SETD8/SET8/Pr-SET7/KMT5A is the sole protein lysine methyltransferase (PKMT) known to monomethylate lysine 20 of histone H4 monomethylation of histone H4 lysine 20 (H4K20me1). in the downregulation of apoptosis either by antagonizing p53 acetylation which is required for p53-mediated transcriptional activation or promoting p53 ubiquitination for degradation.14 15 These findings associate the functions of SETD8 with transcriptional regulation and DNA damage response. Inhibition of SETD8 is thus expected to show a proapoptotic phenotype through the depletion of H4K20 monomethylation Phlorizin (Phloridzin) which leads to cell cycle arrest or p53/Numb-mediated methylation which results in the upregulation of p53 target genes.14 15 SETD8 has been further implicated in cancer invasiveness and metastasis through its interaction with TWIST 17 a master regulator in epithelial-mesenchymal transition. The sheer scope of SETD8-associated biology highlights the importance of accessing SETD8 inhibitors which enable convenient dissection of the functions of SETD8-mediated methylation. Despite such need Phlorizin (Phloridzin) few inhibitors of high quality have been reported so far for SETD8 (also see Note) 18 19 as well as for other PKMTs implicated in epigenetics and disease.20 Development of PKMT inhibitors aiming at both specificity and potency can be challenging because most PKMTs contain highly similar pockets for binding the SAM cofactor and less-structured regions for binding protein substrates.20 A few examples of potent selective PKMT inhibitors with demonstrated cellular activities include the chemical probes of G9a/GLP (e.g. UNC0638 and BRD4770) DOT1L (e.g. EPZ000477) and EZH1/2 (e.g. GSK126 EPZ-005687/6438 and EI1).21?26 Prior efforts aimed at SETD8 inhibition have also led to several compounds such as nahuoic acid A18 and bis(bromo/dibromo-methoxylphenol) derivatives19 as SETD8 inhibitors. However these compounds have not demonstrated high selectivity or cellular activity against SETD8. The state of the field thus prompted us to explore other small-molecule scaffolds for SETD8 inhibition. We recently formulated a radioactivity-based scintillation proximity imaging assay (SPA) in a high throughput screening (HTS) format with the purpose of identifying novel SETD8 inhibitors.27 This assay relies on SETD8 to transfer the radioactive [3H-methyl] group from IC50 and selectivity of SETD8 inhibitors SPS8I1-3. (a) Chemical structures of the three HTS hits with quinonic moieties highlighted in red. SPS8I1 (NSC663284) SPS8I2 (ryuvidine) and SPS8I3 (BVT948) were identified … Among Mmp8 the compounds identified in the SPA-based HTS assays of SETD8 SETD7 SETD2 and GLP we focused on validating the 4 compounds that were identified solely in the HTS of SETD8.27 The dose-response curves of these compounds against SETD8 were determined by a secondary radiometric filter paper assay.27 Here the assay parameters including the concentrations of [3H-methyl]-SAM the H4K20 peptide substrate and SETD8 (a low ratio of SAM/peptide/enzyme = 0.75:1.5:1) are similar to those used in the primary SPA-based HTS (see Supporting Information). Three compounds (SPS8I1-3) were confirmed as potent inhibitors of SETD8 with apparent IC50 values of 0.21 ± 0.03 μM 0.5 Phlorizin (Phloridzin) ± 0.2 μM and 0.7 ± 0.2 μM respectively (NSC95397 was triaged because of its high IC50 value of 82 μM) (Figure ?(Figure1b).1b). The IC50 values largely reflect the interaction between SETD8 and the inhibitors because the concentrations of SAM (0.75 μM) and the H4K20 peptide (1.5 μM) in the assay Phlorizin (Phloridzin) are far below the values of IC50 values of SPS8I1-3 may alter according to the assay parameters such as the concentrations of reactants and preincubation/reaction time (see discussion later) and the unknown ratio of active versus misfolded SETD8 used in the assay. To evaluate the selectivity of SPS8I1-3 on SETD8 versus other PMTs dose-response curves of these compounds were compared among a phylogenic panel of representative human methyltransferases including 6 PKMTs (SETD2 GLP G9a SETD8 SMYD2 and SETD7) and 3 protein arginine methyltransferases (CARM1 PRMT1 and PRMT3) (Figure ?(Figure1c;1c; Supplmentary Tables S1 and S2). According to the 3 × 9 array of IC50 values SPS8I1 (see discussion for its non-PMT targets) was identified as the most potent and selective SETD8 inhibitor with an apparent IC50 of 0.21 ± 0.03 μM for SETD8 which is 2.5-fold lower than that of its next Phlorizin (Phloridzin) hit SMYD2 (0.5 ± 0.2 μM) and >6-fold lower than those of other examined PMTs (from 1.3 to >100 μM) (Figure ?(Figure1c1c and Supplementary Table S1). With the 2 2.5-fold ratio of IC50 values as a threshold SPS8I2 also demonstrates desired.