17th International HIV Drug Resistance Workshop 10-14 June 2008, Sitges, Spain

DELAYED CHAIN-TERMINATION PROTECTS THE HEPATITIS B VIRUS DRUG ENTECAVIR FROM EXCISION BY HIV-1 REVERSE TRANSCRIPTASE

Antivir Ther. 2008; 13(Suppl. 3):A48 (abstract no. 43)

E Tchesnokov¹, A Obikhod², RF Schinazi² and M Götte^1
1McGill University, Montreal, Quebec, Canada; ²Emory University School of Medicine/VA Medical Center, Atlanta, Georgia, USA

BACKGROUND: Entecavir (ETV) is a potent antiviral drug used to treat infection with the hepatitis B virus (HBV). Recent studies have shown that ETV has anti-HIV activity and can select for the M184V mutation in HIV-1 reverse transcriptase (RT), which limits its clinical use in HIV–HBV-co-infected individuals. However, the mechanism of drug action remains elusive. ETV is a guanosine nucleoside analogue that contains a 3′-hydroxyl group. Thus, the incorporated ETV-5′-monophosphate (ETVMP) may not act as a classic chain-terminator. Inhibitory effects of ETV-MP can occur at a certain distance from its site of incorporation.

METHODS: We utilized various biochemical tools to elucidate the anti-HIV mechanism of ETV and its implications with respect to major resistance pathways against established nucleoside reverse transcriptase inhibitors (NRTIs).

RESULTS: Incorporation of ETV-MP at position n causes RT pausing at positions n and n+3. Increasing concentrations of natural dNTP pools at positions n+1 and n+4 can eventually overcome enzymatic pausing; however, incorporation of the natural nucleotide at position n+4 is severely compromised. Kinetic measurements revealed a subtle eightfold decrease in efficiency of nucleotide incorporation at position n+1 when ETV-terminated primers are compared with the natural counterpart, whereas nucleotide incorporation at position n+4 is reduced by three orders of magnitude (1,230-fold). High-resolution footprinting experiments show that complexes with HIV-1 RT and a primer/template that mimics the latter situation are as stable as complexes that contain natural primers. Rather, ETV-MP forces the enzyme to slide away from the 3′-end of the primer at position n+3, which provides a plausible mechanism for such ‘delayed chain-termination’. RT enzymes with thymidine analogue mutations (TAMs) can efficiently excise the incorporated ETV-MP at position n, as demonstrated for several established NRTIs. However, ETV is fully sensitive against TAM-containing HIV variants, which shows that the additional nucleotides at positions n+1 to n+3 protect the inhibitor from excision.

CONCLUSION: The results of this study demonstrate that ‘delayed chain-termination’ at position n+3 is the dominant mechanism of action of ETV. The combined data provide a rationale for the development of ETV-like, delayed chain-terminators as anti-HIV compounds that can evade the excision mechanism.

2008-06-10
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