HSL Enzyme Assay: HSL enzyme activity was measured by a colorimetric assay using 2,3-dimercapto-1-propanol tributyrate (Aldrich, St. Louis, Mo.) as a substrate. Typically, 1.5 mM 2,3-dimercapto-1-propanol tributyrate (DMPT) in 100 mM MOPS, pH 7.2, 0.2 mg/ml fatty acid-free BSA was prepared by sonication at 4° C. to homogenous suspension. Test compounds (2 mM stock in DMSO) were diluted 3 fold in series in DMSO. Compound solutions were diluted 24 fold in 1.5 mM DMPT containing solution and 18 ul per well was added to 384-well microplates (Corning Costar). Twelve microliters per well of human HSL (15 ug/ml) was added and the reaction mixture was incubated at 37° C. for 20 minutes. Six microliters of 12 mM dithio-bis-(2-nitrobenzoic acid) (DTNB) in DMSO plus 1.2% SDS and 0.6% Triton X-100 were added and the mixture was incubated at room temperature for 15 minutes. Product production was monitored by reading absorbance at 405 nm on an Envision Reader (PerkinElmer Life and Analytical Sciences, Shelton, Conn.).
Alphascreen Assay: The AlphaScreen assay was performed according to the manufacturer's protocol (Perkin Elmer, Benelux). Reactions were performed in 25 ul final volume in 384-well Optiwell microtiter plates (Perkin Elmer). The reaction buffer contained 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM MgCl2, 0.01% (v/v) Tween-20 and 0.1% (w/v) bovine serum albumin. His6-tagged integrase (300 nM final concentration) was incubated with the compounds for 30 min at 4C. The compounds were added at varying concentrations spanning a wide range from 0.1 up to 100 uM. Afterwards 100 nM flag-LEDGF/p75 was added and incubation was prolonged for an additional hour at 4C. Subsequently 5 ul of Ni-chelate-coated acceptor beads and 5 ul anti-flag donor beads were added to a final concentration of 20 ug/ml of both beads. Proteins and beads were incubated for 1 h at 30C. in order to allow association to occur.
Enzyme Inhibition Assay: The enzyme inhibitory activity (Ki) was determined according to an assay protocol reported by Toth and Marshall (Toth, M. V.; Marshall, G. R. Int. J. Pep. Protein Res. 1990, 36, 544-550).