AAC Accepts, published online ahead of print on 13 November 2006

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Antimicrob. Agents Chemother. doi:10.1128/AAC.00400-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Mechanism of activation of -D-2'-deoxy-2'-fluoro-2'-C-methylcytidine and inhibition of hepatitis C virus NS5B RNA polymerase

Eisuke Murakami^*, Haiying Bao, Mangala Ramesh, Tamara R. McBrayer, Tony Whitaker, Holly M. Micolochick Steuer, Raymond F. Schinazi, Lieven J. Stuyver, Aleksandr Obikhod, Michael J. Otto, and Phillip A. Furman

Pharmasset, Inc., 303A College Road East, Princeton, NJ 08540, USA; Emory University VA Medical Center, Medical Research-151, 1670 Clairmont Road, Decatur, GA 30033, USA

^* To whom correspondence should be addressed. Email: eisuke.murakami{at}pharmasset.com,

-D-2'-Deoxy-2'-fluoro-2'-C-methyl-cytidine (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. Phosphorylation of PSI-6130 by recombinant human 2'-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK with a K_m of 81 µM and k_cat of 0.007 sec^-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 (YMPK) and nucleoside diphosphate kinase (NDPK), respectively. The inhibition of wild-type and S282T mutated HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (K_i) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 µM. Similar results were obtained with 2'-C-Me-adenosine-TP (K_i = 1.5 µM) and 2'-C-Me-cytidine-TP (K_i = 1.6 µM). NS5B with the S282T mutation, which is known to confer resistance to 2'-C-Me-adenosine, was inhibited by PSI-6130-TP as efficiently as the wild-type. Incorporation of PSI-6130 monophosphate (PSI-6130-MP) into RNA catalyzed by purified NS5B RNA polymerase resulted in chain-termination.

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