| This project explores the hypothesis that enhancing efferent modulation of cochlear outer
hair cells by targeting α9α10 nAChRs with unconventional ligands, such as positive allosteric
modulators (PAMs), could prevent and treat hidden hearing loss. Based on a previously
identified lead compound, L-Ascorbic Acid (AA), this project is focused on unraveling the
structural requirements for a molecule to function as a PAM for α9α10 nAChRs through a
structure-activity relationship (SAR) study.
AA is not a potent modulator of α9α10 nAChRs, so, while retaining its higher efficacy
and selectivity for this receptor subtype, we aim to create analogs with greater potency and
stability. However, given the challenges of AA as a lead molecule, the development of α9α10
PAMs also involves identifying new potential lead molecules. The effect of known modulators
of other nAChR subtypes, such as α7 and α4β2 PAMs, on α9α10 nAChRs has also been explored
in this study. Functional testing of both AA analogs and known nAChR modulators was
performed using standard two-electrode voltage-clamp (TEVC) electrophysiological techniques
in X. laevis oocytes expressing nAChR subtypes. New, highly potent PAMs were identified in
this study, including a commercially available AA analog, 3-O-Ethyl L-Ascorbic Acid (EA), and
lab-synthesized 3-O-Propargyl L-Ascorbic Acid Acetonide (PAA).
To explore potential binding sites for α9α10 PAMs, we constructed a homology model of
the α9α10 nAChRs and performed molecular docking of active PAMs. In order to enable highsensitivity binding assays in addition to TEVC functional assays, we designed an α9/LsAChBP chimera and expressed it in Cos-7 mammalian cell lines. This soluble amino-terminal α9-based
chimera is amenable to analysis using Surface Plasmon Resonance (SPR) techniques and
provides a higher throughput platform for binding interactions, including the determination of on
and off rates for test compounds. The purified chimera was immobilized in SPR sensor chips to
study the binding kinetics of nAChR antagonist Tubocurarine (dTC) and potential modulators.
The soluble protein may also enable crystallography studies. Altogether, these studies add
substantially to our fundamental understanding of the molecular basis of ascorbate-positive
allosteric modulation of the α9α10 nAChRs and provide new approaches to future studies of this
receptor.
Keywords: α9α10 nicotinic acetylcholine receptors, hearing loss, Ascorbate, positive allosteric
modulators, chimera, homology modeling |