Figure 3: Kinetic analysis of HIV-1 PR reactivation by inhibitor photodegradation.

(a) A non-linear fit of Morrison equation of inhibition of HIV-1 PR by compound 1. The activity of purified recombinant HIV-1 PR was determined in vitro as described in the experimental section in the presence of the indicated inhibitor concentrations. Two independent experiments yielded very similar results; (b,c) Reactivation of purified recombinant HIV-1 PR in buffer (100 mM sodium acetate, 300 mM NaCl, 4 mM EDTA, pH 4.7) by photodegradation of 1 using either the cuvette set up (b) or the capillary set up (c): (b) Purified recombinant HIV-1 PR (8 nM) incubated with compound 1 at the indicated concentrations was irradiated with two 405 nm lasers (combined output of 300 mW) for various time intervals. The PR activity was then measured using a chromogenic substrate. The plot shows relative PR activity as a function of time. (c) Purified recombinant HIV-1 PR (160 nM) incubated with 2 μM compound 1 was pumped at different flow rates through a thin glass capillary onto which two 405 nm lasers (combined output of 300 mW) were focused (for set up see Supplementary Fig. 4). Relative PR activity was determined as in b after 20-fold dilution into cleavage buffer using the same chromogenic substrate (for details, see Experimental section) and plotted against the flow rate of the sample through the capillary. Flow rate 0 represents a non-irradiated sample. The graph shows mean values and s.d. from three independent experiments.