Developmental arrest in Caenorhabditis elegans dauer larvae causes high expression of enzymes involved in thymidylate biosynthesis, similar to that found in Trichinella muscle larvae.

Crude extract specific activities of thymidylate synthase, dUTPase, thymidine kinase and dihydrofolate reductase were high during the development of Caenorhabditis elegans, the dauer larva activities being similar to those previously determined in Trichinella spiralis and T. pseudospiralis muscle larvae (with the exception of thymidine kinase, not detected in Trichinella). High thymidylate synthase expression in developmentally arrested larvae, demonstrated also at the mRNA and protein levels, is in agreement with a global cell cycle arrest of dauer larvae and indicates this unusual cell cycle regulation pattern can be shared by developmentally arrested larvae of C. elegans and the two Trichnella species. Hence, the phenomenon may be characteristic for developmentally arrested larvae of different nematodes, rather than specific for the parasitic Trichinella muscle larvae. Endogenous C. elegans thymidylate synthase was purified and its molecular properties compared with those of the recombinant protein, expression of the latter in E. coli cells confirming the NCBI database sequence identity.

Trichinella spiralis thymidylate synthase: cDNA cloning and sequencing, and developmental pattern of mRNA expression.

The persistent expression of thymidylate synthase activity has previously been demonstrated not only in adult forms, but also in non-developing muscle larvae of Trichinella spiralis and T. pseudospiralis, pointing to an unusual pattern of cell cycle regulation, and prompting further studies on the developmental pattern of T. spiralis thymidylate synthase gene expression. The enzyme cDNA was cloned and sequenced, allowing the characterization of a single open reading frame of 307 amino acids coding for a putative protein of 35,582 Da molecular weight. The amino acid sequence of the parasite enzyme was analysed, the consensus phylogenetic tree built and its stability assessed. The aa sequence identity with thymidylate synthase was confirmed by the enzymatic activity of the recombinant protein expressed in E. coli. As compared with the enzyme purified from muscle larvae, it showed apparently similar Vmax value, but higher Km(app) values desscribing interactions with dUMP (28.8 microM vs. 3.9 microM) and (6RS,alphaS)-N(5,10)-methylenetetrahydrofolate (383 microM vs. 54.7 microM). With the coding region used as a probe, thymidylate synthase mRNA levels, relative to 18S rRNA, were found to be similar in muscle larvae, adult forms and newborn larvae, in agreement with muscle larvae cells being arrested in the cell cycle.

Interaction of thymidylate synthase with the 5'-thiophosphates, 5'-dithiophosphates, 5'-H-phosphonates and 5'-S-thiosulfates of 2'-deoxyuridine, thymidine and 5-fluoro-2'-deoxyuridine.

New analogs of dUMP, dTMP and 5-fluoro-dUMP, including the corresponding 5'-thiophosphates (dUMPS, dTMPS and FdUMPS), 5'-dithiophosphates (dUMPS2, dTMPS2 and FdUMPS2), 5'-H-phosphonates (dUMP-H, dTMP-H and FdUMP-H) and 5'-S-thiosulfates (dUSSO3, dTSSO3 and FdUSSO3), have been synthesized and their interactions studied with highly purified mammalian thymidylate synthase. dUMPS and dUMPS2 proved to be good substrates, and dTMPS and dTMPS2 classic competitive inhibitors, only slightly weaker than dTMP. Their 5-fluoro congeners behaved as potent, slow-binding inhibitors. By contrast, the corresponding 5'-H-phosphonates and 5'-S-thiosulfates displayed weak activities, only FdUMP-H and FdUSSO3 exhibiting significant interactions with the enzyme, as weak competitive slow-binding inhibitors versus dUMR The pH-dependence of enzyme time-independent inhibition by FdUMP and FdUMPS was found to correlate with the difference in pKa values of the phosphate and thiophosphate groups, the profile of FdUMPS being shifted (approximately 1 pH unit) toward lower pH values, so that binding of dUMP and its analogs is limited by the phosphate secondary hydroxyl ionization. Hence, together with the effects of 5'-H-phosphonate and 5'-S-thiosulfate substituents, the much weaker interactions of the nucleotide analogs (3-5 orders of magnitude lower than for the parent 5'-phosphates) with the enzyme is further evidence that the enzyme's active center prefers the dianionic phosphate group for optimum binding.

