Tony Whitaker

Ribonucleoside Analogue That Blocks Replication of Bovine Viral Diarrhea and Hepatitis C Viruses in Culture

ABSTRACT A base-modified nucleoside analogue, β-d-N4-hydroxycytidine (NHC), was found to have antipestivirus and antihepacivirus activities. This compound inhibited the production of cytopathic bovine viral diarrhea virus (BVDV) RNA in a dose-dependant manner with a 90% effective concentration (EC90) of 5.4 μM, an observation that was confirmed by virus yield assays (EC90 = 2 μM). When tested for hepatitis C virus (HCV) replicon RNA reduction in Huh7 cells, NHC had an EC90 of 5 μM on day 4. The HCV RNA reduction was incubation time and nucleoside concentration dependent. The in vitro antiviral effect of NHC was additive with recombinant alpha interferon-2a and could be prevented by the addition of exogenous cytidine and uridine but not of other natural ribo- or 2′-deoxynucleosides. When HCV RNA replicon cells were cultured in the presence of increasing concentrations of NHC (up to 40 μM) for up to 45 cell passages, no resistant replicon was selected. Similarly, resistant BVDV could not be selected after 20 passages. NHC was phosphorylated to the triphosphate form in Huh7 cells, but in cell-free HCV NS5B assays, synthetic NHC-triphosphate (NHC-TP) did not inhibit the polymerization reaction. Instead, NHC-TP appeared to serve as a weak alternative substrate for the viral polymerase, thereby changing the mobility of the product in polyacrylamide electrophoresis gels. We speculate that incorporated nucleoside analogues with the capacity of changing the thermodynamics of regulatory secondary structures (with or without introducing mutations) may represent an important class of new antiviral agents for the treatment of RNA virus infections, especially HCV.

Mechanism of Activation of β-d-2′-Deoxy-2′-Fluoro-2′-C-Methylcytidine and Inhibition of Hepatitis C Virus NS5B RNA Polymerase

ABSTRACT β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) is a potent specific inhibitor of hepatitis C virus (HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be phosphorylated to the 5′-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B were investigated. The phosphorylation of PSI-6130 by recombinant human 2′-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK, with a Km of 81 μM and a kcat of 0.007 s−1, but was not a substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphosphate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphosphate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (Ki) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 μM. Similar results were obtained with 2′-C-methyladenosine triphosphate (Ki = 1.5 μM) and 2′-C-methylcytidine triphosphate (Ki = 1.6 μM). NS5B with the S282T mutation, which is known to confer resistance to 2′-C-methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination.

The Mechanism of Action of β-d-2′-Deoxy-2′-Fluoro-2′-C-Methylcytidine Involves a Second Metabolic Pathway Leading to β-d-2′-Deoxy-2′-Fluoro-2′-C-Methyluridine 5′-Triphosphate, a Potent Inhibitor of the Hepatitis C Virus RNA-Dependent RNA Polymerase

ABSTRACT β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) RNA replication in an HCV replicon assay. The 5′-triphosphate of PSI-6130 is a competitive inhibitor of the HCV RNA-dependent RNA polymerase (RdRp) and acts as a nonobligate chain terminator. Recently, it has been shown that the metabolism of PSI-6130 also results in the formation of the 5′-triphosphate of the uridine congener, β-d-2′-deoxy-2′-fluoro-2′-C-methyluridine (PSI-6206; RO2433). Here we show that the formation of the 5′-triphosphate of RO2433 (RO2433-TP) requires the deamination of PSI-6130 monophosphate and that RO2433 monophosphate is subsequently phosphorylated to the corresponding di- and triphosphates by cellular UMP-CMP kinase and nucleoside diphosphate kinase, respectively. RO2433-TP is a potent inhibitor of the HCV RdRp; however, both enzymatic and cell-based assays show that PSI-6130 triphosphate is a more potent inhibitor of the HCV RdRp than RO2433-TP.

Inhibition of the Subgenomic Hepatitis C Virus Replicon in Huh-7 Cells by 2′-Deoxy-2′-Fluorocytidine

ABSTRACT 2′-Deoxy-2′-fluorocytidine (FdC) is a potent inhibitor of the hepatitis C virus RNA replicon in culture, and FdC-5′-triphosphate is an effective inhibitor of the NS5B polymerase. Dynamic profiling of cell growth in an antiviral assay showed that FdC caused cytostasis due to an S-phase arrest. These observations demonstrate that FdC treatment is affecting both a viral target and a cellular target.

