Biomedical Engineering Reference
In-Depth Information
OH
N
O
OH
HO
N
O
N
N
S
O
N
N
O
O
O
O
NH
NH
2
Indinavir
Amprenavir
N
N
S
O
O
O
O
O
HO
HO
H
N
N
H
H
OH
OH
S
H
KNI-764
Nelinavir
H
CONH
2
N
O
OH
H
N
CF
3
H
O
O
N
O
NH
O
HN
S
N
O
O
Saquinavir
Tipranavir
FIGURE 2.8
Examples on HIV-1 protease inhibitors. Indinavir, neli navir, and saquinavir are examples
of i rst generation, amprenavir and KNI-764 of second generation, and tipranavir of third-generation HIV-1
protease inhibitors.
In order to circumvent this problem, inhibitors binding not only primarily due to hydrophobic
effects but also by hydrogen bonding to the enzyme backbone atoms (amide NH group and amide
carbonyl oxygen atom) were designed. These hydrogen-bonding contacts are not sensitive to muta-
tions, since they do not involve the side chains but only the backbone atoms. These second-generation
inhibitors like, e.g., amprenavir (Agenerase) and KNI-764 (Figure 2.8) showed signii cantly differ-
ent binding characteristics.
Thermodynamic determination of the enthalpy and entropy components to the free energy of
binding (
S
) can be determined by isothermal titration calorimetry (ITC), and this
method has led to a much more detailed understanding of the energetics associated with the process
of binding a ligand to a protein. By using ITC to guide the design of new inhibitors it has been pos-
sible to optimize the binding characteristics of these new inhibitors.
For the i rst-generation HIV-1 protease inhibitors, the majority of the free energy of binding
is due to an entropy gain associated with i lling hydrophobic pockets in the HIV-1 protease with
hydrophobic substituents on the inhibitor. The second-generation inhibitors were characterized by
both enthalpy and entropy now contributing to the free energy of binding, making the enzyme less
likely to develop resistance.
Recently, third-generation HIV-1 protease inhibitors have been developed based on the careful
optimization of the structural and energetic contributions to binding. Tipranavir (Aptivus) (Figure
2.8) is an example of an HIV-1 protease inhibitor with unique binding characteristics. It is a highly
potent inhibitor (
K
i
= 19 pM), which primarily binds to the wild-type HIV-1 protease due to entropy
effects. The unusually high binding entropy is most likely caused by release of buried water mol-
ecules from the active site of the HIV-1 protease. When binding to the multidrug-resistant HIV
mutants tipranavir only looses little in potency, because the reduction in binding entropy is compen-
sated by a gain in binding enthalpy.
In 1994, researchers at the DuPont Merck Pharmaceutical Company reported an important obser-
vation. They had realized that in most of the complexes between HIV-1 protease and the peptide-
like inhibitors a structural water molecule bridged the ligand and enzyme. They concluded that by
Δ
G
=
Δ
H − T
Δ
