(R)-PFI-2 is a potent and selective inhibitor of SETD7 methyltransferase activity in cells

SET domain containing (lysine methyltransferase) 7 (SETD7) is implicated in multiple signaling and disease related pathways with a broad diversity of reported substrates. Here, we report the discovery of ( R )-PFI-2—a first-in-class, potent ( K i app = 0.33 nM), selective, and cell-active inhibitor of the methyltransferase activity of human SETD7—and its 500-fold less active enantiomer, ( S )-PFI-2. ( R )-PFI-2 exhibits an unusual cofactor-dependent and substrate-competitive inhibitory mechanism by occupying the substrate peptide binding groove of SETD7, including the catalytic lysine-binding channel, and by making direct contact with the donor methyl group of the cofactor, S -adenosylmethionine. Chemoproteomics experiments using a biotinylated derivative of ( R )-PFI-2 demonstrated dose-dependent competition for binding to endogenous SETD7 in MCF7 cells pretreated with ( R )-PFI-2. In murine embryonic fibroblasts, ( R )-PFI-2 treatment phenocopied the effects of Setd7 deficiency on Hippo pathway signaling, via modulation of the transcriptional coactivator Yes-associated protein (YAP) and regulation of YAP target genes. In confluent MCF7 cells, ( R )-PFI-2 rapidly altered YAP localization, suggesting continuous and dynamic regulation of YAP by the methyltransferase activity of SETD7. These data establish ( R )-PFI-2 and related compounds as a valuable tool-kit for the study of the diverse roles of SETD7 in cells and further validate protein methyltransferases as a druggable target class.