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35 results on '"Topalis, Dimitri"'

3. A Herpes Simplex Virus 1 DNA Polymerase Multidrug Resistance Mutation Identified in a Patient With Immunodeficiency and Confirmed by Gene Editing.

Author

Schalkwijk, Hanna Helena, Georgala, Aspasia, Gillemot, Sarah, Temblador, Arturo, Topalis, Dimitri, Wittnebel, Sebastian, Andrei, Graciela, and Snoeck, Robert

Subjects
MULTIDRUG resistance, GENOME editing, DNA polymerases, GAIN-of-function mutations, DNA viruses, SEVERE combined immunodeficiency, STEM cell transplantation
Abstract

Background Herpes simplex virus 1 can cause severe infections in individuals who are immunocompromised. In these patients, emergence of drug resistance mutations causes difficulties in infection management. Methods Seventeen herpes simplex virus 1 isolates were obtained from orofacial/anogenital lesions in a patient with leaky severe combined immunodeficiency over 7 years, before and after stem cell transplantation. Spatial/temporal evolution of drug resistance was characterized genotypically—with Sanger and next-generation sequencing of viral thymidine kinase (TK) and DNA polymerase (DP)—and phenotypically. CRISPR/Cas9 was used to introduce the novel DP Q727R mutation, and dual infection-competition assays were performed to assess viral fitness. Results Isolates had identical genetic backgrounds, suggesting that orofacial/anogenital infections derived from the same virus lineage. Eleven isolates proved heterogeneous TK virus populations by next-generation sequencing, undetectable by Sanger sequencing. Thirteen isolates were acyclovir resistant due to TK mutations, and the Q727R isolate additionally exhibited foscarnet/adefovir resistance. Recombinant Q727R mutant virus showed multidrug resistance and increased fitness under antiviral pressure. Conclusions Long-term follow-up of a patient with severe combined immunodeficiency revealed virus evolution and frequent reactivation of wild-type and TK mutant strains, mostly as heterogeneous populations. The DP Q727R resistance phenotype was confirmed with CRISPR/Cas9, a useful tool to validate novel drug resistance mutations. [ABSTRACT FROM AUTHOR]

Published
2023
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16. Nucleotide binding to human UMP-CMP kinase using fluorescent derivatives -- a screening based on affinity for the UMP-CMP binding site

Author

Topalis, Dimitri, Kumamoto, Hiroki, Amaya Velasco, Maria-Fernanda, Dugué, Laurence, Haouz, Ahmed, Alexandre, Julie Anne C, Gallois-Montbrun, Sarah, Alzari, Pedro Maria, Pochet, Sylvie, Agrofoglio, Luigi André, Deville-Bonne, Dominique, Enzymologie Moléculaire et Fonctionnelle, Université Pierre et Marie Curie - Paris 6 (UPMC), Institut de Chimie Organique et Analytique (ICOA), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Orléans (UO)-Institut de Chimie du CNRS (INC), Biochimie Structurale, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Chimie Organique, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Cristallogenèse et Diffraction des Rayons X (Plate-forme/PF6), Régulation Enzymatique des Activités Cellulaires, Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, [SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], Biological Products, Binding Sites, Cytidine Diphosphate, Protein Structure, Tertiary, Substrate Specificity, carbohydrates (lipids), Kinetics, Spectrometry, Fluorescence, Cytidine Monophosphate, Animals, Humans, ortho-Aminobenzoates, Nucleoside-Phosphate Kinase, Uridine Monophosphate, Fluorescent Dyes
Abstract

Methylanthraniloyl derivatives of ATP and CDP were used in vitro as fluorescent probes for the donor-binding and acceptor-binding sites of human UMP-CMP kinase, a nucleoside salvage pathway kinase. Like all NMP kinases, UMP-CMP kinase binds the phosphodonor, usually ATP, and the NMP at different binding sites. The reaction results from an in-line phosphotransfer from the donor to the acceptor. The probe for the donor site was displaced by the bisubstrate analogs of the Ap5X series (where X = U, dT, A, G), indicating the broad specificity of the acceptor site. Both CMP and dCMP were competitors for the acceptor site probe. To find antimetabolites for antivirus and anticancer therapies, we have developed a method of screening acyclic phosphonate analogs that is based on the affinity of the acceptor-binding site of the human UMP-CMP kinase. Several uracil vinylphosphonate derivatives had affinities for human UMP-CMP kinase similar to those of dUMP and dCMP and better than that of cidofovir, an acyclic nucleoside phosphonate with a broad spectrum of antiviral activities. The uracil derivatives were inhibitors rather than substrates of human UMP-CMP kinase. Also, the 5-halogen-substituted analogs inhibited the human TMP kinase less efficiently. The broad specificity of the enzyme acceptor-binding site is in agreement with a large substrate-binding pocket, as shown by the 2.1 A crystal structure.

