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. 2014 Sep 2;111(35):12853-8.
doi: 10.1073/pnas.1407358111. Epub 2014 Aug 18.

(R)-PFI-2 Is a Potent and Selective Inhibitor of SETD7 Methyltransferase Activity in Cells

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Free PMC article

(R)-PFI-2 Is a Potent and Selective Inhibitor of SETD7 Methyltransferase Activity in Cells

Dalia Barsyte-Lovejoy et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

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 (Ki (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.

Keywords: chemical biology; chemical probe; epigenetics.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
(R)-PFI-2 is a potent inhibitor of SETD7. (A) Chemical structures of SETD7 inhibitors (R)-PFI-2 and its less-active enantiomer (S)-PFI-2. (B) The effect of (R)-PFI-2 (●) and (S)-PFI-2 (▲) on methyltransferase activity of SETD7. Compounds inhibited SETD7 activity with IC50 values of 2.0 ± 0.2 nM (Hill slope, 0.8) and 1.0 ± 0.1 µM (Hill slope: 0.7), respectively. All experiments were performed in quadruplicate. (C) Effect of (R)-PFI-2 on activity of 18 different protein methyltransferases [(red filled circle) G9a, (blue filled square) EZH2, (green filled triangle) EHMT1, SUV39H2, EZH1, SUV420H1, SUV420H2, SETD8, SETD2, PRMT1, PRMT3, PRMT5, PRMT8, SETDB1, MLL1, DOT1L, WHSC1, and SMYD2] and DNMT1 was assessed using as high as 50 µM (R)-PFI-2. Experiments were performed in triplicate.
Fig. 2.
Fig. 2.
Structure and binding mode of (R)-PFI-2. (A) Molecular graphics of the 1.9-Å crystal structure of (R)-PFI-2 (magenta) bound within the substrate peptide-binding groove of human SETD7 (surface representation in gray). The cofactor, SAM, is in yellow. (B) Detailed interactions between SETD7 (green) and (R)-PFI-2 (magenta). Hydrogen bonds are shown as dashed lines. (C) Superimposition of (R)-PFI-2 with the SETD7-bound conformation of a substrate histone peptide (PDB ID code 1O9S) (28), showing partial occupation of the peptide-binding site by (R)-PFI-2. (D) Superimposition of (R)-PFI-2bound SETD7 (green) with all 23 SETD7 structures in complex with cofactor (or cofactor mimic, sinefungin) available from the PDB (gray), showing the conformational variability of the post-SET loop. SETD7 residues that form H-bonds to (R)-PFI-2 are shown in green and are located in the less conformationally variable region of the protein. (E) Surface representation of (R)-PFI-2–bound SETD7 highlighting an induced conformation of the post-SET loop (green) that is not seen in any of the other SETD7 structures. (F) Molecular surface representation of SAM (yellow) and (R)-PFI-2 (purple), highlighting hydrophobic interactions between the methyl group of SAM and the pyrrolidine moiety of the inhibitor.
Fig. 3.
Fig. 3.
(R)-PFI-2 is a SAM-dependent inhibitor of SETD7. (A) Biacore SPR sensorgram of (R)-PFI-2 and SAM binding to SETD7. Three single-cycle kinetics runs with five concentrations were performed. Five injections of 20 µM SAM show consistent binding with rapid on and off kinetics (green); 20 µM SAM is expected to be saturating as the KD of SAM for SETD7 was determined to be 1.1 µM under these conditions. In the absence of SAM, a five-point dilution series of (R)-PFI-2 from 7.8 nM to 125 nM exhibited no binding to SETD7 (red). When the (R)-PFI-2 injections included 20 µM SAM, a visually biphasic binding curve was observed, reflecting fast SAM binding with subsequent slower (R)-PFI-2 binding (blue). At the highest (R)-PFI-2 concentration (rightmost blue curve), the dissociation curve was single phase whereas, at lower (R)-PFI-2 concentrations, the dissociation curves were biphasic. Under these latter conditions, SETD7 is essentially saturated with SAM, but the (R)-PFI-2 concentrations were not fully saturating and the protein is a mix of SAM-bound and SAM plus (R)-PFI-2 bound. This situation gives rise to two visibly different off rates, one for SAM alone and one for the combination of SAM plus (R)-PFI-2. A single-cycle kinetics methodology was used. The analysis was done in triplicate using a 1:1 kinetic fitting model and the data were processed separately and values were averaged. (B) IC50 values were determined for (R)-PFI-2 at varying concentrations of SAM, 5 µM peptide, and 20 nM enzyme. (C) IC50 values at varying concentrations of substrate H3(1–25) peptide and at SAM concentrations of 0.125 (●), 0.25 (■), 0.5 (▲), and 2 µM (▼) using 20 nM enzyme as described in SI Appendix.
Fig. 4.
Fig. 4.
(R)-PFI-2 binds to SETD7 in cells. (A) Structure of PFI-766. (B) Schematic of the experiment using PFI-766 to pull down endogenous SETD7. (C) PFI-766 pulls down endogenous SETD7 from washed MCF7 cells, and the amount of pulled-down material decreased in a dose-dependent manner in cells treated with increasing doses of (R)-PFI-2. The control lane of 0 (R)-PFI-2 indicates the solvent DMSO control. The gels shown are representative of at least three independent experiments.
Fig. 5.
Fig. 5.
Inhibition of Setd7 affects YAP in MEFs and MCF7 cells. (A) MEFs derived from Setd7+/+ or Setd7−/− mice were grown at low density (LD) or high density (HD) in the presence of 10 μM (S)-PFI-2 or (R)-PFI-2. The amount of nuclear YAP was quantified from images as described in SI Appendix. Data from three experiments were pooled (n > 30; *P < 0.001). (B) (R)-PFI-2 treatment induces YAP target genes in high-density Setd7+/+ MEF cultures. Relative expression is displayed as the expression levels of Ctgf after (R)-PFI-2 treatment over that with (S)-PFI-2 treatment prenormalized to the levels of Actb housekeeping gene (n = 6; *P < 0.05). (C) Confluent MCF7 cultures were treated with (S)-PFI-2 or (R)-PFI-2 for 2 h at 1 µM, and stained for the Hippo pathway transducer YAP (red) and DAPI (blue). Representative confocal images of equal magnification (150 × 150 μm) are shown. (D) The amount of nuclear YAP quantified from images in C. Data are from two pooled experiments (n > 30; *P < 0.001). (E) Confluent MCF7 cells were treated with the indicated concentrations of (R)-PFI-2 for 2 h and show a clear decrease in cytosolic YAP with simultaneous increase in nuclear YAP with increasing (R)-PFI-2. (F) Confluent MCF7 cultures were incubated with 1 µM (R)-PFI-2 for the indicated times. Western blots of nuclear extracts were probed for YAP, SETD7, and ac-H3.

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