Mechanism of inhibition of mammalian tumor and other thymidylate synthases by N4-hydroxy-dCMP, N4-hydroxy-5-fluoro-dCMP, and related analogs

Abstract

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)

DerivFit:A Program for Rate Equation Parameter Fitting Using Derivatives

“…This example demonstrates the behavior of gradient of ⌽ in the scheme of slow, tight-binding inhibition (10,11,13):…”

“…[1]. If the reaction satisfies some mass-balance conditions of the form of linear equations n jϭ1 c kj y j ϭ Y k , k ϭ 1, ..., n b [13] (n b is the number of mass-balance equations), it is possible to use them in the reduction of the number of ODEs from n to n Ϫ n b . Such a reduction leads, however, to a significant complication in the elegant algorithm elaborated originally to solve [1].…”

“…Such a reduction leads, however, to a significant complication in the elegant algorithm elaborated originally to solve [1]. The reduced ODEs, i.e., including explicit mass balances of [13], can be written as…”

Different Activities of 5-Hydroxy-dUMP and 5-Hydroxymethyl-dUMP in Thymidylate Synthase-Catalyzed Reaction in View of Molecular Modeling and Structural Studies

“…Structures tending to promote dissociation of the proton from the hydroxylic group are shown in bold (6,(9)(10)(11)(12).…”

In vitro antitumour activity of cis- and trans-5-fluoro-5,6-dihydro-6-alkoxy-uracils; effects on thymidylate synthesis

“…This has promoted the development of potent analogues of the folate cofactor (Harrap et al, 1989). Research concentrated to a lesser extent on analogues of FdUMP (Rode et al, 1990) or nucleotides of FU analogues (Heidelberger et al, 1983).…”