19 research outputs found
Targeted Gene Repression Using Novel Bifunctional Molecules to Harness Endogenous Histone Deacetylation Activity
Epigenome
editing is a powerful method for life science research
and could give rise to new therapies for diseases initiated or maintained
by epigenetic dysregulation, including several types of cancers and
autoimmune disorders. In addition, much is still unknown about the
mechanisms by which histone-modifying proteins work in concert to
properly regulate gene expression. To investigate and manipulate complex
epigenetic interactions in live cells, we have developed a small molecule
platform for specifically inducing gene repression and histone deacetylation
at a reporter gene. We synthesized bifunctional ligands, or chemical
epigenetic modifiers (CEMs), that contain two functional groups: a
FK506 derivative capable of binding to a FKBP-Gal4 fusion transcription
factor, and a histone deacetylase (HDAC) inhibitor that recruits HDAC-containing
corepressor complexes. In our reporter cell line, which contains a
GFP reporter allele upstream of a Gal4 DNA binding array in the murine <i>Oct4</i> locus, our lead CEM repressed GFP expression by 50%.
We also show that CEM recruitment of deacetylation activity causes
marked deacetylation along our target loci. This system allowed us
to detail the direct results of deacetylation to chromatin and measure
the resulting gene expression in a chemically dependent and reversible
manner. The CEMs system provides new insights into epigenetic gene
regulation and has the potential to control disease-relevant gene
regulation. The CEMs are derived from FDA-approved epigenetic modulator
drugs, and use their pharmacology in a gene-specific way that avoids
the toxicities and off-target effects caused by whole-cell application
of these drugs
Discovery of G Protein-Biased D2 Dopamine Receptor Partial Agonists
Biased ligands (also known as functionally
selective ligands) of
G protein-coupled receptors are valuable tools for dissecting the
roles of G protein-dependent and independent signaling pathways in
health and disease. Biased ligands have also been increasingly pursued
by the biomedical community as promising therapeutics with improved
efficacy and reduced side effects compared with unbiased ligands.
We previously discovered first-in-class β-arrestin-biased agonists
of dopamine D2 receptor (D2R) by extensively exploring multiple regions
of aripiprazole, a balanced D2R agonist. In our continuing efforts
to identify biased agonists of D2R, we unexpectedly discovered a G
protein-biased agonist of D2R, compound <b>1</b>, which is the
first G protein-biased D2R agonist from the aripiprazole scaffold.
We designed and synthesized novel analogues to explore two regions
of <b>1</b> and conducted structure–functional selectivity
relationship (SFSR) studies. Here we report the discovery of <b>1</b>, findings from our SFSR studies, and characterization of
novel G protein-biased D2R agonists
Structure-Based Design of a Covalent Inhibitor of the SET Domain-Containing Protein 8 (SETD8) Lysine Methyltransferase
Selective
inhibitors of protein lysine methyltransferases, including
SET domain-containing protein 8 (SETD8), are highly desired, as only
a fraction of these enzymes are associated with high-quality inhibitors.
From our previously discovered SETD8 inhibitor, we developed a more
potent analog and solved a cocrystal structure, which is the first
crystal structure of SETD8 in complex with a small-molecule inhibitor.
This cocrystal structure allowed the design of a covalent inhibitor
of SETD8 (MS453), which specifically modifies a cysteine residue near
the inhibitor binding site, has an IC<sub>50</sub> value of 804 nM,
reacts with SETD8 with near-quantitative yield, and is selective for
SETD8 against 28 other methyltransferases. We also solved the crystal
structure of the covalent inhibitor in complex with SETD8. This work
provides atomic-level perspective on the inhibition of SETD8 by small
molecules and will help identify high-quality chemical probes of SETD8
Discovery of a Selective, Substrate-Competitive Inhibitor of the Lysine Methyltransferase SETD8
The
lysine methyltransferase SETD8 is the only known methyltransferase
that catalyzes monomethylation of histone H4 lysine 20 (H4K20). Monomethylation
of H4K20 has been implicated in regulating diverse biological processes
including the DNA damage response. In addition to H4K20, SETD8 monomethylates
non-histone substrates including proliferating cell nuclear antigen
(PCNA) and promotes carcinogenesis by deregulating PCNA expression.
