590] INHIBITION OF HCV REPLICATION BY PSI-6130: MECHANISM OF BIOCHEMICAL ACTIVATION AND INHIBITION

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Background: Nearly 2% of the U S . population and an estimated 170 million people worldwide are HCV carriers. The current standard of care, a combination of pegylated interferon and ribavirin, has limited efficacy. We have identified a nucleoside analog, (3-D-2’-deoxy-2’-fluoro2’-C-methylcytidine (PSI-61 30), which is a specific, potent and noncytotoxic inhibitor of HCV in the subgenomic replicon assay. To inhibit the HCV NS5B polymerase, PSI-6 130 must be phosphorylated to the 5’triphosphate. The enzymes involved in the phosphorylation of PSI-61 30 and inhibition of HCV NS5B were investigated. Methods: Anti-HCV activity was measured using subgenomic-HCV replicon cells and a real-time PCR assay. Spectrophotometric assays and recombinant deoxycytidine kinase, uridine-cytidine kinase, UMP-CMP kinase, and nucleoside diphosphate kinase (NDPK) were used to investigate the phosphorylation pathway of PSI-6130 to the corresponding 5’-triphosphate. The NS5B RNA polymerase reaction was performed in the presence of PSI-6 130-TP or other 2’-C-Me-nucleoside triphosphates using 32P-labeled UTP and the amount of radioactivity in the product was measured. Results: An EC90 (concentration producing I loglo reduction in viral RNA) value of 4.6f2.0pM was determined for PSI-6130 and was essentially inactive against other viruses including BVDV Little or no cytotoxicity or mitochondrial toxicity was observed with cells treated with PSI-6 130. Purified dCK, UMP-CMP kinase, and NDPK phosphorylated PSI-61 30 to the 5’-mono-, di-, and triphosphate derivatives respectively. PSI-6130-TP was an alternative substrate inhibitor of purified NSSB. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B resulted in chain-termination. The steady-state inhibition constant for PSI6 130-TP was Ki = 4.3 pM. Against the S282T NS5B enzyme PSI-6 130-TP showed only a 3-fold resistance compared to a 12-fold resistance for 2‘C-Me-cytidine-TP and a 150-fold resistance for 2’-C-Me-adenosine-TP. Conclusion: PSI-6 130 demonstrated potent and specific anti-HCV activity in cell-based assays with little or no cytotoxicity and mitochondrial toxicity. PSI-6 130 was efficiently phosphorylated to its active 5’-triphosphate form. Deoxycytidine kinase, UMP-CMP kinase, and NDPK were identified as enzymes involve in the phosphorylation pathway of PSI-6130. PSI6130-TP inhibited HCV NSSB RNA polymerase and acted as a chainterminator upon incorporation into the elongating RNA strand by HCV NS5B.