Eleven different recombinant, drug-resistant HIV-1 protease (HIV PR) mutants--R8Q, V32I, M46I, V82A, V82F, V82I, I84V, V32I/I84V, M46I/V82F, M46I/I84V, and V32I/K45I/F53L/A71V/I84V/L89M--were generated on the basis of results of in vitro selection experiments using the inhibitors A-77003, A-84538, and KNI-272. Kinetic parameters of mutant and wild-type (WT) enzymes were measured along with inhibition constants (Ki) toward the inhibitors A-77003, A-84538, KNI-272, L-735,524, and Ro31-8959. The catalytic efficiency, kcat/Km, for the mutants decreased relative to WT by a factor of 1.2-14.8 and was mainly due to the elevation of Km. The effects of specific mutations on Ki values were unique with respect to both inhibitor and mutant enzyme. A new property, termed vitality, defined as the ratio (Kikcat/Km)mutant/(Kikcat/Km)WT was introduced to compare the selective advantage of different mutants in the presence of a given inhibitor. High vitality values were generally observed with mutations that emerged during in vitro selection studies. The kinetic model along with the panel of mutants described here should be useful for evaluating and predicting patterns of resistance for HIV PR inhibitors and may aid in the selection of inhibitor combinations to combat drug resistance.

The catalytic activity and inhibitor binding energetics of enzymes are often pH-dependent properties. Aspartic proteases comprise an important class of enzyme targets for structure-based drug design. We have performed a complete thermodynamic study of pepstatin binding to plasmepsin II, an aspartic proteinase found in Plasmodium falciparum, using isothermal titration calorimetry and circular dichroism. Thermodynamic parameters (DeltaG, DeltaH, DeltaCp, and DeltaS) were measured as functions of both pH and temperature. In the pH range from 4.5 to 7.0, pepstatin binding is accompanied by proton transfer between the solvent and the complex. We used thermodynamic proton linkage theory to derive both the pH-independent binding energetics for pepstatin and the number and pKa values of ionizable residues whose pKa values change during ligand binding. These residues were identified as the two catalytic aspartates, with pKas of 6.5 and 3.0, and His 164, with a pKa of 7.5, based on the three-dimensional structure of the pepstatin-plasmepsin II complex. At pH 5.0, where the protease has optimum activity, the proton transfer process contributes almost 40% of the total binding free energy change and the total charge of the active-site aspartic acid residues is -1. These experimental results provide direct measurement for the protonation states of the catalytic aspartates in the presence of bound ligands. Comparison of the thermodynamic and structural data for pepstatin binding with human cathepsin D, a lysosomal aspartic protease that shares 35% sequence identity with plasmepsin II, suggests that the energetic differences between these two proteins are due to a higher interdomain flexibility in plasmepsin II.

Cathepsin D is a lysosomal aspartic proteinase that has been implicated in several pathological processes such as breast cancer and Alzheimer's disease. We designed and synthesized a number of quenched fluorogenic substrates with P2 variations in the series AcEE(EDANS)KPIXFFRLGK(DA-BCYL)E-NH2, where X = cysteine, methylcysteine, ethylcysteine, fert-butylcysteine, carboxymethylcysteine, methionine, valine or isoleucine. Most of the fluorogenic substrates exhibited greater k cat IK m ratios than the best cathepsin D substrates described so far. Differences in kinetic constants, which were rationalized using structure-based modeling, might make certain substrates useful for particular applications, such as active site titrations or initial velocity determination using a fluorescent plate reader.

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