5-Substituted N(4)-hydroxy-2'-deoxycytidines and their 5'-monophosphates: synthesis, conformation, interaction with tumor thymidylate synthase, and in vitro antitumor activity.

Convenient procedures are described for the synthesis of 5-substituted N(4)-hydroxy-2'-deoxycytidines 5a,b,d-h via transformation of the respective 5-substituted 3', 5'-di-O-acetyl-2'-deoxyuridines 1a-c,e-h. These procedures involved site-specific triazolation or N-methylimidazolation at position C(4), followed by hydroxylamination and deblocking with MeOH-NH(3). Nucleosides 5a,b,d-h were selectively converted to the corresponding 5'-monophosphates 6a,b,d-h with the aid of the wheat shoot phosphotransferase system. Conformation of each nucleoside in D(2)O solution, deduced from (1)H NMR spectra and confirmed by molecular mechanics calculations, showed the pentose ring to exist predominantly in the conformation S (C-2'-endo) and the N(4)-OH group as the cis rotamer. Cell growth inhibition was studied with two L5178Y murine leukemia cell lines, parental and 5-fluoro-2'-deoxyuridine (FdUrd)-resistant, the latter 70-fold less sensitive toward FdUrd than the former. With FdUrd-resistant L5178Y cells, 5-fluoro-N(4)-hydroxy-2'-deoxycytidine (5e) caused almost 3-fold stronger growth inhibition than FdUrd; 5e was only some 3-fold weaker growth inhibitor of the resistant cells than of the parental cells. Thymidylate synthase inhibition was studied with two forms of the enzyme differing in sensitivities toward 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), isolated from parental and FdUrd-resistant L1210 cell lines. All N(4)-hydroxy-dCMP (6a,b,d-h) and dUMP analogues studied were competitive vs dUMP inhibitors of the enzyme. Analogues 6b,d-h and 5-hydroxymethyl-dUMP, similar to N(4)-hydroxy-dCMP (6a) and FdUMP, were also N(5), N(10)-methylenetetrahydrofolate-dependent, hence mechanism-based, slow-binding inhibitors. 5-Chloro-dUMP, 5-bromo-dUMP, and 5-iodo-dUMP, similar to dTMP, did not cause a time-dependent inactivation of the enzyme. Instead, they behaved as classic inhibitors of tritium release from [5-(3)H]dUMP. 5-Bromo-dUMP and 5-iodo-dUMP showed substrate activity independent of N(5), N(10)-methylenetetrahydrofolate in the thymidylate synthase-catalyzed dehalogenation reaction. The =N-OH substituent of the pyrimidine C(4) prevented the enzyme-catalyzed release from the C(5) of Br(-) and I(-) (the same shown previously for H(+)). While FdUMP and 6a showed a higher affinity and greater inactivation power with the parental cell than FdUrd-resistant cell enzyme, an opposite relationship could be seen with 5-hydroxymethyl-dUMP.

Trichinella spiralis and Trichinella pseudospiralis: developmental patterns of enzymes involved in thymidylate biosynthesis and pyrimidine salvage.

