1st International Workshop on HIV Drug Resistance & Treatment Strategies 25-28 June 1997, St. Petersburg, Florida, USA

Antiviral Therapy 1997;2 (Suppl 5):9 (abstract no. 14)

J Ermolieff, L Hong, X Lin, S Foundling, JA Hartsuck and J Tang
Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA

HIV-1 protease (PR) and its saquinavir-resistant mutants G48V, L90M and G48V/L90M were studied kinetically and mutant G48V was studied structurally in order to understand the basis of PR mutation resistance. In the kinetic experiments, among peptides derived from the eight gag-pol cleavage sites, results from the hydrolysis of sites A, C and E (MA/CA, p2/NC and p6/Pr respectively) were most consistent with respect to resistance of these mutants against saquinavir. Both steady-state and ‘competitive hydrolysis’ kinetic methods were used to ensure the accuracy of the k_cat, K_m and K_i values. From these data, the intravirion processing activities, called ‘mutation modulated activity’ (MMA) of the wild-type PR and three mutants were calculated [1] for different saquinavir concentrations. The following conclusions were drawn: (i) G48V and L90M are important resistant mutants in vivo, but mutant G48V/L90M is much less significant because it contributes only a small fraction of total processing activity in the saquinavir concentration range where HIV propagation takes place. (ii) The in vivo population ratios of these four mutants at different times during clinical trials [2] correlate strongly with the predicted MMA ratios of the wild-type HIV PR and mutants at certain saquinavir concentrations. For example, in the 12th week of clinical trial V13330(m), the percentage in the population of wild-type, single mutants and double mutant were 63, 31 and 6%, while the percentage of total MMA for the same three groups at a saquinavir concentration of 0.8 × 10⁹ M were 62, 32 and 6%. This correlation suggests that the in vivo population ratio of the mutants is driven by saquinavir concentration via the processing activity of PR mutants and that intravirion processing by PR is rate limiting for HIV propagation. The latter has been separately verified in an in vitro experiment in which the propagation rates of wild-type HIV and protease mutants correlated with the k_cat/K_m values of the PR species. The crystal structure of PR mutant G48H bound to inhibitor U89360E was determined and a model for G48V bound to saquinavir was built. These structures show that these mutations at G48 result in a new interaction of Phe-53 with Val-48 or His-48 and result in increased rigidity of the flap region of PR. For the mutant enzymes there was contact of residue 48 with P2, P3 or P2´, P3´ inhibitor subsites. For both complexes, van der Waals and electrostatic interactions combine to destabilize the mutant complex as compared to that of the wild-type and predict the observed increase of K_i for these mutants.

1. Tang J and Hartsuck JA. A kinetic model for comparing proteolytic processing activity and inhibitor resistance potential of mutant HIV-1 proteases. FEBS Lett. 1995 Jun 26;367(2):112-6.

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1997-06-25
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