Optimized Chemical Proteomics Assay for Kinase Inhibitor Profiling

Médard, Guillaume; Pachl, Fiona; Ruprecht, Benjamin; Klaeger, Susan; Heinzlmeir, Stephanie; Helm, Barbara; Qiao, Huichao; Ku, Xin; Wilhelm, Mathias; Kuehne, Thomas; Wu, Zhixiang; Dittmann, Antje; Hopf, Carsten; Kramer, Karl; Küster, Bernhard

doi:10.1021/pr5012608

Cited by 114 publications

(163 citation statements)

References 65 publications

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“…1a). 5, 24, 28, 33–35 The inhibitor analogs 1 and 3–5 were described previously and the affinity reagents 2 , 6 and 7 were newly developed in our laboratory. Compounds 1–7 were immobilized on carboxy-derivatized sepharose using amide coupling chemistry to yield the corresponding affinity matrices.…”

Section: Resultsmentioning

confidence: 99%

Kinobead and Single-Shot LC-MS Profiling Identifies Selective PKD Inhibitors

et al. 2017

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ATP-competitive protein kinase inhibitors are important research tools and therapeutic agents. Because there are >500 human kinases that contain highly conserved active sites, the development of selective inhibitors is extremely challenging. Methods to rapidly and efficiently profile kinase inhibitor targets in cell lysates are urgently needed to discover selective compounds and to elucidate the mechanisms of action for polypharmacological inhibitors. Here, we describe a protocol for microgram-scale chemoproteomic profiling of ATP-competitive kinase inhibitors using kinobeads. We employed a gel-free in situ digestion protocol coupled to nanoflow liquid chromatography-mass spectrometry to profile ~200 kinases in single analytical runs using as little as 5 μl of kinobeads and 300 μg of protein. With our kinobead reagents, we obtained broad coverage of the kinome, monitoring the relative expression levels of 312 kinases in a diverse panel of 11 cancer cell lines. Further, we profiled a set of pyrrolopyrimidine- and pyrazolopyrimidine-based kinase inhibitors in competition-binding experiments with label-free quantification, leading to the discovery of a novel selective and potent inhibitor of protein kinase D (PKD) 1, 2 and 3. Our protocol is useful for rapid and sensitive profiling of kinase expression levels and ATP-competitive kinase inhibitor selectivity in native proteomes.

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Section: Resultsmentioning

confidence: 99%

Kinobead and Single-Shot LC-MS Profiling Identifies Selective PKD Inhibitors

et al. 2017

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“…Kinobeads gamma (KBγ) pulldowns (biological triplicates) were performed in 96‐well filter plates (Porvair Sciences) as described elsewhere (Medard et al , ), with minor modifications. For each pulldown, 3 ml of 2.2 mg/ml TCL was cleared by ultracentrifugation at 167,000 × g and 4°C for 20 min.…”

Section: Methodsmentioning

confidence: 99%

Pharmacoproteomic characterisation of human colon and rectal cancer

Frejno

Chiozzi

Wilhelm

et al. 2017

Molecular Systems Biology

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Most molecular cancer therapies act on protein targets but data on the proteome status of patients and cellular models for proteome‐guided pre‐clinical drug sensitivity studies are only beginning to emerge. Here, we profiled the proteomes of 65 colorectal cancer (CRC) cell lines to a depth of > 10,000 proteins using mass spectrometry. Integration with proteomes of 90 CRC patients and matched transcriptomics data defined integrated CRC subtypes, highlighting cell lines representative of each tumour subtype. Modelling the responses of 52 CRC cell lines to 577 drugs as a function of proteome profiles enabled predicting drug sensitivity for cell lines and patients. Among many novel associations, MERTK was identified as a predictive marker for resistance towards MEK1/2 inhibitors and immunohistochemistry of 1,074 CRC tumours confirmed MERTK as a prognostic survival marker. We provide the proteomic and pharmacological data as a resource to the community to, for example, facilitate the design of innovative prospective clinical trials.

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“…Kinase inhibitor profiling with kinobeads : Kinobead selectivity profiling was performed as described previously . Briefly, 5 mg mL −1 protein of a four cell line mix (Colo205, K562, OVCAR8, SKNBE2) were incubated with compound dilutions in DMSO (control, 3 n m , 10 n m , 30 n m , 100 n m , 300 n m , 1 μ m , 3 μ m , 30 μ m ) for 45 min at 4 °C on an end‐over‐end shaker.…”

Section: Methodsmentioning

confidence: 99%

Identification of Highly Potent Protein Kinase C‐Related Kinase 1 Inhibitors by Virtual Screening, Binding Free Energy Rescoring, and in vitro Testing

et al. 2016

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Despite the considerable interest in protein kinase C-related kinase 1 (PRK1) as a target in cancer research, there is still a lack of PRK1 inhibitors with suitable selectivity profiles and physicochemical properties. To identify new PRK1 inhibitors we applied a virtual screening approach, which combines ensemble docking, minimization of the protein-ligand complex, binding free energy calculations, and application of quantitative structure-activity relationship (QSAR) models for predicting in vitro activity. The developed approach was then applied in a prospective manner to screen available libraries of kinase inhibitors from Selleck and GlaxoSmithKline (GSK). Compounds that showed favorable prediction were then tested in vitro for PRK1 inhibition. Some of the hits were found to inhibit PRK1 in the low-nanomolar range. Three in vitro hits were additionally tested in a mass-spectrometry-based cellular kinase profiling assay to examine selectivity. Our findings show that nanomolar and drug-like inhibitors can be identified by the virtual screening approach presented herein. The identified inhibitors are valuable tools for gaining a better understanding of PRK1 inhibition, and the identified hits can serve as starting points for further chemical optimization.

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Optimized Chemical Proteomics Assay for Kinase Inhibitor Profiling

Cited by 114 publications

References 65 publications

Kinobead and Single-Shot LC-MS Profiling Identifies Selective PKD Inhibitors

Kinobead and Single-Shot LC-MS Profiling Identifies Selective PKD Inhibitors

Pharmacoproteomic characterisation of human colon and rectal cancer

Identification of Highly Potent Protein Kinase C‐Related Kinase 1 Inhibitors by Virtual Screening, Binding Free Energy Rescoring, and in vitro Testing

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