Thymidylate synthase, dihydrofolate reductase and dUTPase specific activities were found to remain at a high and constant level in crude extracts from adult worms of Trichinella spiralis, as well as from muscle larvae of both Trichinella spiralis (isolated 1-24 months after infection) and Trichinella pseudospiralis (isolated 5.5-13 months after infection). The results obtained with Trichinella pseudospiralis muscle larvae isolated with the use of pepsin did not differ from those obtained when pepsin was not used. No thymidine kinase activity could be detected in muscle larvae of either species and thymidine phosphorylase could be found only in T. pseudospiralis larvae isolated without the use of pepsin. Muscle larvae of both species contained orotidylate phosphoribosyl transferase activity, pointing to a possibility of 5-fluorouracil activation. Uridine phosphorylase, another enzyme involved in 5-fluorouracil anabolism, was also present in T. pseudospiralis muscle larvae. Results of comparative studies on inhibition of purified T. spiralis and rat thymidylate synthases by substrate (4-thio-5-fluoro-dUMP, 2-thio-5-fluoro-dCMP and N4-hydroxy-dCMP) and cofactor (ZD 9331) analogues indicated only dUMP analogues to show feeble selectivity towards the parasite enzyme. A hypothesis is discussed, assuming high expression of thymidylate synthase in muscle larvae to be connected with their cells being arrested in the cell cycle.

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

In order to explain different activities shown by 5-hydroxy-dUMP (substrate) and its close analogue 5-hydroxymethyl-dUMP (slow-binding inhibitor) in the reaction catalyzed by thymidylate synthase, studies have been undertaken involving (i) ab initio RHF simulations, (ii) comparative analysis of crystallographic structures available from CSD, and (iii) QSAR analysis of experimental results describing thymidylate synthase interaction with various 5-substituted dUMP analogues. Assuming substrate activity of 5-hydroxy-dUMP to be associated with proton release from the C(5) hydroxyl in the enzyme-catalyzed reaction, acidities of 5-hydroxy and 5-hydroxymethyl substituents in dUMP molecule were compared. The results indicate the 5-hydroxyl deprotonation to be easier and supported by resonance electronic effect, pointing to a probable mechanism of different activities of the two dUMP analogues in thymidylate synthase reaction. The possibility is discussed that 5-mercapto-dUMP and 5-hydroseleno-dUMP, previously assumed to be inhibitors, could be also substrates for thymidylate synthase, as the 5-mercaptyl and 5-hydroselenidyl appear to be deprotonated even more easily than the 5-hydroxyl. Copyright 2000 Academic Press.

Crystal structures of rat thymidylate synthase inhibited by Tomudex, a potent anticancer drug.

Two crystal structures of rat thymidylate synthase (TS) complexed with dUMP and the anticancer drug Tomudex (ZD1694) have been determined to resolutions of 3.3 and 2.6 A. Tomudex is one of several new antifolates targeted to TS and the first to be approved for clinical use. The structures represent the first views of any mammalian TS bound to ligands and suggest that the rat protein undergoes a ligand-induced conformational change similar to that of the Escherichia coli protein. Surprisingly, Tomudex does not induce the "closed" conformation in rat TS that is seen on binding to E. coli TS, resulting in inhibitor atoms that differ in position by more than 1.5 A. Several species-specific differences in sequence may be the reason for this. Phe 74 shifts to a new position in the rat complex and is in van der Waals contact with the inhibitor, while in the E. coli protein the equivalent amino acid (His 51) hydrogen bonds to the glutamate portion of the inhibitor. Amino acids Arg 101, Asn 106, and Met 305 make no contacts with the inhibitor in the open conformation, unlike the equivalent residues in the E. coli protein (Thr 78, Trp 83, and Val 262). dUMP binding is similar in both proteins, except that there is no covalent adduct to the active site cysteine (Cys 189) in the rat structures. Two insertions in the rat protein are clearly seen, but the N-termini (residues 1-20) and C-termini (residues 301-307) are disordered in both crystal forms.

An isotopic assay of dUTPase activity based on coupling with thymidylate synthase.

A new rapid, sensitive and convenient procedure is presented allowing determination of dUTPase activity. With [5-(3)H]dUTP used as the substrate, dUTPase, converts it to the corresponding monophosphate and is coupled with thymidylate synthase-catalyzed reaction, resulting in tritium release from [5-(3)H]dUMP. Following charcoal absorption of the labeled nuleotides, radioactivity of tritiated water is determined. The new assay was tested to show comparable results with a previously described assay, based on measuring dUTPase-catalyzed [5-(3)H]dUMP production.

