In our efforts to identify agents that would specifically inhibit ALDH3A1, we had previously studied extensively the effect of an N(1)-alkyl, an N(1)-methoxy, and several N(1)-hydroxy-substituted
ester derivatives of
chlorpropamide on the catalytic activities of ALDH3A1s derived from human normal stomach mucosa (nALDH3A1) and human
tumor cells (tALDH3A1), and of two recombinant
aldehyde dehydrogenases, viz. human rALDH1A1 and rALDH2. The N(1)-methoxy analogue of
chlorpropamide, viz. 4-chloro-N-methoxy-N-[(propylamino)carbonyl]
benzenesulfonamide (API-2), was found to be a relatively selective and potent inhibitor of tALDH3A1-catalyzed oxidation as compared to its ability to inhibit nALDH3A-catalyzed oxidation, but even more potently inhibited ALDH2-catalyzed oxidation, whereas an
ester analogue, viz. (acetyloxy)[(4-chlorophenyl)sulfonyl]
carbamic acid 1,1-dimethylethyl
ester (NPI-2), selectively inhibited tALDH3A1-catalyzed oxidation as compared to its ability to inhibit nALDH3A1-, ALDH1A1- and ALDH2-catalyzed oxidations, and this inhibition was apparently irreversible. Three additional
chlorpropamide analogues, viz. 4-chloro-N,O-bis(ethoxycarbonyl)-N-hydroxybenzenesulfonamide (NPI-4), N,O-bis(carbomethoxy)
methanesulfohydroxamic acid (NPI-5), and 2-[(ethoxycarbonyl)oxy]-1,2-benzisothiazol-3(2H)-one 1,1-dioxide (NPI-6), were evaluated in the present investigation. Quantified were
NAD-linked oxidation of
benzaldehyde catalyzed by nALDH3A1 and tALDH3A1, and
NAD-linked oxidation of
acetaldehyde catalyzed by rALDH1A1 and rALDH2, all at 37 degrees C and pH 8.1, and in the presence and absence of inhibitor. NPI-4, NPI-5 and NPI-6 were not substrates for the oxidative reactions catalyzed by any of the ALDHs studied. Oxidative reactions catalyzed by the ALDH3A1s, rALDH1A1 and rALDH2 were each inhibited by NPI-4 and NPI-5. NPI-6 was a poor inhibitor of nALDH3A1- and tALDH3A1-catalyzed oxidations, but was a relatively potent inhibitor of rALDH1A1- and rALDH2-catalyzed oxidations. In all cases, inhibition of ALDH-catalyzed oxidation was directly related to the product of inhibitor concentration and preincubation (enzyme+inhibitor) time. As judged by the product values (microMxmin) required to effect 50% inhibition (IC(50)): (1) nALDH3A1 and tALDH3A1 were essentially equisensitive to inhibition by NPI-4 and NPI-5, and both
enzymes were poorly inhibited by NPI-6; (2) rALDH1A1 was, relative to the ALDH3A1s, slightly more sensitive to inhibition by NPI-4 and NPI-5, and far more sensitive to inhibition by NPI-6; and (3) rALDH1A1 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.