Asymmetric Binding to NS5A by Daclatasvir (BMS-790052) and Analogs Suggests Two Novel Modes of HCV Inhibition

Symmetric, dimeric daclatasvir (BMS-790052) is the clinical lead for a class of picomolar inhibitors of HCV replication. While specific, resistance-bearing mutations at positions 31 and 93 of domain I strongly suggest the viral NS5A as target, structural mechanism(s) for the drugs’ activities and resistance remains unclear. Several previous models suggested symmetric binding modes relative to the homodimeric target; however, none can fully explain SAR details for this class. We present semiautomated workflows to model potential receptor conformations for docking. Surprisingly, ranking docked hits with our library-derived 3D-pharmacophore revealed two distinct asymmetric binding modes, at a conserved poly-proline region between 31 and 93, consistent with SAR. Interfering with protein–protein interactions at this membrane interface can explain potent inhibition of replication–complex formation, resistance, effects on lipid droplet distribution, and virion release. These detailed interaction models and proposed mechanisms of action will allow structure-based design of new NS5A directed compounds with higher barriers to HCV resistance.

Synthesis and biological evaluation of new potent and selective HCV NS5A inhibitors.

NS5A inhibitors are a new class of direct-acting antiviral agents which display very potent anti-HCV activity in vitro and in humans. Rationally designed modifications to the central biphenyl linkage of a known NS5A series led to selection of several compounds that were synthesized and evaluated in a HCV genotype 1b replicon. The straight triphenyl linked compound 11a showed similar anti-HCV activity to the clinical compound BMS-790052 and a superior cytotoxicity profile in three different cell lines, with an EC(50) value of 26 pM and a therapeutic index of over four million in an HCV replicon assay. This triphenyl analog warrants further preclinical evaluation as an anti-HCV agent.

Synthesis and evaluation of non-dimeric HCV NS5A inhibitors

Based on the symmetrical bidentate structure of the NS5A inhibitor BMS-790052, a series of new monodentate molecules were designed. The synthesis of 36 new non-dimeric NS5A inhibitors is reported along with their ability to block HCV replication in an HCV 1b replicon system. Among them compound 5a showed picomolar range activity along with an excellent selectivity index (SI > 90,000).

Anti-hepatitis C Virus Activity of Novel β-D-2′-C-methyl-4′-azido Pyrimidine Nucleoside Phosphoramidate Prodrugs:

Background: 2′-C-methyl and 4′-azido nucleosides have previously demonstrated inhibition of hepatitis C virus (HCV) replication by targeting the RNA-dependent RNA polymerase NS5B. In an effort to discover new and more potent anti-HCV agents, we envisioned synthesizing nucleoside analogues by combining the 2′-C-methylmoiety with the 4′-azido-moiety into one molecule. Methods: 2′-C-methyl-4′-azido pyrimidine nucleosides were synthesized by first converting 2′-C-methyl ribonucleosides to the corresponding 4′-exocyclic methylene nucleosides. Treatment with iodine azide, benzoylation of the 2′- and 3′-hydroxy groups, oxidative displacement of the 5′-iodo group with meta-chloroperoxybenzoic acid, and debenzoylation gave the desired 2′-C-methyl-4′-azido uridine and thymidine analogues in good yield. Standard conversion of uridine to cytidine via the 4-triazole yielded 2′-C-methyl-4′-azido cytidine. In addition, 5′-phosphoramidate derivatives of 2′-C-methyl-4′-azido uridine and cytidine were synthesized to bypass the initial phosphorylation step. Results: The prepared nucleosides and their 5′-monophosphate prodrugs were evaluated for their ability to inhibit replication of the hepatitis C virus in a subgenomic replicon cell based assay. Cytotoxicity in Huh7 cells was determined simultaneously with anti-HCV activity by extraction and amplification of both HCV RNA and ribosomal RNA. Among the newly synthesized compounds, only the 5′-monophosphate nucleoside prodrugs had modest and selective anti-HCV activity. All prepared pyrimidine nucleosides and 5′-monophosphate nucleoside prodrugs displayed no evidence of cytotoxicity at high concentrations. Conclusions: This work is the first example of both inactive uridine and cytidine analogues of a nucleoside being converted to active anti-HCV nucleosides via 5′-monophosphate prodrugs.

Azetidines and spiro azetidines as novel P2 units in hepatitis C virus NS3 protease inhibitors

Herein, we report the synthesis and structure-activity relationship studies of new analogs of boceprevir 1 and telaprevir 2. Introduction of azetidine and spiroazetidines as a P2 substituent that replaced the pyrrolidine moiety of 1 and 2 led to the discovery of a potent hepatitis C protease inhibitor 37c (EC50=0.8 μM).

SYNTHESIS OF N3,5′-CYCLO-4-(β-D-RIBOFURANOSYL)-VIC-TRIAZOLO[4,5-b]PYRIDIN-5-ONE AND ITS 3′-DEOXYSUGAR ANALOGUE AS POTENTIAL ANTI-HEPATITIS C VIRUS AGENTS

We recently discovered a novel compound, identified as N3,5′-cyclo-4-(β-D-ribofuranosyl)-vic-triazolo[4,5-b]pyridinin-5-one, with anti-hepatitis C virus (HCV) activity in vitro. The structure was confirmed by chemical synthesis from 2-hydroxy-5-nitropyridine. It showed anti-HCV activity with EC50=19.7 μM in replicon cells. Its 3′-deoxy sugar analogue was also synthesized, but was inactive against HCV in vitro.