Flavin-dependent thymidylate synthase: A novel pathway towards thymine

For several decades only one chemical pathway was known for the de novo biosynthesis of the essential DNA nucleotide, thymidylate. This reaction catalyzed by thyA or TYMS encoded thymidylate synthases is the last committed step in the biosynthesis of thymidylate and proceeds via the reductive methylation of uridylate. However, many microorganisms have recently been shown to produce a novel, flavin-dependent thymidylate synthase encoded by the thyX gene. Preliminary structural and mechanistic studies have shown substantial differences between these deoxyuridylate-methylating enzymes. Recently, both the chemical and kinetic mechanisms of FDTS have provided further insight into the distinctions between thyA and thyX encoded thymidylate synthases. Since FDTSs are found in several severe human pathogens their unusual mechanism offers a promising future for the development of antibiotic and antiviral drugs with little effect on human thymidylate biosynthesis.     

Design, synthesis and evaluation of potent thymidylate synthase X inhibitors

Three synthesized series of compounds based on a thiazolidine core allowed identification of potent inhibitors of thymidylate synthase X. The evaluation of the catalytic activity of the enzyme in the presence of these molecules revealed two distinct classes of compounds that inhibit ThyX with submicromolar concentrations, which could lead, after optimization, to effective inhibitors with potential biomedical interest.     

Complementing thymidylate synthase

The structure of thymidylate synthase complementing protein with substrates dUMP and FAD, presented in this issue of Structure, sheds light on a fascinating new catalytic mechanism, suggests a strategy for the design of new antimicrobial compounds, and highlights the promise of proteomics in medicine.     

5-propynylpyrimidine nucleoside derivatives: rationally designed mechanism-based inactivators of thymidylate synthase

A novel series of 5-propynyl-dUMP derivatives, with a variety of leaving groups on the side-chain, was designed as potential mechanism-based inhibitors of thymidylate synthase (TS), and synthesized from 5-iodo-2'-deoxyuridine by Pd(0)-catalyzed coupling, followed by direct phosphorylation with POCl3. All members of the series inhibited TS competitively with Ki-values of 0.015-18 microM. Analogs with fluorine or imidazole-based leaving groups caused rapid, irreversible inactivation of TS.     

Structure of human thymidylate synthase suggests advantages of chemotherapy with noncompetitive inhibitors

Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms. The emergence of resistance to the treatment is often related to the increased levels of TS in cancer cells, which have been linked to the elimination of TS binding to its own mRNA upon drug binding, a feedback regulatory mechanism, and/or to the increased stability to intracellular degradation of TS.drug complexes (versus unliganded TS). The active site loop of human TS (hTS) has a unique conformation resulted from a rotation by 180 degrees relative to its orientation in bacterial TSs. In this conformation, the enzyme must be inactive, because the catalytic cysteine is no longer positioned in the ligand-binding pocket. The ordered solvent structure obtained from high resolution crystallographic data (2.0 A) suggests that the inactive loop conformation promotes mRNA binding and intracellular degradation of the enzyme. This hypothesis is supported by fluorescence studies, which indicate that in solution both active and inactive forms of hTS are present. The binding of phosphate ion shifts the equilibrium toward the inactive conformation; subsequent dUMP binding reverses the equilibrium toward the active form. Thus, TS inhibition via stabilization of the inactive conformation should lead to less resistance than is observed with presently used drugs, which are analogs of its substrates, dUMP and CH(2)H(4)folate, and bind in the active site, promoting the active conformation. The presence of an extension at the N terminus of native hTS has no significant effect on kinetic properties or crystal structure.     

Molecular docking studies on quinazoline antifolate derivatives as human thymidylate synthase inhibitors

We have performed molecular docking on quinazoline antifolates complexed with human thymidylate synthase to gain insight into the structural preferences of these inhibitors. The study was conducted on a selected set of one hundred six compounds with variation in structure and activity. The structural analyses indicate that the coordinate bond interactions, the hydrogen bond interactions, the van der Waals interactions as well as the hydrophobic interactions between ligand and receptor are responsible simultaneously for the preference of inhibition and potency. In this study, fast flexible docking simulations were performed on quinazoline antifolates derivatives as human thymidylate synthase inhibitors. The results indicated that the quinazoline ring of the inhibitors forms hydrophobic contacts with Leu192, Leu221 and Tyr258 and stacking interaction is conserved in complex with the inhibitor and cofactor.     