Published
2007
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23. Novel Antiviral C5-Substituted Pyrimidine Acyclic Nucleoside Phosphonates Selected as Human Thymidylate Kinase Substrates

Author

Topalis, Dimitri, primary, Pradère, Ugo, additional, Roy, Vincent, additional, Caillat, Christophe, additional, Azzouzi, Ahmed, additional, Broggi, Julie, additional, Snoeck, Robert, additional, Andrei, Graciela, additional, Lin, Jay, additional, Eriksson, Staffan, additional, Alexandre, Julie A. C., additional, El-Amri, Chahrazade, additional, Deville-Bonne, Dominique, additional, Meyer, Philippe, additional, Balzarini, Jan, additional, and Agrofoglio, Luigi A., additional

Published
2010
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29. Alkyne-Azide Click Chemistry Mediated Carbanucleosides Synthesis

Author

Broggi, Julie, primary, Joubert, Nicolas, additional, Aucagne, Vincent, additional, Berteina-Raboin, Sabine, additional, Diez-Gonzalez, S., additional, Nolan, Steve, additional, Topalis, Dimitri, additional, Deville-Bonne, Dominique, additional, Balzarini, Jan, additional, Neyts, Johan, additional, Andrei, Graciela, additional, Snoeck, Robert, additional, and Agrofoglio, Luigi A., additional

Published
2007
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31. Cross-Metathesis Mediated Synthesis of New Acyclic Nucleoside Phosphonates

Author

Roy, Vincent, primary, Kumamoto, Hiroki, additional, Berteina-Raboin, Sabine, additional, Nolan, Steven P., additional, Topalis, Dimitri, additional, Deville-Bonne, Dominique, additional, Balzarini, Jan, additional, Neyts, Johan, additional, Andrei, Gracelia, additional, Snoeck, Robert, additional, and Agrofoglio, Luigi A., additional

Published
2007
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32. Crystal structure of poxvirus thymidylate kinase: An unexpected dimerization has implications for antiviral therapy.

Author

CaiIIat, Christophe, Topalis, Dimitri, Agrofoglio, Luigi A., Pochet, Sylvie, Balzarini, Jan, DeviIle-Bonne, Dominique, and Meyer, Philippe

Subjects
*HERPESVIRUS diseases, *THYMIDINE, *ORTHOPOXVIRUSES, *PHOSPHORYLATION, *IMMUNOSPECIFICITY, *BINDING sites, *MONOMERS
Abstract

Unlike 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. [ABSTRACT FROM AUTHOR]

Published
2008

33. KAY-2-41, a Novel Nucleoside Analogue Inhibitor of Orthopoxviruses In Vitroand In Vivo

Author

Duraffour, Sophie, Drillien, Robert, Haraguchi, Kazuhiro, Balzarini, Jan, Topalis, Dimitri, van den Oord, Joost J., Andrei, Graciela, and Snoeck, Robert

Abstract

ABSTRACTThe availability of adequate treatments for poxvirus infections would be valuable not only for human use but also for veterinary use. In the search for novel antiviral agents, a 1′-methyl-substituted 4′-thiothymidine nucleoside, designated KAY-2-41, emerged as an efficient inhibitor of poxviruses. In vitro, KAY-2-41 was active in the micromolar range against orthopoxviruses (OPVs) and against the parapoxvirus orf. The compound preserved its antiviral potency against OPVs resistant to the reference molecule cidofovir. KAY-2-41 had no noticeable toxicity on confluent monolayers, but a cytostatic effect was seen on growing cells. Genotyping of vaccinia virus (VACV), cowpox virus, and camelpox virus selected for resistance to KAY-2-41 revealed a nucleotide deletion(s) close to the ATP binding site or a nucleotide substitution close to the substrate binding site in the viral thymidine kinase (TK; J2R) gene. These mutations resulted in low levels of resistance to KAY-2-41 ranging from 2.7- to 6.0-fold and cross-resistance to 5-bromo-2′-deoxyuridine (5-BrdU) but not to cidofovir. The antiviral effect of KAY-2-41 relied, at least in part, on activation (phosphorylation) by the viral TK, as shown through enzymatic assays. The compound protected animals from disease and mortality after a lethal challenge with VACV, reduced viral loads in the serum, and abolished virus replication in tissues. In conclusion, KAY-2-41 is a promising nucleoside analogue for the treatment of poxvirus-induced diseases. Our findings warrant the evaluation of additional 1′-carbon-substituted 4′-thiothymidine derivatives as broad-spectrum antiviral agents, since this molecule also showed antiviral potency against herpes simplex virus 1 in earlier studies.

