In vitro activity: (R)-PFI-2 shows 500-fold more inhibitory activity against human SETD7 than its enantiomer, (S)-PFI-2. In a cellular environment, through direct interactions with SETD7, affects Yes-Associated protein localization and phenocopies Setd7 genetic deletion on Hippo pathway signaling.

Kinase Assay: The reaction mixtures containing 2 nM SETD7, 2 uM biotinylated peptide H3 (1-25) and 2 uM SAM in 20 mM Tris, pH 8, 5 mM DTT and 0.01% Triton X-100 were incubated for 1 hour at 23°C. Compound concentration was varied from 0.3 nM to 5 uM. All reactions (in 10 ul) started by adding 3H-SAM and stopped by addition of 10 ul of 7.5 M guanidine hydrochloride. 60 ul of buffer (20 mM Tris, pH 8) was added, mixed and transferred to a 384-well FlashPlate (Cat. # SMP410A001PK; Perkin Elmer; www.perkinelmer.com). The reaction mixtures in Flash plate were incubated for 1 hour and the CPM counts were measured using Topcount plate reader ((Perkin Elmer, www.perkinelmer.com). The CPM counts in the absence of compound for each data set were defined as 100% activity. In the absence of the enzyme, the CPM counts in each data set were defined as background (0%). The IC50 values were determined using GraphPad Prism 6 with Sigmoidal 4PL equation. Cell assay [1] Compound stability was checked in the media with and without cell. Samples were analyzed using Waters Synapt G2 S Quadruple Time-of-flight (Q-Tof) hybrid mass spectrometer (MS) system coupled with second generation ACQUITY ultra-performance liquid chromatography (UPLC-II). Chromatographic separations were carried out on an ACQUITY UPLC BEH C18 (2.1 X 50 mm, 1.7 um) column. The mobile phase was 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Compound concentrations were measured as relative peak areas at various time points.

Proc Natl Acad Sci U S A. 2014 Sep 2;111(35):12853-8.