3rd International Workshop on HIV Drug Resistance 2-5 August 1994, Kauai, Hawaii, USA

MUTATIONS CONFERRING VIRAL RESISTANCE TO HIV-1 PROTEASE INHIBITORS CAN LIE OUTSIDE, AS WELL AS WITHIN, THE ENZYME ACTIVE SITE & BINDING POCKETS

Int Wkshop HIV Drug Res 1994 Aug 2-5;3:9 (abstract no. 8)

B. Maschera, C. Blance, D. Brown & E.D. Blair
Wellcome Research Labs, Beckenham, UK BR3 3BS

HIV-l (HXBc2) was passaged, in parallel, with increasing concentrations of two protease inhibitors A75925 (Abbott; C₂ diol) and RoXI (Roche; transition state mimetic) and the mutations associated with resistance development identified. With A-75925, a mutation V32I arose as early as pass 1 (about 50% wt, 50% mutant) and was homogeneous in the virus population by pass 3, conferring a 40-fold decrease in sensitivity to A-75925. With RoXI, a mutation L90M arose by pass 2 to comprise about 30% of virus population and then remained as a subpopulation of up to 95% but never becoming homogeneous. Consequently, release from RoXI selection resulted in the reversion of the viral population to predominantly wild-type sequence. G48V, a change seen together with L90M, under selection with Ro31-8959 (see Tisdale et al, Int Wkshop HIV Drug Res 1994 Aug 2-5;3:14 (abstract no. 13), this symposium) was not found. L90M conferred a 8-10 fold decrease in sensitivity to RoXI on the mixed population. Infectious molecular clones with protease genes bearing these individual mutations, in a background otherwise identical to a clone containing the wild-type protease gene, confirmed the role of V32I and L90M in drug resistance. The A-75^R and RoXI^R viruses were not cross-resistant, but an infectious clone carrying both mutations was viable and showed dual-resistance, suggesting that combined therapy with such inhibitors could give rise to viable, multi-resistant virus.

Modelling based on the crystal structure of RoXI with wild-type HIV-l protease (determined at Wellcome Research Labs) suggests that the L90M mutation affects the structure of the active site, since the S-linked methyl on M90 is approximately O.6Å closer to the main chains for D25 and D25´ than either methyl on the branched side-chain of L90. The crystal structure of wild-type HIV-l protease with A75925 (Brookhaven Database) shows that the V32I change affects the interaction of enzyme with the valine side chains of the inhibitor in P2 and P2´, and allows us to predict that V32I would also be cross-resistant to similar C₂ inhibitors, e.g. P9941 (DuPont Merck), A-77003. Proteases containing V32I and others mutations have been expressed in E. coli and appear to self-process as well as wild-type enzyme. We await biochemical and structural data to confirm our modelling of the atomic basis of HIV drug resistance.

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1994-08-02
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