However, selective inhibitors of SETD8 are scarce. The only known
selective inhibitor of SETD8 to date is nahuoic acid A, a marine natural
product, which is competitive with the cofactor. Here, we report the
discovery of the first substrate-competitive inhibitor of SETD8, UNC0379
(<b>1</b>). This small-molecule inhibitor is active in multiple
biochemical assays. Its affinity to SETD8 was confirmed by ITC (isothermal
titration calorimetry) and SPR (surface plasmon resonance) studies.
Importantly, compound <b>1</b> is selective for SETD8 over 15
other methyltransferases. We also describe structure–activity
relationships (SAR) of this series
Image_4_MS0621, a novel small-molecule modulator of Ewing sarcoma chromatin accessibility, interacts with an RNA-associated macromolecular complex and influences RNA splicing.jpeg
Ewing sarcoma is a cancer of children and young adults characterized by the critical translocation-associated fusion oncoprotein EWSR1::FLI1. EWSR1::FLI1 targets characteristic genetic loci where it mediates aberrant chromatin and the establishment of de novo enhancers. Ewing sarcoma thus provides a model to interrogate mechanisms underlying chromatin dysregulation in tumorigenesis. Previously, we developed a high-throughput chromatin-based screening platform based on the de novo enhancers and demonstrated its utility in identifying small molecules capable of altering chromatin accessibility. Here, we report the identification of MS0621, a molecule with previously uncharacterized mechanism of action, as a small molecule modulator of chromatin state at sites of aberrant chromatin accessibility at EWSR1::FLI1-bound loci. MS0621 suppresses cellular proliferation of Ewing sarcoma cell lines by cell cycle arrest. Proteomic studies demonstrate that MS0621 associates with EWSR1::FLI1, RNA binding and splicing proteins, as well as chromatin regulatory proteins. Surprisingly, interactions with chromatin and many RNA-binding proteins, including EWSR1::FLI1 and its known interactors, were RNA-independent. Our findings suggest that MS0621 affects EWSR1::FLI1-mediated chromatin activity by interacting with and altering the activity of RNA splicing machinery and chromatin modulating factors. Genetic modulation of these proteins similarly inhibits proliferation and alters chromatin in Ewing sarcoma cells. The use of an oncogene-associated chromatin signature as a target allows for a direct approach to screen for unrecognized modulators of epigenetic machinery and provides a framework for using chromatin-based assays for future therapeutic discovery efforts.</p
Table_1_MS0621, a novel small-molecule modulator of Ewing sarcoma chromatin accessibility, interacts with an RNA-associated macromolecular complex and influences RNA splicing.xlsx
Ewing sarcoma is a cancer of children and young adults characterized by the critical translocation-associated fusion oncoprotein EWSR1::FLI1. EWSR1::FLI1 targets characteristic genetic loci where it mediates aberrant chromatin and the establishment of de novo enhancers. Ewing sarcoma thus provides a model to interrogate mechanisms underlying chromatin dysregulation in tumorigenesis. Previously, we developed a high-throughput chromatin-based screening platform based on the de novo enhancers and demonstrated its utility in identifying small molecules capable of altering chromatin accessibility. Here, we report the identification of MS0621, a molecule with previously uncharacterized mechanism of action, as a small molecule modulator of chromatin state at sites of aberrant chromatin accessibility at EWSR1::FLI1-bound loci. MS0621 suppresses cellular proliferation of Ewing sarcoma cell lines by cell cycle arrest. Proteomic studies demonstrate that MS0621 associates with EWSR1::FLI1, RNA binding and splicing proteins, as well as chromatin regulatory proteins. Surprisingly, interactions with chromatin and many RNA-binding proteins, including EWSR1::FLI1 and its known interactors, were RNA-independent. Our findings suggest that MS0621 affects EWSR1::FLI1-mediated chromatin activity by interacting with and altering the activity of RNA splicing machinery and chromatin modulating factors. Genetic modulation of these proteins similarly inhibits proliferation and alters chromatin in Ewing sarcoma cells. The use of an oncogene-associated chromatin signature as a target allows for a direct approach to screen for unrecognized modulators of epigenetic machinery and provides a framework for using chromatin-based assays for future therapeutic discovery efforts.</p
