External ID: FECH 20K screen

Protocol: We screened 20,231 compounds from two small molecule libraries as ferrochelatase inhibitors. Screening was done at 1 concentration (10microM) for this primary screen.
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Assay components:
Buffer: 166.7mM Tris-HCl pH 8.0 with 0.05% octyl glucoside (final concentrations)

FECH/NiCl solution: 3.33mM NiCl solution (final concentration in assay is 100microM) with 5microM FECH enzyme (final concentration in assay is 0.75 microM) in buffer.

Mesoporphyrin solution: 333microM Mesoporphyrin (final concentration in assay is 100microM), diluted from 1mM stock in buffer

Added compounds or DMSO dilutions to plate first, then added FECH/NiCl solution to wells. Finally, added the mesoporphyrin solution to start the reaction, mixed by tapping, spun plate and read absorbance at 550nm after 20 minutes. Ni2+ insertion into mesoporphyrin IX by ferrochelatase can be monitored as an increase in absorbance at 550nm.

Hits were scored as compounds that reduced the slope by more than 3 SD from DMSO controls on each plate.

Comment:

External ID: ST2_IL33_Inhibitors_Primary_Screening_77700

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.