Heymann, Tim (2023)
Synthesis and mass spectrometry based characterization of chemical tools to explore FK506 binding proteins.
Technische Universität Darmstadt
doi: 10.26083/tuprints-00021441
Dissertation, Erstveröffentlichung, Verlagsversion
Kurzbeschreibung (Abstract)
The FK506 binding proteins (FKBPs) are a protein family belonging to the immunophilins and are interesting drug targets. FKBP51 is associated with stress-related diseases, metabolic diseases and chronic pain which makes it an interesting drug target. Microbial or parasitic FKBPs (Mips) are important for the replication of pathogens and thus inhibitors for Mips are anti-infective. While the effects of addressing FKBPs by knockout or with FKBP ligands are well characterized, the underlying mechanisms leading to the observed effects on the cell level are unclear and the literature is often controversial. To address this problem, several chemical tools and MS based methods for their characterization and later use have been developed in this thesis. The development of a gram scale SAFit2 synthesis makes this so far best chemical tool for FKBP51 available for extensive in vivo experiments, allowing further study of FKBP51 inhibition in animals. The development of a synthesis for a diazirine building block allows coupling of this photoreactive warhead to almost any given ligand or molecule of choice via a linker. This allows a broad spectrum of new photoreactive ligands with versatile use, as demonstrated by the photoreactive ligands and cysteine reactive photo labels synthesized and used for photoaffinity labeling in this thesis. Development of a simple protein intact mass LC-MS analysis supplemented by a top-down approach allows assessment of in vitro experiments performed with chemical crosslinkers, facilitating rapid identification of labeled proteins with high confidence. A more sophisticated bottom-up proteomics approach allows the use and analysis of photoreactive FKBP ligands in cells, enabling the identification of protein binding partners and ligand off-targets otherwise not possible by targeted methods such as western blotting. Both LC-MS based methods in combination with the photoreactive chemical tools make up a powerful toolbox for the analysis of FKBPs.
| Typ des Eintrags: | Dissertation | ||||
|---|---|---|---|---|---|
| Erschienen: | 2023 | ||||
| Autor(en): | Heymann, Tim | ||||
| Art des Eintrags: | Erstveröffentlichung | ||||
| Titel: | Synthesis and mass spectrometry based characterization of chemical tools to explore FK506 binding proteins | ||||
| Sprache: | Englisch | ||||
| Referenten: | Hausch, Prof. Dr. Felix ; Meyer-Almes, Prof. Dr. Franz-Josef | ||||
| Publikationsjahr: | 2023 | ||||
| Ort: | Darmstadt | ||||
| Kollation: | xii, 169 Seiten | ||||
| Datum der mündlichen Prüfung: | 9 Mai 2022 | ||||
| DOI: | 10.26083/tuprints-00021441 | ||||
| URL / URN: | https://tuprints.ulb.tu-darmstadt.de/21441 | ||||
| Kurzbeschreibung (Abstract): | The FK506 binding proteins (FKBPs) are a protein family belonging to the immunophilins and are interesting drug targets. FKBP51 is associated with stress-related diseases, metabolic diseases and chronic pain which makes it an interesting drug target. Microbial or parasitic FKBPs (Mips) are important for the replication of pathogens and thus inhibitors for Mips are anti-infective. While the effects of addressing FKBPs by knockout or with FKBP ligands are well characterized, the underlying mechanisms leading to the observed effects on the cell level are unclear and the literature is often controversial. To address this problem, several chemical tools and MS based methods for their characterization and later use have been developed in this thesis. The development of a gram scale SAFit2 synthesis makes this so far best chemical tool for FKBP51 available for extensive in vivo experiments, allowing further study of FKBP51 inhibition in animals. The development of a synthesis for a diazirine building block allows coupling of this photoreactive warhead to almost any given ligand or molecule of choice via a linker. This allows a broad spectrum of new photoreactive ligands with versatile use, as demonstrated by the photoreactive ligands and cysteine reactive photo labels synthesized and used for photoaffinity labeling in this thesis. Development of a simple protein intact mass LC-MS analysis supplemented by a top-down approach allows assessment of in vitro experiments performed with chemical crosslinkers, facilitating rapid identification of labeled proteins with high confidence. A more sophisticated bottom-up proteomics approach allows the use and analysis of photoreactive FKBP ligands in cells, enabling the identification of protein binding partners and ligand off-targets otherwise not possible by targeted methods such as western blotting. Both LC-MS based methods in combination with the photoreactive chemical tools make up a powerful toolbox for the analysis of FKBPs. |
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| Alternatives oder übersetztes Abstract: |
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| Status: | Verlagsversion | ||||
| URN: | urn:nbn:de:tuda-tuprints-214415 | ||||
| Sachgruppe der Dewey Dezimalklassifikatin (DDC): | 500 Naturwissenschaften und Mathematik > 540 Chemie | ||||
| Fachbereich(e)/-gebiet(e): | 07 Fachbereich Chemie 07 Fachbereich Chemie > Clemens-Schöpf-Institut > Fachgebiet Biochemie |
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| Hinterlegungsdatum: | 09 Mai 2023 12:03 | ||||
| Letzte Änderung: | 10 Mai 2023 07:46 | ||||
| PPN: | |||||
| Referenten: | Hausch, Prof. Dr. Felix ; Meyer-Almes, Prof. Dr. Franz-Josef | ||||
| Datum der mündlichen Prüfung / Verteidigung / mdl. Prüfung: | 9 Mai 2022 | ||||
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| Suche nach Titel in: | TUfind oder in Google | ||||
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