A two-fold (C2) symmetric inhibitor of the protease of human immunodeficiency virus type-1 (HIV-1) has been designed on the basis of the three-dimensional symmetry of the enzyme active site. The symmetric molecule inhibited both protease activity and acute HIV-1 infection in vitro, was at least 10,000-fold more potent against HIV-1 protease than against related enzymes, and appeared to be stable to degradative enzymes. The 2.8 angstrom crystal structure of the inhibitor-enzyme complex demonstrated that the inhibitor binds to the enzyme in a highly symmetric fashion.

Inhibitors of the human immunodeficiency virus type 1 protease represent a promising class of antiviral drugs for the treatment of AIDS, and several are now in clinical trials. Here, we report the in vitro selection of viral variants with decreased sensitivity to a C2-symmetric protease inhibitor (A-77003). We show that a single amino acid substitution (Arg to Gln or Lys) at position 8 of the protease results in a substantial decrease in the inhibitory activity of the drug on the enzyme and a comparable increase in viral resistance. These findings, when analyzed by using the three-dimensional structure of the protease-drug complex, provide a strategic guide for the future development of inhibitors of the human immunodeficiency virus type 1 protease.

The structure-activity studies leading to the potent and clinically efficacious HIV protease inhibitor ritonavir are described. Beginning with the moderately potent and orally bioavailable inhibitor A-80987, systematic investigation of peripheral (P3 and P2') heterocyclic groups designed to decrease the rate of hepatic metabolism provided analogues with improved pharmacokinetic properties after oral dosing in rats. Replacement of pyridyl groups with thiazoles provided increased chemical stability toward oxidation while maintaining sufficient aqueous solubility for oral absorption. Optimization of hydrophobic interactions with the HIV protease active site produced ritonavir, with excellent in vitro potency (EC50 = 0.02 microM) and high and sustained plasma concentrations after oral administration in four species. Details of the discovery and preclinical development of ritonavir are described.

The incorporation of C2 symmetry has become a useful paradigm in the design of active site inhibitors for HIV-I protease (HIV PR) and has led to the design of a series of highly potent, C2 symmetry-based, diol-containing inhibitors of HIV PR, one of which, A-77003, has reached clinical trials. However, the stereochemistry of the diol core influences protease inhibition and antiviral activity in a manner that is not well understood. We analyzed the crystal structures of a diastereomeric series of C2 symmetry-based diol inhibitors, along with a deshydroxy analogue, bound to HIV PR and found that the stereochemistry of the diol core influences the mode of binding to the active site aspartic acids. Diasteromers with similar binding affinity can bind in different, asymmetric and symmetric, modes, while those with different binding affinities can bind in a similar manner. The positional symmetry of an inhibitor with respect to the enzyme C2 axis may be distinguished from its conformational symmetry. The structural differences between the inhibitor complexes were mainly confined to the central core portion of the diols, can be described by torsional differences about the central three bonds, and primarily affect interactions within the active site pocket formed by Asp 25/125 and Gly 271127. Some flexibility in the enzyme backbone at Gly 127 was also apparent. Based on these results, we suggest that the binding mode for central hydroxy-bearing, Cz-symmetric inhibitors will be determined by how well the inhibitor can simultaneously optimize hydrogen bonding with the active site carboxylate groups and van der Waals contacts with the neighboring backbone atoms of the active site "+"-loops. A symmetric hydrogenbonding arrangement with either one or two symmetrically positioned hydroxy groups appears to be preferred over less symmetric configurations.