Protocol:
High-throughput screening was performed at Indiana University screening facility (Indiana University, Indianapolis, IN).
In summary, a mixture of ST2 and IL-33 was prepared by adding 0.30 microL of 14.1 microM recombinant human ST2-Fc Chimera and 0.30 microL of 55.25 biotinylated recombinant human IL-33 to 1.4 mL of assay buffer. In the HTS, each well of a 384-well Proxi-plate was first blocked by 100 microL of assay buffer for 1 h at room temperature. After removing the blocking buffer, 20 microL of ST2/IL-33 mixture was pipetted into each well and incubated at room temperature for 1 h followed by addition of compounds at 17 microM (0.8 microL) to each well and incubated at room temperature for 1 h. Ten microliters of 60 microg/mL anti-6xHis-conjugated AlphaLISA acceptor beads were added to each well and incubated at room temperature for 1 h before addition of 10 microL of 60 microg/mL of streptavidin-labeled AlphaLISA donor beads for incubation at room temperature for 30 min. Incubation of acceptor and receptor beads was conducted in darkness. The well with 3 nM ST2 alone was used as a negative control, whereas the ST2/IL-33 mixture with a human ST2 antibody added at 0.45 ng/microL was used as the positive control. Plates were read using the Envision Plate Reader. The buffer used in the assay contains 14.37 mL PBS, 30 microL Tween 20, and 600 microL of 5% bovine serum albumin in PBS.
Comment:
PubChem active indicates >= 30% inhibition of ST2/IL-33 at 17 uM of the compound. Inconclusive: >=10% and < 30% inhibition.
% Inhibition
98
97
97
97
97
97
96
96
96
96
95
95
94
94
93
92
92
89
88
85
HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader -
7071-02_Inhibitor_Dose_DryPowder_Activity_Set16
来源：Center for Chemical Genomics, University of Michigan靶标：
External ID: MScreen:TargetID_600
Protocol:
C-terminally 6xHis tagged CDK2/Cyclin A complex and N-terminally Flag tagged CDC25B C473S (372-566) were expressed and purified. Proteins were incubated together at a final concentration of 125 nM each for 1 hr prior to incubation with compound for 1 hr, followed by addition of anti-6xHis europium cryptate donor beads (Cisbio) and anti-Flag XL-665 acceptor beads (Cisbio) at a final dilution of 1:350 for 1 hr. 20mM potassium fluoride was added 10 minute prior to plate reading. Assay reagents were dispensed using a multidrop liquid dispenser (Thermo Scientific) onto uncoated, black, low-volume, 384-well plates (Corning). Assay plates were quantified using an Envision plate reader (Perkin-Elmer) with excitation of the europium crytate donor at 337 nm wavelength and emission of the donor at 620 nm and emission of the XL-665 acceptor at 665 nm in 18 uL volumes. Assays were performed in a buffer containing 50 mM Tris (pH 7.5), 50 mM NaCl, 10mM MgCl2, 1mM TCEP, with addition of and 1mM ATP, 0.05% BSA, and 0.05% Tween-20 immediately prior to the start of the assay.
Comment:
The activity outcome is based on a Z-score (number of standard deviations from the negative control mean) of 3 or higher on at least 50% times that the sample was screened. For instance, if the sample was screened in n=4 runs, it would be considered active only if it had a Z-score of 3 or above in at least 2 runs.
This screen was funded by NIH grant number: R01CA181185