Leuenberger, Michele; Jack, Thomas; Ruepp, Marc-David; Lagache, Sophie; Heller, Manfred; Mühlemann, Oliver; Thompson, Andrew J.; Lochner, Martin (15 June 2015). Synthesis of photo-crosslinking probes and their application for the site-selective chemical modification of the 5-HT3 receptor (Unpublished). In: Challenges in Chemical Biology (ISACS16). Book of Abstracts. ETH Zürich, Hönggerberg, Switzerland. 15.06.-18.06.2015.
The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the CNS and PNS that is activated by the endogenous agonist serotonin (5-hydroxytryptamine, 5-HT). 5-HT3R is the only serotonin receptor belonging to the Cys-loop superfamily of neurotransmitter receptors. Different structural biology approaches can be applied, such as crystallization and x-ray analysis. Nonetheless, characterizing the exact ligand binding site(s) of these dynamic receptors is still challenging. The use of photo-crosslinking probes is an alternative validated approach allowing identification of regions in the protein that are important for the binding of small molecules. We designed our probes based on the core structure of the 5-HT3R antagonist granisetron, a FDA approved drug used for the treatment of chemotherapy-induced nausea and vomiting. We synthesized a small library of photo-crosslinking probes by conjugating diazirines and benzophenones via various linkers to granisetron. We were able to obtain several compounds with diverse linker lengths and different photo-crosslinking moieties that show nanomolar binding affinity for the orthosteric binding site. Furthermore we established a stable h5-HT3R expressing cell line and a purification protocol to yield the receptor in a high purity. Several experiments showed unambiguously that we are able to photo-crosslink our probes with the receptor site-specifically. The functionalised protein was analysed by Western blot and MS-analysis. This yielded the exact covalent modification site, corroborating current ligand binding models derived from mutagenesis and docking studies.
Item Type: |
Conference or Workshop Item (Poster) |
|---|---|
Division/Institute: |
08 Faculty of Science > Department of Chemistry, Biochemistry and Pharmaceutical Sciences (DCBP) 04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > DCR Services > Core Facility Massenspektrometrie- und Proteomics-Labor |
UniBE Contributor: |
Leuenberger, Michele, Jack, Thomas, Ruepp, Marc-David, Braga, Sophie Marie-Pierre, Heller, Manfred, Mühlemann, Oliver, Lochner, Martin |
Subjects: |
500 Science > 570 Life sciences; biology 500 Science > 540 Chemistry |
Publisher: |
Royal Society of Chemistry (RSC) |
Language: |
English |
Submitter: |
Martin Lochner |
Date Deposited: |
22 Jun 2015 14:45 |
Last Modified: |
02 Mar 2023 23:26 |
URI: |
https://boris.unibe.ch/id/eprint/69680 |
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