Mechanism of inhibition of mammalian tumor and other thymidylate synthases by N4-hydroxy-dCMP, N4-hydroxy-5-fluoro-dCMP, and related analogues.
N4-Hydroxy-dCMP (N4-OH-dCMP), N4-methoxy-dCMP (N4-OMe-dCMP), and their 5-fluoro congeners (syntheses of which are described) were all slow-binding inhibitors of Ehrlich carcinoma thymidylate synthase (TS), competitive with respect to dUMP, and had differing kinetic constants describing interactions with the two TS binding sites. N4-OH-dCMP was not a substrate (no dihydrofolate produced; no tritium released with 5-3H-labeled molecule), and its inactivation of TS was methylenetetrahydrofolate-dependent, hence mechanism-based, with arrest of a step posterior to addition of cofactor and blocking abstraction of the C(5) hydrogen. Ki values for N4-OH-dCMP and its 5-fluoro analogue were in the range 10(-7) - 10(-8) M, 2-3 orders of magnitude higher for the corresponding N4-OMe analogues. The 5-methyl analogue of N4-OH-dCMP was 10(4)-fold less potent, pointing to the anti rotamer of the imino form of exocyclic N4-OH, relative to the ring N(3), as the active species. This is consistent with weaker slow-binding inhibition of the altered enzyme from 5-FdUrd-resistant, relative to parent, L1210 cells by both FdUMP and N4-OH-dCMP, suggesting interaction of both N4-OH and C(5)-F groups with the same region of the active center. Kinetic studies with purified enzyme from five sources, viz., Ehrlich carcinoma, L1210 parental, and 5-FdUrd-resistant cells, regenerating rat liver, and the tapeworm Hymenolepis diminuta, demonstrated that addition of a 5-fluoro substituent to N4-OH-dCMP increased its affinity from 2- to 20-fold for the enzyme from different sources.(ABSTRACT TRUNCATED AT 250 WORDS)
Rode
W
W
Nencki Institute of Experimental Biology, Academy of Sciences, Warszawa, Poland.
Zieliński
Z
Z
Dzik
J M
JM
Kulikowski
T
T
Bretner
M
M
Kierdaszuk
B
B
Cieśla
J
J
Shugar
D
D
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology; D003843:Deoxycytidine Monophosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D003856:Deoxyuracil Nucleotides / Q000494:pharmacology; D005468:Fluorodeoxyuridylate / Q000494:pharmacology; D006926:Hymenolepis / Q000201:enzymology; D007700:Kinetics; D007939:Leukemia L1210 / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008115:Liver Regeneration; D051379:Mice; D009374:Neoplasms, Experimental / Q000201:enzymology*; D051381:Rats; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D003856:Deoxyuracil Nucleotides; D003843:Deoxycytidine Monophosphate; C067000:N(4)-hydroxy-5-fluorodeoxycytidine monophosphate; D005468:Fluorodeoxyuridylate; C042920:N-4-hydroxy-2'-deoxycytidylic acid; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase