22 research outputs found
The L3MBTL3 Methyl-Lysine Reader Domain Functions As a Dimer
L3MBTL3
recognizes mono- and dimethylated lysine residues on histone
tails. The recently reported X-ray cocrystal structures of the chemical
probe UNC1215 and inhibitor UNC2533 bound to the methyl-lysine reading
MBT domains of L3MBTL3 demonstrate a unique and flexible 2:2 dimer
mode of recognition. In this study, we describe our <i>in vitro</i> analysis of L3MBTL3 dimerization via its MBT domains and additionally
show that this dimerization occurs within a cellular context in the
absence of small molecule ligands. Furthermore, mutations to the first
and second MBT domains abrogated L3MBTL3 dimerization both <i>in vitro</i> and in cells. These observations are consistent
with the hypothesis that L3MBTL3 engages methylated histone tails
as a dimer while carrying out its normal function and provides an
explanation for the presence of repeated MBT domains within L3MBTL3
A Novel Family of Small Molecules that Enhance the Intracellular Delivery and Pharmacological Effectiveness of Antisense and Splice Switching Oligonucleotides
The pharmacological effectiveness
of oligonucleotides has been hampered by their tendency to remain
entrapped in endosomes, thus limiting their access to cytosolic or
nuclear targets. We have previously reported a group of small molecules
that enhance the effects of oligonucleotides by causing their release
from endosomes. Here, we describe a second novel family of oligonucleotide
enhancing compounds (OECs) that is chemically distinct from the compounds
reported previously. We demonstrate that these molecules substantially
augment the actions of splice switching oligonucleotides (SSOs) and
antisense oligonucleotides (ASOs) in cell culture. We also find enhancement
of SSO effects in a murine model. These new compounds act by increasing
endosome permeability and causing partial release of entrapped oligonucleotides.
While they also affect the permeability of lysosomes, they are clearly
different from typical lysosomotropic agents. Current members of this
compound family display a relatively narrow window between effective
dose and toxic dose. Thus, further improvements are necessary before
these agents can become suitable for therapeutic use
Structure–Activity Relationships and Kinetic Studies of Peptidic Antagonists of CBX Chromodomains
To better understand the contribution
of methyl-lysine (Kme) binding proteins to various disease states,
we recently developed and reported the discovery of <b>1</b> (UNC3866), a chemical probe that targets two families of Kme binding
proteins, CBX and CDY chromodomains, with selectivity for CBX4 and
-7. The discovery of <b>1</b> was enabled in part by the use
of molecular dynamics simulations performed with CBX7 and its endogenous
substrate. Herein, we describe the design, synthesis, and structure–activity
relationship studies that led to the development of <b>1</b> and provide support for our model of CBX7–ligand recognition
by examining the binding kinetics of our antagonists with CBX7 as
determined by surface-plasmon resonance
Discovery of Peptidomimetic Ligands of EED as Allosteric Inhibitors of PRC2
The
function of EED within polycomb repressive complex 2 (PRC2)
is mediated by a complex network of protein–protein interactions.
Allosteric activation of PRC2 by binding of methylated proteins to
the embryonic ectoderm development (EED) aromatic cage is essential
for full catalytic activity, but details of this regulation are not
fully understood. EED’s recognition of the product of PRC2
activity, histone H3 lysine 27 trimethylation (H3K27me3), stimulates
PRC2 methyltransferase activity at adjacent nucleosomes leading to
H3K27me3 propagation and, ultimately, gene repression. By coupling
combinatorial chemistry and structure-based design, we optimized a
low-affinity methylated jumonji, AT-rich interactive domain 2 (Jarid2)
peptide to a smaller, more potent peptidomimetic ligand (<i>K</i><sub>d</sub> = 1.14 ± 0.14 μM) of the aromatic cage of
EED. Our strategy illustrates the effectiveness of applying combinatorial
chemistry to achieve both ligand potency and property optimization.
Furthermore, the resulting ligands, UNC5114 and UNC5115, demonstrate
that targeted disruption of EED’s reader function can lead
to allosteric inhibition of PRC2 catalytic activity
Gut Microbial β-Glucuronidase Inhibition via Catalytic Cycle Interception
Microbial
β-glucuronidases (GUSs) cause severe gut toxicities that limit
the efficacy of cancer drugs and other therapeutics. Selective inhibitors
of bacterial GUS have been shown to alleviate these side effects.
Using structural and chemical biology, mass spectrometry, and cell-based
assays, we establish that piperazine-containing GUS inhibitors intercept
the glycosyl-enzyme catalytic intermediate of these retaining glycosyl
hydrolases. We demonstrate that piperazine-based compounds are substrate-dependent
GUS inhibitors that bind to the GUS–GlcA catalytic intermediate
as a piperazine-linked glucuronide (GlcA, glucuronic acid). We confirm
the GUS-dependent formation of inhibitor–glucuronide conjugates
by LC–MS and show that methylated piperazine analogs display
significantly reduced potencies. We further demonstrate that a range
of approved piperazine- and piperidine-containing drugs from many
classes, including those for the treatment of depression, infection,
and cancer, function by the same mechanism, and we confirm through
gene editing that these compounds selectively inhibit GUS in living
bacterial cells. Together, these data reveal a unique mechanism of
GUS inhibition and show that a range of therapeutics may impact GUS
activities in the human gut
Table_3_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_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_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
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