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

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 alpha 4 beta 2*, alpha 2 beta 2*, alpha 2 beta 4*, and (alpha 4 beta 4)(2)alpha 4 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 alpha 4. 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 (alpha 4(E282Q)beta 2)(2)alpha 5 nAChRs, we discovered that the trifluoromethylated derivatives of Br-PBTC can potentiate channel opening of alpha 5-containing nAChRs. Mutating Tyr-430 in the alpha 5 M4 domain changed alpha 5-selectivity among Br-PBTC derivatives. There are two kinds of alpha 4 subunits in alpha 4 beta 2 nAChRs. Primary alpha 4 forms an agonist-binding site with another beta 2 subunit. Accessory alpha 4 forms an agonist-binding site with another alpha 4 subunit. The pharmacological effect of Br-PBTC depends both on its own and agonists' occupancy of primary and accessory alpha 4 subunits. Br-PBTC reactivates desensitized (alpha 4 beta 2)(2)alpha 4 nAChRs. Its full efficacy requires intact Br-PBTC sites in at least one accessory and one primary alpha 4 subunit. PAM potency increases with higher occupancy of the agonist sites. Br-PBTC and its derivatives should prove useful as alpha subunit-selective nAChR PAMs.