TY - JOUR

T1 - Discovery and functional characterization of N-(thiazol-2-yl)-benzamide analogs as the first class of selective antagonists of the Zinc-Activated Channel (ZAC)

AU - Madjroh, Nawid

AU - Mellou, Eleni

AU - Davies, Paul A.

AU - Söderhielm, Pella Cecilia

AU - Jensen, Anders A.

PY - 2021

Y1 - 2021

N2 - The Zinc-Activated Channel (ZAC) is an atypical member of the Cys-loop receptor (CLR) superfamily of pentameric ligand-gated ion channels, with its very different endogenous agonists and signalling properties. In this study, a compound library screening at ZAC resulted in the identification of 2-(5-bromo-2-chlorobenzamido)-4-methylthiazole-5-methyl ester (1) as a novel ZAC antagonist. The structural determinants for ZAC activity in 1 were investigated by functional characterization of 61 analogs at ZAC expressed in Xenopus oocytes by two-electrode voltage clamp electrophysiology, and couple of analogs exerting more potent ZAC inhibition than 1 were identified (IC50 values: 1–3 μM). 1 and N-(4-(tert-butyl)thiazol-2-yl)-3-fluorobenzamide (5a, TTFB) were next applied in studies of the functional properties and the mode of action of this novel class of ZAC antagonists. TTFB was a roughly equipotent antagonist of Zn+- and H+-evoked ZAC signaling and of spontaneous ZAC activity, and the slow on-set of its channel block suggested that its ZAC inhibition is state-dependent. TTFB was found to be a selective ZAC antagonist, exhibiting no significant agonist, antagonist or modulatory activity at 5-HT3A, α3β4 nicotinic acetylcholine, α1β2γ2S GABAA or α1 glycine receptors at 30 μM. 1 displayed largely non-competitive antagonism of Zn2+-induced ZAC signalling, and TTFB was demonstrated to target the transmembrane and/or intracellular domains of the receptor, which collectively suggests that the N-(thiazol-2-yl)-benzamide analog acts a negative allosteric modulator of ZAC. We propose that this first class of selective ZAC antagonists could constitute useful pharmacological tools in future explorations of the presently poorly elucidated physiological functions governed by this CLR.

AB - The Zinc-Activated Channel (ZAC) is an atypical member of the Cys-loop receptor (CLR) superfamily of pentameric ligand-gated ion channels, with its very different endogenous agonists and signalling properties. In this study, a compound library screening at ZAC resulted in the identification of 2-(5-bromo-2-chlorobenzamido)-4-methylthiazole-5-methyl ester (1) as a novel ZAC antagonist. The structural determinants for ZAC activity in 1 were investigated by functional characterization of 61 analogs at ZAC expressed in Xenopus oocytes by two-electrode voltage clamp electrophysiology, and couple of analogs exerting more potent ZAC inhibition than 1 were identified (IC50 values: 1–3 μM). 1 and N-(4-(tert-butyl)thiazol-2-yl)-3-fluorobenzamide (5a, TTFB) were next applied in studies of the functional properties and the mode of action of this novel class of ZAC antagonists. TTFB was a roughly equipotent antagonist of Zn+- and H+-evoked ZAC signaling and of spontaneous ZAC activity, and the slow on-set of its channel block suggested that its ZAC inhibition is state-dependent. TTFB was found to be a selective ZAC antagonist, exhibiting no significant agonist, antagonist or modulatory activity at 5-HT3A, α3β4 nicotinic acetylcholine, α1β2γ2S GABAA or α1 glycine receptors at 30 μM. 1 displayed largely non-competitive antagonism of Zn2+-induced ZAC signalling, and TTFB was demonstrated to target the transmembrane and/or intracellular domains of the receptor, which collectively suggests that the N-(thiazol-2-yl)-benzamide analog acts a negative allosteric modulator of ZAC. We propose that this first class of selective ZAC antagonists could constitute useful pharmacological tools in future explorations of the presently poorly elucidated physiological functions governed by this CLR.

U2 - 10.1016/j.bcp.2021.114782

DO - 10.1016/j.bcp.2021.114782

M3 - Journal article

C2 - 34560054

VL - 193

JO - Biochemical Pharmacology

JF - Biochemical Pharmacology

SN - 0006-2952

M1 - 114782

ER -