| Assay Method Information | |
| | Ki Determination for Genotypes 1b and 3a NS3 Protease |
| Description: | Purified NS3 protease domain (amino acids 1-181) of the genotype 1b and 3a virus were generated as above. The internally quenched fluorogenic depsipeptide substrate Ac-DED(Edans)-EEAbuΨ[COO]ASK(Dabcyl)-NH2 and a synthetic peptide containing the hydrophobic core residues of the NS4A protein cofactor (KKGSVVIVGRIILSGRKK; NS4A peptide) were obtained from Anaspec, Inc. (San Jose, Calif.). Other chemicals and biochemicals were of reagent grade or better and were purchased from standard suppliers.Reactions were run at room temperature in buffer consisting of 50 mM HEPES, 40% glycerol, 0.05% Triton X-100, 10 mM DTT, and 10% DMSO. The final assay solutions contained 50 pM NS3 genotype 1 b protease or 200 pM genotype 3a protease, 20 μM NS4A peptide, and 4 μM substrate (genotype 1b) or 2 μM substrate (genotype 3a). Inhibitor concentrations varied from 100 nM to 5 pM in 3-fold dilutions, and no-inhibitor controls were included.Compound dilutions were made in DMSO at 20× final concentration. Reaction mixtures were prepared in 96-well assay plates. A solution of enzyme and NS4A peptide in assay buffer (25 μL volume with both reagents at 4× final concentration) was mixed with 45 μL assay buffer and 5 μL of either inhibitor or DMSO, and pre-incubated at room temperature for 1 hour. The reaction was started by addition of 25 μL substrate solution at 4× final concentration. Plates were mixed vigorously for 5-10 seconds and reactions were allowed to proceed for 90 minutes, fluorescence was measured every 30 s between 90 and 120 minutes reaction time using a Tecan InfiniTe M1000 or PerkinElmer Envision multimode plate reader with an excitation wavelength of 340 nm and an emission wavelength of 490 nm.Rates were calculated from the progress curves at steady state, in the time frame of 90-120 minutes after addition of substrate. To determine the Ki, rates were plotted as a function of inhibitor concentration, and the data were fit with equation 1 (Morrison, J. F., Biochimica et Biophysica Acta 1969, 185, 269-286) to calculate Ki app using GraphPad Prism 5. Active fraction of enzyme was determined by active site titration with known potent inhibitors. Ki was calculated from Ki app/(1+[[S]/Km]). |
| Affinity data for this assay | |
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