Novel competitive irreversible inhibitors of aldehyde dehydrogenase (ALDH1):: restoration of chemosensitivity of L1210 cells overexpressing ALDH1 and induction of apoptosis in BAF3 cells overexpressing bcl2

4-Amino-4-methyl-pent-2-ynthioc acid S-methyl ester (ampal thiolester: ATE) was used as a lead compound to synthesise new aminosubstituted derivatives of alpha,beta acetylenic thiolester compounds as inhibitors of aldehyde dehydrogenase 1, (ALDH1). Of these compounds, the dimethyl derivative (DIMATE) was a competitive irreversible inhibitor (K-i similar to 280 muM) of baker's yeast ALDH1 in vitro showing 80% inhibition at 400 muM when preincubated with the enzyme for 30 min, whereas the trimethyl ammonium and the morpholine derivatives showed only 15% inhibition at 600 muM even after 60 min preincubation. ATE inhibited ALDH1 activity in ALDH1-transfected L1210 T cells resistant to hydroperoxycyclophosphamide (HCPA) and inhibited growth synergistically in the presence of HCPA. In non-transfected L1210 counterparts ATE did not potentiate growth inhibition by HCPA. DIMATE was a 30-100-fold more effective growth inhibitor than ATE. Endogenous ALDH1 activities of BAF(3) cells over-expressing different levels of bcl(2) (0-100%) were similar (16-20 mU/mg protein) and were all inhibited by DIMATE, reaching 20-30% at 4 muM. Up to 4 muM no apoptosis, as measured by DNA-fragmentation was observed, but at 8 and 10 muM DIMATE, DNA-fragmentation increased concomitantly with ALDH1 inhibition. No DNA-fragmentation was observed with ALDH1 irreversible inhibitors devoid of a thiolester group or with thiolesters which were not inhibitors of ALDH1. It was seen only with competitive irreversible inhibitors having the methanethiol and enzyme-inhibitory moieties. The methanethiol putatively released from DIMATE by ALDH1 esterase activity plays a role, albeit undefined, in lowering intramitochondrial glutathione levels which decreased by 47% as DNA-fragmentation increased. (C) 2002 Elsevier Science Inc. All rights reserved.