Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Hydrogen-deuterium exchange mass spectrometry for molecular glue characterisation

ABSTRACT: Molecular glues are powerful bioactive molecules that stabilize protein-protein interactions. Yet, the precise mechanisms by which many molecular glues exert their adhesive effects are still not well understood. Native mass spectrometry is an established technique used to monitor the stoichiometry and binding equilibria of molecular glue-induced protein-protein interactions. However, knowledge is lacking on how protein interaction dynamics change upon molecular glue-induced stabilization, and what conformational changes occur that enhance the stability of the resulting protein-protein-glue ternary complex. Here, we showcase hydrogen-deuterium exchange mass spectrometry (HDX-MS) as an analytical tool for molecular glue characterization. Using a broadly applicable molecular glue system involving the eukaryotic regulatory protein 14-3-3, its binding partners, and the molecular glue fusicoccin A, we show the power of HDX-MS in revealing not only molecular glue binding sites, but also conformational changes upon glue binding that result in differentially stabilized protein-protein complexes. Overall, we envisage our HDX-MS approach will become highly valuable for the characterization of molecular glues, guiding their optimization towards successful design.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Aneika Leney

LAB HEAD: Aneika C. Leney

PROVIDER: PXD065689 | Pride | 2025-07-30

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	13Jan25_14-3-3_10minD20.raw	Raw
	13Jan25_14-3-3_10minD20_repeat2.raw	Raw
	13Jan25_14-3-3_5hrD20.raw	Raw
	13Jan25_14-3-3_5hrD20_repeat2.raw	Raw
	13Jan25_14-3-3_LRRK2_10minD20.raw	Raw

Items per page:

1 - 5 of 93

Similar Datasets

Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger Cyclin K degradation [WES]

Project description:Molecular glues are small molecules that exert their biologic or therapeutic activities by inducing gain-of-function interactions between pairs of proteins. In particular, molecular-glue degraders, which mediate interactions between target proteins and components of the ubiquitin proteasome system to cause targeted protein degradation, hold great promise as a unique modality for therapeutic targeting of proteins that are currently intractable. Here, we report a new molecular glue HQ461 discovered by high-throughput screening of small molecules that inhibited NRF2 activity. Using unbiased loss-of-function and gain-of-function genetic screening followed by biochemical reconstitution, we show that HQ461 acts by promoting interaction between CDK12 and DDB1-CUL4-RBX1 E3 ubiquitin ligase, leading to polyubiquitination and proteasomal degradation of CDK12’s interacting protein Cyclin K (CCNK). Degradation of CCNK mediated by HQ461 compromised CDK12 function, leading to reduced phosphorylation of CDK12 substrate, downregulation of DNA damage response genes, and cell death. Structure-activity relationship analysis of HQ461 revealed the importance of a 5-methylthiazol-2-amine pharmacophore and resulted in an HQ461 derivate with improved potency. Our studies reveal a new molecular glue that engages its target protein directly with DDB1 to bypass the requirement of a substrate-specific receptor, presenting a new strategy for targeted protein degradation.

2020-07-03 | GSE153707 | GEO

Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger Cyclin K degradation [CRISPR]

2020-07-03 | GSE153700 | GEO

Allosteric inhibition of PPM1D serine/threonine phosphatase via an altered conformational state

Project description:Detailed characterization of PPM1D phosphatase by HDX-MS: conformational dynamics of the pathogenic truncated form (1-420) found in various cancers, the effect of different truncations and mutations and mapping an inhibitor binding site.

2022-01-20 | MSV000088711 | MassIVE

Covalent 14-3-3 Molecular Glues and Heterobifunctional Molecules Against Nuclear Transcription Factors and Regulators

Project description:14-3-3 proteins have the unique ability to bind and sequester a multitude of diverse phosphorylated signaling proteins and transcription factors. Many previous studies have shown that 14-3-3 interactions with specific phosphorylated substrate proteins can be enhanced through small-molecule natural product or fully synthetic molecular glue interactions. However, enhancing 14-3-3 interactions with both therapeutically intractable transcription factor substrates as well as potential neo-substrates to sequester and inhibit their function has remained elusive. One of the 14-3-3 proteins, 14-3-3or SFN, has a cysteine C38 at the substrate binding interface near sites where previous 14-3-3 molecular glues have been found to bind. In this study, we screened a fully synthetic cysteine-reactive covalent ligand library to identify molecular glues that enhance interaction of 14-3-3with not only druggable transcription factors such as estrogen receptor (ER), but also challenging oncogenic transcription factors such as YAP and TAZ that are part of the Hippo transducer pathway. We identified a hit EN171 that covalently targets both C38 and C96 on 14-3-3 to enhance 14-3-3 interactions with ERYAP, and TAZ leading to impaired estrogen receptor and Hippo pathway transcriptional activity. We further demonstrate that EN171 could not only be used as a molecular glue to enhance native protein interactions, but also could be used as a covalent 14-3-3 recruiter in heterobifunctional molecules to sequester nuclear neo-substrates such as BRD4 and BLC6 into the cytosol. Overall, our study reveals a covalent ligand that acts as a novel 14-3-3 molecular glue for challenging transcription factors such as YAP and TAZ and demonstrates that these glues can be potentially utilized in heterobifunctional molecules to sequester nuclear neo-substrates out of the nucleus and into the cytosol to enable targeted protein localization.

