Thymidylate synthases from Hymenolepis diminuta and regenerating rat liver: purification, properties, and inhibition by substrate and cofactor analogues.

Comparative studies of thymidylate synthases, isolated from the tapeworm, Hymenolepis diminuta, and regenerating liver of its host, rat, aimed at a possibility of specific inhibition of the helminthic enzyme, are presented. While similar in structure (dimers with monomer molecular masses of 33.7 kDa and 34.9 kDa, respectively) and parameters describing interactions with substrates and products, the tapeworm and rat enzymes differed in the dependences of reaction velocity on temperature (Arrhenius plots biphasic and linear, respectively). The tapeworm, compared with the host, enzyme was less sensitive to the competitive slow-binding inhibition by 5-fluoro-dUMP and its 2-thio congener, but equally sensitive to inhibition by 4-thio-5-fluoro-dUMP, N4-hydroxy-dCMP and N4-hydroxy-5-fluoro-dCMP, the latter being more potent inhibitor of the parasite enzyme than 5-fluoro-dUMP. alpha-Anomer of 5-fluoro-dUMP behaved as a very weak competitive slow-binding inhibitor of both enzymes. Both enzymes differed markedly in sensitivity to inhibition by 10-propargyl-5,8-dideazafolate and its di- and triglutamates (pddPteGlu1-3), with pddPteGlu1 being stronger inhibitor of the mammalian enzyme, but pddPteGlu3 showing opposite specificity. Sulfonamidobenzoylglutamate analogue of pddPteGlu (pddPteSO2Glu) and 2-desamino-2-methyl derivative of this analogue (CH3pddPteSO2Glu) were weaker inhibitors of both enzymes than the parent compound. Substitution of the glutamyl residue in CH3pddPteSO2Glu with either norvaline or alanine increased inhibition potency, whereas similar substitutions with glycine, valine or phenylglycine were without a distinct effect with the host enzyme but weakened inhibition of the tapeworm enzyme.

Syntheses and thymidylate synthase inhibitory activity of the poly-gamma-glutamyl conjugates of N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino ]-2-thenoyl]-L-glutamic acid (ICI D1694) and other quinazoline antifolates.

Thirteen poly-gamma-glutamates derived from several novel antifolates have been synthesized by a convergent route. The syntheses of poly-gamma-glutamyl conjugates of N-[5-[N-(3,4-dihydro-2- methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-theno yl]-L-glutamic acid (8) (ICI D1694), 2-desamino-N10-propargyl-5,8-dideazafolic acid (6), 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (7), 2-desamino-2-methyl-N10-propargyl-2'-fluoro-5,8-dideazafolic acid (9), and 2-desamino-2-methyl-4-chloro-N10-propargyl-2'-fluoro-3,5,8-trideazafo lic acid (11) are described. A key step in the route involves coupling of an alpha-tert-butyl-protected poly-gamma-glutamate of the required chain length to the appropriate 5,8-dideazapteroic acid, obtained by carboxypeptidase G2 cleavage of the parent monoglutamate, if available, or by chemical synthesis. Deprotection with trifluoroacetic acid in the final step gave the desired poly-gamma-glutamyl antifolates as their trifluoroacetate salts. As inhibitors of thymidylate synthase, these polyglutamates were more potent in every case than the corresponding non-polyglutamylated drug.

Quinazoline antifolates inhibiting thymidylate synthase: synthesis of four oligo(L-gamma-glutamyl) conjugates of N10-propargyl-5,8-dideazafolic acid and their enzyme inhibition.

The synthesis is described of four oligo(gamma-glutamyl) conjugates of N10-propargyl-5,8-dideazafolic acid containing a total of two, three, four, and five L-glutamic acid residues. The tert-butyl group was chosen as the carboxyl protecting group in order to obviate the use of alkali and thus the possibility of gamma----alpha transpeptidation. The starting material, di-tert-butyl glutamate, was coupled to N-(benzyloxycarbonyl)-L-glutamic acid alpha-tert-butyl ester via a mixed anhydride with isobutyl chloroformate. Hydrogenolysis of the benzyloxycarbonyl group in the product gave a carboxyl-protected diglutamate, which either was acylated with 4-[(benzyloxycarbonyl)amino] benzoyl chloride to give a protected aminobenzamide or was cycled further by using the above mixed anhydride/hydrogenolysis sequence into tri-, tetra-, and pentaglutamates. Each of the last named was also acylated, as above, to give a benzamide. The benzyloxycarbonyl group in the benzamides was removed by hydrogenolysis and the amino groups thus exposed were N-alkylated with propargyl bromide. The resulting proparglyamines were further alkylated with 2-amino-6-(bromomethyl)-4-hydroxyquinazoline hydrobromide to give the antifolate poly(t-Bu) esters. Deprotection with trifluoroacetic acid in the final step delivered the desired antifolates as their trifluoroacetate salts. The di- to pentaglutamates were, respectively, 31-, 97-, 171-, and 167-fold more inhibitory to WI-L2 human thymidylate synthase than the parent compound.

