Enantiospecific Positive Allosteric Modulation of α4β2 Nicotinic Receptor Subtypes

Nicotinic acetylcholine receptors (nAChRs) are integral to brain function, playing critical roles in cognition and reward pathways. Among these, α4β2 nAChRs are key targets for developing therapeutics to address nicotine addiction and cognitive disorders. Here, we report the synthesis, stereochemical resolution, and pharmacological evaluation of GAT2800, a racemic compound identified as a positive allosteric modulator (PAM) of α4β2 nAChRs. Enantiomeric resolution yielded the inactive <i>S</i>-enantiomer (GAT2801) and the pharmacologically active <i>R</i>-enantiomer (GAT2802), with their configurations confirmed via X-ray crystallography. Two-electrode voltage-clamp recordings from <i>Xenopus oocytes</i> expressing α4β2 nAChRs revealed that GAT2802, but not GAT2801, significantly potentiates current responses of both high-sensitivity (HS) (α4)2(β2)3 and low-sensitivity (LS) (α4)3(β2)2 isoforms, with EC₅₀ values of ∼1 and ∼0.8 μM, respectively. Notably, GAT2802 enhanced ACh efficacy more profoundly in HS (α4)2(β2)3 nAChR while showing minimal activity at α3-containing nAChRs. Computational docking analyses provided insight into potential binding sites of GAT2802 at subunit interfaces within the transmembrane domain. Mutational analyses identified α4Cys233 located in the first transmembrane helix and projecting to the β2:α4 subunit transmembrane interface, as a molecular determinant for selectivity of GAT2802 for α4- over α3-containing nAChRs. Safety evaluation demonstrated negligible cellular toxicity of GAT2802 in HEK cells expressing α4β2 nAChRs and no significant developmental effects in zebrafish larvae at concentrations up to 100 μM. These findings establish GAT2802 as a promising lead compound for the development of selective α4β2 nAChR PAMs, with significant therapeutic potential for nicotine addiction and cognitive disorders.