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

Abstract

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, α₁β₂γ_2S GABA_A 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.

Keywords: Cys-loop receptor (CLR); N-(thiazol-2-yl)-benzamide analogs; Negative allosteric modulator (NAM); Pentameric ligand-gated ion channel (pLGIC); State-dependent inhibition; Zinc-Activated Channel (ZAC).

Fig. 1.. Identification of a novel ZAC antagonist.

A. Concentration-response curve for Cu²⁺ at ZAC-HEK293 cells in the FMP assay. Data are from a single representative experiment performed in duplicate (of a total of 4) and are given as ΔRFU (mean ± S.D.) values. B. Data obtained for one 96-well plate (out of of a total of 21 96-well plates) tested in the screening at the ZAC-HEK293 cell line in the FMP assay using Cu²⁺ (200 μM) as agonist, more spefically the 96-well plate containing compound 1. The data for the 16 wells in the 96-well plate containing controls [where the cells in the absence of any test compound were challenged with either pure buffer (red) or buffer supplemented with Cu²⁺ (200 μM) (blue)] and for the 80 wells in the plate containing various test compounds that either displayed negligible effects on the Cu²⁺ (200 μM)-induced response (black), were fluorescent or mediated non-specific effects (brown), mediated non-specific or toxic effects (purple), exhibited significant inhibition that not be reproduced in subsequent experiments (grey), and compound 1 (green) are indicated. C. Chemical structure of compound 1. D. Concentration-inhibition curve for compound 1 at ZAC-HEK293 cells in the FMP assay using Cu²⁺ (200 μM) as agonist. Data are from a single representative experiment performed in duplicate (of a total of 3) and are given as ΔRFU (mean ± S.D.) values.

Fig. 2.. Antagonist properties displayed by compound 1 at ZAC expressed in Xenopus oocytes in TEVC recordings.

A. Compound 1 inhibits Zn²⁺ -evoked currents through ZAC in oocytes. Representative traces of Zn²⁺ (1 mM) evoked currents in ZAC-expressing oocytes in the absence and in the presence of increasing concentrations of 1 (left), and averaged concentration-inhibition relationships for 1 and TC at Zn²⁺ (1 mM)-induced currents in the oocytes (right). Data for 1 are given as mean ± S.E.M. values (n = 6), and the tubocurarine (TC) data given for comparison are from a recent study [25], B. Compound 1 inhibits the spontaneous ZAC activity in oocytes. Representative traces of the effects of increasing concentrations of 1 on the leak current in ZAC-expressing oocytes (left), and averaged concentration-inhibition relationships for 1 and TC at tire spontaneous currents of ZAC in the oocytes (right). Data for 1 are given as mean ± S.E.M. values (n = 6), and the tubocurarine (TC) data given here for comparison are from a recent study [25]. C. Delineation of the mode of antagonism exerted by compound 1 at Zn²⁺ -evoked ZAC currents in oocytes. Left: Averaged concentration–response relationships displayed by Zn²⁺ at ZAC in tire absence and in the presence of various concentrations of 1. Data are given as mean ± S.E.M. values in % of the current evoked by 10 mM Zn²⁺ in the absence of 1 (I_{10 mM Zn2+}) in the specific oocyte (n = 5–8). Right: The relative degrees of inhibition mediated by different concentration of 1 of the responses evoked by 10 mM, 3 mM, 1 mM and 0.3 mM Zn²⁺ through ZAC. Data (extracted from the data in Fig. 2C, left) are given as mean values in % of the current evoked by the specific Zn²⁺ concentration in the absence of 1 (I_Zn2+).

Fig. 3.. N-(thiazol-2-yl)-benzamide analogs with modifications to the thiazole ring.

A. Chemical structures of Series 2 analogs (2a-i) and the functional properties exhibited by them as antagonists at ZAC in oocytes using 1 mM Zn²⁺ as agonist. Data ate given as mean ± S.E.M. values (n = 4–8). B. Chemical structures of Series 3 analogs (3a-k) and the functional properties exhibited by them as antagonists at ZAC in oocytes using 1 mM Zn²⁺ as agonist. Data are given as mean ± S.E.M. values (n = 3–8).

Fig. 4.. N-(thiazol-2-yl)-benzamide analogs with modifications to the phenyl ring.

A. Chemical structures of Series 4 analogs (4a-o) and the functional properties exhibited by them as antagonists at ZAC in oocytes using 1 mM Zn⁺ as agonist. Data are given as mean ± S.E.M. values (n = 4–8). B. Chemical structures of Series 5 analogs (5a-i) and the functional properties exhibited by them as antagonists at ZAC in oocytes using 1 mM Zn²⁺ as agonist. Data are given as mean ± S.E.M. values (n = 4–8).

Fig. 5.. Miscelleneous N-(thiazol-2-yl)-benzamide analogs.

Chemical structures of Series 6 analogs (6a-q) and the functional properties exhibited by them as antagonists at ZAC in oocytes using 1 mM Zn²⁺ as agonist. Data are given as mean ± S.E.M. values (n = 5–8).

Fig. 6.. Antagonist properties exhibited by five N-(thiazol-2-yl)-benzamide analogs at ZAC expressed in Xenopus oocytes in TEVC recordings.

Averaged concentration-inhibition curves for analogs 1, 2b, 3f, 4c and TTFB (5a) at Zn²⁺ (1 mM)-induced currents in ZAC-expressing oocytes. Data are given as mean ± S.E.M. values (n = 5–8). Averaged IC₅₀, pIC₅₀, n_H, range of inhibition and n values for the five analogs are given in Table 1.

