Entwicklung, Synthese und Charakterisierung niedermolekularer Liganden G-Protein gekoppelter Rezeptoren
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Abstract
With more than 800 members, G-protein-coupled receptors (GPCRs) represent one of the largest protein superfamilies in humans. GPCRs can be activated by a wide variety of extracellular stimuli and due to their diverse signal transduction, they are associated with many diseases such as cancer, inflammation, immune diseases and the metabolic syndrome. Accordingly, around 30% of approved drugs target GPCRs. They consist of a seven-transmembrane domain with an orthosteric binding pocket in the extracellular region and an intracellular G-protein binding pocket. Because of the often conserved orthosteric binding pocket of GPCR subtypes, the development of selective drugs is challenging. Consequently, the identification and targeting of allosteric binding sites is considered a potential strategy to obtain drugs with increased receptor selectivity and reduced side effects. As part of the GLUE project (G-protein-coupled receptor ligands of underexplored epitopes), this strategy was pursued in the present work, in which the pharmacologically relevant receptor families of endothelin receptors (ETRs) and free fatty acid receptors (FFARs) as well as the orphan receptor GPRC5b served as targets.
In the first part of this thesis, the peptidic ETBR-ligand 4Ala-ET-1 was synthesized using automated solid-phase peptide synthesis. Afterwards, the N-terminus of the resulting peptide was coupled to biotin via a linker in order to obtain a suitable ligand for protein purification of the ETBR by affinity chromatography. Furthermore, a fluorescence labeled and three truncated 4Ala ET 1 derivatives were synthesized. After the completion of the synthesis, the obtained compounds were handed over to the cooperating working groups of the GLUE project for further investigations.
In addition, a fluorescence-labeled FFAR2 ligand, which should serve as a tool compound for a BRET binding assay, was synthesized according to an 11-step literature procedure with slight modifications. Moreover, the core structure of this FFAR2 ligand was linked to biotin via an ethylene glycol derived linker segment in order to obtain an analogous biotinylated derivative for this GLUE target.
In the third part of the work, possible allosteric ligands were evaluated based on docking studies by the working group of Prof. Kolb. The most promising ligands were selected for synthesis, which was then planned and carried out. All resulting compounds were analytically characterized. Consequently, ligands from three ETBR docking studies were prepared, with two campaigns addressing the intracellular G-protein binding pocket and one addressing the known site 5. These docking studies were used to select seven hits, on the basis of which a total of 41 derivatives were synthesized. Furthermore, two well known allosteric GPCR modulators and six derivatives of them were synthesized. Subsequently, all compounds were handed over to the working group of Prof. Bünemann for analysis in a FRET-based G-protein binding and receptor sensor assay on the ETBR at a concentration of 100 µM. In addition, selected compounds were examined on the structurally related GLUE target FFAR3 using analogous assays. In this in vitro characterization, no biological activity could be determined for any of the synthesized target compounds, neither on the ETBR nor the FFAR3.
In a further docking study, the extracellular binding pocket of the orphan receptor GPRC5b was investigated. Based on the docking results, two hits and 42 derivatives were synthesized and analytically characterized. Afterwards, these target compounds should be examined in vitro in the working group of Prof. Bünemann with regard to their biological activity on GPRC5b. However, this has not yet been possible due to the lack of an established test system. Therefore, the biological characterization is still pending.
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Created: 2024Issued: 2024-06-20Updated: 2024-06-20
Faculty
Fachbereich Pharmazie
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
medicinal chemistry, organic chemistrychemistry
DFG-subjects
Organische ChemieSyntheseMedizinische ChemieGPCRPharmazieArzneistoffentwicklungChemie
DDC-Numbers
540
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Neumann, Sebastian (M. Sc.): Entwicklung, Synthese und Charakterisierung niedermolekularer Liganden G-Protein gekoppelter Rezeptoren. : Philipps-Universität Marburg 2024-06-20. DOI: https://doi.org/10.17192/z2024.0121.