Sensing cell states induced by chemical probes and approved drugs using fluorescent based biological reporters
- Bioactive small molecules are used in many research areas as important tools to uncover biological pathways, interpret phenotypic changes, deconvolute protein functions and explore new therapeutic strategies in disease relevant cellular model systems. To unlock the full potential of these small molecules and to ensure reliability of results obtained in cellular assays, it is crucial to understand the properties of these small molecules. These properties encompass their activity and potency on their designated target(s), their selectivity towards unintended off-targets and their phenotypic effects in a cellular system. Approved drugs often engage with multiple targets, which can be beneficial for some applications such as treatment of cancer where several pathways need to be inhibited for treatment efficacy. However, targeting multiple key proteins in diverse pathways also increases the possibility for unspecific or unwanted side effects. For many drugs the entire target space that they modulate is not known. This makes it difficult to use these drugs for target deconvolution or functional assays with the aim to understand the underlying biological processes. In contrast to drugs, for mechanistic studies, a good alternative are chemical tool compounds so called chemical probes that are usually exclusively selective as well as chemogenomic compounds, that inhibit several targets but have narrow selectivity profiles. Because they are mechanistic tools, chemical tool compounds must meet stringent quality criteria and they are therefore well characterized in terms of their potency, selectivity and cellular on-target activity. To ensure that an observed phenotypic effect caused by a compound can be attributed to the described target(s), it is essential to study also properties of chemical tools leading to unspecific cellular effects. There are a variety of unspecific effects that can be caused by physiochemical compound properties that can interfere with phenotypic assays as well as functional compound evaluations. One of these effects is low solubility causing toxicity or intrinsic fluorescence potentially interfering with assay readouts. But unanticipated cellular responses can also arise from unspecific binding, accumulation in cellular compartments or damage caused to organelles such as mitochondria or the cytoskeleton that can result in the induction of diverse forms of cell death. In this study, we investigated the influence of a variety of small molecules on distinct cell states, by establishing and validating high-content imaging assays, which we called Multiplex assay. This assay portfolio enabled us to detect different cellular responses using diverse fluorescent reporters, such as the influence of a compound on cell viability, induction of cell death programs and modulation of the cell cycle. Additionally, general compound properties such as precipitation and intrinsic fluorescence were simultaneously detected. The assay is adaptable to assess other cellular properties of interest, such as mitochondrial health, changes in cytoskeletal morphology or phospholipidosis. A significant advantage of the assay is that we are using live cells, so we can capture dynamic cellular changes and fluctuations that can be crucial for the understanding of cellular responses.
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| Author: | Amelie MengeGND |
|---|---|
| URN: | urn:nbn:de:hebis:30:3-844453 |
| DOI: | https://doi.org/10.21248/gups.84445 |
| Place of publication: | Frankfurt am Main |
| Referee: | Stefan KnappORCiD, Achim SchmidtkoORCiDGND |
| Document Type: | Doctoral Thesis |
| Language: | English |
| Date of Publication (online): | 2024/05/16 |
| Year of first Publication: | 2023 |
| Publishing Institution: | Universitätsbibliothek Johann Christian Senckenberg |
| Granting Institution: | Johann Wolfgang Goethe-Universität |
| Date of final exam: | 2024/05/07 |