Published
2013
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34. Structural basis for the specificity of thymidylate kinases from human pathogens: implications for nucleotide analogues activation

Author

Meyer, Philippe, Caillat, Christophe, Topalis, Dimitri, Balzarini, Jan, and Deville-Bonne, Dominique

Abstract

Several human pathogens possess nucleoside or nucleotide kinases with large substrate specificity compared to their human counterparts. This phenomenon has been successfully exploited for the specific targeting of prodrugs such as Acyclovir against herpes virus. Combined structural and biochemical studies of these enzymes can thus provide essential information for the rational design of specific antimicrobial agents. Here we studied the structural basis for the specificity of a thymidylate kinase from the poxvirus family. Poxvirus thymidylate kinase has unusual substrate specificity and can accept bulky analogues such as 5-bromo-vinyl-dUMP (BVdUMP). The 2 Å crystal structure of the thymidylate kinase bound to this compound now gives the structural basis for its specific molecular recognition.

Published
2009
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35. Novel antiviral C5-substituted pyrimidine acyclic nucleoside phosphonates selected as human thymidylate kinase substrates.

Author

Topalis D, Pradère U, Roy V, Caillat C, Azzouzi A, Broggi J, Snoeck R, Andrei G, Lin J, Eriksson S, Alexandre JA, El-Amri C, Deville-Bonne D, Meyer P, Balzarini J, and Agrofoglio LA

Subjects
Antiviral Agents chemistry, Antiviral Agents pharmacology, Catalytic Domain, Cells, Cultured, Crystallography, X-Ray, Herpesviridae drug effects, Humans, Ligands, Models, Molecular, Molecular Structure, Nucleoside-Phosphate Kinase antagonists & inhibitors, Organophosphonates chemistry, Organophosphonates pharmacology, Prodrugs chemistry, Prodrugs pharmacology, Pyrimidine Nucleosides chemistry, Pyrimidine Nucleosides pharmacology, Stereoisomerism, Structure-Activity Relationship, Substrate Specificity, Thymidine chemical synthesis, Thymidine chemistry, Thymidine pharmacology, Thymidine Kinase antagonists & inhibitors, Antiviral Agents chemical synthesis, Nucleoside-Phosphate Kinase metabolism, Organophosphonates chemical synthesis, Prodrugs chemical synthesis, Pyrimidine Nucleosides chemical synthesis, Thymidine analogs & derivatives
Abstract

Acyclic nucleoside phosphonates (ANPs) are at the cornerstone of DNA virus and retrovirus therapies. They reach their target, the viral DNA polymerase, after two phosphorylation steps catalyzed by cellular kinases. New pyrimidine ANPs have been synthesized with unsaturated acyclic side chains (prop-2-enyl-, but-2-enyl-, pent-2-enyl-) and different substituents at the C5 position of the uracil nucleobase. Several derivatives in the but-2-enyl- series 9d and 9e, with (E) but not with (Z) configuration, were efficient substrates for human thymidine monophosphate (TMP) kinase, but not for uridine monophosphate-cytosine monophosphate (UMP-CMP) kinase, which is in contrast to cidofovir. Human TMP kinase was successfully crystallized in a complex with phosphorylated (E)-thymidine-but-2-enyl phosphonate 9e and ADP. The bis-pivaloyloxymethyl (POM) esters of (E)-9d and (E)-9e were synthesized and shown to exert activity against herpes virus in vitro (IC(50) = 3 μM) and against varicella zoster virus in vitro (IC(50) = 0.19 μM), in contrast to the corresponding inactive (Z) derivatives. Thus, their antiviral activity correlates with their ability to act as thymidylate kinase substrates.

Published
2011
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