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Encarnacao, Joao Crispim, MasterORCID iD iconorcid.org/0000-0002-4509-4106
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Publications (2 of 2) Show all publications
Encarnação, J. C. (2019). Towards time-resolved molecular interaction assays in living bacteria. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Towards time-resolved molecular interaction assays in living bacteria
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Rare and neglected diseases such as multidrug resistant (MDR) tuberculosis, malaria and trypanosomiasis are re-emerging in Europe. New strategies are needed to accelerate drug discovery to fight these pathogens. AEGIS is a Pan-European project that combines different technologies to accelerate the discovery of molecules suitable for drug development in selected neglected diseases. This thesis is part of the AEGIS research area that considers time in a multidisciplinary approach, combining biology, physics and mathematics to provide tools to characterize biological events for improving drug development and information about the target diseases and lead compounds.

Real-time cell binding assays (RT-CBA) of receptor-ligand interactions are fundamental in basic research and drug discovery. However, this kind of assays are still rare on living cells, especially in the microbiology field. In this project, we apply the same high-precision assay type on bacterial systems and explored the interior of the cell with a time resolved assay.

The effect of temperature was evaluated in the RT-CBA using LigandTracer to ensure that it was possible to use the technology in a range of temperatures suitable for bacteria. A method for attaching Gram positive and negative bacteria on the surface of a normal Petri dish, showing a high reproducibly and a high cellular viability after 16 h. With these two key steps, an RT-CBA fit for microbiology is available.

Next, to answer biological questions, intracellular interactions were explored by expression and validation of intracellular proteins with fluorescent tags suitable for RT-CBAs. First, we used the subunit B from the Shiga toxin (STxB) as a model to understand different aspects about the internalization processes. RT-CBAs allowed to discovery new features of STxB binding and mechanism to deliver small molecules or small proteins into cancer cells. Then, for exploring intracellular interactions, insect cells were bioengineered for evaluating the ability of small molecules to internalize and bind to its target. Using Carbonic anhydrase II – sulfonamides as a model system, the molecular interaction in the cytoplasm could be measured using a quencher label approach. The development of this kind of novel RT-CBA tools provide new information about drug candidates for targets that are not properly expressed in bacterial cells.

The assays in this project can make drug design more efficient. Furthermore, the evaluation of binding activity of the new compounds developed by AEGIS, focusing on rare/neglected diseases, in a biological environment has the potential to accelerate drug discovery for the targeted emerging diseases.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 63
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1618
Keywords
Intracellular Molecular Interactions, Real-Time Cell Binding Assays, Bacteria, LigandTracer, Kinetics
National Category
Medical Laboratory and Measurements Technologies Pharmaceutical Sciences
Research subject
Biochemistry; Biopharmaceutics; Medical Science
Identifiers
urn:nbn:se:uu:diva-396008 (URN)978-91-513-0819-7 (ISBN)
Public defence
2020-01-24, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2019-12-18 Created: 2019-11-22 Last updated: 2020-01-13
Encarnacao, J. C., Schulte, T., Achour, A., Bjorkelund, H. & Andersson, K. (2018). Detecting ligand interactions in real time on living bacterial cells. Applied Microbiology and Biotechnology, 102(9), 4193-4201
Open this publication in new window or tab >>Detecting ligand interactions in real time on living bacterial cells
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2018 (English)In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 102, no 9, p. 4193-4201Article in journal (Refereed) Published
Abstract [en]

Time-resolved analysis assays of receptor-ligand interactions are fundamental in basic research and drug discovery. Adequate methods are well developed for the analysis of recombinant proteins such as antibody-antigen interactions. However, assays for time-resolved ligand-binding processes on living cells are still rare, in particular within microbiology. In this report, the real-time cell-binding assay (RT-CBA) technology LigandTracerA (R), originally designed for mammalian cell culture, was extended to cover Gram-positive and Gram-negative bacteria. This required the development of new immobilization methods for bacteria, since LigandTracer depends on cells being firmly attached to a Petri dish. The evaluated Escherichia coli CJ236 and BL21 as well as Staphylococcus carnosus TM300 strains were immobilized to plastic Petri dishes using antibody capture, allowing us to depict kinetic binding traces of fluorescently labeled antibodies directed against surface-displayed bacterial proteins for as long as 10-15 h. Interaction parameters, such as the affinity and kinetic constants, could be estimated with high precision (coefficient of variation 9-44%) and the bacteria stayed viable for at least 16 h. The other tested attachment protocols were inferior to the antibody capture approach. Our attachment protocol is generic and could potentially also be applied to other assays and purposes.

Place, publisher, year, edition, pages
SPRINGER, 2018
Keywords
Real-time interactions, Drug kinetics, Living bacteria, Antibodies
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-352571 (URN)10.1007/s00253-018-8919-3 (DOI)000429800600027 ()29550990 (PubMedID)
Funder
Swedish Research CouncilEU, European Research Council, 675555
Available from: 2018-08-07 Created: 2018-08-07 Last updated: 2019-11-22Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-4509-4106

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