Acyclic analogues of 5-fluoro-dUMP and 5-fluoro-2'-deoxyuridine: synthesis and inhibition of thymidylate synthase and tumour cell growth.

1-[(2-Hydroxyethoxy)methyl]-5-fluorouracil (HEMFU) and 1-[(1,3-dihydroxy-2-propoxy)methyl]-5-fluorouracil (DHPFU) were prepared by alkylation of the di-O-TMS derivative of 5-fluorouracil and phosphorylated with the use of the wheat shoot phosphotransferase system to their monophosphates, HEMFUMP and DHPFUMP. 1-(2-Phosphonylmethoxyethyl)-5-fluorouracil (PMEFU) was obtained by condensation of diethyl-2-chloroethoxymethanephosphonate with 5-fluorouracil and cleavage of the alkylphosphoester with trimethylbromosilane. Inhibition of highly purified thymidylate synthase from mouse tumour Ehrlich carcinoma and leukemia L1210 cells by each of the nucleotide analogues, DHPFUMP, PMEFU and HEMFUMP, and of L5178Y mouse leukemia cell growth by the nucleoside (HEMFU) analogue, were studied. DHPFUMP proved to be the strongest inhibitor, non-competitive vs dUMP, with K(i)app 2.8 microM for time-independent interaction with the enzyme and N5,N10-methylenetetrahydrofolate (CH2H4PteGlu). In the presence of CH2H4PteGlu, DHPFUMP exhibited time-dependent inactivation of the enzyme, the inactivation rate plots being biphasic and pointing to Ki values in the microM range (10(3)-fold higher than for 5-fluoro-dUMP). HEMFUMP and PMEFU were much weaker inhibitors of the enzyme, with K(i)app values of 0.26 mM (non-competitive vs dUMP) and 30 mM (non-competitive vs dUMP), respectively. HEMFU, despite the weak interaction of its nucleotide analogue with the enzyme, proved to be a strong cell (L5178Y) growth inhibitor, with IC50 in the range 10(-5) M.

Synthesis and interaction with thymidylate synthase of 5'-dithiophosphate and 5'-fluorothiophosphate of thymidine.

Thymidine-5'-fluorothiophosphate, dTMP(S)-F, was synthesized by the oxathiaphospholane, and thymidine 5'-dithiophosphate, dTMPS2, by the dithiaphospholane, method. To estimate the role of 5'-phosphate group ionization in binding of pyrimidine nucleotides by thymidylate synthase, dTMP(S)-F was studied as an inhibitor of mouse tumour (L1210) enzyme, and its inhibitory properties were compared with those of dTMPS2, a close dTMP analogue. While dTMPS2 proved to be an inhibitor, competitive vs dUMP, with K(i)app = 94 microM, the 5'-fluorothiophosphate congener displayed no activity, indicating that the enzyme requires for binding the presence of a dianionic 5'-phosphate group in a nucleotide.

DerivFit: a program for rate equation parameter fitting using derivatives.

A C program for fitting parameters in enzymatic rate equations is presented. The DerivFit program employs the reaction scheme in the form of ordinary differential equations (ODEs). The kinetic parameters are fitted to the experimental data by minimizing the sum of squared deviations of experimental points from theoretically predicted progress curves. In the minimization process we use the Gradient, Newton, and Marquardt algorithms. The gradients are calculated explicitly by solving a set of additional ODEs that are automatically attached by the program, taking advantage of a general formulation of the basic ODEs that determine the reaction's time course. The program is applied to simple enzymatic systems including slow tight-binding inhibition.

Crystal structures of 5-fluoro-dUrd and its 2 and/or 4-thio analogues: models of substituted dUMP pyrimidine ring interacting with thymidylate synthase.

