Studies towards chemical probes for short chain fatty acid GPCRs FFAR2 and FFAR3
File(s)
Author(s)
Nikoloudaki, Roxani
Type
Thesis
Abstract
The pivotal role of gut microbiota in health and disease has drawn interest in deorphanisation of FFAR2 and FFAR3 as metabolite-sensing GPCRs, paired with SCFAs as endogenous ligands. This established crosstalk between diet, the human microbiome, and pathogenesis of metabolic and inflammatory disorders. However, a significant challenge remains the lack of comprehensive structural data for the receptors and selective, potent tools to dissect their signalling pathways and define their physiological roles.
Two strategies were developed in this thesis to address these gaps: (1) chemical biology and (2) medicinal chemistry. The chemical biology approach featured two key principles: (I) A non-invasive strategy mimicking SCFA signalling as the physiological receptor response while maintaining receptor function. (II) A system enabling selective receptor targeting through bioorthogonal labelling using Halo and SNAP self-labelling protein tags, allowing isolated and spatially precise examination of downstream signalling. To support this, three tools were developed: (i) Stable Flp-In T-REx HEK293 cell lines expressing tagged receptors, demonstrating normal downstream signalling by endogenous and synthetic agonists using the TR-FRET LANCE Ultra cAMP assay. (ii) Complementary bioorthogonal multifunctional chemical tools incorporating SCFA as a pharmacophore, masked with a PPG for optically controlled release to ensure spatiotemporal precision or the synthetic FFAR2 agonist Cmp1 to enhance potency. (iii) A workflow for implementing the cAMP assay to assess probe functionality within the cell-based system. Iterative cycles of probe design, synthesis, and biological evaluation identified two potential probe scaffolds accommodating a free pharmacophore that elicited promising receptor response.
The medicinal chemistry strategy focused on developing spirocyclic ligands for FFAR2 by identifying promising scaffolds as novel chemical space for the receptor with potential allosteric and antagonist activity across human and murine orthologs. Further investigation is required to confirm the suitability of the bioorthogonal probe scaffolds and SAR exploration of the spirocyclic ligands for robust receptor activation.
Two strategies were developed in this thesis to address these gaps: (1) chemical biology and (2) medicinal chemistry. The chemical biology approach featured two key principles: (I) A non-invasive strategy mimicking SCFA signalling as the physiological receptor response while maintaining receptor function. (II) A system enabling selective receptor targeting through bioorthogonal labelling using Halo and SNAP self-labelling protein tags, allowing isolated and spatially precise examination of downstream signalling. To support this, three tools were developed: (i) Stable Flp-In T-REx HEK293 cell lines expressing tagged receptors, demonstrating normal downstream signalling by endogenous and synthetic agonists using the TR-FRET LANCE Ultra cAMP assay. (ii) Complementary bioorthogonal multifunctional chemical tools incorporating SCFA as a pharmacophore, masked with a PPG for optically controlled release to ensure spatiotemporal precision or the synthetic FFAR2 agonist Cmp1 to enhance potency. (iii) A workflow for implementing the cAMP assay to assess probe functionality within the cell-based system. Iterative cycles of probe design, synthesis, and biological evaluation identified two potential probe scaffolds accommodating a free pharmacophore that elicited promising receptor response.
The medicinal chemistry strategy focused on developing spirocyclic ligands for FFAR2 by identifying promising scaffolds as novel chemical space for the receptor with potential allosteric and antagonist activity across human and murine orthologs. Further investigation is required to confirm the suitability of the bioorthogonal probe scaffolds and SAR exploration of the spirocyclic ligands for robust receptor activation.
Version
Open Access
Date Issued
2025-02-11
Date Awarded
01/10/2025
License URL
Advisor
Tate, Edward
Frost, Gary
Hanyaloglu, Aylin
Blanchard , Carine
Le Coutre, Johannes
Sponsor
Engineering and Physical Sciences Research Council
Nestlé
Publisher Department
Department of Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)