title: Structures of human thymidylate kinase in complex with prodrugs: implications for the structure-based design of novel compounds.
abstract: Nucleoside analogue prodrugs are dependent on efficient intracellular stepwise phosphorylation to their triphosphate form to become therapeutically active. In many cases it is this activation pathway that largely determines the efficacy of the drug. To gain further understanding of the determinants for efficient conversion by the enzyme thymidylate kinase (TMPK) of clinically important thymidine monophosphate analogues to the corresponding diphosphates, we solved the crystal structures of the enzyme, with either ADP or the ATP analogue AppNHp at the phosphoryl donor site, in complex with TMP, AZTMP (previous work), NH2TMP, d4TMP, ddTMP, and FLTMP (this work) at the phosphoryl acceptor site. In conjunction with steady-state kinetic data, our structures shed light on the effect of 3'-substitutions in the nucleoside monophosphate (NMP) sugar moiety on the catalytic rate. We observe a direct correlation between the rate of phosphorylation of an NMP and its ability to induce a closing of the enzyme's phosphate-binding loop (P-loop). Our results show the drastic effects that slight modifications of the substrates exert on the enzyme's conformation and, hence, activity and suggest the type of substitutions that are compatible with efficient phosphorylation by TMPK.
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
authors.forename: Nils
authors.initials: N
authors.lastname: Ostermann
authors.forename: Dario
authors.initials: D
authors.lastname: Segura-Peña
authors.forename: Chris
authors.initials: C
authors.lastname: Meier
authors.forename: Thomas
authors.initials: T
authors.lastname: Veit
authors.forename: Christian
authors.initials: C
authors.lastname: Monnerjahn
authors.forename: Manfred
authors.initials: M
authors.lastname: Konrad
authors.forename: Arnon
authors.initials: A
authors.lastname: Lavie
mesh_terms: D019380:Anti-HIV Agents / Q000138:chemical synthesis / Q000737:chemistry; D018360:Crystallography, X-Ray; D054306:Dideoxynucleotides; D015195:Drug Design; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry*; D011355:Prodrugs / Q000138:chemical synthesis / Q000737:chemistry*; D011487:Protein Conformation; D018119:Stavudine / Q000031:analogs & derivatives* / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013936:Thymidine / Q000031:analogs & derivatives* / Q000737:chemistry; D013938:Thymidine Monophosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D013942:Thymine Nucleotides
chemical_list: C113124:2',3'-dideoxy-2',3'-didehydrothymidine monophosphate; D019380:Anti-HIV Agents; C415254:Cyclosaligenyl-2',3'-didehydro-2',3'-dideoxythymidine monophosphate; D054306:Dideoxynucleotides; D046911:Macromolecular Substances; D011355:Prodrugs; D013942:Thymine Nucleotides; D013938:Thymidine Monophosphate; C080992:3'-amino-3'-deoxythymidine 5'-monophosphate; D018119:Stavudine; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase; D013936:Thymidine