Novel 2-amino-4-oxo-5-arylthio-substituted-pyrrolo[2,3-d]pyrimidines as nonclassical antifolate inhibitors of thymidylate synthase

A series of 17 novel 2-amino-4-oxo-5-[(substituted phenyl)thio]pyrrolo[2,3-d]pyrimidines were synthesized as potential inhibitors of thymidylate synthase (TS) and as antitumor agents. The analogues contain a variety of electron withdrawing substituents on the phenyl ring of the side chain and were evaluated as inhibitors of human TS (hTS) and Escherichia coli TS and of human and E. coli dihydrofolate reductase (DHFR). The analogues 14, 17, and 18 were potent inhibitors of hTS with IC50 values of 0.28, 0.21, and 0.22 microM, respectively, and were more potent than the clinically used ZD1694, 2 and LY231514, 3 against human TS.     

Crystallization of human thymidylate synthase

Human thymidylate synthase has been crystallized in the absence of ligands and diffracts beyond 3.0 A. The protein was cloned and expressed in Escherichia coli and then crystallized from ammonium sulfate in the presence of beta-mercaptoethanol at a variety of pH values. The crystals are trigonal in the space-group P3(1)21; the unit cell dimensions are a = b = 96.7 A, c = 84.1 A.     

Substituted pyrrolo[2,3-d]pyrimidines as Cryptosporidium hominis thymidylate synthase inhibitors

Cryptosporidiosis, a gastrointestinal disease caused by a protozoan Cryptosporidium hominis is often fatal in immunocompromised individuals. There is little clinical data to show that the existing treatment by nitazoxanide and paromomycin is effective in immunocompromised individuals.1, 2 Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer and malaria. A novel series of classical antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines have been evaluated as Cryptosporidium hominis thymidylate synthase (ChTS) inhibitors. Crystal structure in complex with the most potent compound, a 2′-chlorophenyl with a sulfur bridge with a K_i of 8.83 ± 0.67 nM is discussed in terms of several Van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate. Of these interactions, two interactions with the non-conserved residues (A287 and S290) offer an opportunity to develop ChTS specific inhibitors. Compound 6 serves as a lead compound for analog design and its crystal structure provides clues for the design of ChTS specific inhibitors.     

2,4-Diamino-5-methyl-6-substituted arylthio-furo[2,3-d]pyrimidines as novel classical and nonclassical antifolates as potential dual thymidylate synthase and dihydrofolate reductase inhibitors

A novel classical antifolate N-{4-[(2,4-diamino-5-methyl-furo[2,3-d]pyrimidin-6-yl)thio]-benzoyl}-l-glutamic acid 5 and 11 nonclassical antifolates 6–16 were designed, synthesized, and evaluated as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS). The nonclassical compounds 6–16 were synthesized from 20 via oxidative addition of substituted thiophenols using iodine. Peptide coupling of the intermediate acid 21 followed by saponification gave the classical analog 5. Compound 5 is the first example, to our knowledge, of a 2,4-diamino furo[2,3-d]pyrimidine classical antifolate that has inhibitory activity against both human DHFR and human TS. The classical analog 5 was a nanomolar inhibitor and remarkably selective inhibitor of Pneumocystis carinii DHFR and Mycobacterium avium DHFR at 263-fold and 2107-fold, respectively, compared to mammalian DHFR. The nonclassical analogs 6–16 were moderately potent against pathogen DHFR or TS. This study shows that the furo[2,3-d]pyrimidine scaffold is conducive to dual human DHFR-TS inhibitory activity and to high potency and selectivity for pathogen DHFR.     

Targeting the alpha-folate receptor with cyclopenta[g]quinazoline-based inhibitors of thymidylate synthase

The alpha-FR has been reported to be overexpressed in many carcinomas, in particular those of the ovary and uterus. The high expression of alpha-FR in some tumours compared with normal tissues has been exploited over the last decade for folate-mediated targeting of macromolecules, anticancer drugs, imaging agents and nucleic acids to cancer cells. CB300638, a cyclopenta[g]quinazoline-based inhibitor of thymidylate synthase (TS), has been reported to have high affinity for the receptor and selectivity for alpha-FR overexpressing tumour cell lines. In this study, the structural features of the molecule, in particular modifications at the 2-position, have been investigated with respect to TS inhibition, affinity for the alpha-FR and reduced folate carrier (RFC) and activity in A431-FBP cells (transfected with human alpha-FR) compared with neo-transfected A431 cells. Compounds 1a,b, 2a,b and 3a,b were synthesised utilising multistep sequences. It was found that the 2-substituent does not affect the affinity for the alpha-FR; however, it greatly affects selectivity for A431-FBP cells, and suggests that there are factors other than TS inhibition and alpha-FR affinity that are important for the activity of these compounds. Compound 2b (2-CH2OH derivative) displayed the highest selectivity for the A431-FBP cells compared with A431 cells.     

