466   Molecular Mechanism of I50V HIV-1 Protease Resistance and Cross-Resistance to Protease Inhibitors.

R. Xu, W. Andrews, A. Spaltenstein, D. Danger, W. Dallas, L. Carter, L. Wright, and E. Furfine*.
Glaxo Wellcome Inc., Res. Triangle Park, NC.

Background:I50V mutations in the HIV-1 protease gene mediate the primary pathway of resistance to amprenavir in vitro and in vivo, though multiple mutations in addition to I50V are required for high-level amprenavir resistance. I50V is partially cross-resistant to ritonavir and minimally cross-resistant to nelfinavir. We have determined that I50V-protease has increased K_ivalues for amprenavir, ritonavir and nelfinavir compared to WT enzyme (140-, 50-, and 10-fold, respectively), which correlate with the rank order of cross- resistance of the virus.

Methods and Results:X-ray structure data suggested a molecular mechanism for the level of resistance. Comparing amprenavir, ritonavir, and nelfinavir bound to WT protease, the most dense protease conformation is the amprenavir complex, followed by ritonavir and then nelfinavir. In the ritonavir and nelfinavir complexes, shifts in the conformation of the peptide loop containing P81 cause the decreased density of these complexes. The change "I50V" is the loss of a methyl group in the active site that creates a "vacuum" because of lost hydrophobic contacts with the inhibitors. Two events occur to recapture the interaction of the lost methyl group or "fill the vacuum". First, the protein condenses by shifting the P81 loop. Second, there are conformational changes in the inhibitor and V32 that better "fill" the space. Because amprenavir starts with the most condensed conformation of the P81 loop, it is not possible to collapse the I50V protease structure further, and minor shifts in the inhibitor conformation can not make up the lost interaction with the methyl group. An intermediate shift of the P81 loop occurs in the ritonavir mutant structure that partially compensates for the lost interaction. The thiazole ring of ritonavir also rotates to better fill the space, but this conformation is partially penalized by loss of a hydrogen bond. The largest shift of the P81 loop occurs with the nelfinavir complex, thus best filling the space lost from the missing methyl group but resulting in a slight lengthening of the H-bond between the cresol OH and Asp 30.

Conclusion:Therefore, greater compensation by the protease for the I50V mutation by compacting the P81 loop correlates with less loss of affinity and less resistance.