The synthesis and thymidylate synthase inhibitory activity of L-gamma-L-linked dipeptide and L-gamma-amide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583).

Sixteen gamma-linked dipeptide and four L-Glu-gamma-amide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) have been synthesized and evaluated as inhibitors of thymidylate synthase (TS). Z-blocked L-Glu-gamma-L-linked dipeptides and L-Glu-gamma-amides were prepared by condensing alpha-tert-butyl-N-(benzyloxycarbonyl)-L-glutamic acid with the appropriate tert-butyl-protected L-amino acid or amine. The Z group was removed by catalytic hydrogenolysis, and the resulting dipeptides or L-Glu-gamma-amides were condensed with the appropriate pteroic acid analogue trifluoroacetate salt using diethyl cyanophosphoridate as coupling reagent. Deprotection with trifluoroacetic acid in the final step gave the desired quinazoline gamma-linked dipeptides and L-Glu-gamma-amides as their trifluoroacetate salts. Nearly all the dipeptide analogues were potent inhibitors of TS, the best being ICI 198583-gamma-L-2-aminoadipate (IC50 = 2 nM). Several of these dipeptides were found to be susceptible to enzymatic hydrolysis in mice. The quinazoline monocarboxylate L-Glu-gamma-amides, lacking an alpha'-carboxyl group, are less active against TS and L1210 cell growth but are also not susceptible to enzymatic hydrolysis in mice.

Synergistic effect of 5-fluorodeoxyuridine and quinazoline antifolates on murine leukemia self-cultured in vitro.

The effect of thymidylate synthase inhibitors, fluorodeoxyuridine (FdUrd) and its two sulphonamide derivatives was examined in the culture of murine leukemia cells -- 5178Y (parental subline) and its fluorodeoxyuridine resistant subline 5178Y/F. A synergistic effect of the antimetabolites on cell survival was observed on exposure of the culture of either line to a slightly inhibitory concentration of FdUrd (1 nM) in combination with 2-desamino-2-methyl-10-propargyl-5,8-dideaza-pteroylsulphogluta mate or 2-desamino-2-methyl-10-propargyl-5,8-dideaza-pteroylsulphoglyci ne. This effect was accompanied by a marked reduction, in both cell lines of intracellular concentration of 5,10-methylenetetrahydro-pteroyl-polyglutamate, although its concentration in the resistant subline was 3 times as high as in the parental line. The inhibitory effect of combined drugs on the cellular pool of folates in 5178Y line depended also on the sequence of drug addition, whereas in the FdUrd resistant line this sequence was without any effect. The results obtained strongly suggest that under certain conditions inhibition of thymidylate synthesis by antifolates is intensified by a prior use of FdUrd.

The effects of combined antifolates on inhibition of growth of murine leukemia cells cultured in vitro.

The synergistic effect of trimetrexate (TMTX) and sulphonamide derivatives of quinazoline on the cultured 5178Y murine leukemia cells was examined. On exposure to the slightly inhibitory concentrations of TMTX (0.1 nM) in combination with 2-desamino-2-methyl-10-propargyl-5,8-dideaza-pteroyl-sulphoglyc ine (DMPDDSF) (0.02 microM) a synergistic inhibitory effect of the antifolates on cell growth was observed. These two drugs in the same combination caused also synergistic inhibition of de novo synthesis of thymidylate in intact cells as measured by tritium release from [5-(3)H]deoxyuridylate. This was accompanied by a marked reduction in intracellular concentration of 5,10-methylenetetrahydro-pteroyl-polyglutamate (5,10CH2H4PteGlu(n)) (0.2 microM) and dihydropteroyl-polyglutamate (0.12 microM). In these conditions de novo biosynthesis of purine was decreased by 50%. These observations show that growth inhibition by combined antifolates is mediated by intracellular depletion of the substrate of thymidylate synthase -- 5,10CH2H4PteGlu(n). The results obtained strongly suggest that under certain conditions inhibition of thymidylate synthesis by DMPDDSF is intensified by prior application of TMTX -- an inhibitor of dihydrofolate reductase.

Effect of combined inhibitors of thymidylate synthase-5-fluorodeoxyuridine and quinazoline antifolates on murine leukemia cells cultured in vitro.

