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.

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