Figure 7.

A comparison of 20 different hits ranked by Δδ₅₀ by PrOF NMR and different assay data (signal reduction in ¹H CPMG in panel A or PrOF NMR chemical shift perturbation in panel B). A correlation is noticeable between Δδ₅₀ and ¹H CPMG signal drop, as well as Δδ₅₀ and W81 chemical shift perturbation. While one could expect broadening of W81 to also correlate well, with Brd4 this is not the case. Using a protein with a lower ligandability could result in better rank-ordering with broadening, but with Brd4 many of the tighter binders are already coalescing at 400 μM ligand.