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High content screening for inhibitors of protein interactions and post-translational modifications in primary cells by proximity ligation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology. (Cancer Pharmacology and Informatics/Rolf Larsson)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology. (Cancer Pharmacology and Informatics/Rolf Larsson)
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2010 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, no 1, 178-183 p.Article in journal (Refereed) Published
Abstract [en]

The cost of developing new drugs is a major obstacle for pharmaceutical companies and academia with many drugs identified in the drug discovery process failing approval for clinical use due to lack of intended effect or because of severe side effects. Since the early 1990 s, high throughput screening of drug compounds has increased enormously in capacity but has not resulted in a higher success rate of the identified drugs. Thus, there is a need for methods that can identify biologically relevant compounds and more accurately predict in vivo effects early in the drug discovery process. To address this, we developed a proximity ligation-based assay for high content screening of drug effects on signaling pathways. As a proof of concept, we used the assay to screen through a library of previously identified kinase inhibitors, including six clinically used tyrosine kinase inhibitors, to identify compounds that inhibited the platelet-derived growth factor (PDGF) receptor beta signaling pathway in stimulated primary human fibroblasts. Thirteen of the 80 compounds were identified as hits, and the dose responses of these compounds were measured. The assay exhibited a very high Z' factor (0.71) and signal to noise ratio (11.7), demonstrating excellent ability to identify compounds interfering with the specific signaling event. A comparison with regular immunofluorescence detection of phosphorylated PDGF receptor demonstrated a far superior ability by the in situ proximity ligation assay to reveal inhibition of receptor phosphorylation. In addition, inhibitor-induced perturbation of protein-protein interactions of the PDGF signaling pathway could be quantified, further demonstrating the usefulness of the assay in drug discovery.

Place, publisher, year, edition, pages
2010. Vol. 9, no 1, 178-183 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-112102DOI: 10.1074/mcp.M900331-MCP200ISI: 000275505900014PubMedID: 19864249OAI: oai:DiVA.org:uu-112102DiVA: diva2:284913
Available from: 2010-01-08 Created: 2010-01-08 Last updated: 2012-03-14Bibliographically approved
In thesis
1. High Content Analysis of Proteins and Protein Interactions by Proximity Ligation
Open this publication in new window or tab >>High Content Analysis of Proteins and Protein Interactions by Proximity Ligation
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fundamental to all biological processes is the interplay between biomolecules such as proteins and nucleic acids. Studies of interactions should therefore be more informative than mere detection of expressed proteins. Preferably, such studies should be performed in material that is as biologically and clinically relevant as possible, i.e. in primary cells and tissues. In addition, to be able to take into account the heterogeneity of such samples, the analyses should be performed in situ to retain information on the sub-cellular localization where the interactions occur, enabling determination of the activity status of individual cells and allowing discrimination between e.g. tumor cells and surrounding stroma. This requires assays with an utmost level of sensitivity and selectivity.

Taking these issues into consideration, the in situ proximity-ligation assay (in situ PLA) was developed, providing localized detection of proteins, protein-protein interactions and post-translational modifications in fixed cells and tissues. The high sensitivity and selectivity afforded by the assay's requirement for dual target recognition in combination with powerful signal amplification enables visualization of single protein molecules in intact single cells and tissue sections.

To further increase the usefulness and application of in situ PLA, the assay was adapted to high content analysis techniques such as flow cytometry and high content screening. The use of in situ PLA in flow cytometry offers the possibility for high-throughput analysis of cells in solution with the unique characteristics offered by the assay. For high content screening, it was demonstrated that in situ PLA can enable cell-based drug screening of compounds affecting post-translational modifications and protein-protein interactions in primary cells, offering superior abilities over current assays.

The methods presented in this thesis provide powerful new tools to study proteins in genetically unmodified cells and tissues, and should offer exciting new possibilities for molecular biology, diagnostics and drug discovery.



Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 56 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 530
Keyword
in situ, proximity ligation, flow cytometry, high content screening, rolling circle amplification, in situ PLA, single-cell, single-molecule, protein interactions, drug screening, post-translational modifications
National Category
Cell and Molecular Biology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-119530 (URN)978-91-554-7739-4 (ISBN)
Public defence
2010-04-16, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2010-03-25 Created: 2010-02-26 Last updated: 2010-03-25Bibliographically approved
2. Visualization of Protein Activity Status in situ Using Proximity Ligation Assays
Open this publication in new window or tab >>Visualization of Protein Activity Status in situ Using Proximity Ligation Assays
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In 2001 the human proteome organization (HUPO) was created with the ambition to identify and characterize all proteins encoded in the human genome according to several criteria; their expression levels in different tissues and under different conditions; the sub-cellular localization; post-translational modifications; interactions, and if possible also the relationship between their structure and function.When the knowledge of different proteins and their potential interactions increases, so does the need for methods able to unravel the nature of molecular processes in cells and organized tissues, and ultimately for clinical use in samples obtained from patients.

The in situ proximity ligation assay (in situ PLA) was developed to provide localized detection of proteins, post-translational modifications and protein-protein interactions in fixed cells and tissues. Dual recognition of the target or interacting targets is a prerequisite for the creation of a circular reporter DNA molecule, which subsequently is locally amplified for visualization of individual protein molecules in single cells. These features offer the high sensitivity and selectivity required for detection of even rare target molecules.

Herein in situ PLA was first established and then employed as a tool for detection of both interactions and post-translational modifications in cultured cells and tissue samples. In situ PLA was also adapted to high content screening (HCS) for therapeutic effects, where it was applied for cell-based drug screening of inhibitors influencing post-translational modifications. This was performed using primary cells, paving the way for evaluation of drug effects on cells from patient as a diagnostic tool in personalized medicine.

In conclusion, this thesis describes the development and applications of in situ PLA as a tool to study proteins, post-translational modifications and protein-protein interactions in genetically unmodified cells and tissues, and for clinical interactomics.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 44 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 609
Keyword
proximity ligation, in situ, high content screening, platelet-derived growth factor receptor, rolling circle amplification, protein interactions, in situ PLA, post-translational modifications, drug screening
National Category
Cell and Molecular Biology Medical Genetics
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-131934 (URN)978-91-554-7919-0 (ISBN)
Public defence
2010-12-04, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2010-11-11 Created: 2010-10-11 Last updated: 2011-01-13Bibliographically approved

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Landegren, UlfLarsson, RolfSöderberg, OlaFryknäs, MårtenJarvius, Jonas

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