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

BIOCHEMICAL BASIS AND CONSEQUENCES OF ACTIVE SITE MUTATIONS IN THE HIV-l ASPARTYL PROTEASE

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

D.J. Livingston, S. Pazhanisamy, J.A. Partaledis, C. Stuver, A. Cullinan, O. Futer, S.P. Chambers, J.R. Fulghum and R.D. Tung
Vertex Pharmaceuticals Incorporated, Cambridge, MA 02139, USA

We have synthesized a new class of potent, low molecular weight HIV protease inhibitors based on an N,N-disubstituted sulfonamide scaffold. Many of these compounds display high antiviral potency, with IC₅₀ for both laboratory strains and clinical isolates ranging from 5-100 nM. Additionally, several of these compounds, including VX-478 now in development, display good oral availability in rodents and mammals. As viral resistance to these and other protease inhibitors may arise in vivo, we have been studying the effects of active site protease mutations on affinity for inhibitors of different chemical classes, as well as the consequences of altered catalytic efficiency to viral replication.

We have expressed HIV-l protease mutant enzymes in E.coli and purified them to homogeneity. The mutations were selected from those observed to occur in resistant variants to the compounds VX-478, Ro31-8959 (Roche) and L-735524 (Merck). The HIV-1 protease mutants studied include G48V/L90M, I47V, I50V, M46I/I47V/I50V, V82A, V82F and I84V. Affinity (K_i) decreases 10-1,000-fold for inhibitors within the chemical class of compound selected for in viral resistance experiments. Changes in K_i for compounds of a different chemical class are lower, ranging from no change to 20-fold. Structure-activity observations at P₂-P₂´ will be described for the effects of mutations on a range of sulfonamide inhibitors. Additionally, we have separated the effects on K_i into association (k_on) and disassociation rate (k_off) by direct determination of the latter rate constant. Effects of mutations on the catalytic parameters k_cat and K_m are more modest, resulting in decresases of up to 25-fold in the catalytic efficiency (k_cat/K_m). The changes in kinetic constants, as well as their activation energy, are dependent on the peptide substrate used in the assay, where each peptide substrate corresponds to a different gag-pol cleavage sequence. Finally, the effect of alterations on protease catalytic efficiency on HIV-l viral infectivity and replication rate have been examined in collaboration with Dr R. Byrn at the New England Deaconess Hospital, Boston, MA, USA

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1994-08-02
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