Binding kinetics of darunavir to human immunodeficiency virus type 1 protease explain the potent antiviral activity and high genetic barrier

Abstract

The high incidence of cross-resistance between human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) limits their sequential use. This necessitates the development of PIs with a high genetic barrier and a broad spectrum of activity against PI-resistant HIV, such as tipranavir and darunavir (TMC114). We performed a surface plasmon resonance-based kinetic study to investigate the impact of PI resistance-associated mutations on the protease binding of five PIs used clinically: amprenavir, atazanavir, darunavir, lopinavir, and tipranavir. With wild-type protease, the binding affinity of darunavir was more than 100-fold higher than with the other PIs, due to a very slow dissociation rate. Consequently, the dissociative half-life of darunavir was much higher (>240 h) than that of the other PIs, including darunavir's structural analogue amprenavir. The influence of protease mutations on the binding kinetics was tested with five multidrug-resistant (MDR) proteases derived from clinical isolates harboring 10 to 14 PI resistance-associated mutations with a decreased susceptibility to various PIs. In general, all PIs bound to the MDR proteases with lower binding affinities, caused mainly by a faster dissociation rate. For amprenavir, atazanavir, lopinavir, and tipranavir, the decrease in affinity with MDR proteases resulted in reduced antiviral activity. For darunavir, however, a nearly 1,000-fold decrease in binding affinity did not translate into a weaker antiviral activity; a further decrease in affinity was required for the reduced antiviral effect. These observations provide a mechanistic explanation for darunavir's potent antiviral activity and high genetic barrier to the development of resistance.

FIG. 1.

Overlay of the experimental sensorgrams of the interaction between WT protease and its inhibitors at a concentration of 40 nM (ATV, IDV, LPV, NFV, RTV, SQV, and TPV) or 50 nM (APV and DRV). The sensorgrams were normalized to a maximum response of 1. For clarity, sensorgrams are spread over two graphs, with DRV included in both graphs as a reference point.

FIG. 2.

WT protease interaction kinetic map for all currently used PIs shown as k_on and k_off values and the combinations of k_on and k_off that result in the same K_D values (diagonal lines). The area beyond the detection limits due to biophysical and instrument limitations is shaded.

FIG. 3.

Interaction kinetic map of binding studies between APV, ATV, DRV, LPV, and TPV on WT and MDR proteases. The area beyond the detection limits due to biophysical and instrument limitations is shaded.

FIG. 4.

Relationship between binding affinity (K_D) and antiviral activity (EC₅₀) of PIs to HIV-1 MDR proteases.