27 research outputs found
Tenofovir Activating Kinases May Impact the Outcome of HIV Treatment and Prevention
No full textstatus: Publishe
Thymidine kinase and protein kinase in drug-resistant herpesviruses: heads of a Lernaean hydra
No full textHerpesviruses thymidine kinase (TK) and protein kinase (PK) allow the activation of nucleoside analogues used in anti-herpesvirus treatments. Mutations emerging in these two genes often lead to emergence of drug-resistant strains responsible for life-threatening diseases in immunocompromised populations. In this review, we analyze the binding of different nucleoside analogues to the TK active site of the three α-herpesviruses [Herpes Simplex Virus 1 and 2 (HSV-1 and HSV-2) and Varicella-Zoster Virus (VZV)] and present the impact of known mutations on the structure of the viral TKs. Furthermore, models of β-herpesviruses [Human cytomegalovirus (HCMV) and human herpesvirus-6 (HHV-6)] PKs allow to link amino acid changes with resistance to ganciclovir and/or maribavir, an investigational chemotherapeutic used in patients with multidrug-resistant HCMV. Finally, we set the basis for the understanding of drug-resistance in γ-herpesviruses [Epstein-Barr virus (EBV) and Kaposi's sarcoma associated herpesvirus (KSHV)] TK and PK through the use of animal surrogate models.status: Publishe
Mechanisms of substrate selectivity for Bacillus anthracis thymidylate kinase
No full textBacillus anthracis is well known in connection with biological warfare. The search for new drug targets and antibiotics is highly motivated because of upcoming multiresistant strains. Thymidylate kinase is an ideal target since this enzyme is at the junction of the de novo and salvage synthesis of dTTP, an essential precursor for DNA synthesis. Here the expression and characterization of thymidylate kinase from B. anthracis (Ba-TMPK) is presented. The enzyme phosphorylated deoxythymidine-5'-monophosphate (dTMP) efficiently with K (m) and V (max) values of 33 microM and 48 micromol mg(-1) min(-1), respectively. The efficiency of deoxyuridine-5'-monophosphate phosphorylation was approximately 10% of that of dTMP. Several dTMP analogs were tested, and D-FMAUMP (2'-fluoroarabinosyl-5-methyldeoxyuridine-5'-monophosphate) was selectively phosphorylated with an efficiency of 172% of that of D-dTMP, but L-FMAUMP was a poor substrate as were 5-fluorodeoxyuridine-5'-monophosphate (5FdUMP) and 2',3'-dideoxy-2',3'-didehydrothymidine-5'-monophosphate (d4TMP). No activity could be detected with 3'-azidothymidine-5'-monophosphate (AZTMP). The corresponding nucleosides known as efficient anticancer and antiviral compounds were also tested, and d-FMAU was a strong inhibitor with an IC(50) value of 10 microM, while other nucleosides--L-FMAU, dThd, 5-FdUrd, d4T, and AZT, and 2'-arabinosylthymidine--were poor inhibitors. A structure model was built for Ba-TMPK based on the Staphylococcus aureus TMPK structure. Docking with various substrates suggested mechanisms explaining the differences in substrate selectivity of the human and the bacterial TMPKs. These results may serve as a start point for development of new antibacterial agents.sponsorship: This work was supported by grants from the Swedish Research Council for the Environment, Agricultural Sciences and Spatial Planning (FORMAS), and the Swedish Research Council. We thank Dominique Devill-Bonne for providing us with the human TMPK plasmid. (Swedish Research Council for the Environment, Agricultural Sciences and Spatial Planning (FORMAS), Swedish Research Council)status: Publishe
Substrate specificity of vaccinia virus thymidylate kinase.
No full textInternational audienceAnti-poxvirus therapies are currently limited to cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine], but drug-resistant strains have already been characterized. In the aim of finding a new target, the thymidylate (TMP) kinase from vaccinia virus, the prototype of Orthopoxvirus, has been overexpressed in Escherichia coli after cloning the gene (A48R). Specific inhibitors and alternative substrates of pox TMP kinase should contribute to virus replication inhibition. Biochemical characterization of the enzyme revealed distinct catalytic features when compared to its human counterpart. Sharing 42% identity with human TMP kinase, the vaccinia virus enzyme was assumed to adopt the common fold of nucleoside monophosphate kinases. The enzyme was purified to homogeneity and behaves as a homodimer, like all known TMP kinases. Initial velocity studies showed that the Km for ATP-Mg2+ and dTMP were 0.15 mm and 20 microM, respectively. Vaccinia virus TMP kinase was found to phosphorylate dTMP, dUMP and also dGMP from any purine and pyrimidine nucleoside triphosphate. 5-Halogenated dUMP such as 5-iodo-2'-deoxyuridine 5'-monophosphate (5I-dUMP) and 5-bromo-2'-deoxyuridine 5'-monophosphate (5Br-dUMP) were also efficient alternative substrates. Using thymidine-5'-(4-N'-methylanthraniloyl-aminobutyl)phosphoramidate as a fluorescent probe of the dTMP binding site, we detected an ADP-induced conformational change enhancing the binding affinity of dTMP and analogues. Several thymidine and dTMP derivatives were found to bind the enzyme with micromolar affinities. The present study provides the basis for the design of specific inhibitors or substrates for poxvirus TMP kinase
Heterogeneity and evolution of thymidine kinase and DNA polymerase mutants of herpes simplex virus type 1: Implications for antiviral therapy
