Overcoming drug resistance in HIV-1 chemotherapy: the binding thermodynamics of Amprenavir and TMC-126 to wild-type and drug-resistant mutants of the HIV-1 protease

Abstract

Amprenavir is one of six protease inhibitors presently approved for clinical use in the therapeutic treatment of AIDS. Biochemical and clinical studies have shown that, unlike other inhibitors, Amprenavir is severely affected by the protease mutation I50V, located in the flap region of the enzyme. TMC-126 is a second-generation inhibitor, chemically related to Amprenavir, with a reported extremely low susceptibility to existing resistant mutations including I50V. In this paper, we have studied the thermodynamic and molecular origin of the response of these two inhibitors to the I50V mutation and the double active-site mutation V82F/I84V that affects all existing clinical inhibitors. Amprenavir binds to the wild-type HIV-1 protease with high affinity (5.0 x 10(9) M(-1) or 200 pM) in a process equally favored by enthalpic and entropic contributions. The mutations I50V and V82F/I84V lower the binding affinity of Amprenavir by a factor of 147 and 104, respectively. TMC-126, on the other hand, binds to the wild-type protease with extremely high binding affinity (2.6 x 10(11) M(-1) or 3.9 pM) in a process in which enthalpic contributions overpower entropic contributions by almost a factor of 4. The mutations I50V and V82F/I84V lower the binding affinity of TMC-126 by only a factor of 16 and 11, respectively, indicating that the binding affinity of TMC-126 to the drug-resistant mutants is still higher than the affinity of Amprenavir to the wild-type protease. Analysis of the data for TMC-126 and KNI-764, another second-generation inhibitor, indicates that their low susceptibility to mutations is caused by their ability to compensate for the loss of interactions with the mutated target by a more favorable entropy of binding.

Fig. 1.

The structure of the HIV-1 protease indicating the location of the I50V (yellow) and V82F/I84V (red) inhibitor resistant mutations.

Fig. 2.

The chemical structures of Amprenavir and TMC-126.

Fig. 3.

Calorimetric titrations of wild-type HIV-1 protease with Amprenavir (left panel), acetyl pepstatin (center panel), and with Amprenavir in the presence of acetyl pepstatin (displacement titration; right panel). These experiments were performed at 25°C in 10 mM acetate, pH 5, 2% DMSO. In all experiments the protease was in the calorimeter reaction cell and the inhibitors in the injection syringe. The inhibitor was added in stepwise injections of 10 μL. For each experiment the reactant concentrations were: (left panel) 23.7 μM protease, 250 μM Amprenavir; (center panel) 18.9 μM protease, 300 μM acetyl pepstatin; (right panel) 19.1 μM protease, 300 μM acetyl pepstatin, 250 μM Amprenavir.

Fig. 4.

Calorimetric titrations of wild-type HIV-1 protease with the inhibitor TMC-126 (left panel), the inhibitor acetyl pepstatin (center), and with TMC-126 in the presence of acetyl pepstatin (displacement titration; right panel). These experiments were performed at 25°C in 10 mM acetate, pH 5, 2% DMSO. In all experiments the protease was in the calorimeter reaction cell and the inhibitors in the injection syringe. The inhibitor was added in stepwise injections of 10 μL. For each experiment the reactant concentrations were: (left panel) 6.4 μM protease, 84 μM TMC-126; (center panel) 18.9 μM protease, 300 μM acetyl pepstatin; (right panel) 6.6 μM protease, 400 μM acetyl pepstatin, 53 μM TMC-126.

Fig. 5.

Dissection of the binding energetics (ΔG = ΔH − TΔS) of five HIV-1 protease inhibitors presently in clinical use (Indinavir, Nelfinavir, Saquinavir, Ritonavir, Amprenavir) and two second-generation inhibitors (KNI-764 and TMC-126) to wild-type HIV-1 protease. In this figure, (solid bars) ΔG; (hatched bars) −TΔS; (cross-hatched bars) ΔH. The data for Indinavir, Nelfinavir, Saquinavir, and Ritonavir were reported in Todd et al. (2000), and the data for KNI-764 in Velazquez-Campoy et al. (2001a).

Fig. 6.

Thermodynamic dissection of the effects of the inhibitor-resistant mutation V82F/I84V on the binding affinity of five HIV-1 protease inhibitors currently in clinical use (Indinavir, Nelfinavir, Saquinavir, Ritonavir, Amprenavir) and two second-generation inhibitors KNI-764 and TMC-126. In this figure, solid bars, ΔΔG; hatched bars, −TΔΔS; cross-hatched bars, ΔΔH. ΔΔX values are in reference to values obtained with the wild-type HIV-1 protease. The data for Indinavir, Nelfinavir, Saquinavir, and Ritonavir were reported in Todd et al. (2000), and the data for KNI-764 in Velazquez-Campoy et al. (2001a).

Fig. 7.

The binding-site cavity of the wild-type (left) and V82F/I84V (right) drug-resistant mutation. Indicated by a yellow arrow is the region where the bulge created by the phenylalanine ring at the edge of the cavity is located. The V82F/I84V mutation as well as the I50V mutation do not change the polarity or hydrophobic character of the binding site as indicated by the color scheme: (white) nonpolar, (green) polar, (blue) positive charge, (red) negative charge. These mutations only affect the geometry of the binding site. The figure was made with the program VMD (Humphrey et al. 1996).