The Km and Ki Assays were carried out as previously described40 (link), 41 (link). In the Km assay, a 10-amino acid substrate containing the natural MA/CA cut site with an EDANS/DABCYL FRET pair was dissolved in 8% DMSO at 40nM and 6% DMSO at 30 nM. The 30 nM of substrate was 4/5th serially diluted from 30 nM to 6 nM, including a 0 nM control. HIV protease was diluted to 120 nM and, using a PerkinElmer Envision plate reader, 5 μL was added to the 96-well plate to obtain a final concentration of 10 nM. The fluorescence was observed with an excitation at 340 nm and emission at 492 nm and monitored for 200 counts, for approximately 23 minutes. FRET inner filter effect correction was applied as previously described56 (link). Corrected data was analyzed with Prism7.
To determine the Ki, in a 96-well plate, each inhibitor was 2/3 serially diluted from 3000 pM to 52 pM, including a 0 pM control, and incubated with 0.35 nM protein for 1 hour. A 10-amino acid substrate containing an optimized protease cut site with an EDANS/DABCYL FRET pair was dissolved in 4% DMSO at 120 μM. Using the Envision plate reader, 5 μL of the 120 μM substrate was added to the 96-well plate to a final concentration of 10 μM. The fluorescence was observed with an excitation at 340 nm and emission at 492 nm and monitored for 200 counts, for approximately 60 minutes. Data was analyzed with Prism7.
To determine the Ki, in a 96-well plate, each inhibitor was 2/3 serially diluted from 3000 pM to 52 pM, including a 0 pM control, and incubated with 0.35 nM protein for 1 hour. A 10-amino acid substrate containing an optimized protease cut site with an EDANS/DABCYL FRET pair was dissolved in 4% DMSO at 120 μM. Using the Envision plate reader, 5 μL of the 120 μM substrate was added to the 96-well plate to a final concentration of 10 μM. The fluorescence was observed with an excitation at 340 nm and emission at 492 nm and monitored for 200 counts, for approximately 60 minutes. Data was analyzed with Prism7.
