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Sensitive Measurement of Drug-Target Engagement Using Cellular Thermal Shift Assays with Multiplex Proximity Extension Assay Readout
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Science for Life Laboratory, SciLifeLab, Science for Life Laboratory, SciLifeLab. (Molekylära verktyg, Molecular tools)ORCID iD: 0000-0002-0762-9034
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
Department of Oncology-Pathology, Karolinska Institutet.
Department of Medicine, Karolinska Institutet.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The ability to measure target engagement in cellular contexts is key for successful drug discovery and clinical care. The cellular thermal shift assay (CETSA) provides realistic information about drug binding in cells and tissues, revealing drug-target engagement in clinically relevant samples. CETSA combined with mass spectrometry (MS) readout can be applied in the early hit identification phase to generate target engagement data for large sets of proteins. However, the analysis low-throughput and requires substantial amounts of sample material. Here, we combined CETSA and the multiplex proximity extension assay (PEA) for analysis of target engagement of 184 proteins from minimal sample material treated with kinase inhibitors. PEA allows analyses of large numbers of specific target proteins at high sensitivity in small sample aliquots. We observed concordant results for proteins measured by MS or PEA. This highly sensitive CETSA-PEA procedure is promising for monitoring drug-target engagement in small aliquots of patient material for analysis of drug binding in drug development and in clinical settings. 

National Category
Natural Sciences
Research subject
Medical Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-374264OAI: oai:DiVA.org:uu-374264DiVA, id: diva2:1280488
Available from: 2019-01-18 Created: 2019-01-18 Last updated: 2019-01-21
In thesis
1. Molecular Approaches to Explore Drug-Target Interactions
Open this publication in new window or tab >>Molecular Approaches to Explore Drug-Target Interactions
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Improved means to assess the clinical potential of drug candidates can critically influence development of new therapeutic entities, a central aim in medical life science. Drug discovery and development relies on construction and selection of small organic compounds or biological agents that bind targets of interest. This thesis includes new methodology to investigate target engagement - that is the tendency for these drugs and drug candidates to bind their intended target molecules versus any off-targets. This is a matter of great importance and current strong interest in the pharmaceutical industry as well as academically and an important aim for precision medicine. Paper I describes the target engagement-mediated amplification (TEMA) technique, an accurate, selective and physiological relevant techniques to monitor target binding by DNA-conjugated low molecular weight drug molecules. The DNA conjugated forms of the drugs are uniquely suited to accurately and sensitively reveal the binding characteristics of drugs directly in relevant tissues. Paper II describes the evaluation of cellular thermal shift assays (CETSA) by multiplex proximity extension assays (PEA), to sensitively measure binding of drugs to their proper targets and off-targets in minimal samples of cells and tissues, and for many targets and samples in parallel. The technique provides valuable advantages during drug development, and potentially also in clinical care. Paper III describes a high-throughput approach to use in situ proximity ligation assays to investigate protein interactions or modifications along with phenotypic responses to drugs or cytokines. The technique allows responses by large numbers of cells to be evaluated by automated microscopy and computer-based analysis. Our approach expands the scope for combined molecular and morphological profiling, offering an information-rich means to profile cellular responses to drugs and other agents at the single cell level.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 46
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1533
Keywords
Drug discovery, target engagement, target engagement-mediated amplification, cellular thermal shift assay, proximity extension assay, in situ PLA, high-content imaging
National Category
Medical and Health Sciences
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-374329 (URN)978-91-513-0560-8 (ISBN)
Public defence
2019-03-08, Svedbergsalen (B8), Biomedicinskt centrum, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2019-02-11 Created: 2019-01-21 Last updated: 2019-02-19

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Al-Amin, Rasel A.Gallant, Caroline J.Landegren, Ulf
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