3rd International Workshop on HIV Drug Resistance


2-5 August 1994, Kauai, Hawaii, USA


PROBING THE MECHANISM FOR THE IN VITRO DRUG RESISTANCE-SENSITIZATION MEDIATED BY THE PROLINE TO LEUCINE CHANGE AT 236 OF HIV-1 REVERSE TRANSCRIPTASE

Int Wkshop HIV Drug Res 1994 Aug 2-5;3:41 (abstract no. 40)

S.K. Sharma, N. Fan, D.B. Evans, K.B. Rank and W.G. Tarpley*
Biochemistry and *Cancer and Infectious Diseases Research, The Upjohn Company, Kalamazoo, USA

Bisheteroarylpiperazines (BHAPs) are potent inhibitors of HIV-1 reverse transcriptase (RT), but are inactive against HIV-2 RT and other polymerases. BHAP-resistant HIV-1 is sensitized to other classes of nonnucleoside RT inhibitors and this has been primarily attributed to a proline-to-leucine substitution at amino acid 236 (P236L) of HIV-1 RT. To understand the basis for the in vitro sensitization-resistance phenomenon, we used site directed mutagenesis to generate HIV-1 RT mutants with amino acid changes at and around position P236.

Active HIV-1 RT mutants H235W, D237T, and H235W/D237T/T240K, containing substitutions from HIV-2 RT, were cloned, expressed, and purified. Single base pair mutations at amino acid P236 in HIV-1 RT were also introduced to obtain P236L, P236T, P236R, P236H, and P236A HIV-1 RT mutants. Three BHAPs (U-88204E, U-87201E, and U-90125S) and the pyridinone L-697,661 were selected to quantitatively assess the effects of these amino acid substitutions on sensitization to L-697,661 and resistance to the BHAPs. The studies with the P236 HIV-1 RT mutants demonstrate a direct correlation between sensitization to L-697,661 and resistance to the BHAPs. It is concluded that susceptibility and resistance to these RT inhibitors are the consequence of the precise geometry of the inhibitor-binding pocket.

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1994-08-02
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