Relative free energies of binding to thymidylate synthase of 2- and/or 4-thio and/or 5-fluoro analogues of dUMP

Free energy perturbation calculations have been applied to evaluate the relative free energies of binding of 2′-deoxyuridine-5′-monophosphate (dUMP) and its 2- and/or 4-thio and/or 5-fluoro analogues to the wild-type E. coli thymidylate synthase (ecTS). The results accurately reproduce experimentally measured differences in the free energy of binding of dUMP versus 5-fluoro-dUMP to thymidylate synthase. They indicate that preferred binding of dUMP compared to 5-fluoro-dUMP in the binary complex is equally related to (i) more favorable electrostatic interactions of the dUMP molecule in the enzyme active site, and (ii) its less favorable solvation in the aqueous solution. The relative free energies of binding in the binary complex show moderate and qualitatively indistinguishable discrimination among the studied fluorinated and non-fluorinated 2- and/or 4-thio analogues of dUMP. The binding free energies of monothio analogues of dUMP and 5-fluoro-dUMP correspond quite well with experimentally measured activities of these nucleotides in the thymidylate synthase reaction. On the other hand, the binding free energies of both dithio analogues, 2,4-dithio-dUMP and 2,4-dithio-FdUMP, show lack of such correlation. The latter suggests that very low activities of the dithio analogues of dUMP and 5-fluoro-dUMP may relate more to the covalent reaction of these nucleotides within the ternary complex with TS and 5,10-methylenetetrahydrofolate, than to their pre-covalent binding. We speculate that a lack of substrate activity of 2,4-dithio-dUMP is related to the high aromaticity of its pyrimidine ring that prevents the Michael addition of the active site cysteine thiol to the pyrimidine C6 atom. A stronger affinity of the fluorinated analogues of dUMP to thymidylate synthase, compared to the non-fluorinated congeners, results from the fluorine substituent producing a local strain in the C6 region in the pyrimidine ring, thus sensitizing C6 to the Michael addition of the cysteine thiol.

[1]  Wolfram Saenger,et al.  Principles of Nucleic Acid Structure , 1983 .

[2]  J. Apfeld,et al.  Contributions of orientation and hydrogen bonding to catalysis in Asn229 mutants of thymidylate synthase. , 1998, Journal of molecular biology.

[3]  R. Stroud,et al.  Structure, multiple site binding, and segmental accommodation in thymidylate synthase on binding dUMP and an anti-folate. , 1990 .

[4]  J. Åqvist,et al.  Ligand binding affinities from MD simulations. , 2002, Accounts of chemical research.

[5]  Peter A. Kollman,et al.  INSIGHT INTO THE SPECIFICITY OF THYMIDYLATE SYNTHASE FROM MOLECULAR DYNAMICS AND FREE ENERGY PERTURBATION CALCULATIONS , 1995 .

[6]  C. Heidelberger,et al.  Studies on fluorinated pyrimidines. XIII. Inhibition of thymidylate synthetase. , 1961, The Journal of biological chemistry.

[7]  Henry A. Bent,et al.  An Appraisal of Valence-bond Structures and Hybridization in Compounds of the First-row elements. , 1961 .

[8]  R. Stroud,et al.  Conformational dynamics along an enzymatic reaction pathway: thymidylate synthase, "the movie". , 2003, Biochemistry.

[9]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[10]  N. Xuong,et al.  Crystal structure of Escherichia coli thymidylate synthase containing bound 5-fluoro-2'-deoxyuridylate and 10-propargyl-5,8-dideazafolate. , 1990, Journal of molecular biology.

[11]  J. Dzik,et al.  2-Thio derivatives of dUrd and 5-fluoro-dUrd and their 5'-monophosphates: synthesis, interaction with tumor thymidylate synthase, and in vitro antitumor activity. , 1993, Journal of medicinal chemistry.

[12]  Peter A. Kollman,et al.  FREE ENERGY CALCULATIONS : APPLICATIONS TO CHEMICAL AND BIOCHEMICAL PHENOMENA , 1993 .

[13]  L. Adamowicz,et al.  Crystal structures of 5-fluoro-dUrd and its 2 and/or 4-thio analogues: models of substituted dUMP pyrimidine ring interacting with thymidylate synthase. , 1998, Biochimica et biophysica acta.

[14]  W. L. Jorgensen,et al.  Comparison of simple potential functions for simulating liquid water , 1983 .

[15]  P. Kollman,et al.  A well-behaved electrostatic potential-based method using charge restraints for deriving atomic char , 1993 .

