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Quantitative Interpretation of Intracellular Drug Binding and Kinetics Using the Cellular Thermal Shift Assay
Karolinska Inst, Sci Life Labs, Chem Biol Consortium Sweden, SE-17165 Solna, Sweden.;Karolinska Inst, Dept Med Biochem & Biophys, SE-17165 Solna, Sweden..
Karolinska Inst, Sci Life Labs, Chem Biol Consortium Sweden, SE-17165 Solna, Sweden.;Karolinska Inst, Dept Med Biochem & Biophys, SE-17165 Solna, Sweden..
Karolinska Inst, Sci Life Labs, Chem Biol Consortium Sweden, SE-17165 Solna, Sweden.;Karolinska Inst, Dept Med Biochem & Biophys, SE-17165 Solna, Sweden..
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry.ORCID iD: 0000-0003-0596-0222
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2018 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 57, no 48, p. 6715-6725Article in journal (Refereed) Published
Abstract [en]

Evidence of physical interaction with the target protein is essential in the development of chemical probes and drugs. The cellular thermal shift assay (CETSA) allows evaluation of drug binding in live cells but lacks a framework to support quantitative interpretations and comparisons with functional data. We outline an experimental platform for such analysis using human kinase p38 alpha. Systematic variations to the assay's characteristic heat challenge demonstrate an apparent loss of compound potency with an increase in duration or temperature, in line with expectations from the literature for thermal shift assays. Importantly, data for five structurally diverse inhibitors can be quantitatively explained using a simple model of linked equilibria and published binding parameters. The platform further distinguishes between ligand mechanisms and allows for quantitative comparisons of drug binding affinities and kinetics in live cells and lysates. We believe this work has broad implications in the appropriate use of the CETSA for target and compound validation.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2018. Vol. 57, no 48, p. 6715-6725
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-240736DOI: 10.1021/acs.biochem.8b01057ISI: 000452693000009PubMedID: 30418016Scopus ID: 2-s2.0-85057521293OAI: oai:DiVA.org:kth-240736DiVA, id: diva2:1276368
Funder
Swedish Research CouncilScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20190108

Available from: 2019-01-08 Created: 2019-01-08 Last updated: 2019-01-08Bibliographically approved

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Dahlgren, BjörnJonsson, Mats

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