Hit Confirmation and Data comparison with Novartis Diverse Library HTRF Screening results. We tested this 2029 compound set by 40 μM dose in WDR5-MYC PPI biochemical HTRF assay, with the similar assay condition used in the previous biochemical screening (Novartis diverse library HTRF screen) which led to the finding of WM-662 and Compound 1 , the known WBM pocket binding molecules. The compound activity distribution was presented in a dot plot (Figure ) that shows 7 compounds inhibited the assay signal greater than 40%, which was the same criteria we applied to select primary hits in the early experimental HTRF screen using the library of around 1.1 million compounds. WM-662 showing 70% inhibition was used as the positive control. Notably, our efforts leveraging Pocket Crafter on WDR5 have yielded encouraging outcome for the hit rate. Through the implementation of this integrated workflow, we have achieved a substantial 12.8-fold increase in the hit rate compared to the early diversity library HTRF screening of 1,101,793 compounds (Table ). This significant improvement highlights the effectiveness of our tailored compound generation strategy in enhancing the chance of identifying compounds as primary hits against the target protein. Biological Activity Profiling for WDR5 Hit Compounds. Next, we further conducted WDR5-MYC HTRF dose-response curve (DRC) study to analyze the single dose activity from the primary hits more quantitatively. Three out of the 7 primary hits (Figure ) showed good dose response curve fitting and the IC50s of 35.6, 27.5 and 28.5 μM respectively for compound PC-1, PC-2 and PC-3 (Figure ), reaffirming the in vitro potency of these hit compounds biochemically from single dose testing. Even though their activities are a little weaker than WM-662 or Compound 1 (18 or 14 μM) in this WDR5-MYC HTRF assay , they don't hit the HTRF counter assay as the flat black curves indicated in Figure , suggesting the true specificity of these compounds in disrupting WDR5 and MYC interaction. Moreover, we tested the binding of these hit compounds to WDR5 in an orthogonal biophysical assay. The direct binding of PC-1 and PC-2 to WDR5 was demonstrated by Differential Scanning Fluorimetry (DSF) assay (Figure ). Thermal shift analysis showed that adding PC-1 or PC-2 to WDR5 resulted in a negative Tm (melting temperature) shift of 4.2 °C or 1.2 °C in compound treated groups, compared to DMSO control. WM-662 was tested together as a binder control with the validated shift, which triggered the Tm shift of 3.2 °C. This Tm shift in DSF indicates that the PC-1 or PC-2 compound can work on WDR5 protein as the true binder from this biophysical readout. The negative Tm shift indicates that there might be an interaction between the compound and the protein that is destabilizing to the protein's tertiary structure , since positive or negative Tm shift is dependent on the binding energy of the ligand for the protein.