Biology subjects

Stashko, M. A.

Publications and source records attributed to Stashko, M. A..

3 recordsLinked to original sources

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↗

bioRxiv2024

Discovery of a cell-active chikungunya virus nsP2 protease inhibitor using a covalent fragment-based screening approach

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has been responsible for numerous large-scale outbreaks in the last twenty years. Currently, there are no FDA-approved therapeutics for any alphavirus infection. CHIKV non-structural protein 2 (nsP2), which contains a cysteine protease domain, is essential for viral replication, making it an attractive target for a drug discovery campaign. Here, we optimized a CHIKV nsP2 protease (nsP2pro) biochemical assay for the screening of a 6,120-compound cysteine-directed covalent fragment library. Using a 50% inhibition threshold, we identified 153 hits (2.5% hit rate). In dose-response follow up, RA-0002034, a covalent fragment that contains a vinyl sulfone warhead, inhibited CHIKV nsP2pro with an IC50 of 58 {+/-} 17 nM, and further analysis with time-dependent inhibition studies yielded a kinact/KI of 6.4 x 103 M-1s-1. LC-MS/MS analysis determined that RA-0002034 covalently modified the catalytic cysteine in a site-specific manner. Additionally, RA-0002034 showed no significant off-target reactivity against a panel of cysteine proteases. In addition to the potent biochemical inhibition of CHIKV nsP2pro activity and exceptional selectivity, RA-0002034 was tested in cellular models of alphavirus infection and effectively inhibited viral replication of both CHIKV and related alphaviruses. This study highlights the discovery and characterization of the chemical probe RA-0002034 as a promising hit compound from covalent fragment-based screening for development toward a CHIKV or pan-alphavirus therapeutic. Significance StatementChikungunya virus is one of the most prominent and widespread alphaviruses and has caused explosive outbreaks of arthritic disease. Currently, there are no FDA-approved drugs to treat disease caused by chikungunya virus or any other alphavirus-caused infection. Here, we report the discovery of a covalent small molecule inhibitor of chikungunya virus nsP2 protease activity and viral replication of four diverse alphaviruses. This finding highlights the utility of covalent fragment screening for inhibitor discovery and represents a starting point towards the development of alphavirus therapeutics targeting nsP2 protease.

biochemistry↗

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↗