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CellEKT: A chemical proteomics assay to profile cellular target engagement of kinase inhibitors

ABSTRACT: Activity-based protein profiling is a powerful tool to study target engagement in complex biological environments. XO44, ALX005 and ALX011 are chemical probes based on the scaffold of a promiscuous kinase binder, which covalently reacts with a conserved lysine in the ATP-binding pocket of kinases. Pretreatment of cells with a drug (candidate) results in competition of XO44/ALX005/ALX011 binding, which can be interpreted as in situ target engagement. Here CellEKT is presented, a workflow which is optimized for kinome coverage, reproducibility and data processing. This dataset pertains to the selectivity testing of Dasatinib in MV4-11 cells using a probe cocktail of XO44, ALX005 and ALX011.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Joel Rüegger

LAB HEAD: Mario van der Stelt

PROVIDER: PXD053911 | Pride | 2025-04-08

REPOSITORIES: Pride

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			Action	DRS
	240611-JR-021_Slot2-21_1_5898.d.zip	Other
	240611-JR-022_Slot2-22_1_5899.d.zip	Other
	240611-JR-023_Slot2-23_1_5913.d.zip	Other
	240611-JR-024_Slot2-24_1_5914.d.zip	Other
	240611-JR-025_Slot2-25_1_5915.d.zip	Other

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CellEKT: A chemical proteomics assay to profile cellular target engagement of kinase inhibitors

Project description:Activity-based protein profiling is a powerful tool to study target engagement in complex biological environments. XO44 is a chemical probe based on the scaffold of a promiscuous kinase binder, which covalently reacts with a conserved lysine in the ATP-binding pocket of kinases. Pretreatment of cells with a drug (candidate) results in competition of XO44 binding, which can be interpreted as in situ target engagement. Here, a workflow is presented which is optimized for kinome coverage, reproducibility and data processing. This dataset pertains to the kinome coverage of XO44 in 19 human cell lines as determined by chemical proteomics.

2025-04-08 | PXD035544 | Pride

CellEKT: A chemical proteomics assay to profile cellular target engagement of kinase inhibitors

Project description:Activity-based protein profiling is a powerful tool to study target engagement in complex biological environments. XO44, ALX005 and ALX011 are chemical probes based on the scaffold of a promiscuous kinase binder, which covalently reacts with a conserved lysine in the ATP-binding pocket of kinases. Pretreatment of cells with a drug (candidate) results in competition of XO44/ALX005/ALX011 binding, which can be interpreted as in situ target engagement. Here CellEKT is presented, a workflow which is optimized for kinome coverage, reproducibility and data processing. This dataset contains the kinome coverage assessment of ALX005 and ALX011 in comparison to the kinome coverage of XO44 which was determined in the selectivity screening of Midostaurin (PXD035619, PXD035549)

2025-04-08 | PXD054026 | Pride

CellEKT: A chemical proteomics assay to profile cellular target engagement of kinase inhibitors

Project description:Activity-based protein profiling is a powerful tool to study target engagement in complex biological environments. XO44 is a chemical probe based on the scaffold of a promiscuous kinase binder, which covalently reacts with a conserved lysine in the ATP-binding pocket of kinases. Pretreatment of cells with a drug (candidate) results in competition of XO44 binding, which can be interpreted as in situ target engagement. Here, a workflow is presented which is optimized for kinome coverage, reproducibility and data processing. This dataset pertains to the selectivity testing of BTK inhibitors in MV4-11 cells.

2025-04-08 | PXD035550 | Pride

CellEKT: A chemical proteomics assay to profile cellular target engagement of kinase inhibitors

Project description:Activity-based protein profiling is a powerful tool to study target engagement in complex biological environments. XO44 is a chemical probe based on the scaffold of a promiscuous kinase binder, which covalently reacts with a conserved lysine in the ATP-binding pocket of kinases. Pretreatment of cells with a drug (candidate) results in competition of XO44 binding, which can be interpreted as in situ target engagement. Here, a workflow is presented which is optimized for kinome coverage, reproducibility and data processing. This dataset pertains to the selectivity testing of BTK inhibitors in HEK293T cells.

2025-04-08 | PXD035551 | Pride

CellEKT: A chemical proteomics assay to profile cellular target engagement of kinase inhibitors

Project description:Activity-based protein profiling is a powerful tool to study target engagement in complex biological environments. XO44 is a chemical probe based on the scaffold of a promiscuous kinase binder, which covalently reacts with a conserved lysine in the ATP-binding pocket of kinases. Pretreatment of cells with a drug (candidate) results in competition of XO44 binding, which can be interpreted as in situ target engagement. Here, a workflow is presented which is optimized for kinome coverage, reproducibility and data processing. This dataset pertains to the selectivity testing of Midostaurin in HEK293T cells.

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CellEKT: A chemical proteomics assay to profile cellular target engagement of kinase inhibitors

Project description:Activity-based protein profiling is a powerful tool to study target engagement in complex biological environments. XO44 is a chemical probe based on the scaffold of a promiscuous kinase binder, which covalently reacts with a conserved lysine in the ATP-binding pocket of kinases. Pretreatment of cells with a drug (candidate) results in competition of XO44 binding, which can be interpreted as in situ target engagement. Here, a workflow is presented which is optimized for kinome coverage, reproducibility and data processing. This dataset pertains to the selectivity testing of Midostaurin in MV4-11 cells.

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CellEKT: A chemical proteomics assay to profile cellular target engagement of kinase inhibitors

Project description:Activity-based protein profiling is a powerful tool to study target engagement in complex biological environments. XO44 is a chemical probe based on the scaffold of a promiscuous kinase binder, which covalently reacts with a conserved lysine in the ATP-binding pocket of kinases. Pretreatment of cells with a drug (candidate) results in competition of XO44 binding, which can be interpreted as in situ target engagement. Here, a workflow is presented which is optimized for kinome coverage, reproducibility and data processing. This dataset pertains to the difference in kinome coverage when using different protocols and (strept)avidin beads.

2025-04-08 | PXD035542 | Pride

CellEKT: A chemical proteomics assay to profile cellular target engagement of kinase inhibitors

Project description:Activity-based protein profiling is a powerful tool to study target engagement in complex biological environments. XO44 is a chemical probe based on the scaffold of a promiscuous kinase binder, which covalently reacts with a conserved lysine in the ATP-binding pocket of kinases. Pretreatment of cells with a drug (candidate) results in competition of XO44 binding, which can be interpreted as in situ target engagement. Here, a workflow is presented which is optimized for kinome coverage, reproducibility and automated data processing. This dataset pertains to the use of high probe dose samples as peptide library in MaxQuant.

2025-04-08 | PXD035540 | Pride

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2025-04-09 | PXD055387 | Pride

Site-Specific Competitive Kinase Inhibitor Target Profiling Using Phosphonate Affinity Tags

Project 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.

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