Extreme entropy-enthalpy compensation in a drug-resistant variant of HIV-1 protease

Abstract

The development of HIV-1 protease inhibitors has been the historic paradigm of rational structure-based drug design, where structural and thermodynamic analyses have assisted in the discovery of novel inhibitors. While the total enthalpy and entropy change upon binding determine the affinity, often the thermodynamics are considered in terms of inhibitor properties only. In the current study, profound changes are observed in the binding thermodynamics of a drug-resistant variant compared to wild-type HIV-1 protease, irrespective of the inhibitor bound. This variant (Flap+) has a combination of flap and active site mutations and exhibits extremely large entropy-enthalpy compensation compared to wild-type protease, 5-15 kcal/mol, while losing only 1-3 kcal/mol in total binding free energy for any of six FDA-approved inhibitors. Although entropy-enthalpy compensation has been previously observed for a variety of systems, never have changes of this magnitude been reported. The co-crystal structures of Flap+ protease with four of the inhibitors were determined and compared with complexes of both the wild-type protease and another drug-resistant variant that does not exhibit this energetic compensation. Structural changes conserved across the Flap+ complexes, which are more pronounced for the flaps covering the active site, likely contribute to the thermodynamic compensation. The finding that drug-resistant mutations can profoundly modulate the relative thermodynamic properties of a therapeutic target independent of the inhibitor presents a new challenge for rational drug design.

Figure 1

Structure of inhibitors and HIV-1 protease. (a) Chemical structures of inhibitors. (b) Overview of the mutation sites of Flap+ and Act mutants mapped on an HIV-1 protease dimer. The monomers are distinguished in cyan and magenta, while the inhibitor ATV is shown in yellow stick model. The mutation sites of Flap+ (L10I/G48V/I54V/V82A) and Act (V82T/I84V) along with the site of the natural polymorphism L63P are highlighted in red and green stick models.

Figure 2

Thermodynamics of inhibitor binding. Differences in binding energetics between (a) WT and Flap+ and (b) WT and Act variants. The differences in ΔG, TΔS and ΔH are shown in green, red and blue, respectively.

Figure 3

Structural deviation between different inhibitor complexes. (a) Distribution of root mean squared deviations (RMSD) in Cα coordinates between the four inhibitor complexes of Flap+ (black), WT (blue) and Act (green) variants. The RMSD in Cα coordinates in (b) Flap+, (c) WT and (d) Act mapped on an HIV-1 protease dimer model. The color code for distinguishing the RMSD values are: blue, 0–0.25 Å; purple, 0.25–0.5 Å; red, 0.5–0.65 Å; yellow, 0.65–0.8 Å; white, 0.8 Å and above.

Figure 3

Structural deviation between different inhibitor complexes. (a) Distribution of root mean squared deviations (RMSD) in Cα coordinates between the four inhibitor complexes of Flap+ (black), WT (blue) and Act (green) variants. The RMSD in Cα coordinates in (b) Flap+, (c) WT and (d) Act mapped on an HIV-1 protease dimer model. The color code for distinguishing the RMSD values are: blue, 0–0.25 Å; purple, 0.25–0.5 Å; red, 0.5–0.65 Å; yellow, 0.65–0.8 Å; white, 0.8 Å and above.

Figure 4

Binding of inhibitors in HIV-1 protease complex structures. The active site region (Asp25–Asp30), flaps (Lys45–Lys55) and the inhibitors of (a) Flap+, (b) WT and (c) Act are superposed and illustrated as stereo pairs. The complexes involving APV, ATV, DRV and SQV are distinguished in red, green, cyan and magenta, respectively.

Figure 5

Comparison of mutant complex structures with WT HIV-1 protease. Double difference plots (see Methods) illustrating the WT vs Flap+ structural changes in (a) APV, (b) ATV, (c) DRV and (d) SQV, and WT vs Act structural changes in (e) APV, (f) ATV, (g) DRV, (h) SQV. The key for contours: (i) black −5.0 to −1.0 Å and green −1.0 to −0.5Å (Corresponding residue distances in the mutant structures have increased); (ii) blue 0.5 to 1.0 Å and magenta 1.0 to 5.0 Å (Corresponding distances in the mutant structures have decreased).

Figure 6

Packing around the bound inhibitor in WT and MDR protease variants. (a) Total inhibitor-protease van der Waals interaction energies for WT (gray), Act (white) and Flap+ (black). Residue-wise distribution of interaction energy is shown in (b) APV, (c) ATV, (d) DRV and (e) SQV. The distribution of energies in the WT complexes are shown in the upper panels while the lower panels illustrate the WT vs Flap+ (black) and WT vs Act (white) differences in energy distribution.