ABSTRACT
Selective and potent inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa) have the potential to increase endogenous and therapeutic fibrinolysis and to behave like profibrinolytic agents without the risk of major hemorrhage, since they do not interfere either with platelet activation or with coagulation during blood hemostasis. Therefore, TAFIa inhibitors could be used in at-risk patients for the treatment, prevention, and secondary prevention of stroke, venous thrombosis, and pulmonary embolisms. In this paper, we describe the design, the structure-activity relationship (SAR), and the synthesis of novel, potent, and selective phosphinanes and azaphosphinanes as TAFIa inhibitors. Several highly active azaphosphinanes display attractive properties suitable for further in vivo efficacy studies in thrombosis models.
Subject(s)
Aza Compounds/pharmacology , Carboxypeptidase B2/antagonists & inhibitors , Cyclic P-Oxides/pharmacology , Fibrinolytic Agents/pharmacology , Phosphinic Acids/pharmacology , Protease Inhibitors/pharmacology , Animals , Aza Compounds/chemical synthesis , Aza Compounds/metabolism , Carboxypeptidase B2/metabolism , Catalytic Domain , Cyclic P-Oxides/chemical synthesis , Cyclic P-Oxides/metabolism , Fibrinolysis/drug effects , Fibrinolytic Agents/chemical synthesis , Fibrinolytic Agents/metabolism , Humans , Male , Molecular Docking Simulation , Molecular Structure , Phosphinic Acids/chemical synthesis , Phosphinic Acids/metabolism , Protease Inhibitors/chemical synthesis , Protease Inhibitors/metabolism , Protein Binding , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
PSI-352938 is a novel cyclic phosphate prodrug of ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methylguanosine-5'-monophosphate with potent anti-HCV activity. In order to inhibit the NS5B RNA-dependent RNA polymerase, PSI-352938 must be metabolized to the active triphosphate form, PSI-352666. During in vitro incubations with PSI-352938, significantly larger amounts of PSI-352666 were formed in primary hepatocytes than in clone A hepatitis C virus (HCV) replicon cells. Metabolism and biochemical assays were performed to define the molecular mechanism of PSI-352938 activation. The first step, removal of the isopropyl group on the 3',5'-cyclic phosphate moiety, was found to be cytochrome P450 (CYP) 3A4 dependent, with other CYP isoforms unable to catalyze the reaction. The second step, opening of the cyclic phosphate ring, was catalyzed by phosphodiesterases (PDEs) 2A1, 5A, 9A, and 11A4, all known to be expressed in the liver. The role of these enzymes in the activation of PSI-352938 was confirmed in primary human hepatocytes, where prodrug activation was reduced by inhibitors of CYP3A4 and PDEs. The third step, removal of the O(6)-ethyl group on the nucleobase, was shown to be catalyzed by adenosine deaminase-like protein 1. The resulting monophosphate was consecutively phosphorylated to the diphosphate and to the triphosphate PSI-352666 by guanylate kinase 1 and nucleoside diphosphate kinase, respectively. In addition, formation of nucleoside metabolites was observed in primary hepatocytes, and ecto-5'-nucleotidase was able to dephosphorylate the monophosphate metabolites. Since CYP3A4 is highly expressed in the liver, the CYP3A4-dependent metabolism of PSI-352938 makes it an effective liver-targeted prodrug, in part accounting for the potent antiviral activity observed clinically.
Subject(s)
Antiviral Agents/metabolism , Cyclic P-Oxides/metabolism , Hepacivirus/drug effects , Nucleosides/metabolism , Cells, Cultured , Cytochrome P-450 CYP3A/metabolism , Guanylate Kinases/metabolism , Hepatocytes/metabolism , Humans , Nucleoside-Diphosphate Kinase/metabolism , Phosphoric Diester Hydrolases/metabolismABSTRACT
We previously used in vitro selection to identify Mg(2+)-dependent deoxyribozymes that mediate the ligation reaction of an RNA 5'-hydroxyl group with a 2',3'-cyclic phosphate. In these efforts, all of the deoxyribozymes were identified via a common in vitro selection strategy, and all of the newly formed RNA linkages were non-native 2'-5' phosphodiester bonds rather than native 3'-5' linkages. Here we performed several new selections in which the relative arrangements of RNA and DNA were different as compared with the earlier studies. In all cases, we again find deoxyribozymes that create only 2'-5' linkages. This includes deoxyribozymes with an arrangement that favors 3'-5' linkages for a different chemical reaction, that of a 2',3'-diol plus 5'-triphosphate. These data indicate a strong and context-independent chemical preference for creating 2'-5' RNA linkages upon opening of a 2',3'-cyclic phosphate with a 5'-hydroxyl group. Preliminary assays show that some of the newly identified deoxyribozymes have promise for ligating RNA in a sequence-general fashion. Because 2',3'-cyclic phosphates are the products of uncatalyzed RNA backbone cleavage, their ligation reactions may be of direct relevance to the RNA World hypothesis.
Subject(s)
Cyclic P-Oxides/metabolism , DNA, Catalytic/metabolism , Directed Molecular Evolution , RNA/chemistry , RNA/metabolism , Base Sequence , Catalysis , Cloning, Molecular , DNA, Catalytic/genetics , Kinetics , Molecular Sequence Data , Molecular Structure , RNA/genetics , RNA Ligase (ATP)/metabolism , Selection, GeneticABSTRACT
A new species of lysophosphatidic acid was isolated from myxoamoebae of a true slime mold, Physarum polycephalum, and structural studies were performed. The purified substance was subjected to nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR), fast atom bombardment mass spectroscopy (FAB/MS), alkaline hydrolysis and tandem mass spectroscopy (MS/MS), and the results suggested this substance to be lysophosphatidic acid composed of a cyclic phosphate and cis-11,12-methylene octadecanoic acid. The effects of the LPA on DNA polymerases were studied and compared with the effects of PHYLPA, which had been isolated as a specific inhibitor of eukaryotic DNA polymerase alpha (6). It showed a specific inhibitory activity on eukaryotic DNA polymerase alpha, but no activity on the repair-type, or mitochondrial DNA polymerases.