Distribution of mutations in human thymidylate synthase yielding resistance to 5-fluorodeoxyuridine

Thymidylate synthase (TS) catalyzes methylation of dUMP to dTMP and is the target of cancer chemotherapeutic agents (e.g. 5-fluorouracil). Here, we used error-prone PCR to mutagenize the full-length human TS cDNA and then selected mutants resistant to 5-fluorodeoxyuridine in a bacterial complementation system. We found that resistant mutants contained 1-5 amino acid substitutions and that these substitutions were located along the entire length of the polypeptide. Mutations were frequent near the active site Cys(195) and in the catalytically important Arg(50) loop; however, many mutations were also distributed throughout the remainder of the cDNA. Mutants containing a single amino acid replacement identified the following 14 residues as unreported sites of resistance: Glu(23), Thr(51), Thr(53), Val(84), Lys(93), Asp(110), Asp(116), Pro(194), Ser(206), Met(219), His(250), Asp(254), Tyr(258), and Lys(284). Many of these residues are distant from the active site and/or have no documented function in catalysis or resistance. We conclude that mutations distributed throughout the linear sequence and three-dimensional structure of human TS can confer resistance to 5-fluorodeoxyuridine. Our findings imply that long range interactions within proteins affect catalysis at the active site and that mutations at a distance can yield variant proteins with desired properties.     

Synthesis of 5-ethynyl-2'-deoxyuridine-5'-boranomono phosphate as a potential thymidylate synthase inhibitor

The 5-ethynyl-2'-deoxyuridine nucleoside and the 5'-boranomonophosphate nucleotide were synthesized as analogs of 5-fluoro-2'-deoxyuridine monophosphate (5-FdUMP), a widely used mechanism-based inhibitor of thymidylate synthase. Synthesis was carried out from protected 5-iodo-2'-deoxyuridine and trimethylsilylacetylene by Sonogashira palladium-catalyzed cross coupling reaction followed by selective phosphorylation and finally boronation.     

Saturation site-directed mutagenesis of thymidylate synthase

We have subjected 12 different codons of a synthetic Lactobacillus casei thymidylate synthase (TS) gene to saturation site-directed mutagenesis to create amino acid "replacement sets" at each of those positions. The target residues were chosen because they are highly conserved and because they are important for the structure and function of the protein as indicated by solution and structural studies. The mutagenesis procedure involved excision of a fragment of the synthetic gene containing the target codon, followed by its replacement with a mixture of oligonucleotides which code for all 20 amino acids and the amber stop codon. TS mutants were identified by DNA sequencing, and catalytically active mutants were identified by genetic complementation using a Thy- strain of Escherichia coli. Only 3 of the 12 target amino acids examined were essential for TS activity; and of the 125 total mutants identified, 57 were catalytically active. These results point to a high degree of plasticity of TS in accommodating function with structural change.     

Subunit complementation of thymidylate synthase.

Each of the two active sites of thymidylate synthase contains amino acid residues contributed by the other subunit. For example, Arg-178 of one monomer binds the phosphate group of the substrate dUMP in the active site of the other monomer [Hardy et al. (1987) Science 235, 448-455]. Inactive mutants of such residues should combine with subunits of other inactive mutants to form heterodimeric hybrids with one functional active site. In vivo and in vitro approaches were used to test this hypothesis. In vivo complementation was accomplished by cotransforming plasmid mixtures encoding pools of inactive Arg-178 mutants and pools of inactive Cys-198 mutants into a host strain deficient in thymidylate synthase. Individual inactive mutants of Arg-178 were also cotransformed with the C198A mutant. Subunit complementation was detected by selection or screening for transformants which grew in the absence of thymidine, and hence produced active enzyme. Many mutants at each position representing a wide variety of size and charge supported subunit complementation. In vitro complementation was accomplished by reversible dissociation and unfolding of mixtures of purified individual inactive Arg-178 and Cys-198 mutant proteins. With the R178F + C198A heterodimer, the Km values for dUMP and CH2H4folate were similar to those of the wild-type enzyme. By titrating C198A with R178F under unfolding-refolding conditions, we were able to calculate the kcat value for the active heterodimer. The catalytic efficiency of the single wild-type active site of the C198A + R178F heterodimer approaches that of the wild-type enzyme.     

