title: CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil.
abstract: Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery.
authors.affiliation: Laboratories for Chemical Biology, Karolinska Institutet, Science for Life Laboratory Stockholm, Division of Translational Medicine &Chemical Biology, Department of Medical Biochemistry &Biophysics, Karolinska Institutet, Tomtebodavägen 23A, Solna 171 65, Sweden.
authors.forename: Helena
authors.initials: H
authors.lastname: Almqvist
authors.affiliation: Laboratories for Chemical Biology, Karolinska Institutet, Science for Life Laboratory Stockholm, Division of Translational Medicine &Chemical Biology, Department of Medical Biochemistry &Biophysics, Karolinska Institutet, Tomtebodavägen 23A, Solna 171 65, Sweden.
authors.forename: Hanna
authors.initials: H
authors.lastname: Axelsson
authors.affiliation: Department of Medical Biochemistry &Biophysics, Division of Biophysics, Karolinska Institutet, Scheeles väg 2, Stockholm 171 77, Sweden.
authors.forename: Rozbeh
authors.identifier: http://orcid.org/0000-0002-3396-4709
authors.initials: R
authors.lastname: Jafari
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 61 Biopolis Drive (Proteos), Singapore 138673, Singapore.
authors.forename: Chen
authors.initials: C
authors.lastname: Dan
authors.affiliation: Department of Pharmacy, Uppsala University, BMC, Box 580, Uppsala SE-751 23, Sweden.
authors.forename: André
authors.initials: A
authors.lastname: Mateus
authors.affiliation: Laboratories for Chemical Biology, Karolinska Institutet, Science for Life Laboratory Stockholm, Division of Translational Medicine &Chemical Biology, Department of Medical Biochemistry &Biophysics, Karolinska Institutet, Tomtebodavägen 23A, Solna 171 65, Sweden.
authors.forename: Martin
authors.initials: M
authors.lastname: Haraldsson
authors.affiliation: School of Biological Sciences, Nanyang Technological University, SBS-04s-45, 60 Nanyang Drive, Singapore 639798, Singapore.
authors.forename: Andreas
authors.initials: A
authors.lastname: Larsson
authors.affiliation: Department of Medical Biochemistry &Biophysics, Division of Biophysics, Karolinska Institutet, Scheeles väg 2, Stockholm 171 77, Sweden.
authors.forename: Daniel
authors.initials: D
authors.lastname: Martinez Molina
authors.affiliation: Department of Pharmacy, Uppsala University, BMC, Box 580, Uppsala SE-751 23, Sweden.|Uppsala University Drug Optimization and Pharmaceutical Profiling Platform (UDOPP), Department of Pharmacy, Uppsala University, BMC, Box 580, Uppsala SE-751 23, Sweden.|Science for Life Laboratory Drug Discovery and Development platform, Uppsala University, Uppsala SE-751 23, Sweden.
authors.forename: Per
authors.initials: P
authors.lastname: Artursson
authors.affiliation: Laboratories for Chemical Biology, Karolinska Institutet, Science for Life Laboratory Stockholm, Division of Translational Medicine &Chemical Biology, Department of Medical Biochemistry &Biophysics, Karolinska Institutet, Tomtebodavägen 23A, Solna 171 65, Sweden.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lundbäck
authors.affiliation: Department of Medical Biochemistry &Biophysics, Division of Biophysics, Karolinska Institutet, Scheeles väg 2, Stockholm 171 77, Sweden.|School of Biological Sciences, Nanyang Technological University, 61 Biopolis Drive (Proteos), Singapore 138673, Singapore.|Institute of Cellular and Molecular Biology, ASTAR, 61 Biopolis Drive (Proteos), Singapore 138673, Singapore.
authors.forename: Pär
authors.initials: P
authors.lastname: Nordlund
mesh_terms: D065767:Activation, Metabolic / Q000187:drug effects; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001681:Biological Assay; D003641:Deamination / Q000187:drug effects; D000077209:Decitabine; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005472:Fluorouracil / Q000378:metabolism*; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D020014:K562 Cells; D007700:Kinetics; D010766:Phosphorylation / Q000187:drug effects; D054852:Small Molecule Libraries / Q000032:analysis / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries; D000077209:Decitabine; D013940:Thymidylate Synthase; D001374:Azacitidine; D005472:Fluorouracil