Thomas I. Kalman

Rapid determination of thymidylate synthase activity and its inhibition in intact L1210 leukemia cells in vitro

A rapid and convenient tritium release assay for measuring thymidylate (dTMP) synthase activity and its inhibition within intact mammalian cells is described in detail. Short-term incubation of murine leukemia L1210 cells with an appropriately labeled substrate precursor, either deoxyuridine ([5-3H]dUrd) or deoxycytidine ([5-3H]dCyd), allowed for: (1) uptake and intracellular conversion to the substrate deoxyuridylate ([5-3H]dUMP); and (2) the obligatory displacement of tritium from [5-3H]-dUMP during the dTMP synthase catalyzed reaction. Tritium released into the aqueous environment was quantitated after a quick one-step separation of tritiated H2O from other radiolabeled materials and cell debris. The amount of tritium released was evaluated as a function of a number of variables, including the concentration of labeled substrate precursors, cell number, and incubation time. Tritium from [5-3H]dCyd was released significantly faster than from [5-3H]dUrd under a variety of conditions. Both 5-fluorodeoxyuridine (1 microM) and methotrexate (10 microM), which effectively block intracellular dTMP synthesis, completely inhibited the release of tritium from either [5-3H]dCyd or [5-3H]dUrd demonstrating that the release of tritium is mediated exclusively by the dTMP synthase catalyzed reaction. In addition, there was a good correlation between tritium release, cellular uptake, and incorporation of [2-14C]dUrd into DNA. The inhibitory effects of antifolates such as methotrexate were independent of the type of labeled precursor used. In contrast, preferential interference with the release of tritium from [5-3H]-dCyd by dCyd derivatives and from [5-3H]dUrd by dUrd derivatives was observed, suggesting that competition for uptake and/or phosphorylation may contribute to the overall effects of certain nucleoside analogues on cellular dTMP synthase activity measured using the tritium release assay.

Inhibition of thymidylate synthetase by showdomycin and its 5′-phosphate

Abstract Thymidylate synthetase of dichloromethotrexate resistant Lactobacillus casei was found to be rapidly and irreversibly inactivated by showdomycin (3-β-D-ribofuranosylmaleimide). Prior treatment of the enzyme with hydroxyethyldisulfide prevented the inhibitory effect of showdomycin. The enzyme could also be protected by its substrate, deoxyuridylate, against inactivation by the antibiotic. Reduction of the 3, 4-double bond by catalytic hydrogenation destroyed the enzyme inhibitory activity of showdomycin. Phosphorylation of its 5′-hydroxyl group greatly enhanced the inhibitory potency of the antibiotic, presumably by conferring substrate-like specificity. These results suggest that the maleimide moiety of showdomycin alkylates a reactive sulfhydryl group at the active site of thymidylate synthetase, and are consistent with a sulfhydryl addition-elimination mechanism of the enzyme catalyzed reaction.

5-Mercaptodeoxyuridine—Its enzymatic synthesis and mode of action in microbiological systems☆

5-Mercapto-2′-deoxyuriCline (MUdR) was synthesized by enzymatic transfer of the deoxyribosyl group of thymidine (TdR) to 5-mercaptouracil (MU), by using the trans-N-deoxyribosylase from Lactobacillus helveticus. The acceptor and donor activities of MU, MUdR and their respective disulfides were studied in this enzyme system; the disulfides were found inactive as substrates, but they showed the same growth inhibitory activities as the corresponding thiols in the microbiological assays. MUdR was nearly as active as 5-fluoro-2′-deoxyuridine (FUdR) in the Lactobacillus leichmannii assay, but was considerably less active than FUdR in the Lactobacillus arabinosus and Streptococcus faecalis systems. Inhibition analysis studies indicated that MUdR, like FUdR, acts via inhibition of thymidylate synthetase. The significant differences found in the spectra of activities of MU and MUdR, as compared to those of 5-fluorouracil (FU) and FUdR, are related to differences in the routes and efficiency of their metabolic activation in which the mercapto analogs, due to the size of their 5-S− group, are restricted to the pathways available for the metabolic transformations of thymine and TdR, while the fluoro analogs may be converted to the nucleotide via the uracil pathway. Some synergism and cross-resistance studies are also reported.

Formation of 5- and 6-Aminocytosine Nucleosides and Nucleotides from the Corresponding 5-Bromocytosine Derivatives: Synthesis and Reaction Mechanism

Abstract An improved method for the synthesis of 5-aminocytidine (3a), 5-amino-2′-deoxycytidine (3b), and their 5′-monophosphates (3c,d) from the corresponding 5-bromo pyrimidines, using liquid ammonia, is described. The respective 6-aminocytosine derivatives (4a,b,c,d), minor products of the amination reaction, were isolated and characterized. A plausible mechanism is proposed to account for the formation of both 5-and 6-substituted products.

6-substituted 5-fluorouracil derivatives as transition state analogue inhibitors of thymidine phosphorylase.

A combination of mechanism-based and structure-based design strategies led to the synthesis of a series of 5- and 6-substituted uracil derivatives as potential inhibitors of thymidine phosphorlase/platelet derived endothelial cell growth factor (TP/PD-ECGF). Among those tested, 6-imidazolylmethyl-5-fluorouracil was found to be the most potent inhibitor with a Ki-value of 51 nM, representing a new class of 5-fluoropyrimidines with a novel mechanism of action.

