HUMAN-IMMUNODEFICIENCY-VIRUS - MUTATIONS IN THE VIRAL PROTEASE THAT CONFER RESISTANCE TO SAQUINAVIR INCREASE THE DISSOCIATION RATE-CONSTANTOF THE PROTEASE-SAQUINAVIR COMPLEX

Mutations in the human immunodeficiency virus (HIV) protease (L90M, G4 8V, and L90M/G48V) arise when HIV is passaged in the presence of the H IV protease inhibitor saquinavir. These mutations yield a virus with l ess sensitivity to the drug (L90M > G48V >> L90M/ G48V). L90M, G48V, a nd L90M/G48V proteases have 1/20, 1/160, and 1/1000 the affinity for s aquinavir compared to WT protease, respectively. Therefore, the affini ty of mutant protease for saquinavir decreased as the sensitivity of t he virus to saquinavir decreased. Association rate constants for WT an d mutant proteases with saquinavir were similar, ranging from 2 to 4 x 10(7) m(-1) s(-1). In contrast, the dissociation rate constants for W T, L90M, G48V, and L90M/G48V proteases complexed with saquinavir were 0.0014, 0.019, 0.128, and 0.54 s(-1), respectively. This indicated tha t the reduced affinity for mutant proteases and saquinavir is primaril y the result of larger dissociation rate constants. The increased diss ociation rate constants may be the result of a decrease in the interna l equilibrium between the bound inhibitor with the protease flaps up a nd the bound inhibitor with the flaps down, Interestingly, the affinit y of these mutant proteases for VX-478, ABT-538, AG-1343, or L-735,524 was not reduced as much as that for saquinavir. Finally, the catalyti c constants of WT and mutant proteases were determined for eight small peptide substrates that mimic the viral cleavage sites in vivo. WT an d L90M proteases had similar catalytic constants for these substrates. In contrast, G48V and L90M/G48V proteases had catalytic efficiency (k (cat)/K-m) values with TLNF-PISP, RKIL-FLDG, and AETF-YVDG that were 1 /10 to 1/20 the value of WT protease, The decreased catalytic efficien cies were primarily the result of increased K-m values. Thus, mutation s in the protease decrease the affinity of the enzyme for saquinavir a nd the catalytic efficiency with peptide substrates.