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J. Biol. Chem., Vol. 283, Issue 9, 5452-5459, February 29, 2008

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Pre-steady-state Kinetic Studies Establish Entecavir 5'-Triphosphate as a Substrate for HIV-1 Reverse Transcriptase^*

Robert A. Domaoal, Moira McMahon, Chloe L. Thio, Christopher M. Bailey, Julian Tirado-Rives^¶, Aleksander Obikhod^||, Mervi Detorio^||, Kimberly L. Rapp^||, Robert F. Siliciano, Raymond F. Schinazi^||, and Karen S. Anderson¹

From the Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066, Department of Medicine, Johns Hopkins University School of Medicine and Howard Hughes Medical Institute, Baltimore Maryland 21205, ^||Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033, and ^¶Department of Chemistry, Yale University, New Haven, Connecticut 06510

The novel 2'-deoxyguanosine analog Entecavir (ETV) is a potent inhibitor of hepatitis B virus (HBV) replication and is recommended for treatment in human immunodeficiency virus type 1 (HIV-1) and HBV-co-infected patients because it had been reported that ETV is HBV-specific. Recent clinical observations, however, have suggested that ETV may indeed demonstrate anti-HIV-1 activity. To investigate this question at a molecular level, kinetic studies were used to examine the interaction of 5'-triphosphate form of ETV with wild type (WT) HIV-1 reverse transcriptase (RT) and the nucleoside reverse transcriptase inhibitor-resistant mutation M184V. Using single turnover kinetic assays, we found that HIV-1 WT RT and M184V RT could use the activated ETV triphosphate metabolite as a substrate for incorporation. The mutant displayed a slower incorporation rate, a lower binding affinity, and a lower incorporation efficiency with the 5'-triphosphate form of ETV compared with WT RT, suggesting a kinetic basis for resistance. Our results are supported by cell-based assays in primary human lymphocytes that show inhibition of WT HIV-1 replication by ETV and decreased susceptibility of the HIV-1 containing the M184V mutation. This study has important therapeutic implications as it establishes ETV as an inhibitor for HIV-1 RT and illustrates the mechanism of resistance by the M184V mutant.

Received for publication, September 19, 2007 , and in revised form, October 24, 2007.

Note Added in Proof—The print version differs from the earlier Papers in Press version due to our finding that our Entecavir triphosphate (ETVTP) sample was contaminated with pyrophosphate. The presence of pyrophosphate affected the rate, affinity, and incorporation efficiency of ETVTP. The K_d and k_pol values for ETVTP incorporation by WT and M184V are modified in Table 1, Fig. 2, and text to reflect the values determined after removal of contaminating pyrophosphate.

^* This work was supported in part by National Institutes of Health Grants GM-49551 (to K. S. A.), and AI-44616 (to W. Jorgensen in support of J. T.-R.), AI-51178, a grant from AI-43222, Howard Hughes Medical Institute (to R. S.), Emory's Center for AIDS Research National Institutes of Health Grants 5P30-AI-50409 and 5R37-AI-041980, and a grant from the Department of Veterans Affairs (to R. F. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Pharmacology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520. Tel.: 203-785-4526; Fax: 203-785-7670; E-mail: karen.anderson{at}yale.edu.

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