34 research outputs found
Disease Association and Druggability of WD40 Repeat Proteins
WD40
repeat (WDR) domains are protein interaction scaffolds that
represent one of the largest protein families in human, and a first
WDR inhibitorî—¸an allosteric antagonist of polycomb repressive
complex 2î—¸just entered the clinic. A systematic analysis of
the CORUM database of protein complexes shows that WDR is the most
represented domain in transcriptional regulation and one of the most
prevalent in the ubiquitin proteasome system, two pathways of high
relevance to drug discovery. Parsing the literature and the vulnerability
of cancer cell lines to CRISPR knockout indicates that WDR proteins
are targets of interest in oncology and other disease areas. A quantitative
analysis of WDR structures reveals that druggable binding pockets
can be found on multiple surfaces of these multifaceted protein interaction
platforms. These data support the development of chemical probes to
further interrogate WDR proteins as an emerging therapeutic target
class
An Integrative Proteomic Approach Identifies Novel Cellular SMYD2 Substrates
Protein
methylation is a post-translational modification with important roles
in transcriptional regulation and other biological processes, but
the enzyme–substrate relationship between the 68 known human
protein methyltransferases and the thousands of reported methylation
sites is poorly understood. Here, we propose a bioinformatic approach
that integrates structural, biochemical, cellular, and proteomic data
to identify novel cellular substrates of the lysine methyltransferase
SMYD2. Of the 14 novel putative SMYD2 substrates identified by our
approach, six were confirmed in cells by immunoprecipitation: MAPT,
CCAR2, EEF2, NCOA3, STUB1, and UTP14A. Treatment with the selective
SMYD2 inhibitor BAY-598 abrogated the methylation signal, indicating
that methylation of these novel substrates was dependent on the catalytic
activity of the enzyme. We believe that our integrative approach can
be applied to other protein lysine methyltransferases, and help understand
how lysine methylation participates in wider signaling processes
An Integrative Proteomic Approach Identifies Novel Cellular SMYD2 Substrates
Protein
methylation is a post-translational modification with important roles
in transcriptional regulation and other biological processes, but
the enzyme–substrate relationship between the 68 known human
protein methyltransferases and the thousands of reported methylation
sites is poorly understood. Here, we propose a bioinformatic approach
that integrates structural, biochemical, cellular, and proteomic data
to identify novel cellular substrates of the lysine methyltransferase
SMYD2. Of the 14 novel putative SMYD2 substrates identified by our
approach, six were confirmed in cells by immunoprecipitation: MAPT,
CCAR2, EEF2, NCOA3, STUB1, and UTP14A. Treatment with the selective
SMYD2 inhibitor BAY-598 abrogated the methylation signal, indicating
that methylation of these novel substrates was dependent on the catalytic
activity of the enzyme. We believe that our integrative approach can
be applied to other protein lysine methyltransferases, and help understand
how lysine methylation participates in wider signaling processes
Strategy to Target the Substrate Binding site of SET Domain Protein Methyltransferases
Protein
methyltransferases (PMTs) are a novel gene family of therapeutic
relevance involved in chromatin-mediated signaling and other biological
mechanisms. Most PMTs are organized around the structurally conserved
SET domain that catalyzes the methylation of a substrate lysine. A
few potent chemical inhibitors compete with the protein substrate,
and all are anchored in the channel recruiting the methyl-accepting
lysine. We propose a novel strategy to design focused chemical libraries
targeting the substrate binding site, where a limited number of warheads
each occupying the lysine-channel of multiple enzymes would be decorated
by different substituents. A variety of sequence and structure-based
approaches used to analyze the diversity of the lysine channel of
SET domain PMTs support the relevance of this strategy. We show that
chemical fragments derived from published inhibitors are valid warheads
that can be used in the design of novel focused libraries targeting
other PMTs
Identification and Structure–Activity Relationship of HDAC6 Zinc-Finger Ubiquitin Binding Domain Inhibitors
HDAC6
plays a central role in the recruitment of protein aggregates
for lysosomal degradation and is a promising target for combination
therapy with proteasome inhibitors in multiple myeloma. Pharmacologically
displacing ubiquitin from the zinc-finger ubiquitin-binding domain
(ZnF-UBD) of HDAC6 is an underexplored alternative to catalytic inhibition.