No full textBackground. Infections caused by acyclovir-resistant isolates of herpes simplex virus (HSV) after hematopoietic stem cell transplantation (HSCT) are an emerging concern. An understanding of the evolutionary aspects of HSV infection is crucial to the design of effective therapeutic and control strategies.Methods. Eight sequential HSV-1 isolates were recovered from an HSCT patient who suffered from recurrent herpetic gingivostomatitis and was treated alternatively with acyclovir, ganciclovir, and foscavir. The diverse spectra and temporal changes of HSV drug resistance were determined phenotypically (drug-resistance profiling) and genotypically (sequencing of the viral thymidine kinase and DNA polymerase genes).Results. Analysis of 60 clones recovered from the different isolates demonstrated that most of these isolates were heterogeneous mixtures of variants, indicating the simultaneous infection with different drug-resistant viruses. The phenotype/genotype of several clones associated with resistance to acyclovir and/or foscavir were identified. Two novel mutations (E798K and I922T) in the viral DNA polymerase could be linked to drug resistance.Conclusions. The heterogeneity within the viral populations and the temporal changes of drug-resistant viruses found in this HSCT recipient were remarkable, showing a rapid evolution of HSV-1. Drug-resistance surveillance is highly recommended among immunocompromised patients to manage the clinical syndrome and to avoid the emergence of multidrug-resistant isolates. © 2013 The Author.SCOPUS: ar.jinfo:eu-repo/semantics/publishe
Enantioselectivity of human AMP, dTMP and UMP-CMP kinases
No full textInternational audiencel-Nucleoside analogues such as lamivudine are active for treating viral infections. Like d-nucleosides, the biological activity of the l-enantiomers requires their stepwise phosphorylation by cellular or viral kinases to give the triphosphate. The enantioselectivity of NMP kinases has not been thoroughly studied, unlike that of deoxyribonucleoside kinases. We have therefore investigated the capacity of l-enantiomers of some natural (d)NMP to act as substrates for the recombinant forms of human uridylate-cytidylate kinase, thymidylate kinase and adenylate kinases 1 and 2. Both cytosolic and mitochondrial adenylate kinases were strictly enantioselective, as they phosphorylated only d-(d)AMP. l-dTMP was a substrate for thymidylate kinase, but with an efficiency 150-fold less than d-dTMP. Both l-dUMP and l-(d)CMP were phosphorylated by UMP-CMP kinase although much less efficiently than their natural counterparts. The stereopreference was conserved with the 2'-azido derivatives of dUMP and dUMP while, unexpectedly, the 2'-azido-d-dCMP was a 4-fold better substrate for UMP-CMP kinase than was CMP. Docking simulations showed that the small differences in the binding of d-(d)NMP to their respective kinases could account for the differences in interactions of the l-isomers with the enzymes. This in vitro information was then used to develop the in vivo activation pathway for l-dT
Resistance to the nucleotide analogue cidofovir in HPV(+) cells: a multifactorial process involving UMP/CMP kinase 1
No full textInternational audienceHuman papillomavirus (HPV) is responsible for cervical cancer, and its role in head and neck carcinoma has been reported. No drug is approved for the treatment of HPV-related diseases but cidofovir (CDV) exhibits selective antiproliferative activity.In this study, we analyzed the effects of CDV-resistance (CDVR) in two HPV(+) (SiHaCDV and HeLaCDV) and one HPV(−) (HaCaTCDV) tumor cell lines. Quantification of CDV metabolites and analysis of the sensitivity profile to chemotherapeutics was performed. Transporters expression related to multidrug-resistance (MRP2, P-gp, BCRP) was also investigated.Alterations of CDV metabolism in SiHaCDV and HeLaCDV, but not in HaCaTCDV, emerged via impairment of UMP/CMPK1 activity. Mutations (P64T and R134M) as well as down-regulation of UMP/CMPK1 expression were observed in SiHaCDV and HeLaCDV, respectively. Altered transporters expression in SiHaCDV and/or HeLaCDV, but not in HaCaTCDV, was also noted.Taken together, these results indicate that CDVR in HPV(+) tumor cells is a multifactorial process
Crystal structure of poxvirus thymidylate kinase: an unexpected dimerization has implications for antiviral therapy.
No full textInternational audienceUnlike most DNA viruses, poxviruses replicate in the cytoplasm of host cells. They encode enzymes needed for genome replication and transcription, including their own thymidine and thymidylate kinases. Some herpes viruses encode only 1 enzyme catalyzing both reactions, a peculiarity used for prodrug activation to obtain maximum specificity. We have solved the crystal structures of vaccinia virus thymidylate kinase bound to TDP or brivudin monophosphate. Although the viral and human enzymes have similar sequences (42% identity), they differ in their homodimeric association and active-site geometry. The vaccinia TMP kinase dimer arrangement is orthogonal and not antiparallel as in human enzyme. This different monomer orientation is related to the presence of a canal connecting the edge of the dimer interface to the TMP base binding pocket. Consequently, the pox enzyme accommodates nucleotides with bulkier bases, like brivudin monophosphate and dGMP; these are efficiently phosphorylated and stabilize the enzyme. The brivudin monophosphate-bound structure explains the structural basis for this specificity, opening the way to the rational development of specific antipox agents that may also be suitable for poxvirus TMP kinase gene-based chemotherapy of cancer
Microwave-assisted silylation-amination of uracil acyclonucleosides to 4-alkylamino-2(1H)-pyrimidinone analogues
No full textThe synthesis of acyclic N(4)-substituted cytosine nucleoside phosphonates using a microwave-assisted N(4)-silylation-amination and olefin cross-metathesis as key assembly steps is described. The microwave-assisted silylation-arnination is compared to a conventional heating protocol. While yields and purities of the coupling products were comparable under both conditions, microwave heating allows a significant acceleration of the reaction (from 48 h to 5 h)
ChemInform Abstract: Microwave‐Assisted Silylation‐Amination of Uracil Acyclonucleosides to 4‐Alkylamino‐2(1H)‐pyrimidinone Analogues.
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