Thermodynamic Basis of Resistance to HIV-1 Protease Inhibition: Calorimetric Analysis of the V82F/I84V Active Site Resistant Mutant ² Matthew J. Todd, Irene Luque, Adrian Vela ´zquez-Campoy, and Ernesto Freire* The Johns Hopkins UniVersity, Department of Biology and Biocalorimetry Center, Baltimore, Maryland 21218 ReceiVed May 3, 2000; ReVised Manuscript ReceiVed July 11, 2000 ABSTRACT: One of the most serious side effects associated with the therapy of HIV-1 infection is the appearance of viral strains that exhibit resistance to protease inhibitors. The active site mutant V82F/ I84V has been shown to lower the binding affinity of protease inhibitors in clinical use. To identify the origin of this effect, we have investigated the binding thermodynamics of the protease inhibitors indinavir, ritonavir, saquinavir, and nelfinavir to the wild-type HIV-1 protease and to the V82F/I84V resistant mutant. The main driving force for the binding of all four inhibitors is a large positive entropy change originating from the burial of a significant hydrophobic surface upon binding. At 25 °C, the binding enthalpy is unfavorable for all inhibitors except ritonavir, for which it is slightly favorable (-2.3 kcal/mol). Since the inhibitors are preshaped to the geometry of the binding site, their conformational entropy loss upon binding is small, a property that contributes to their high binding affinity. The V82F/I84V active site mutation lowers the affinity of the inhibitors by making the binding enthalpy more positive and making the entropy change slightly less favorable. The effect on the enthalpy change is, however, the major one. The predominantly enthalpic effect of the V82F/I84V mutation is consistent with the idea that the introduction of the bulkier Phe side chain at position 82 and the Val side chain at position 84 distort the binding site and weaken van der Waals and other favorable interactions with inhibitors preshaped to the wild-type binding site. Another contribution of the V82F/I84V to binding affinity originates from an increase in the energy penalty associated with the conformational change of the protease upon binding. The V82F/I84V mutant is structurally more stable than the wild-type protease by about 1.4 kcal/mol. This effect, however, affects equally the binding affinity of substrate and inhibitors. The HIV-1 protease has been the most important target for drug development against HIV-1 infection due to its key role in viral maturation. The HIV-1 protease is a dimer composed of identical subunits of 99 residues each. The crystallographic structure of the free enzyme as well as the enzyme bound to many inhibitors, including those in clinical use, have been obtained at high resolution (see for example, refs 1-11). Several HIV-1 protease inhibitors are used in antiretroviral therapies and have shown significant promise in combination regimes that include reverse transcriptase inhibitors or several protease inhibitors. A major limiting factor in the treatment of HIV-1 infection has been the emergence of viral strains that exhibit resistance to protease inhibitors (5, 12-18). The loss of sensitivity usually occurs because the resistant viral strains encode for protease molecules containing specific amino acid mutations that lower the affinity for the inhibitors, yet maintain sufficient affinity for the substrate. For some mutations, the affinity toward the inhibitor might decrease by up to 3 orders of magnitude, while the K m for the substrate changes by less than 1 order of magnitude (5, 7, 19). More than 87 mutations have been observed in at least 47 positions within the HIV-1 protease monomer and shown to express resistance toward one or more inhibitors (20). These mutations have been classified as active site and nonactive site (21). Active site mutations usually do not involve residues that are directly involved in catalysis, whereas nonactive site mutations are usually located at the hinge of the flap region, the dimer interface, and the beta sheet region (20, 22). Nonactive site mutations may interfere with binding through long-range interactions rather than direct interactions with inhibitors. The double mutation V82F/I84V has been shown to affect the protease inhibitors in clinical use: ritonavir, saquinavir, nelfinavir, indinavir, and amprenavir (22-24). This double mutation is located at the edges of the active site, distorting its wild-type geometry without changing its polarity or chemical composition. It is therefore important to investigate the thermodynamic origin of the mutation effect on the binding affinity of inhibitors. A detailed knowledge of this effect will provide important information and general rules for the design of inhibitors that are less susceptible to the effects of mutations. EXPERIMENTAL PROCEDURES Protease Purification. HIV-1 protease was prepared ac- cording to the following procedure optimized for the high yield, activity, and stability required for calorimetric analysis. ² Supported by grants from the National Institutes of Health GM 57144 and GM 51362. I.L. is a recipient of a postdoctoral fellowship from the Fundacio ´n Ramo ´n Areces, Madrid (Spain). A.V.C. was partially supported by a postdoctoral fellowship from the Universidad de Granada, Spain (Plan Propio 1,999). * To whom correspondence should be addressed. Phone: (410) 516- 7743; fax: (410) 516-6469; e-mail: ef@jhu.edu. 11876 Biochemistry 2000, 39, 11876-11883 10.1021/bi001013s CCC: $19.00 © 2000 American Chemical Society Published on Web 08/31/2000