Inhibition of ALDH3A1-catalyzed oxidation by chlorpropamide analogues

In our efforts to identify agents that would specifically inhibit ALDH3Al, we had previously studied extensively the effect of an N-1-alkyl, an N-1-me thoxy, and several N-1-hydroxy-substituted ester derivatives of chlorpropam ide on the catalytic activities of ALDH3Als derived from human normal stoma ch mucosa (nALDH3Al) and human tumor cells (tALDH3Al), and of two recombina nt aldehyde dehydrogenases, viz. human rALDH1Al and rALDH2. The N-1-methoxy analogue of chlorpropamide, viz. 4-chloro-N- methoxy-N-[(propylamino)carbo nyl]benzenesulfonamide (API-2), was found to be a relatively selective and potent inhibitor of tALDH3Al-catalyzed oxidation as compared to its ability to inhibit nALDH3A-catalyzed oxidation, but even more potently inhibited A LDH2-catalyzed oxidation, whereas an ester analogue, viz. (acetyloxy)[(4-ch lorophenyl)-sulfonyl]carbamic acid 1,1-dimethylethyl ester (NPI-2), selecti vely inhibited tALDH3Al-catalyzed oxidation as compared to its ability to i nhibit nALDH3Al-, ALDH1Al- and ALDH2-catalyzed oxidations, and this inhibit ion was apparently irreversible. Three additional chlorpropamide analogues, viz. 4-chloro-N,O-bis(ethoxycarbonyl)-N-hydroxybenzene-sulfonamide (NPI-4) , N,O-bis(carbomethoxy)methanesulfohydroxamic acid (NPI-5), and 2-[(ethoxyc arbonyl)oxy]-1,2-benzisothiazol,3(2H)-one 1,1-dioxide (NPI-6), were evaluat ed in the present investigation. Quantified were NAD-linked oxidation of be nzaldehyde catalyzed by nALDH3Al and tALDH3Al, and NAD-linked oxidation of acetaldehyde catalyzed by rALDH1Al and rALDH2, all at 37 degreesC and pH 8. 1, and in the presence and absence of inhibitor. NPI-4, NPI-5 and NPI-6 wer e not substrates for the oxidative reactions catalyzed by any of the ALDHs studied. Oxidative reactions catalyzed by the ALDH3Als, rALDH1Al and rALDH2 were each inhibited by NPI-4 and NPI-5. NPI-6 was a poor inhibitor of nALD H3Al and tALDH3Al-catalyzed oxidations, but was a relatively potent inhibit or of rALDH1A1- and rALDH2-catalyzed oxidations. In all cases, inhibition o f ALDH-catalyzed oxidation was directly related to the product of inhibitor concentration and preincubation (enzyme + inhibitor) time. As judged by th e product values (muM x min) required to effect 50% inhibition (IC50): (1) nALDH3Al and tBLDI13A1 were essentially equisensitive to inhibition by NPI- 4 and NPI-5, and both enzymes were poorly inhibited by NPI-6; (2) rALDH1Al was, relative to the ALDH3Als, slightly more sensitive to inhibition by NPI -4 and NPI-5, and far more sensitive to inhibition by NPI-6; and (3) rALDH1 Al was, relative to rALDH2, essentially equisensitive to inhibition by NPI- 5, whereas, it was slightly more sensitive to inhibition by NPI-4 and NPI-6 . (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.