Madjroh N , Mellou E , Davies PA , Söderhielm PC , Jensen AA .

R01 MH097446 NIMH NIH HHS, R21 NS111064 NINDS NIH HHS

	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 (I10 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 (IZn2+).
	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, nH, 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 antagonistA. TTFB is a selective ZAC antagonist. The modulation exerted by TTFB (30 μM) at agonist-induced signalling through ZAC, m5-HT3AR, hα3β4 nAChR, hα1β2γ2s GABAAR, 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 (Iagonist) (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/hα1-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 (Iagonist) (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 [52–59].

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