KNI-272 is a powerful HIV-1 protease inhibitor
with a reported inhibition constant in the picomolar range.
In this paper, a complete experimental dissection of the
thermodynamic forces that define the binding affinity of
this inhibitor to the wild-type and drug-resistant mutant
V82F/I84V is presented. Unlike other protease inhibitors,
KNI-272 binds to the protease with a favorable binding
enthalpy. The origin of the favorable binding enthalpy
has been traced to the coupling of the binding reaction
to the burial of six water molecules. These bound water
molecules, previously identified by NMR studies, optimize
the atomic packing at the inhibitor/protein interface enhancing
van der Waals and other favorable interactions. These interactions
offset the unfavorable enthalpy usually associated with
the binding of hydrophobic molecules. The association constant
to the drug resistant mutant is 100–500 times weaker.
The decrease in binding affinity corresponds to an increase
in the Gibbs energy of binding of 3–3.5 kcal/mol,
which originates from less favorable enthalpy (1.7 kcal/mol
more positive) and entropy changes. Calorimetric binding
experiments performed as a function of pH and utilizing
buffers with different ionization enthalpies have permitted
the dissection 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 aspartates in the catalytic
aspartyl dyad in the protease and the isoquinoline nitrogen
in the inhibitor molecule. The binding affinity is maximal
between pH 5 and pH 6. At those pH values the affinity
is close to 6 × 1010 M−1
(Kd = 16 pM). Global analysis of the
data yield a buffer- and pH-independent binding enthalpy
of −6.3 kcal/mol. Under conditions in which the exchange
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 KNI-272 is both enthalpically
and entropically favorable. The structure-based thermodynamic
analysis indicates that the allophenylnorstatine nucleus
of KNI-272 provides an important scaffold for the design
of inhibitors that are less susceptible to resistant mutations.