2024-04-26 | PXD051762 | Pride

Template-assisted covalent modification of DCAF16 underlies activity of BRD4 molecular glue degraders

Project description:Small molecules that induce protein-protein interactions to exert proximity-driven pharmacology such as targeted protein degradation are a powerful class of therapeutics1-3. Molecular glues are of particular interest given their favorable size and chemical properties and represent the only clinically approved degrader drugs4-6. The discovery and development of molecular glues for novel targets, however, remains challenging. Covalent strategies could in principle facilitate molecular glue discovery by stabilizing the neo-protein interfaces. Here, we present structural and mechanistic studies that define a trans-labeling covalent molecular glue mechanism, which we term “template-assisted covalent modification”. We found that a novel series of BRD4 molecular glue degraders act by recruiting the CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). BRD4BD2, in complex with DCAF16, serves as a structural template to facilitate covalent modification of DCAF16, which stabilizes the BRD4-degrader-DCAF16 ternary complex formation and facilitates BRD4 degradation. A 2.2 Å cryo-electron microscopy structure of the ternary complex demonstrates that DCAF16 and BRD4BD2 have pre-existing structural complementarity which optimally orients the reactive moiety of the degrader for DCAF16Cys58 covalent modification. Systematic mutagenesis of both DCAF16 and BRD4BD2 revealed that the loop conformation around BRD4His437, rather than specific side chains, is critical for BD2 selectivity. Supporting a general applicability, we find that a subset of compounds leads to a drug-induced GAK-BRD4 interaction stabilized by covalent modification of GAK. Together our work establishes “template-assisted covalent modification” as a mechanism for covalent molecular glues, which opens a new path to proximity driven pharmacology.

2024-08-09 | PXD047138 | Pride

Template-assisted covalent modification of DCAF16 underlies activity of BRD4 molecular glue degraders

2024-08-09 | PXD047141 | Pride

Template-assisted covalent modification of DCAF16 underlies activity of BRD4 molecular glue degraders

2024-08-09 | PXD047137 | Pride

Targeting of disease-linked mutations in PI3K gamma

Project description:Using hydrogen deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics we have identified mutation specific conformational changes, and provide a molecular framework for how they alter kinase activity.Many of the activating conformational changes mimicked previously described changes that occur upon membrane binding and allosteric inhibitor binding. We surveyed a diverse panel of PI3K inhibitors using HDX-MS and X-ray crystallography, and find that inhibitors that interact with the activation loop lead to allosteric conformational changes that partially mimic the R1021C mutation. Intriguingly, inhibitors that showed similar conformational changes to the activating mutant showed increased potency (~3 fold) for the mutant over wild type PI3K. Overall this work provides unique insight into the regulatory mechanisms that control PI3K kinase activity, and reveals a framework for the potential design of PI3K isoform and mutant selective inhibitors.

2021-03-26 | PXD021132 | Pride

Design principles for cyclin K molecular glue degraders

Project description:Molecular glue degraders are an effective therapeutic modality, but their design principles are not well understood. Recently, several unexpectedly diverse compounds were reported to deplete cyclin K by linking CDK12-cyclin K to the DDB1-CUL4-RBX1 E3 ligase. To investigate how chemically dissimilar small molecules trigger cyclin K degradation, we evaluated 91 candidate degraders in structural, biophysical, and cellular studies and reveal all compounds acquire glue activity via simultaneous CDK12 binding and engagement of DDB1 interfacial residues, in particular Arg928. While we identify multiple published kinase inhibitors as cryptic degraders, we also show that these glues do not require pronounced inhibitory properties for activity and that the relative degree of CDK12 inhibition versus cyclin K degradation is tuneable. We further demonstrate cyclin K degraders have transcriptional signatures distinct from CDK12 inhibitors, thereby offering unique therapeutic opportunities. The systematic structure-activity relationship analysis presented herein provides a conceptual framework for rational molecular glue design.

2023-10-20 | PXD041836 | Pride

An engineered Cereblon optimized for high throughput screening and molecular glue discovery

Project description:The majority of clinical degraders utilize an immunomodulatory imide drug (IMiD)-based derivative, that directs their target to the E3 ligase receptor Cereblon (CRBN), however, identification of IMiD molecular glue substrates has remained underexplored. To tackle this, we design human CRBN constructs which retain all features for ternary complex formation, whilst allowing generation of homogenous and cost-efficient expression in E.coli. Extensive profiling of the construct, shows it to be the “best of both worlds” in terms of binding activity and ease of production. We next designed the ‘Enamine focused IMiD library’ and demonstrated applicability of the construct to high-throughput screening, identifying binders with high potency, ligand efficiency and specificity. Finally, we adapt our construct for proof of principle glue screening approaches enabling IMiD cellular interactome determination. Coupled with our IMiD binding landscape, the methods described here should serve as valuable tools to assist discovery of next generation CRBN glues.

2024-12-06 | PXD057393 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data