INHIBITION OF HUMAN IMMUNODEFICIENCY VIRUS-1 PROTEASE BY A C2-SYMMETRICAL PHOSPHINATE - SYNTHESIS AND CRYSTALLOGRAPHIC ANALYSIS

The human immunodeficiency virus type 1 (HIV-1) protease is a potential target of acquired immune deficiency syndrome (AIDS) therapy. A highly potent, perfectly symmetrical phosphinate inhibitor of this enzyme, SB204144, has been synthesized. It is a competitive inhibitor of HIV-1 protease, with an apparent inhibition constant of 2.8 nM at pH 6.0. The three-dimensional structure of SB204144 bound to the enzyme has been determined at 2.3-angstrom resolution by X-ray diffraction techniques and refined to a crystallographic discrepancy factor, R (= SIGMA parallel-to F(o)\-\F(c) parallel-to/SIGMA\F(o)\), of 0.178. The inhibitor is held in the enzyme active site by a set of hydrophobic and hydrophilic interactions, including an interaction between Arg8 and the center of the terminal benzene rings of the inhibitor. The phosphinate establishes a novel interaction with the two catalytic aspartates; each oxygen of the central phosphinic acid moiety interacts with a single oxygen of one aspartic acid, establishing a very short (2.2-2.4 angstrom) oxygen-oxygen contact. As with the structures of penicillopepsin bound to phosphinate and phosphonate inhibitors [Fraser, M. E., Strynadka, N. C., Bartlett, P. A., Hanson, J. E., & James, M. N. (1992) Biochemistry 31, 5201-14], we interpret this short distance and the stereochemical environment of each pair of oxygens in terms of a hydrogen bond that has a symmetric single-well potential energy curve with the proton located midway between the two atoms. Under identical assay conditions, SB204144 binds approximately 2 orders of magnitude more tightly than the monohydroxy analog A74704 [Erickson, J., Neidhart, D. J., VanDrie, J., Kempf, D. J., Wang, X. C., Norbeck, D. W., Plattner, J. J., Rittenhouse, J. W., Turon, M., Wideburg, N., Kohlbrenner, W. E., Simmer, R., Helfrich, R., Paul, D., & Knigge, M. (1990) Science 249, 527-33], apparently as a consequence of the stronger hydrogen bonds between the phosphinate oxygens and the catalytic aspartates. Implications for the catalytic mechanism of the novel mode of binding of the phosphinate group are discussed.