In vitro activity: PFI-3 shows a significant selectivity for PB1(5) and SMARCA2/4 over 36 other tested kinases.

Kinase Assay: Using recombinant purified bromodomains, we discovered that PFI-3 binds with similar avidity to both the short and long isoform of SMARCA2 revealing that the alternatively-spliced 18 amino acid insert does not impair PFI-3 binding. Moreover, profiling against 32 bromodomains at DiscoveRx confirmed exquisite selectivity vs. other subfamilies expanding the PFI-3 selectivity information obtained using differential scanning fluorimetry (DSF). In summary, we find there is a good concordance between the ligand competition (BROMOScan) and the direct biophysical binding (DSF) assays and note that in addition to targeting SMARCA2/4, PFI-3 also has activity (~70% inhibition at 2 uM) against the structurally related fifth bromodomain from PBRM1, another SWI/SNF subunit.

Cell Assay: In cell-based chromatin binding assays, using in situ cell extraction techniques to remove non-chromatin bound proteins, we observed dose-dependent displacement of GFP-tagged SMARCA2 bromodomain (i.e. 132 amino acid residues) by PFI-3. Notably, the inhibitor showed prolonged cell-target engagement with similar potency (IC50 = 5.78 uM) following 2 and 24 hours treatment. As a negative control, JQ1 did not inhibit the binding of ectopically expressed SMARCA2 bromodomain, but selectively displaced GFP-tagged BRD4 (data not shown). Taken together, our cell-biochemical data cooperate the accelerated fluorescence recovery after photobleaching (FRAP) reported for PFI-3 and we conclude that PFI-3 is a selective, cell-permeable probe suitable to study the inhibition of SMARCA2/4 bromodomains in cells.

Cancer Res. 2015 Sep 15; 75(18): 3865–3878.