Figure 4:

uHTS of a 138k compound library identifies MSN–CD44 small-molecule PPI inhibitors. (A) Scatter plot showing percentage inhibition with 138,214 compound library in the MSN–CD44 TR-FRET assay. Primary hit rate is 0.2%, with 271 compounds giving >50% inhibition. (B) Screening flow chart showing hit-to-lead identification strategy. (C) Structures of lead compounds based upon different chemical scaffolds. (D) Confirmatory TR-FRET dose response curves. (E) Dose response of compounds in GST pull down assay. Cell lysates were obtained from cells transfected with full length constructs of GST-tagged MSN and Flag-tagged CD44, and were incubated with the compounds. Immunoblotting was performed to detect GST and Flag. (F) Thermal shift assay, showing melting temperature (T_m) curves of MSN with compounds. The average change in T_m (av. ΔT_m), compared to DMSO alone, was determined from two experiments (1: 1.1 °C at 20 μM; 2: 1.9 °C at 20 μM). (G) Dose response of compound binding to MSN FERM domain, measured by SPR. K_D measurements were determined for compounds 1 (4.2 μM) and 2 (0.7 μM). RU=response units (H) Dose response of compounds in cell-based NanoBiT bioluminescence assay, with HEK293 cells expressing LgBiT-MSN(T588D) and SmBit-CD44 fusions. Luminescence from reconstituted split luciferase was measured after substrate addition. Activities were determined from 2 independent experiments (1: 0.57±0.17 and 0.69±0.12 μM; 2: 0.18±0.04 and 0.19±0.06 μM). (I) Solubility of compounds (PBS buffer, 1% DMSO), as measured by nephelometry. RNU=relative nephelometric units.