PXD058749

PXD058749 is an original dataset announced via ProteomeXchange.

Title	Site-Specific Competitive Kinase Inhibitor Target Profiling Using Phosphonate Affinity Tags
Description	Protein kinases are prime targets for drug development due to their involvement in various cancers. However, selective inhibition of kinases, while avoiding off-target effects remains a significant challenge for the development of protein kinase inhibitors. Activity-based protein profiling (ABPP), in combination with pan-kinase activity-based probes (ABPs) and mass spectrometry-based proteomics, enables the identification of kinase drug targets. Here, we extend existing ABPP strategies for kinase profiling with a site-specific analysis, allowing for protein kinase inhibitor target engagement profiling with amino acid specificity. The site-specific approach involves highly efficient enrichment of ABP-labeled peptides, resulting in a less complex peptide matrix, straightforward data analysis, and the screening of over ~100 kinase active sites in a single LC-MS run. The use of both trypsin and pepsin in parallel to generate the ABP-labeled peptides considerably expanded the coverage of kinases and exact binding sites. Using the site-specific strategy to examine the on- and off-targets of the Ephrin receptor (Eph) B4 inhibitor NVP-BHG712 showed binding to EphA2 with an IC50 of 17 nM (95% CI 10 - 28 nM) and EphB4 with an IC50 of 20.2 nM (95% CI 13.7 - 30.4 nM). Next to the known targets, EphA2 and EphB4, NVP-BHG712 bound to the discoidin domain-containing receptor 1 (DDR1) with an IC50 of 2.1 nM (95% CI 1.3 - 3.4 nM), suggesting that a DDR1-targeting regioisomer of NVP-BHG712 was analyzed. The promiscuity of XO44 toward ATP-binding pockets on other proteins facilitated the screening of an additional 475 sites, revealing inosine-5’-monophosphate dehydrogenase 2 (IMPDH2) as an off-target. Therefore, the presented approach, which can be fully automated with liquid handling platforms, provides a straightforward, valuable strategy for competitive site-specific kinase inhibitor target profiling.
HostingRepository	PRIDE
AnnounceDate	2025-05-07
AnnouncementXML	Submission_2025-05-07_06:20:19.425.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Wouter van Bergen
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	iodoacetamide derivatized residue
Instrument	timsTOF HT

Revision	Datetime	Status	ChangeLog Entry
0	2024-12-10 15:59:36	ID requested
⏵ 1	2025-05-07 06:20:20	announced

van Bergen W, Nederstigt AE, Heck AJR, Baggelaar MP, Site-Specific Competitive Kinase Inhibitor Target Profiling Using Phosphonate Affinity Tags. Mol Cell Proteomics, 24(2):100906(2025) [pubmed]

10.1016/j.mcpro.2025.100906;

submitter keyword: Chemical proteomics

Activity-based protein profiling

Site-specific

Protein kinase inhibitor profiling

Ephrin receptor inhibitor

NVP-BHG712

Albert J.R. Heck
contact affiliation	Biomolecular Mass Spectrometry and Proteomics
contact email	a.j.r.heck@uu.nl
lab head
Wouter van Bergen
contact affiliation	Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, Utrecht 3584 CH, The Netherlands Netherlands Proteomics Center, Padualaan 8, Utrecht 3584 CH, The Netherlands
contact email	w.vanbergen@uu.nl
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/05/PXD058749

PRIDE project URI

• PRIDE
  1. PXD058749
     1. Label: PRIDE project
     2. Name: Site-Specific Competitive Kinase Inhibitor Target Profiling Using Phosphonate Affinity Tags