In order to understand the influence on thymidylate synthase interactions with dUMP analogues of the pyrimidine ring 2- and/or 4-thio, and 5-fluoro substitutions, X-ray diffractions by crystals of 5-fluoro-dUrd and its 2- and 4-thio, and 2,4-dithio analogues were measured, the four structures solved and refined. The following conclusions were suggested by results of comparative analyses of structural parameters (bond lengths, valence angles), followed by theoretical considerations based on calculated resonance structure distributions and aromaticity indices of the uracil, thiouracil, fluorouracil and fluorothiouracil rings. The effect of 4-thio substitution of FdUMP, altering specificity of inactivation of thymidylate synthases from various sources, is probably due to weaker proton acceptor power of the 4-thio substituent and increasing acidity (enhanced proton-donor power) of the N(3)-H moiety, resulting in an impaired fitness into the network of hydrogen bonds in the enzyme active center cleft. 2,4-Dithio substitution results in (i) impaired pyrimidine ring recognition by the enzyme active center, due to the 4-thio substituent (ii) increased pyrimidine ring aromaticity in dUMP, leading to resistance of C(6) to nucleophilic attack by the enzyme active center cysteine and (iii) altered planarity of the pyrimidine ring and deflections, with respect to the ring plane, of substituents at C(2), C(4) and C(5). 5-Fluoro substitution apparently activates the pyrimidine ring towards the interaction with thymidylate synthase by producing local strain, which results in an increased reactivity as predicted by the Walsh-Bent rule.

Modeling of reaction steps relevant to deoxyuridylate (dUMP) enzymatic methylation and thymidylate synthase mechanism-based inhibition.

Theoretical quantum mechanical ab initio Hartree-Fock calculations on molecular systems, modeling processes related to the specificity of thymidylate synthase inactivation are reported. We considered several steps of the methylation of the substrate dUMP and 4- or 5-mono- and 4,5-bisubstituted dUMP analogs, as well. The following reactions were modeled: the cysteine residue (Cys198 in the L.casei enzyme) nucleophilic attack on the substrate and the substrate C(5)-H proton abstraction. The substrate was modeled by the 1-methyluracil molecule and its structural analogs. The cysteine Cys198 residue was modeled by the methylmercaptane molecule. The substrate-enzyme binary complex was modeled by the 1-methyl-5,6-dihydro-6-thiomethyl-uracil (P1) molecule. The present theoretical calculations suggest that the cysteine nucleophilic attack on the substrate may result in the SH-group addition to the pyrimidine C(5)=C(6) bond in the course of a weakly exothermic reaction. The formerly presumed enolate carbanion appeared to be weakly stable or unstable and it can readily split into the thiol and pyrimidine residues. The s2-thio- (P2) and s2,4-dithio- (P3) substrate analogs should form stable thiolate anions after cysteine residue attachment to the C(6) position of the pyrimidine ring. Studies of the deformed P1 molecule interacting with a water molecule bound to the pyrimidine C(4)=O carbonyl residue allow a suggestion that this water molecule may be directly involved in the C(5)-H proton abstraction and may serve as a proton transmitter between the substrate and the proton acceptor residue, possibly located on the cofactor N10-nitrogen. Interaction of the pyrimidine C(4)=O group, or its modification, with the N5,10-methylenetetrahydrofolate N(10) nitrogen atom is suggested as an additional factor influencing the inhibition process.

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.

Synthesis and interactions with thymidylate synthase of 2,4-dithio analogues of dUMP and 5-fluoro-dUMP.

The 2,4-dithio analogues of 2'-deoxyuridine and 2'-deoxy-5-fluorouridine have been synthesized by thiation of the previously described 2-thio analogues, and then phosphorylated enzymatically or chemically to yield 2,4-dithio-dUMP and 2,4-dithio-5-fluoro-dUMP. In striking contrast to the 2-thio and 4-thio analogues of dUMP, which are good substrates of thymidylate synthase, 2,4-dithio-dUMP is not a substrate. But, surprisingly, it is a competitive inhibitor, relative to dUMP, of the purified enzymes from both parental and FdUrd-resistant L1210 cells, with K(i) values of 32 microM and 55 microM, respectively. Although 2,4-dithio-5-fluoro-dUMP behaved as a typical slow-binding inhibitor of the enzyme, its K(i) value was 10(3)-10(4)-fold higher than those for the corresponding 2-thio and 4-thio congeners. Similarly, 2,4-dithio-FdUrd was a much weaker inhibitor of tumour cell growth (IC50 approximately 10(-5)M) than FdUrd (IC50 approximately 10(-9)M), 2-thio-FdUrd(IC50 approximately 10(-7)M) or 4-thio-FdUrd (IC50 approximately 5x10(-8)M), while with 2,4-dithio-dUrd no influence on cell growth could be observed. Theoretical considerations, based on calculated aromaticities of the uracil and thiouracil rings, suggest that lack of substrate activity of 2,4-dithio-dUMP may result from increased pyrimidine ring aromaticity of the latter, leading to resistance of C(6) to nucleophilic attack by the enzyme active center cysteine.

