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Antimicrobial Agents and Chemotherapy, February 2007, p. 503-509, Vol. 51, No. 2
0066-4804/07/$08.00+0     doi:10.1128/AAC.00400-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Mechanism of Activation of ß-D-2'-Deoxy-2'-Fluoro-2'-C-Methylcytidine and Inhibition of Hepatitis C Virus NS5B RNA Polymerase

Eisuke Murakami,^1* Haiying Bao,¹ Mangala Ramesh,¹ Tamara R. McBrayer,^1, Tony Whitaker,^1, Holly M. Micolochick Steuer,¹ Raymond F. Schinazi,² Lieven J. Stuyver,^1, Aleksandr Obikhod,^1, Michael J. Otto,¹ and Phillip A. Furman¹

Pharmasset, Inc., 303A College Road East, Princeton, New Jersey 08540,¹ Emory University VA Medical Center, Medical Research-151, 1670 Clairmont Road, Decatur, Georgia 30033²

Received 31 March 2006/ Returned for modification 26 May 2006/ Accepted 30 October 2006

ß-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 K_m of 81 µM and a k_cat 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 (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-methyladenosine triphosphate (K_i = 1.5 µM) and 2'-C-methylcytidine triphosphate (K_i = 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.

Published ahead of print on 13 November 2006.

Present address: RFS Pharma LLC., 1860 Montreal Road, Tucker, GA 30084.

Present address: Virco BBA, Mechelen, Belgium.

Present address: Emory University VA Medical Center, Medical Research-151, 1670 Clairmont Road, Decatur, GA 30033.

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