HYDROXYETHYLENE ISOSTERE INHIBITORS OF HUMAN IMMUNODEFICIENCY VIRUS-1 PROTEASE - STRUCTURE ACTIVITY ANALYSIS USING ENZYME-KINETICS, X-RAY CRYSTALLOGRAPHY, AND INFECTED T-CELL ASSAYS

Analogues of peptides ranging in size from three to six amino acids and containing the hy-droxyethylene dipeptide isosteres Phe-PSI-Gly, Phe-PSI-Ala, Phe-PSI-NorVal, Phe-PSI-Leu, and Phe-PSI-Phe, where PSI denotes replacement of CONH by (S)-CH(OH)CH2, were synthesized and studied as HIV-1 protease inhibitors. Inhibition constants (K(i)) with purified HIV-1 protease depend strongly on the isostere in the order Phe-PSI-Gly > Phe-PSI-Ala > Phe-PSI-NorVal > Phe-PSI-Leu > Phe-PSI-Phe and decrease with increasing length of the peptide analogue, converging to a value of 0.4 nM. K(i) values are progressively less dependent on inhibitor length as the size of the Pl' side chain within the isostere increases. The structures of HIV-1 protease complexed with the inhibitors Ala-Ala-X-Val-Val-OMe, where X is Phe-PSI-Gly, Phe-PSI-Ala, Phe-PSI-NorVal, and Phe-PSI-Phe, have been determined by X-ray crystallography (resolution 2.3-3.2 angstrom). The crystals exhibit symmetry consistent with space group P6(1) with strong noncrystallographic 2-fold symmetry, and the inhibitors all exhibit 2-fold disorder. The inhibitors bind in similar conformations, forming conserved hydrogen bonds with the enzyme. The Phe-PSI-Gly inhibitor adopts an altered conformation that places its P3' valine side chain partially in the hydrophobic Sl' pocket, thus suggesting an explanation for the greater dependence of the K(i) value on inhibitor length in the Phe-PSI-Gly series. From the kinetic and crystallographic data, a minimal inhibitor model for tight-binding inhibition is derived in which the enzyme subsites S2-S2' are optimally occupied. The K(i) values for several compounds are compared with their potencies as inhibitors of proteolytic processing in T-cell cultures chronically infected with HIV-1 (MIC values) and as inhibitors of acute infectivity (IC50 values). There is a rank-order correspondence, but a 20-1000-fold difference, between the values of K(i) and those of MIC or IC50. IC50 values can approach those of K(i) but are highly dependent on the conditions of the acute infectivity assay and are influenced by physiochemical properties of the inhibitors such as solubility.