13th International HIV Drug Resistance Workshop 8–12 June 2004, Tenerife Sur-Costa Adeje, Canary Islands, Spain

BACKGROUND: One of the greatest challenges facing prolonged antiretroviral therapy is the development of drug resistance. AZT resistance mutations, 41L, 67N, 70R, 210W, T215F/Y, 219Q, alone, or in combination with a dipeptide insertion in HIV-1 reverse transcriptase (RT) confer resistance to most nucleoside analogues in common use today. It is therefore important to devise strategies to prevent and/or suppress the appearance of AZT resistance mutations.

AZT resistance is associated with increased nucleotide-dependent removal of chain-terminators from blocked DNA ends. The products of this reaction are an unblocked primer terminus and a dinucleoside polyphosphate. It is quite possible that dinucleoside polyphosphates could serve as substrates and/or inhibitors of HIV-1 RT. We therefore prepared several homodimeric dinucleoside tetraphosphates containing dideoxynucleosides (ddNp₄ddN) and tested their ability to inhibit DNA synthesis by WT or mutant HIV-1 RT.

METHODS: Incorporation of radioactive deoxynucleotide into M13 P/T by HIV-1 RT was measured in the absence or presence of varying concentrations of either ddNTP or ddNp₄ddN.

RESULTS: DNA polymerization in the presence of either ddTTP or ddTp₄ddT led to a concentration-dependent decrease in incorporated dNTPs due to incorporation of the inhibitor resulting in chain-termination. WT RT was over 70-fold less sensitive to inhibition by ddTp₄ddT (IC₅₀~10 µM) than by ddTTP (IC₅₀~140 nM). However, RT containing the 67N/70R/215Y/219Q mutations was only fivefold less sensitive to inhibition by ddTp₄ddT (IC₅₀~0.9 µM) than to ddTTP (IC₅₀~0.2 µM) and RT containing the 41L/69S-AG/210W/211K/214F/215Y mutations was equally sensitive to inhibition by ddTp₄ddT (IC₅₀~0.35 µM) and ddTTP (IC₅₀~0.32 µM).

CONCLUSIONS: AZT resistance mutations, alone or in combination with a 69-dipeptide insertion, conferred an increased susceptibility to inhibition by dinucleoside polyphosphates containing chain-terminating nucleoside analogues. Dinucleoside polyphosphates served as substrates for DNA polymerization by HIV- 1 RT and were incorporated much more readily by HIV-1 RT containing AZT resistance mutations. These results are very encouraging for development of novel, specific inhibitors of HIV-1 RT with increased efficacy against AZT-resistant RT which could be used to suppress the appearance of AZT-resistance mutations.

PRESENTING AUTHOR: P Meyer

This work was supported by amfAR fellowship: 70567-31-RF, American Heart Predoctoral Fellowship 0215087B and NIH grant AI 39973.

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2004-06-08
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