| Release Date: | 2024/05/17 |
| Page Number: | 310 |
| Note: | Kumulative Dissertation - enthält de Verlagsversionen (Versions of Record) der folgenden Artikel und Buchkapitel: Tjaden, Amelie; Chaikuad, Apirat; Kowarz, Eric; Marschalek, Rolf; Knapp, Stefan; Schröder, Martin; Müller, Susanne (2022): Image-Based Annotation of Chemogenomic Libraries for Phenotypic Screening. Molecules 2022, 27, Artikel 143, ISSN 1420-3049. DOI 10.3390/molecules27041439 Tjaden, Amelie; Giessmann, Robert T.; Knapp, Stefan; Schröder, Martin; Müller, Susanne (2022): High-Content Live-Cell Multiplex Screen for Chemogenomic Compound Annotation Based on Nuclear Morphology. STAR Protocols 2022, 3 no. 4 (2022/12/16/ 2022), Artikel 101791, eISSN 2666-1667. DOI 10.1016/j.xpro.2022.101791 Elson, Lewis; Tjaden, Amelie; Knapp, Stefan; Müller, Susanne (2023): Characterization of Cellular Viability Using Label-Free Brightfield Live-Cell Imaging, in Chemogenomics: Methods and Protocols. Edited by Daniel Merk and Apirat Chaikuad. New York, NY: Springer US, 2023. https://doi.org/10.1007/978-1-0716-3397-7_6 Tjaden, Amelie; Knapp, Stefan; Müller, Susanne (2023): Annotation of the Effect of Chemogenomic Compounds on Cell Health Using High-Content Microscopy in Live-Cell Mode. In Chemogenomics: Methods and Protocols.Edited by Daniel Merk and Apirat Chaikuad. New York, NY: Springer US, 2023. https://doi.org/10.1007/978-1-0716-3397-7_5 Tredup, Claudia; Ndreshkjana, Bernardina; Schneider, Natalie S.; Tjaden, Amelie; Kemas, Aurino M.; Youhanna, Sonia; Lauschke, Volker M.; Berger, Benedict-Tilmann; Krämer, Andreas; Berger, Lena M.; Röhm, Sandra; Knapp, Stefan; Farin, Henner F.; Müller, Susanne (2023): Deep Annotation of Donated Chemical Probes (Dcp) in Organotypic Human Liver Cultures and Patient-Derived Organoids from Tumor and Normal Colorectum. ACS Chemical Biology 2023, Vol. 18, Iss 4, Seite 822–836, ISSN 1554-8929. DOI 10.1021/acschembio.2c00877 Kurz, Christian G.; Preuss, Franziska; Tjaden, Amelie; Cusack, Martin; Amrhein, Jennifer Alisa; Chatterjee, Deep; Mathea, Sebastian; Berger, Lena Marie; Berger, Benedict-Tilman; Krämer, Andreas; Weller, Michael; Weiss, Tobias; Müller, Susanne; Knapp, Stefan; Hanke, Thomas (2022): Illuminating the Dark: Highly Selective Inhibition of Serine/Threonine Kinase 17a with Pyrazolo[1,5-a]Pyrimidine-Based Macrocycles. Journal of Medicinal Chemistry 2022, 2022, 65, 11, 7799–7817, ISSN 0022-2623. DOI 10.1021/acs.jmedchem.2c00173 Röhm, Sandra; Berger, Benedict-Tilman; Schröder, Martin; Chatterjee, Deep; Mathea, Sebastian; Joerger, Andreas C.; Pinkas, Daniel M.; Bufton, Joshua C.; Tjaden, Amelie; Kovooru, Lohitesh; Kudolo, Mark; Pohl, Christian; Bullock, Alex N.; Müller, Susanne; Laufer, Stefan; Knapp, Stefan (2021): Development of a Selective Dual Discoidin Domain Receptor (Ddr)/P38 Kinase Chemical Probe. Journal of Medicinal Chemistry 2021, 64, 18, 13451–13474, ISSN 0022-2623. DOI 10.1021/acs.jmedchem.1c00868 Amrhein, Jennifer Alisa; Berger, Lena Marie; Tjaden, Amelie; Krämer, Andreas; Elson, Lewis; Tolvanen, Tuomas; Martinez-Molina, Daniel; Kaiser, Astrid; Schubert-Zsilavecz; Manfred; Müller, Susanne; Knapp, Stefan; Hanke, Thomas (2022): Discovery of 3-Amino-1h-Pyrazole-Based Kinase Inhibitors to Illuminate the Understudied Pctaire Family. International Journal of Molecular Sciences 2022, 23 (23), Artikel 14834, eISSN 1000-9035. DOI 10.3390/ijms232314834 Hu, Huabin, Tjaden, Amelie; Knapp, Stefan; Antolin, Albert A.; Müller, Susanne (2023): A Machine Learning and Live-Cell Imaging Tool Kit Uncovers Small Molecules Induced Phospholipidosis. Cell Chemical Biology2 023 Volume 30 (12), Seite 1634-1651, ISSN 2451-9456. DOI 10.1016/j.chembiol.2023.09.003. |
| HeBIS-PPN: | 518404102 |
| Institutes: | Biochemie, Chemie und Pharmazie |
| Dewey Decimal Classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
| Sammlungen: | Universitätspublikationen |
| Sammlung Biologie / Biologische Hochschulschriften (Goethe-Universität) | |
| Licence (German): |  Deutsches Urheberrecht |
