 allosteric sites to reactivate desensitized (α4β2)₂α4 nAChRs. For reactivation of these desensitized AChRs, there must be binding sites for Br-PBTC on the accessory α4 and at least one primary α4. WT and mutant (α4β2)₂α4 were expressed in Xenopus oocytes. nAChRs were desensitized by perfusion of 0.2 μm nicotine for 15 min. Increasing concentrations of Br-PBTC were applied to oocytes being perfused in 0.2 μm nicotine to evaluate reactivation of desensitized nAChRs by Br-PBTC. The Br-PBTC evoked current is normalized by responses of 500 μm ACh applied before nicotine perfusion. Complete desensitization by nicotine was confirmed in a separate experiment by application of 1 μm dihydro-β-erythroidine hydrobromide after nicotine perfusion, which produced no significant reduction of current. Sample size is n = 3–5.