WDR5 Biochemical HTRF (Homogeneous Time Resolved Fluorescence) Assay. The assay was adapted and performed in 384-well white OptiPlate plate (PerkinElmer) for compound single dose (40 μM) or the dose response assay. A mixture of protein and peptide was added into the well and incubated for 20 min. Compounds in DMSO were dispensed at a 14-point, 3.16-fold dilution scheme with the top concentration of 75 μM. A mixture of HTRF detection antibodies was then added and incubated for 1h before plate reading (Perkin Elmer, Envision). The final assay component concentrations are 6 nM WDR5 (N-His, 1-334), 50 nM MYC MbIIIb peptide (256-268 a.a., QEDEEEIDVVSVE-GKK-Biotin-OH), 1.5 nM Eu-anti-His-Ab (PerkinElmer, AD0401) and 3 µg mL-1 Sreptavidin-Surelight APC (PerkinElmer, AD0201) in the assay buffer of 25 mM HEPES (pH = 7.5), 0.05% v/v Tween-20, 100 mM NaCl, 2 mM DTT and 0.1% w/v BSA. For the counter assay, protein pair of WDR5 and MYC was replaced with a biotin-PEG-PEG-6xHis peptide (GenScript), to evaluate if the compound interferes with elements of the assay format. This helps to characterize the specific activity of the compound against the PPI of WDR5 and MYC in HTRF. Dose response curves and half-maximal inhibitory concentration (IC50) values were generated by GraphPad Prism. The representative curves are based on the mean values from at least two independent experiments in triplicates.