Here, we present the discovery of an HDAC6 ZnF-UBD-focused chemical
series and its progression from virtual screening hits to low micromolar
inhibitors. A carboxylate mimicking the C-terminal extremity of ubiquitin,
and an extended aromatic system stacking with W1182 and R1155, are
necessary for activity. One of the compounds induced a conformational
remodeling of the binding site where the primary binding pocket opens
up onto a ligand-able secondary pocket that may be exploited to increase
potency. The preliminary structure–activity relationship accompanied
by nine crystal structures should enable further optimization into
a chemical probe to investigate the merit of targeting the ZnF-UBD
of HDAC6 in multiple myeloma and other diseases
Identification and Structure–Activity Relationship of HDAC6 Zinc-Finger Ubiquitin Binding Domain Inhibitors
HDAC6
plays a central role in the recruitment of protein aggregates
for lysosomal degradation and is a promising target for combination
therapy with proteasome inhibitors in multiple myeloma. Pharmacologically
displacing ubiquitin from the zinc-finger ubiquitin-binding domain
(ZnF-UBD) of HDAC6 is an underexplored alternative to catalytic inhibition.
Here, we present the discovery of an HDAC6 ZnF-UBD-focused chemical
series and its progression from virtual screening hits to low micromolar
inhibitors. A carboxylate mimicking the C-terminal extremity of ubiquitin,
and an extended aromatic system stacking with W1182 and R1155, are
necessary for activity. One of the compounds induced a conformational
remodeling of the binding site where the primary binding pocket opens
up onto a ligand-able secondary pocket that may be exploited to increase
potency. The preliminary structure–activity relationship accompanied
by nine crystal structures should enable further optimization into
a chemical probe to investigate the merit of targeting the ZnF-UBD
of HDAC6 in multiple myeloma and other diseases
Identification and Structure–Activity Relationship of HDAC6 Zinc-Finger Ubiquitin Binding Domain Inhibitors
HDAC6
plays a central role in the recruitment of protein aggregates
for lysosomal degradation and is a promising target for combination
therapy with proteasome inhibitors in multiple myeloma. Pharmacologically
displacing ubiquitin from the zinc-finger ubiquitin-binding domain
(ZnF-UBD) of HDAC6 is an underexplored alternative to catalytic inhibition.
Here, we present the discovery of an HDAC6 ZnF-UBD-focused chemical
series and its progression from virtual screening hits to low micromolar
inhibitors. A carboxylate mimicking the C-terminal extremity of ubiquitin,
and an extended aromatic system stacking with W1182 and R1155, are
necessary for activity. One of the compounds induced a conformational
remodeling of the binding site where the primary binding pocket opens
up onto a ligand-able secondary pocket that may be exploited to increase
potency. The preliminary structure–activity relationship accompanied
by nine crystal structures should enable further optimization into
a chemical probe to investigate the merit of targeting the ZnF-UBD
of HDAC6 in multiple myeloma and other diseases
Structure of the Catalytic Domain of EZH2 Reveals Conformational Plasticity in Cofactor and Substrate Binding Sites and Explains Oncogenic Mutations
<div><p>Polycomb repressive complex 2 (PRC2) is an important regulator of cellular differentiation and cell type identity. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Potent EZH2 inhibitors that reduce levels of H3K27me3 kill mutant lymphoma cells and are efficacious in a mouse xenograft model of malignant rhabdoid tumors. Unlike most SET domain methyltransferases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Ă… crystal structure of the EZH2 methyltransferase domain revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation. We propose that protein interactions within the PRC2 complex modulate the trajectory of the post-SET and I-SET domains of EZH2 in favor of a catalytically competent conformation.</p> </div
The cofactor binding site of EZH2 is incomplete.
<p>(A) Superimposition of the EZH2 structure (colored mesh; post-SET shown as blue ribbon) with a ternary complex of EHMT1/GLP (white ribbon) shows that the cofactor binding site is only partially formed in EZH2, due to an atypical orientation of the post-SET domain. (B) The cofactor site of EZH2 is occupied by the CXC domain of a second molecule within the crystal lattice. (C) Mapping of the location of lysine-mediated cross-links detected in the purified PRC2 complex [53]. Cross-links between Lys735 and Lys569 as well as Lys713 indicate that the post-SET domain of EZH2 (yellow) can project towards the CXC domain in solution, consistent with the conformation seen in our structure.</p
Catalytic activity and substrate/cofactor binding of EZH2 (520-746) and the trimeric (EZH2-EED-SUZ12) complex.
<p>(A) The full-length trimeric complex (●) was active, and the crystallized EZH2 construct (○) was not. Activity assay conditions were optimized for the full length EZH2 in complex with EED and SUZ12 as a control. Kinetic analysis shows that the trimeric complex binds SAM (B) and a histone peptide (C) (K<sub>m</sub> SAM: 900 ± 100 nM; K<sub>m</sub> peptide: 205 ± 25 nM; k<sub>cat</sub>: 24 ± 2 h<sup>-1</sup>). Apparent kinetic parameters are the average of three measurements ± standard deviation. ITC shows that the crystallized construct binds neither SAM (D) nor the peptide substrate (E). </p