High-throughput screening for menin-MBM1 inhibitors

FITC-MBM1 at 15 nM and menin at 150 nM in the FP buffer (Supplementary Table 1) were mixed and incubated for 1h in the dark at room temperature. A collection of 49,000 compounds from the Center for Chemical Genomics, University of Michigan, was used for HTS. For point screening, the 0.2 μL of each compound (20 μM final concentration, 1% DMSO) was added to 20 μL of the aliquot of the protein-peptide mixture and incubated on 384-well plates in the dark at room temperature for 1h. In confirmation screening, the serial dilution plates with compounds in DMSO were prepared and used to titrate the menin-FITC-MBM1 complex. Change in fluorescence polarization was monitored at 525 nm after excitations at 495 nm using the PHERAstar microplate reader (BMG) and applied to determine IC₅₀ values with the Origin 7.0 program.