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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{triangledown}

Eisuke Murakami,1* Haiying Bao,1 Mangala Ramesh,1 Tamara R. McBrayer,1,{dagger} Tony Whitaker,1,{dagger} Holly M. Micolochick Steuer,1 Raymond F. Schinazi,2 Lieven J. Stuyver,1,{ddagger} 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 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.

* Corresponding author. Mailing address: Pharmasset, Inc., 303A College Road East, Princeton, NJ 08640. Phone: (609) 613-4100. Fax: (609) 613-4150. E-mail: eisuke.murakami{at}pharmasset.com.

{triangledown} Published ahead of print on 13 November 2006.

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

{ddagger} Present address: Virco BBA, Mechelen, Belgium.

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

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.



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