Image_1_MS0621, a novel small-molecule modulator of Ewing sarcoma chromatin accessibility, interacts with an RNA-associated macromolecular complex and influences RNA splicing.jpeg
Ewing sarcoma is a cancer of children and young adults characterized by the critical translocation-associated fusion oncoprotein EWSR1::FLI1. EWSR1::FLI1 targets characteristic genetic loci where it mediates aberrant chromatin and the establishment of de novo enhancers. Ewing sarcoma thus provides a model to interrogate mechanisms underlying chromatin dysregulation in tumorigenesis. Previously, we developed a high-throughput chromatin-based screening platform based on the de novo enhancers and demonstrated its utility in identifying small molecules capable of altering chromatin accessibility. Here, we report the identification of MS0621, a molecule with previously uncharacterized mechanism of action, as a small molecule modulator of chromatin state at sites of aberrant chromatin accessibility at EWSR1::FLI1-bound loci. MS0621 suppresses cellular proliferation of Ewing sarcoma cell lines by cell cycle arrest. Proteomic studies demonstrate that MS0621 associates with EWSR1::FLI1, RNA binding and splicing proteins, as well as chromatin regulatory proteins. Surprisingly, interactions with chromatin and many RNA-binding proteins, including EWSR1::FLI1 and its known interactors, were RNA-independent. Our findings suggest that MS0621 affects EWSR1::FLI1-mediated chromatin activity by interacting with and altering the activity of RNA splicing machinery and chromatin modulating factors. Genetic modulation of these proteins similarly inhibits proliferation and alters chromatin in Ewing sarcoma cells. The use of an oncogene-associated chromatin signature as a target allows for a direct approach to screen for unrecognized modulators of epigenetic machinery and provides a framework for using chromatin-based assays for future therapeutic discovery efforts.</p
Image_5_MS0621, a novel small-molecule modulator of Ewing sarcoma chromatin accessibility, interacts with an RNA-associated macromolecular complex and influences RNA splicing.jpeg
Ewing sarcoma is a cancer of children and young adults characterized by the critical translocation-associated fusion oncoprotein EWSR1::FLI1. EWSR1::FLI1 targets characteristic genetic loci where it mediates aberrant chromatin and the establishment of de novo enhancers. Ewing sarcoma thus provides a model to interrogate mechanisms underlying chromatin dysregulation in tumorigenesis. Previously, we developed a high-throughput chromatin-based screening platform based on the de novo enhancers and demonstrated its utility in identifying small molecules capable of altering chromatin accessibility. Here, we report the identification of MS0621, a molecule with previously uncharacterized mechanism of action, as a small molecule modulator of chromatin state at sites of aberrant chromatin accessibility at EWSR1::FLI1-bound loci. MS0621 suppresses cellular proliferation of Ewing sarcoma cell lines by cell cycle arrest. Proteomic studies demonstrate that MS0621 associates with EWSR1::FLI1, RNA binding and splicing proteins, as well as chromatin regulatory proteins. Surprisingly, interactions with chromatin and many RNA-binding proteins, including EWSR1::FLI1 and its known interactors, were RNA-independent. Our findings suggest that MS0621 affects EWSR1::FLI1-mediated chromatin activity by interacting with and altering the activity of RNA splicing machinery and chromatin modulating factors. Genetic modulation of these proteins similarly inhibits proliferation and alters chromatin in Ewing sarcoma cells. The use of an oncogene-associated chromatin signature as a target allows for a direct approach to screen for unrecognized modulators of epigenetic machinery and provides a framework for using chromatin-based assays for future therapeutic discovery efforts.</p
