Interaction of the Positive Allosteric Modulator LY2087101 with α4β2 Nicotinic Acetylcholine Receptor

Abstract

LY2087101 ([2-[(4-Fluorophenyl)amino]-4-methyl-5-thiazolyl]-3-thienylmethanone) was identified as a positive allosteric modulator (PAM) of nicotinic acetylcholine receptors (nAChR) in a high-throughput screen at Eli Lilly and Company. LY2087101 potentiated α7 and α4β2 nAChRs but not α3β2 nAChR (Broad et al. 2008, J. Pharmacol. Exp. Ther., 318: 1108). Mutational analyses and computer docking simulation with α7 nAChR predicted that PNU120596 and LY2087101 bind to a common site within the transmembrane domain (Young et al. 2008, PNAS 105:14686). In this study, we use mutational analyses and two-electrode voltage-clamp recording from Xenopus oocytes to begin pharmacological characterization of LY208101 interaction with α4β2 nAChR and to identify the molecular determinants of LY2087101 selectivity for α4β2 vs. α3β4 nAChRs. LY2078101 potentiated ACh-induced currents of low ACh sensitivity (α4)3(β2)2 nAChR and high ACh sensitivity (α4)2(β2)3 nAChR with similar potency. However, the maximum potentiation at 10 μM LY2087101 was higher in (α4)3(β2)2 than (α4)2(β2)3 nAChR. Mutational analyses of amino acids contributing to extracellular subunits interface (e.g. α4His114, α4Gln122, and α4Thr124, known selectivity determinants for the (α4)3(β2)2 nAChR selective PAM NS9283; Olsen et al 2013, JBC 288: 35977) had no effect on LY2087101 potentiation. Within the transmembrane domain, mutation at α4Ser285 but not α4Thr261 within the M1 helix significantly reduced LY2087101 potentiation of α4β2 nAChR α4Ser285 and α4Thr261 are equivalent to α7Ser222 and α7Ala225, respectively; two important positions for potentiation of α7 nAChR by LY2087101 and PNU120596. α4Thr261 projects to intra-subunit pocket within the α4 transmembrane helix bundle whereas α4Ser285 is exposed to intra-subunit pocket and inter-subunit space. Ongoing mutational analyses within the M1 transmembrane domain will define the contribution of intra-subunit vs. inter-subunit binding sites on LY2087101 potentiation of α4β2 nAChR.