MUTATIONAL ANATOMY OF AN HIV-1 PROTEASE VARIANT CONFERRING CROSS-RESISTANCE TO PROTEASE INHIBITORS IN CLINICAL-TRIALS - COMPENSATORY MODULATIONS OF BINDING AND ACTIVITY

Site-specific substitutions of as few as four amino acids (M46I/L63P/V 82T/I84V) of the human immunodeficiency virus type 1 (HIV-1) protease engenders cross-resistance to a panel of protease inhibitors that are either in clinical trials or have recently been approved for HN therap y (Condra, J. H., Schleif, W. A., Blahy, O. M., Gadryelski, L. J., Gra ham, D. J., Quintero, J. C., Rhodes, A, Robbins, H. L., Roth, E., Shiv aprakash, M., Titus, D., Yang, T., Teppler, H., Squires, K. E., Deutsc h, P. J., and Emini, E, A. (1995) Nature 374, 569-571). These four sub stitutions are among the prominent mutations found in primary HIV isol ates obtained from patients undergoing therapy with several protease i nhibitors. Two of these mutations (V82T/I84V) are located in, while th e other two (M46I/L63P) are away from, the binding cleft of the enzyme . The functional role of these mutations has now been delineated in te rms of their influence on the binding affinity and catalytic efficienc y of the protease. We have found that the double substitutions of M46I and L63P do not affect binding but instead endow the enzyme with a ca talytic efficiency significantly exceeding (110-360%) that of the wild -type enzyme. In contrast, the double substitutions of V82T and I84V a re detrimental to the ability of the protease to bind and, thereby, to catalyze. When combined, the four amino acid replacements institute i n the protease resistance against inhibitors and a significantly highe r catalytic activity than one containing only mutations in its active site. The results suggest that in raising drug resistance, these four site-specific mutations of the protease are compensatory in function; those in the active site diminish equilibrium binding (by increasing K -i), and those away from the active site enhance catalysis (by increas ing k(cat)/K-M. This conclusion is further supported by energy estimat es in that the Gibbs free energies of binding and catalysis for the qu adruple mutant are quantitatively dictated by those of the double muta nts.