RESISTANCE TO HIV PROTEASE INHIBITORS - A COMPARISON OF ENZYME-INHIBITION AND ANTIVIRAL POTENCY

Resistance of HIV-1 to protease inhibitors has been associated with ch anges at residues Val82 and Ile84 of HIV-1 protease (HIV PR). Using bo th an enzyme assay with a peptide substrate and a cell-based infectivi ty assay, we examined the correlation between the inhibition constants for enzyme activity (K-i values) and viral replication (IC90 values) for 5 active site mutants and 19 protease inhibitors. Four of the five mutations studied (V82F, V82A, I84V, and V82F/I84V) had been identifi ed as conferring resistance during in vitro selection using a protease inhibitor. The mutant protease genes were expressed in Escherichia co li for preparation of enzyme, and inserted into the HXB2 strain of HIV for test of antiviral activity. The inhibitors included saquinavir, i ndinavir, nelfinavir, 141W94, ritonavir tall in clinical use), and 14 cyclic ureas with a constant core structure and varying P2, P2' and P3 , P3' groups. The single mutations V82F and I84V caused changes with v arious inhibitors ranging from 0,3- to 86-fold in K-i and from 0.1- to Ii-fold in IC90, Much larger changes compared to wild type were obser ved for the double mutation V82F/I84V both for K-i (10-2000-fold) and for (C-90 (0.7-377-fold). However, there were low correlations (r(2) = 0.017-0.53) between the mutant/wild-type ratio of K-i values (enzyme resistance) and the mutant/wild-type ratio of viral IC90 values (antiv iral resistance) for each of the HIV proteases and the viruses contain ing the identicalenzyme. Assessing enzyme resistance by ''vitality val ues'', which adjust the K-i values with the catalytic efficiencies (k( cat)/K-m), caused no significant improvement in the correlation with a ntiviral resistance. Therefore, our data suggest that measurements of enzyme inhibition with mutant proteases may be poorly predictive of th e antiviral effect in resistant viruses even when mutations are restri cted to the protease gene.