[16]  G. Szekeres,et al.  Methylation of 4-thio-2'-deoxyuridylate by thymidylate synthetase. , 1973, Biochemical and biophysical research communications.

[17]  H. Berendsen,et al.  COMPUTER-SIMULATION OF MOLECULAR-DYNAMICS - METHODOLOGY, APPLICATIONS, AND PERSPECTIVES IN CHEMISTRY , 1990 .

[18]  A. Burgen.  Topics in Molecular Pharmacology , 1982 .

[19]  David E. Smith,et al.  Computer simulations of NaCl association in polarizable water , 1994 .

[20]  D. Santi,et al.  Exclusion of 2'-deoxycytidine 5'-monophosphate by asparagine 229 of thymidylate synthase. , 1993, Biochemistry.

[21]  H. Berendsen,et al.  Molecular dynamics with coupling to an external bath , 1984 .

[22]  J. Åqvist,et al.  Ion-water interaction potentials derived from free energy perturbation simulations , 1990 .

[23]  J. Dzik,et al.  Mechanism of inhibition of mammalian tumor and other thymidylate synthases by N4-hydroxy-dCMP, N4-hydroxy-5-fluoro-dCMP, and related analogues. , 1990, Biochemistry.

[24]  J. Bertino,et al.  Interaction of 2-thio-5-fluoro-dUMP and 4-thio-5-fluoro-dUMP with mammalian normal and tumour and helminthic thymidylate synthases: influence of C(4)-substituents on specificity for enzyme inactivation. , 1993, Biochemical and biophysical research communications.

[25]  Trent Spencer,et al.  Differences in natural ligand and fluoropyrimidine binding to human thymidylate synthase identified by transient-state spectroscopic and continuous variation methods. , 2002, Biochimica et biophysica acta.

[26]  R. Ferone,et al.  Enhanced antitumor activity for the thymidylate synthase inhibitor 1843U89 through decreased host toxicity with oral folic acid. , 1995, Cancer research.

[27]  H. Bent.  Hybridization and bond properties Effects of second-order hybridization on the σ-bonds in thionyl, sulphuryl, phosphorus, phosphoryl and thiophosphoryl halides , 1961 .

[28]  D. Matthews,et al.  Stereochemical mechanism of action for thymidylate synthase based on the X-ray structure of the covalent inhibitory ternary complex with 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate. , 1990, Journal of molecular biology.

[29]  R. Raines,et al.  Kinetics and thermodynamics of the interaction of 5-fluoro-2'-deoxyuridylate with thymidylate synthase. , 1987, Biochemistry.

[30]  R. Stroud,et al.  Pairwise specificity and sequential binding in enzyme catalysis: thymidylate synthase. , 1990, Biochemistry.

[31]  Peter A. Kollman,et al.  Application of the multimolecule and multiconformational RESP methodology to biopolymers: Charge derivation for DNA, RNA, and proteins , 1995, J. Comput. Chem..

[32]  D. Santi,et al.  The catalytic mechanism and structure of thymidylate synthase. , 1995, Annual review of biochemistry.

[33]  C. Shih,et al.  Preclinical Pharmacology Studies and the Clinical Development of a Novel Multitargeted Antifolate, MTA (LY231514) , 1999 .

[34]  C. Bartlett,et al.  AG337, a novel lipophilic thymidylate synthase inhibitor: in vitro and in vivo preclinical studies , 1996, Cancer Chemotherapy and Pharmacology.

[35]  R M Stroud,et al.  Refined structures of substrate-bound and phosphate-bound thymidylate synthase from Lactobacillus casei. , 1993, Journal of molecular biology.

[36]  J. Finer-Moore,et al.  Mechanism-based inhibition of thymidylate synthase by 5-(trifluoromethyl)-2'-deoxyuridine 5'-monophosphate. , 1994, Biochemistry.

[37]  F. Muggia.  Experimental and Clinical Progress in Cancer Chemotherapy , 2011, Cancer Treatment and Research.

[38]  J. Dzik,et al.  Interaction of 5-fluoro-4-thio-2'-deoxyuridine 5'-phosphate with mammalian tumour thymidylate synthase: role of the pyrimidine N(3)-H dissociation. , 1987, Biochemical and biophysical research communications.

[39]  P. Kollman,et al.  A Second Generation Force Field for the Simulation of Proteins, Nucleic Acids, and Organic Molecules , 1995 .

[40]  G. Ciccotti,et al.  Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-Alkanes , 1977 .