Investigation of 2‐Fold Disorder of Inhibitors and Relative Potency by Crystallizations of HIV‐1 Protease in Ritonavir and Saquinavir Mixtures

Abstract

HIV‐1 protease (PR) was cocrystallized in competitive mixtures of saquinavir (SQV) and ritonavir (RTV) in an attempt to compare the relative potencies of inhibitors using a crystallographic approach. The mixture ratio of RTV/SQV was in the range of 1:1 to 50:1. The crystal form obtained with 1:1 and 5:1 ratios of RTV/SQV was monoclinic, while ratios 15:1 and 50:1 gave orthorhombic crystal form. The four crystal structures of PR/RTV/SQV were solved at 1.03, 1.12, 1.25, and 1.72 Å resolutions. The X‐ray crystal structures reveal that the crystal forms are dependent on the occupancy of either SQV or RTV in the active site of PR. At low RTV/SQV concentrations, PR/SQV complex is dominant, and at higher ratios, PR/RTV is found. The absence of a crystal structure having both inhibitors statistically disordered in the catalytic site of PR suggests that the two protein complexes are sufficiently different in properties to be discriminated in crystal growth process. The X‐ray structures of the dimeric enzyme with C2 pseudosymmetry show a 2‐fold‐disorder phenomenon for the SQV, while the RTV inhibitor is detected in a single orientation. The dominancy of PR/SQV crystal form at an equimolar mixture of inhibitor and the presence/absence of the 2‐fold disorder of inhibitors have given new insight into the relative potency of these drugs and both suggest a higher potency of SQV with respect to RTV.Research supported by Schlumberger FFTF.