Uguen, M. | bioRxiv Science

bioRxiv2024

Potent and Selective SETDB1 Covalent Negative Allosteric Modulator Reduces Methyltransferase Activity in Cells

A promising drug target, SETDB1, is a dual Kme reader and methyltransferase, which has been implicated in cancer and neurodegenerative disease progression. To help understand the role of the triple Tudor domain (3TD) of SETDB1, its Kme reader, we first identified a low micromolar small molecule ligand, UNC6535, which occupies simultaneously both the TD2 and TD3 reader binding sites. Further optimization led to the discovery of UNC10013, the first covalent 3TD ligand targeting Cys385 of SETDB1. UNC10013 is potent with a kinact/KI of 1.0 x 106 M-1s-1 and demonstrated proteome-wide selectivity. In cells, negative allosteric modulation of SETDB1-mediated Akt methylation was observed after treatment with UNC10013. Therefore, UNC10013 is a potent, selective and cell-active covalent ligand for the 3TD of SETDB1, demonstrating negative allosteric modulator properties and making it a promising tool to study the biological role of SETDB1 in disease progression. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/615363v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@6db8e2org.highwire.dtl.DTLVardef@1852ee3org.highwire.dtl.DTLVardef@c95bb6org.highwire.dtl.DTLVardef@1a43951_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

SETDB1 Triple Tudor Domain Ligand, (R,R)-59, Promotes Methylation of Akt1 in Cells

Increased expression and hyperactivation of the methyltransferase SETDB1 are commonly observed in cancer and central nervous system disorders. However, there are currently no reported SETDB1-specific methyltransferase inhibitors in the literature, suggesting this is a challenging target. Here, we disclose that the previously reported small-molecule ligand for SETDB1s Triple Tudor Domain, (R,R)-59, is unexpectedly able to increase SETDB1 methyltransferase activity both in vitro and in cells. Specifically, (R,R)-59 promotes in vitro SETDB1-mediated methylation of lysine 64 of the protein kinase Akt1. Treatment with (R,R)-59 also increased Akt1 threonine 308 phosphorylation and activation, a known consequence of Akt1 methylation, resulting in stimulated cell proliferation in a dose-dependent manner. (R,R)-59 is the first SETDB1 small-molecule positive activator for the methyltransferase activity of this protein. Mechanism of action studies show that full-length SETDB1 is required for significant in vitro methylation of an Akt1-K64 peptide, and that this activity is stimulated by (R,R)-59 primarily through an increase in catalytic activity rather than a change in SAM binding. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/539986v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@5f4022org.highwire.dtl.DTLVardef@16ba47borg.highwire.dtl.DTLVardef@1fb8b0borg.highwire.dtl.DTLVardef@16595bc_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