The synergistic effect of two different inhibitors of thymidylate synthase-FdUrd and sulphonamide derivatives on murine leukemia cells-5178Y (parental subline) and 5178Y/F (its fluorodeoxyuridine resistant subline) in culture was examined. Upon the exposure of cultures from both lines to a slightly inhibitory concentration of FdUrd (1 nM) in combination with 2-desamino-2-methyl-10-propargyl-5,8-dideaza-pteroylsulphogluta mine or -glycine a synergistic effect of antimetabolites on cell growth was observed. This was accompanied by a marked reduction in intracellular concentration in both cell lines of 5,10CH2H4PteGlu; the intracellular concentration of 5,10CH2H4PteGlu(n) in the resistant subline was 3 times higher than in parental line. The inhibitory effect of combined drugs on the cellular pool of 5178Y of the two antimetabolites also depends on the sequence of their addition; however in the FdUrd resistant cell-line the dependence on the sequence of the addition was not observed. The results obtained strongly suggest that under certain conditions inhibition of thymidylate synthesis by antifolates is intensified by proprior use of FdUrd.

[Comparison of thymidylate synthase properties isolated from the rat tapeworm, hymenolepis diminuta, with properties of enzyme isolated from the host in regenerating rat liver]. / Porównanie wlasciwosci syntazy tymidylanowej oczyszczonej z tasiemca szczurzego, Hymenolepis diminuta, z wlasciwosciami enzymu zywiciela wyizolowanego z regenerujacej watroby szczura.

Thymidylate synthases (TS) from the tapeworm, Hymenolepis diminuta, and regenerating rat liver have been purified by means of affinity chromatography on immobilized 10-formyl-5,8-dideazafolate and concentrated on immobilized p-aminophenyl-5-fluoro-2'-deoxyuridine monophosphate. Molecular weights of native TS from the tapeworm and regenerating rat liver were 62 kD and 81.5 kD, respectively, and molecular weights of the monomers were 34.4 kD and 34.9 kD, respectively, pointing to dimeric structures of both enzymes. The dependence of TS activity on temperature (Arrhenius plot) was biphasic for the parasite enzyme, with lower activation energy above 32 degrees C, and monophasic for the host enzyme. 2'-deoxyuridine-5'-monophosphate (dUMP) analogues, 5-fluoro-2'-deoxyuridine-5'-monophosphate (5-FdUMP), 2-tio-5-FdUMP,N4-hydroxy-2'-deoxycytidine-5'-monophosphate (N4-hydroxy-dCMP) and N4-hydroxy-5-FdCMP, were competitive with respect to dUMP, slow-binding inhibitors of TS from both sources, with K1 values in 10(-6)-10(-9) M range. 5-FdUMP was distinctly stronger inhibitor of the host than the tapeworm TS, whereas N4-hydroksy-5-FdCMP inhibited stronger the parasite enzyme. Interaction of 5,10-methylenetetrahydrofolate (CH2H4PteGlu) analogue, 10-propargyl-5,8-dideazafolate (pddPteGlu), and its di- and triglutamates with both enzymes were studied. Inhibition of the parasite and host enzymes by pddPteGlu was of mixed-type with respect to CH2H4PteGlu, with K1 values in 10(-8) M range. Introduction of additional glutamate residues changed inhibition type to noncompetitive with respect to Ch2H4PteGlu and lowered K1 values (pddPteGlu3 < pddPteGlu2 < pddPteGlu1).(ABSTRACT TRUNCATED AT 250 WORDS)

Trichinella spiralis thymidylate synthase: developmental pattern, isolation, molecular properties, and inhibition by substrate and cofactor analogues.

Thymidylate synthase specific activity was found to remain at a constant level in crude extracts from muscle larvae, isolated (1-15 months after infection) by pepsin-HCI digestion, as well as from adult worms of Trichinella spiralis. The enzyme was purified and its molecular (monomer mol. wt 35 kD) and kinetic (sequential mechanism with the K(m) values 3.1 and 19 microM for dUMP and N5,10-methylenetetrahydrofolate, respectively) properties determined. 5-Fluoro-dUMP was a competitive, slow-binding inhibitor of the parasite enzyme. N5,10-methylenetetrahydrofolate analogues 10-propargy1-5,8- dideazafolate (CB3717), ZD1694, BW1843U89, and AG337 were weaker inhibitors of the parasite than regenerating rat liver enzyme. Inhibition by 10-propargyl-5,8-dideazafolate was strengthened by an increasing number of glutamate residues. Thymidine kinase activity could not be detected in the muscle larvae crude extracts.