Fig. 7.. Antagonist properties displayed by TTFB (5a) at ZAC expressed in Xenopus oocytes in TEVC recordings.

A. TTFB inhibits Zn²⁺ -evoked currents through ZAC in oocytes. Representative traces of Zn²⁺ (1 mM)-evoked currents in ZAC-expressing oocytes in the absence and in the presence of increasing concentrations of TTFB (left), and averaged concentration-inhibition relationship for TTFB (extracted from recorded saturated and peak responses) at the Zn²⁺ (1 mM)-induced currents in the oocytes (right). Data are given as mean ± S.E.M. values (n = 4–7). B. TTFB inhibits H⁺ -evoked currents through ZAC in oocytes. Representative traces of H⁺ (pH 6.0)-evoked currents in ZAC-expressing oocytes in the absence and in the presence of increasing concentrations of TTFB (left), and averaged concentration-inhibition relationship for TTFB (extracted from recorded saturated and peak responses) at the H⁺ (pH 6.0)-induced currents in the oocytes (right). Data are given as mean ± S. E.M. values (n = 4–5). C. TTFB inhibits the constitutive activity exhibited by ZAC in oocytes. Representative traces of the effects of increasing concentrations of TTFB on the leak current in ZAC-expressing oocytes (left), and averaged concentration-inhibition relationship for TTFB at the spontaneous currents of ZAC in the oocytes (right). Data are given as mean ± S.E.M. values (n = 7).

Fig. 8.. Current-voltage relationship of TTFB (5a)-induced currents and its voltage-independent inhibition of H⁺-evoked ZAC currents in Xenopus oocytes in TEVC recordings.

A. Current-voltage (I-V) relationship of TTFB-mediated inhibition of the spontaneous ZAC currents. Representative traces for currents evoked by applications of TTFB (100 μM) at different holding potentials (left) and the averaged IV-relationship displayed by TTFB (100 μM) at ZAC (right). Data given as leak-subtracted average current amplitudes normalized to the amplitude of currents recorded at −60 mV (mean ± S.E.M., n = 7). The IV curve was fitted with a third order polynomial model. B. Voltage-dependency of the ZAC inhibition mediated by TTFB. Representative traces of currents evoked by H⁺ (pH 6.5) in the absence and presence of TTFB (10 μM) under voltage-clamp of −60 mV and + 60 mV in the same ZAC-expressing oocyte (left) and averaged data for the TTFB (10 μM)-mediated inhibition of H⁺ (pH 6.5)-evoked currents in ZAC-expressing oocytes (mean ± S.E.M., n = 4) (right).

Fig. 9.. Selectivity profile and mode of action of TTFB (5a) as a ZAC antagonist

A. TTFB is a selective ZAC antagonist. The modulation exerted by TTFB (30 μM) at agonist-induced signalling through ZAC, m5-HT₃AR, hα₃β₄ nAChR, hα₁β₂γ₂s GABA_AR, and hα₁ GlyR expressed in oocytes in TEVC recordings. EC₂₀–EC₄₀ concentrations of the agonists for the respective receptors (1 mM Zn²⁺, 2 μM 5-HT, 3 μM (S)-nicotine, 30 μM GABA and 100 μM glycine, respectively) were used for the recordings. Representative traces of agonist-evoked currents in oocytes expressing the respective receptors in the absence and in the presence of 30 μM TTFB (left), and averaged data for the currents measured in oocytes expressing the respective receptors in the presence of TTFB (30 μM), normalized to the current evoked by the agonist in the absence of TTFB (I_agonist) (right). The averaged data are given as mean ± S.E.M. values (n = 5–8). B. TTFB acts through the transmembrane and/or intracellular domains of ZAC. Left: Illustration of the topologies of WT ZAC, WT m5-HT₃A, WT hα₁ GlyR, m5-HT₃A/ZAC and ZAC/hα₁-Gly subunits and the pentameric complexes assembled from them. Middle and right: The modulation exerted by TTFB (30 μM) at the agonist-induced responses through the m5-HT₃A/ZAC or ZAC/hα₁-Gly receptors. EC₂₀–EC₄₀ agonist concentrations for m5-HT₃A/ZAC or ZAC/hα₁-Gly (0.3 μM 5-HT and 3 μM Zn²⁺, respectively) were used for the recordings. Representative traces of agonist-evoked currents in oocytes expressing chimeric m5-HT₃A/ZAC or ZAC/hα_1-Gly receptors in the absence and in the presence of 30 μM TTFB (middle), and averaged data for the Currents measured in oocytes expressing ZAC, m5-HT₃AR, hα₁-Gly, m5-HT₃A/ZAC and ZAC/hα₁-Gly in the presence of TTFB (30 μM), normalized to the current recorded in the absence of TTFB (I_agonist) (right). The averaged data for m5-HT₃A/ZAC and ZAC/hα₁-Gly are given as mean ± S.E.M. values (n = 6–7), and the averaged data for ZAC, m5-HT₃AR and hα₁ GlyR from Fig. 9A are presented for comparison.

Fig. 10.. Examples of previously published ligands comprising the N-(thiazol-2-yl)-benzamide moiety.

The chemical structures, main targets and pharmacological activities reported for eight different ligands comprising the N-(thiazol-2-yl)-benzamide moiety [–59].