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CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil
Laboratories for Chemical Biology Karolinska Institutet Science for Life Laboratory Stockholm, Division of Translational Medicine & Chemical Biology.
Laboratories for Chemical Biology Karolinska Institutet Science for Life Laboratory Stockholm, Division of Translational Medicine & Chemical Biology.
Department of Medical Biochemistry & Biophysics, Division of Biophysics, Karolinska Institutet.
School of Biological Sciences, Nanyang Technological University.
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2016 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 7, 11040Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2016. Vol. 7, 11040
National Category
Pharmacology and Toxicology
URN: urn:nbn:se:uu:diva-276077DOI: 10.1038/ncomms11040ISI: 000372887500001PubMedID: 27010513OAI: diva2:901870
The Karolinska Institutet's Research FoundationSwedish Research CouncilSwedish Cancer SocietyKnut and Alice Wallenberg Foundation
Available from: 2016-02-09 Created: 2016-02-09 Last updated: 2016-05-25Bibliographically approved
In thesis
1. Intracellular unbound drug concentrations: Methodology and application for understanding cellular drug exposure
Open this publication in new window or tab >>Intracellular unbound drug concentrations: Methodology and application for understanding cellular drug exposure
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Most known drug targets and metabolizing enzymes are located inside cells. Interactions with these proteins are determined by intracellular unbound drug concentrations. Assessing intracellular drug exposure is technically challenging, but essential for predicting pharmacokinetic, pharmacological, and toxicological profiles of new drugs.

This thesis aims at establishing and applying a straightforward methodology to measure intracellular unbound drug concentrations. This was achieved by separately measuring cellular drug binding (fu,cell), and total intracellular drug accumulation (Kp). This allowed the calculation of intracellular drug bioavailability (Fic), which represents the fraction of the concentration added to the cells that is unbound in the cell interior.

The methodology was initially developed in HEK293 cells, where the Fic of 189 drug-like compounds was measured. Binding to HEK293 cells was governed by compound lipophilicity and was correlated with binding to more complex systems, such as hepatocytes and brain. Due to negligible expression of drug transporters, Fic in this cell line was consistent with pH-dependent subcellular sequestration of lipophilic cations in low pH compartments.

The methodology was then applied to study the effects of drug transporters on Fic. The uptake transporter OATP1B1 increased the Fic of its substrates in a concentration-dependent manner. In contrast, the Fic of P-gp substrates was decreased when P-gp was present. In human hepatocytes, the methodology allowed the determination of Fic without prior knowledge of transporter mechanisms or metabolic activity.

Finally, the methodology was applied to measure the impact of Fic on target binding and cellular drug response. Intracellular concentrations of active metabolites of pro-drugs targeting the intracellular target thymidylate synthase were in agreement with the level of binding to this target. Further, high Fic was generally required for kinase and protease inhibitors to be active in cellular assays.

In conclusion, the methodology can be used to predict if new drug candidates reach their intracellular targets in sufficient amounts. Furthermore, the methodology can improve in vitro predictions of drug clearance and drug-drug interactions, by measuring the drug available for intracellular enzymes. Finally, this work can be expanded to other xenobiotics, e.g., to predict their intracellular toxicity.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 69 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 211
intracellular unbound drug concentrations, free drug, drug binding, drug transport, drug accumulation, cellular drug response, drug target engagement
National Category
Pharmaceutical Sciences Pharmacology and Toxicology
Research subject
Pharmaceutical Science
urn:nbn:se:uu:diva-276095 (URN)978-91-554-9496-4 (ISBN)
Public defence
2016-04-22, room B21, Biomedical center, Husargatan 3, Uppsala, 09:15 (English)
Available from: 2016-03-31 Created: 2016-02-09 Last updated: 2016-04-04

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