The higher barrier of darunavir and tipranavir resistance for HIV-1 protease

Abstract

Darunavir and tipranavir are two inhibitors that are active against multi-drug resistant (MDR) HIV-1 protease variants. In this study, the invitro inhibitory efficacy was tested against a MDR HIV-1 protease variant, MDR 769 82T, containing the drug resistance mutations of 46L/54V/82T/84V/90M. Crystallographic and enzymatic studies were performed to examine the mechanism of resistance and the relative maintenance of potency. The key findings are as follows: (i) The MDR protease exhibits decreased susceptibility to all nine HIV-1 protease inhibitors approved by the US Food and Drug Administration (FDA), among which darunavir and tipranavir are the most potent; (ii) the threonine 82 mutation on the protease greatly enhances drug resistance by altering the hydrophobicity of the binding pocket; (iii) darunavir or tipranavir binding facilitates closure of the wide-open flaps of the MDR protease; and (iv) the remaining potency of tipranavir may be preserved by stabilizing the flaps in the inhibitor-protease complex while darunavir maintains its potency by preserving protein main chain hydrogen bonds with the flexible P2 group. These results could provide new insights into drug design strategies to overcome multi-drug resistance of HIV-1 protease variants.

Fig. 1. Co-crystal structures of darunavir and tipranavir with the multi-drug resistant protease

(A) The tipranavir-MDR 769 82T structure (magenta) was superimposed on the tipranavir-wild-type (WT) HIV-1 protease structure (green). The I54V mutation is displayed in stick model. The magenta and green dashed lines indicate the distance (Å) between the inhibitor and the MDR HIV-1 protease or the WT HIV-1 protease, respectively. (B) The conformational changes of darunavir binding to MDR 769 82T (magenta) in comparison with the darunavir-WT HIV-1 protease complex (green). (C) The electron density of tipranavir bound to MDR 769 82T protease is shown as an F_o–F_o OMIT map contoured at 2σ. Two tipranavir molecules (yellow and magenta) binding in different orientation are presented. (D) The electron density of darunavir bound to MDR 769 82T protease is shown as an F_o–F_c OMIT map contoured at 2σ.

Fig. 2. Hydrogen bonds and hydrophobic interactions of tipranavir and darunavir with MDR 769 82T protease

Hydrogen bonds are shown in green dashed line. The residues that form hydrophobic interactions with tipranavir are illustrated by spokes. The interaction map was produced using LIGPLOT V4.5.3 [19].

Fig. 3. The tipranavir-MDR 768 82T complex showed higher denaturation temperature compared to the darunavir-MDR 769 82T complex

The X-axis is temperature in Celsius degree, and the Y-axis is fluorescence change in Relative Fluorescence Units (RFU).