Conformational Analysis of Human Dihydrofolate Reductase Inhibitor Complexes: Crystal Structure Determination of Wild Type and F31 Mutant Binary and Ternary Inhibitor Complexes

These structural studies reveal unusual intermolecular interactions for the binding of inhibitors and cofactor in ternary complexes with both wild type and F31 mutant recombinant human DHFR and show that these inhibitors have flexibility in occupying the active site. These studies also possibly indicate the first structural data for a ternary complex with a folate inhibitor and a polyglutamate side chain. However, further refinement of this data is necessary before this can be confirmed. In contrast to the ternary complexes of folate and MTX, the lipophilic antifolate PTX binds with its methoxybenzoyl ring oriented toward the cofactor nicotinamide ring, while that of TMQ it is bound closer to the Phe-31 position. Furthermore, the nicotinamide ring makes a close contact to the N10 amine of TMQ, significantly different from its binding site interactions in MTX complexes. These data also reveal that the conserved contacts between the cofactor carboxyamide with the enzyme backbone residues Ala-9 and Ile-16 are dictated by the enzyme and that changes in the orientation of the structural elements requires only subtle changes in the secondary structural units in which they are contained. Therefore, only by careful analysis of a series of enzyme complexes can the mechanisms of binding action be delineated.

Inhibition of gamma-glutamyl hydrolases in human cells by 2-mercaptomethylglutaric acid

Cultured human lymphocytes and fibroblasts accumulate methotrexate during 24 hours and synthesize methotrexate polyglutamates up to the hexaglutamate, with the triglutamate predominating. In the interval after incubation with methotrexate, drug is lost, metabolites are converted to longer chain-lengths, and methotrexate pentaglutamate predominates. 2-Mercaptomethylglutaric acid, an inhibitor of neutral pH gamma-glutamyl hydrolases in vitro, had little effect on polyglutamate synthesis during incubation of the cells with methotrexate, but maintained for 48 hours almost all the methotrexate as the pentaglutamate when added after the removal of the drug. These findings demonstrate that inhibition of gamma-glutamyl hydrolases is an effective approach to alter the distribution of polyglutamate forms of methotrexate in vivo and indicate that enzymatic hydrolysis may contribute to regulation of polyglutamate chain lengths in human cells.

MOLECULAR ASPECTS OF THE MECHANISM OF ACTION OF 5‐FLUORODEOXYURIDINE*

It was established by Cohen et al.’ and Heidelberger’ in the late fifties and early sixties that the antineoplastic drug 5-fluorodeoxyuridine (FUdR) exerts its effect primarily by interferring with the biosynthesis of DNA-thymine. The active form of FUdR is its 5’-monophosphate, an extremely potent inhibitor of thymidylate synthetase. The unusual potency of 5-fluorodeoxyuridylate (FdUMP) stems from the ability of the analog to block the enzyme-catalyzed reaction in a kinetically irreversible manner. Recent studies suggested that the apparent irreversibility of the inhibition may be due to covalent bond formation between the drug and the enzyme.’p5 By contrast, the inherent reversibility of the inactivation process was demonstrated by the ready regeneration of lost catalytic activity under proper condition^.^.^ Based on these and other studies relating to the mechanism of the thymidylate synthetase catalyzed reaction,’ as well as on the known reactivity of the 5,6-double bond of 5-fl~oropyrimidines,~-’~ we assumed as a working hypothesis5,’ ’ that the covalent interaction of the drug with the enzyme involves the reversible addition of a functional amino acid residue of the enzyme across the 5,6-double bond of the 5fluoropyrimidine ring. As a test of the above hypothesis we investigated the effect ofdeuterium substitution at the 6-position of F d U M P on the reactivation of the FdUMP-inactivated thymidylate synthetase. TABLE 1 shows the results of this experiment. A significant secondary deuterium isotope effect of kH/kD = 1.24 was observed during the reversal of enzyme inactivation. This indicates’ 5.16 that a change of configuration of carbon-6 of FdUMP from tetrahedral (sp’) to trigonal (sp’) must have taken place during the reactivation process; i z . , we have witnessed the reformation of the 5,6-double bond of the 5-fluoropyrimidine ring. This is consistent with the idea that the nucleophilic attack of an enzymic group at position 6 of F d U M P leads to the formation of a covalent bond between the drug and the enzyme with concomitant saturation of the 5,6-double bond, which is regenerated during reactivation upon departure of the enzymic nucleophile: 1 &:Enzyme , Inactivation 1 I H ( D )

Methylation of 4-thio-2'-deoxyuridylate by thymidylate synthetase.

Summary 4-Thio-2′-deoxyuridylate was found to be a substrate for the thymidylate synthetase of a dichloromethotrexate resistant strain of Lactobacillus casei . The enzymic product which contained the carbon derived from formaldehyde, was identified as 4-thiothymidylate by chromatographic analysis. 4-Thio-2′-deoxyuridylate inhibited the methylation of deoxyuridylate with an apparent K i of 7×10 −5 M , reflecting its lower affinity for the enzyme as compared to the natural substrate. 4-Thio-2′-deoxyuridylate is, so far, the only substrate analog modified in the base which is methylated by thymidylate synthetase.

Design and synthesis of 3,5-dialkylamino substituted 8H,10H-3(R),5(R),15b(S)-2,3,6,7-tetrahydro-1,5,3-dioxazepino[3,2-c]indolo[3,2-g]pteridine-7-ones

Abstract 3,5-Dialkylamino substituted 8H,10H,15b(S)-2,3,6,7-tetrahydro-1,5,3-dioxazepino[3,2-c]indolo[3,2-g]pteridine-7-one derivatives 6a-6e were synthesized as potential anticancer agents. Preliminary results showed that they were active as inhibitors of the growth of murine leukemia L1210 cells in vitro with IC50 -values of 4 to 24 μM.