Cooperative inhibition of human thymidylate synthase by mixtures of active site binding and allosteric inhibitors

Thymidylate synthase (TS) is a target in the chemotherapy of colorectal cancer and some other neoplasms. It catalyzes the transfer of a methyl group from methylenetetrahydrofolate to dUMP to form dTMP. On the basis of structural considerations, we have introduced 1,3-propanediphosphonic acid (PDPA) as an allosteric inhibitor of human TS (hTS); it is proposed that PDPA acts by stabilizing an inactive conformer of loop 181-197. Kinetic studies showed that PDPA is a mixed (noncompetitive) inhibitor versus dUMP. In contrast, versus methylenetrahydrofolate at concentrations lower than 0.25 microM, PDPA is an uncompetitive inhibitor, while at PDPA concentrations higher than 1 microM the inhibiton is noncompetive, as expected. At the concentrations corresponding to uncompetitive inhibition, PDPA shows positive cooperativity with an antifolate inhibitor, ZD9331, which binds to the active conformer. PDPA binding leads to the formation of hTS tetramers, but not higher oligomers. These data are consistent with a model in which hTS exists preferably as an asymmetric dimer with one subunit in the active conformation of loop 181-197 and the other in the inactive conformation.     

Sulfonylpiperidines as novel,antibacterial inhibitors of Gram-positive thymidylate kinase (TMK)

Thymidylate kinase (TMK) is an essential enzyme for DNA synthesis in bacteria, phosphorylating deoxythymidine monophosphate (dTMP) to deoxythymidine diphosphate (dTDP), and thus is a potential new antibacterial drug target. Previously, we have described the first potent and selective inhibitors of Gram-positive TMK, leading to in vivo validation of the target. Here, a structure-guided design approach based on the initial series led to the discovery of novel sulfonylpiperidine inhibitors of TMK. Formation of hydrogen bonds with Arg48 in Staphylococcus aureus TMK was key to obtaining excellent enzyme affinity, as verified by protein crystallography. Replacement of a methylene linker in the series by a sulfonamide was accomplished with retention of binding conformation. Further optimization of log D yielded phenol derivative 11, a potent inhibitor of TMK showing excellent MICs against a broad spectrum of Gram-positive bacteria and >10⁵ selectivity versus the human TMK homologue.     

Modification of Escherichia coli thymidylate synthase at tyrosine-94 by 5-imidazolylpropynyl-2'-deoxyuridine 5'-monophosphate

Evidence is presented that 5-imidazolylpropynyl-2'-deoxyuridine 5'-monophosphate (IP-dUMP) is a mechanism-based, irreversible inactivator of Escherichia coli thymidylate synthase (TS), which covalently modifies Tyr94 at the active site of the enzyme. The inactivation of TS was time and concentration dependent and did not require the folate cofactor. Due to the rapidity of the inactivation process, accurate kinetic parameters could be determined only in the presence of saturating concentrations (1000K(M)) of the competing substrate, dUMP. Under these conditions, a K(I) of 0.36 +/- 0.09 microM and an inactivation rate constant (k(inact)) of 0.53 +/- 0.15 min(-1) were obtained from Kitz-Wilson plots. Electrospray ionization-mass spectrometry (ESI-MS) determined a 412 amu mass increase of TS after inhibition by IP-dUMP with no mass difference being detected for the TS mutants Tyr94Phe or Cys146Ala, thus indicating the importance of these residues for complex formation. The change in WT-TS mass was consistent with covalent modification by IP-dUMP, which was confirmed by proteolytic digestion of the modified protein followed by ESI-MS. By these means, a 43-residue trypsin peptide (residues 54-96), a 16-residue endoAspN peptide (residues 89-104), and an 8-residue endoAspN/endoLysC peptide (residues 89-96), each containing the IP-dUMP adduct, were observed. MS/MS analysis of the IP-dUMP-endoAspN peptide identified a modified 3-residue daughter ion, YGK (residues 94-96). A mechanistic scheme requiring the participation of Cys146 is proposed for the covalent modification of IP-dUMP by Tyr94, which, unlike an earlier proposal [Kalman, T. I., Nie, Z., and Kamat, A. (2001) Nucleosides Nucleotides Nucleic Acids 20, 869-871], does not require the release of imidazole for the activation of the inhibitor.