Thermodynamic dissection of the binding energetics of KNT-272, a potent HIV-1 protease inhibitor

KNI-272 is a powerful HTV-1 protease inhibitor with a reported inhibition c onstant in the picomolar range. In this paper, a complete experimental diss ection of the thermodynamic forces that define the binding affinity of this inhibitor to the wild-type and drug-resistant mutant V82F/I84V is presente d. Unlike other protease inhibitors, KNI-272 binds to the protease with a f avorable binding enthalpy. The origin of the favorable binding enthalpy has been traced to the coupling of the binding reaction to the burial of six w ater molecules. These bound water molecules, previously identified by NMR s tudies, optimize the atomic packing at the inhibitor/protein interface enha ncing van der Waals and other favorable interactions. These interactions of fset the unfavorable enthalpy usually associated with the binding of hydrop hobic molecules. The association constant to the drug resistant mutant is 1 00-500 times weaker. The decrease in binding affinity corresponds to an inc rease in the Gibbs energy of binding of 3-3.5 kcal/mol, which originates fr om less favorable enthalpy (1.7 kcal/mol more positive) and entropy changes . Calorimetric binding experiments performed as a function of pH and utiliz ing buffers with different ionization enthalpies have permitted the dissect ion of proton linkage effects. According to these experiments, the binding of the inhibitor is linked to the protonation/deprotonation of two groups. in the uncomplexed form these groups have pKs of 6.0 and 4.8, and become 6. 6 and 2.9 in the complex. These groups have been identified as one of the a spartates in the catalytic aspartyl dyad in the protease and the isoquinoli ne nitrogen in the inhibitor molecule. The binding affinity is maximal betw een pH 5 and pH 6. At those pH values the affinity is close to 6 x 10(10) M -1 (K-d = 16 pM) Global analysis of the data yield a buffer- and pH-indepen dent binding enthalpy of -6.3 kcal/mol. Under conditions in which the excha nge of protons is zero, the Gibbs energy of binding is -14.7 kcal/mol from which a binding entropy of 28 cal/K mol is obtained. Thus, the binding of K NI-272 is both enthalpically and entropically favorable. The structure-base d thermodynamic analysis indicates that the allophenyl-norstatine nucleus o f KNI-272 provides an important scaffold for the design of inhibitors that are less susceptible to resistant mutations.