Table_4_MS0621, a novel small-molecule modulator of Ewing sarcoma chromatin accessibility, interacts with an RNA-associated macromolecular complex and influences RNA splicing.xlsx
Ewing sarcoma is a cancer of children and young adults characterized by the critical translocation-associated fusion oncoprotein EWSR1::FLI1. EWSR1::FLI1 targets characteristic genetic loci where it mediates aberrant chromatin and the establishment of de novo enhancers. Ewing sarcoma thus provides a model to interrogate mechanisms underlying chromatin dysregulation in tumorigenesis. Previously, we developed a high-throughput chromatin-based screening platform based on the de novo enhancers and demonstrated its utility in identifying small molecules capable of altering chromatin accessibility. Here, we report the identification of MS0621, a molecule with previously uncharacterized mechanism of action, as a small molecule modulator of chromatin state at sites of aberrant chromatin accessibility at EWSR1::FLI1-bound loci. MS0621 suppresses cellular proliferation of Ewing sarcoma cell lines by cell cycle arrest. Proteomic studies demonstrate that MS0621 associates with EWSR1::FLI1, RNA binding and splicing proteins, as well as chromatin regulatory proteins. Surprisingly, interactions with chromatin and many RNA-binding proteins, including EWSR1::FLI1 and its known interactors, were RNA-independent. Our findings suggest that MS0621 affects EWSR1::FLI1-mediated chromatin activity by interacting with and altering the activity of RNA splicing machinery and chromatin modulating factors. Genetic modulation of these proteins similarly inhibits proliferation and alters chromatin in Ewing sarcoma cells. The use of an oncogene-associated chromatin signature as a target allows for a direct approach to screen for unrecognized modulators of epigenetic machinery and provides a framework for using chromatin-based assays for future therapeutic discovery efforts.</p
Table_5_MS0621, a novel small-molecule modulator of Ewing sarcoma chromatin accessibility, interacts with an RNA-associated macromolecular complex and influences RNA splicing.xlsx
Ewing sarcoma is a cancer of children and young adults characterized by the critical translocation-associated fusion oncoprotein EWSR1::FLI1. EWSR1::FLI1 targets characteristic genetic loci where it mediates aberrant chromatin and the establishment of de novo enhancers. Ewing sarcoma thus provides a model to interrogate mechanisms underlying chromatin dysregulation in tumorigenesis. Previously, we developed a high-throughput chromatin-based screening platform based on the de novo enhancers and demonstrated its utility in identifying small molecules capable of altering chromatin accessibility. Here, we report the identification of MS0621, a molecule with previously uncharacterized mechanism of action, as a small molecule modulator of chromatin state at sites of aberrant chromatin accessibility at EWSR1::FLI1-bound loci. MS0621 suppresses cellular proliferation of Ewing sarcoma cell lines by cell cycle arrest. Proteomic studies demonstrate that MS0621 associates with EWSR1::FLI1, RNA binding and splicing proteins, as well as chromatin regulatory proteins. Surprisingly, interactions with chromatin and many RNA-binding proteins, including EWSR1::FLI1 and its known interactors, were RNA-independent. Our findings suggest that MS0621 affects EWSR1::FLI1-mediated chromatin activity by interacting with and altering the activity of RNA splicing machinery and chromatin modulating factors. Genetic modulation of these proteins similarly inhibits proliferation and alters chromatin in Ewing sarcoma cells. The use of an oncogene-associated chromatin signature as a target allows for a direct approach to screen for unrecognized modulators of epigenetic machinery and provides a framework for using chromatin-based assays for future therapeutic discovery efforts.</p