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Target Engagement-Mediated Amplification for Monitoring Drug-Target Interactions in Situ
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Science for Life Laboratory, SciLifeLab, Science for Life Laboratory, SciLifeLab. (Molekylära verktyg, Molecular tools)ORCID-id: 0000-0002-0762-9034
Division of Translational Medicine & Chemical Biology, Department of Medical Biochemistry & Biophysics, Karolinska Institutet.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Autoimmunitet. Uppsala universitet, Science for Life Laboratory, SciLifeLab. Department of Medicine (Solna), Karolinska University Hospital, Karolinska Institutet. (Autoimmunitet, Autoimmunity)ORCID-id: 0000-0002-6163-9540
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
Vise andre og tillknytning
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
Abstract [en]

It is important to determine the localization of drugs or drug candidates at cellular and subcellular resolution in relevant clinical specimens. This is necessary to evaluate drug candidates from early stages of drug development to clinical evaluation of mutations potentially causing resistance to targeted therapy. We describe a technology where oligonucleotide-conjugated drug molecules are used to visualize and measure target engagement in situ via rolling-circle amplification (RCA) of circularized oligonucleotide probes (padlock probes). We established this target engagement-mediated amplification (TEMA) technique using kinase inhibitor precursor compounds, and we applied the assay to investigate target interactions by microscopy in pathology tissue sections and using flow cytometry for blood samples from patients, as well as in commercial arrays including almost half of all human proteins.  In the variant proxTEMAtechnique, in situ proximity ligation assays were performed by combining drug-DNA conjugates with antibody-DNA conjugates to specifically reveal drug binding to particular on- or off-targets in pathological tissues sections. In conclusion, the TEMA methods successfully visualize drug-target interaction by experimental and clinically approved kinase inhibitors in situ and with kinases among a large collection of arrayed proteins. 

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Forskningsprogram
Molekylär bioteknik
Identifikatorer
URN: urn:nbn:se:uu:diva-374262OAI: oai:DiVA.org:uu-374262DiVA, id: diva2:1280481
Tilgjengelig fra: 2019-01-18 Laget: 2019-01-18 Sist oppdatert: 2019-01-21
Inngår i avhandling
1. Molecular Approaches to Explore Drug-Target Interactions
Åpne denne publikasjonen i ny fane eller vindu >>Molecular Approaches to Explore Drug-Target Interactions
2019 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2019. s. 46
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1533
Emneord
Drug discovery, target engagement, target engagement-mediated amplification, cellular thermal shift assay, proximity extension assay, in situ PLA, high-content imaging
HSV kategori
Forskningsprogram
Molekylär medicin
Identifikatorer
urn:nbn:se:uu:diva-374329 (URN)978-91-513-0560-8 (ISBN)
Disputas
2019-03-08, Svedbergsalen (B8), Biomedicinskt centrum, Husargatan 3, Uppsala, 13:15 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2019-02-11 Laget: 2019-01-21 Sist oppdatert: 2019-02-19

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