title: Enzymatic synthesis of acyclic nucleoside thiophosphonate diphosphates: effect of the α-phosphorus configuration on HIV-1 RT activity.
abstract: The acyclic nucleosides thiophosphonates (9-[2-(thiophosphonomethoxy)ethyl]adenine (S-PMEA) and (R)-9-[2-(thiophosphonomethoxy)propyl]adenine (S-PMPA), exhibit antiviral activity against HIV-1, -2 and HBV. Their diphosphate forms S-PMEApp and S-PMPApp, synthesized as stereoisomeric mixture, are potent inhibitors of wild-type (WT) HIV-1 RT. Understanding HIV-1 RT stereoselectivity, however, awaits resolution of the diphosphate forms into defined stereoisomers. To this aim, thiophosphonate monophosphates S-PMEAp and S-PMPAp were synthesized and used in a stereocontrolled enzyme-catalyzed phosphoryl transfer reaction involving either nucleoside diphosphate kinase (NDPK) or creatine kinase (CK) to obtain thiophosphonate diphosphates as separated isomers. We then quantified substrate preference of recombinant WT HIV-1 RT toward pure stereoisomers using in vitro steady-state kinetic analyses. The crystal structure of a complex between Dictyostelium NDPK and S-PMPApp at 2.32Å allowed to determine the absolute configuration at the α-phosphorus atom in relation to the stereo-preference of studied enzymes. The RP isomer of S-PMPApp and S-PMEApp are the preferred substrate over SP for both NDPK and HIV-1 RT.
authors.affiliation: Laboratoire d'Architecture et Fonction des Macromolécules Biologiques, UMR CNRS 7257, Team "Viral Replicases: Structure, Mechanism and Drug-Design", Université Aix-Marseille, Parc scientifique de Luminy, 163 av. de Luminy, 13288 Marseille Cedex 9, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Priet
authors.affiliation: Laboratoire d'Architecture et Fonction des Macromolécules Biologiques, UMR CNRS 7257, Team "Antiviral Medicinal Chemistry", Université Aix-Marseille, Parc scientifique de Luminy, 163 av. de Luminy, 13288 Marseille Cedex 9, France.
authors.forename: Loic
authors.initials: L
authors.lastname: Roux
authors.affiliation: Laboratoire d'Architecture et Fonction des Macromolécules Biologiques, UMR CNRS 7257, Team "Antiviral Medicinal Chemistry", Université Aix-Marseille, Parc scientifique de Luminy, 163 av. de Luminy, 13288 Marseille Cedex 9, France.
authors.forename: Magali
authors.initials: M
authors.lastname: Saez-Ayala
authors.affiliation: Laboratoire d'Architecture et Fonction des Macromolécules Biologiques, UMR CNRS 7257, Team "Viral Replicases: Structure, Mechanism and Drug-Design", Université Aix-Marseille, Parc scientifique de Luminy, 163 av. de Luminy, 13288 Marseille Cedex 9, France.
authors.forename: François
authors.initials: F
authors.lastname: Ferron
authors.affiliation: Laboratoire d'Architecture et Fonction des Macromolécules Biologiques, UMR CNRS 7257, Team "Viral Replicases: Structure, Mechanism and Drug-Design", Université Aix-Marseille, Parc scientifique de Luminy, 163 av. de Luminy, 13288 Marseille Cedex 9, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Canard
authors.affiliation: Laboratoire d'Architecture et Fonction des Macromolécules Biologiques, UMR CNRS 7257, Team "Antiviral Medicinal Chemistry", Université Aix-Marseille, Parc scientifique de Luminy, 163 av. de Luminy, 13288 Marseille Cedex 9, France. Electronic address: Karine.Alvarez@afmb.univ-mrs.fr.
authors.forename: Karine
authors.initials: K
authors.lastname: Alvarez
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D019380:Anti-HIV Agents / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D003402:Creatine Kinase / Q000378:metabolism; D003460:Crystallization; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology; D015498:HIV-2 / Q000187:drug effects; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008968:Molecular Conformation; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism; D010758:Phosphorus / Q000737:chemistry; D013237:Stereoisomerism
chemical_list: C120290:9-(2-(phosphonomethoxy)ethyl)adenine diphosphate; C000604246:9-(2-thiophosphonomethoxy)propyl)adenine; D019380:Anti-HIV Agents; D010758:Phosphorus; D003402:Creatine Kinase; D009701:Nucleoside-Diphosphate Kinase; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D000225:Adenine
keywords: Acyclic nucleosides thiophosphonates; Creatine kinase; HIV-1 reverse transcriptase; In vitro drug susceptibility; Nucleotide diphosphate kinase; Phosphorylation