ABSTRACT
Nucleoside phosphonates have been designed as stable 5'-mononucleotide mimics and are nowadays considered a potent class of antiviral agents. Within cells, they must be metabolised to the corresponding diphosphate to exert their biological activity. In this process, the first phosphorylation step, catalysed by nucleoside monophosphate kinases (NMP kinases), has been proposed as a bottleneck. Herein, we report the synthesis of a series of ribonucleoside phosphonate derivatives isosteric to 5'-mononucleotides, with different degrees of flexibility within the 5',6'-C-C bond, as well as different polarities, through the introduction of hydroxy groups. The influence of these modifications on the capacity of the compounds to act as substrates for appropriate human NMP kinases, involved in nucleic acids metabolism, has been investigated. Low flexibility, as well as an absence of hydroxy groups within the ribose-phosphorus architecture, is critical for efficient phosphotransfer. Among the series of pyrimidine analogues, one derivative was shown to be phosphorylated by human UMP-CMP kinase, with rates similar to those of dUMP and even better than dCMP.
Subject(s)
Nucleoside-Phosphate Kinase/metabolism , Organophosphonates/chemistry , Organophosphonates/pharmacology , Ribonucleosides/chemistry , Ribonucleosides/pharmacology , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Humans , Models, Molecular , Organophosphonates/chemical synthesis , Protein Binding , Ribonucleosides/chemical synthesis , Substrate SpecificityABSTRACT
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.
Subject(s)
Antiviral Agents/chemical synthesis , Nucleoside-Phosphate Kinase/metabolism , Organophosphonates/chemical synthesis , Prodrugs/chemical synthesis , Pyrimidine Nucleosides/chemical synthesis , Thymidine/analogs & derivatives , 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 & inhibitorsABSTRACT
Human UMP-CMP kinase is involved in the phosphorylation of nucleic acid precursors and also in the activation of antiviral analogues including cidofovir, an acyclic phosphonate compound that mimicks dCMP and shows a broad antiviral spectrum. The binding of ligands to the enzyme was here investigated using a fluorescent probe and a competitive titration assay. At the acceptor site, the enzyme was found to accommodate any base, purine and pyrimidine, including thymidine. A method for screening analogues based on their affinity for the UMP binding site was developed. The affinities of uracil vinylphosphonate derivatives modified in the 5 position were found similar to (d)UMP and (d)CMP and improved when compared to cidofovir.
Subject(s)
Nucleoside-Phosphate Kinase/chemistry , Pyrimidine Nucleotides/chemistry , Pyrimidine Nucleotides/isolation & purification , Adenosine Diphosphate/analogs & derivatives , Adenosine Diphosphate/chemistry , Binding Sites , Fluorescent Dyes/chemistry , Humans , Organophosphonates/chemistry , Phosphorylation , Vinyl Compounds/chemistryABSTRACT
L-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.
