Novel competitive irreversible inhibitors of aldehyde dehydrogenase (ALDH1): restoration of chemosensitivity of L1210 cells overexpressing ALDH1 and induction of apoptosis in BAF(3) cells overexpressing bcl(2).
4-Amino-4-methyl-pent-2-ynthioc acid S-methyl ester (ampal thiolester: ATE) was used as a lead compound to synthesise new amino-substituted 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) approximately 280 microM) of baker's yeast ALDH1 in vitro showing 80% inhibition at 400 microM when preincubated with the enzyme for 30min, whereas the trimethyl ammonium and the morpholine derivatives showed only 15% inhibition at 600 microM even after 60min 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 (16-20mU/mg protein) and were all inhibited by DIMATE, reaching 20-30% at 4 microM. Up to 4 microM no apoptosis, as measured by DNA-fragmentation was observed, but at 8 and 10 microM 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.
Quash
Gerard
G
Laboratoire d'Immunochimie, INSERM U329, Faculté de Médecine Lyon-Sud, Oullins, France. quash@lyon-sud.univ.lyon1.fr
Fournet
Guy
G
Chantepie
Jacqueline
J
Gore
Jacques
J
Ardiet
Claude
C
Ardail
Dominique
D
Michal
Yvonne
Y
Reichert
Uwe
U
D000444:Aldehyde Dehydrogenase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000080924:Aldehyde Dehydrogenase 1 Family; D000480:Alkynes / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D003520:Cyclophosphamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects; D004952:Esters / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007939:Leukemia L1210 / Q000473:pathology*; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000502:physiology; D050697:Retinal Dehydrogenase; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D014407:Tumor Cells, Cultured
C466358:4-Amino-4-methyl-pent-2-ynthioc acid S-methyl ester; D000480:Alkynes; D004952:Esters; D007527:Isoenzymes; D019253:Proto-Oncogene Proteins c-bcl-2; D013438:Sulfhydryl Compounds; C466360:TRIMATE cpd; C466361:dimethylthioampal; D003520:Cyclophosphamide; D000080924:Aldehyde Dehydrogenase 1 Family; D000444:Aldehyde Dehydrogenase; C510223:ALDH1A1 protein, human; C515028:ALDH1A1 protein, mouse; D050697:Retinal Dehydrogenase; D005978:Glutathione; C011272:perfosfamide