Molecular mechanism of thymidylate synthase-catalyzed reaction and interaction of the enzyme with 2- and/or 4-substituted analogues of dUMP and 5-fluoro-dUMP.

Thymidylate synthase is a target enzyme in anticancer, antiviral, antifungal and antiprotozoan chemotherapy. With two dUMP analogues, 5-fluoro-dUMP (FdUMP) and 5-(trifluoromethyl)-dUMP (CF3dUMP), strong thymidylate synthase inhibitors and active forms of drugs, the inhibition mechanism is based on the reaction mechanism. Recent comparative studies of new dUMP analogues, containing more than one substituent in the pyrimidine ring, showed that substitution of the pyrimidine ring C(4) = O group in FdUMP by either C(4) = N-OH group (in N4-hydroxy-FdCMP) or C(4) = S group (in 4-thio-FdUMP) preserves high inhibitory potency of the drug but may alter its specificity for thymidylate synthases from various sources, which differ in sensitivity to slow-binding inhibition by FdUMP. Informations suggesting mechanisms responsible for the foregoing have been reviewed, including results of molecular modeling studies suggesting interaction of the pyrimidine C(4) = O group, or its modification, with the N5,10-methylene.(ABSTRACT TRUNCATED)

2-Thio derivatives of dUrd and 5-fluoro-dUrd and their 5'-monophosphates: synthesis, interaction with tumor thymidylate synthase, and in vitro antitumor activity.

A convenient synthesis of 5-fluoro-2-thiouracil (11) is based on hydrolytic deamination of 5-fluoro-2-thiocytosine (9). Lewis acid-catalyzed condensation of di-TMS-5-fluoro-2-thiouracil (13) or di-TMS-2-thiouracil (14) with 2-deoxy-3,5-di-O-p-toluyl-D-ribofuranosyl chloride (15) led to mixtures of the beta- and alpha-anomers of 3',5'-toluylated 2'-deoxy-5-fluoro-2-thiouridine (16 and 18) or 2'-deoxy-2-thiouridine (17 and 19), each of which was deblocked with MeOH-NH3 to give the desired free anomeric nucleoside pairs 1, 5 and 3, 7, respectively. These were selectively converted to the corresponding 5'-monophosphates 2, 6 and 4, 8, with the aid of the wheat shoot phosphotransferase system. Conformations of the nucleosides 1, 3, 5, 7 are deduced from 1H NMR spectra, and circular dichroism spectra for nucleotide anomeric pairs 2, 6 and 4, 8 are reported. Whereas beta-2-thio-dUMP (4) was a good substrate (Km approximately 10(-5) M), beta-5-fluoro-2-thio-dUMP (2) proved to be a potent competitive, slow-binding inhibitor (Ki approximately 10(-8) M) of the purified enzymes from Ehrlich ascites carcinoma and L1210 cells. The alpha-anomer 6 was a weak inhibitor, with Ki in the mM range, and its congener 8 hardly interacted with the enzyme. The beta-anomer 1 exhibited antitumor activity in a mouse leukemic cell line L5178Y (IC50 approximately 10(-6) M), hence 40-100-fold weaker than 5-fluoro-dUrd. Its alpha-anomer 5 was 10-fold less active, but exhibited at least 10-fold higher selectivity with respect to the tumor cells than the beta-anomer 1.