Molecular and in-silico approaches to understanding intracellular allosteric modulation at the CXC chemokine receptor 2Tools Casella, Bianca Maria (2024) Molecular and in-silico approaches to understanding intracellular allosteric modulation at the CXC chemokine receptor 2. PhD thesis, University of Nottingham.
AbstractThe CXC chemokine receptor 2 (CXCR2) is a key mediator of the inflammation response, for example it regulates neutrophil migration and activation in a number of inflammatory-based disorders. CXCR2 inhibition is a potential strategy for the treatment of several pulmonary diseases, such as chronic obstructive pulmonary disorder (COPD) and asthma, and through its expression on various tumour cell types CXCR2 is also a candidate cancer target. The interaction of large endogenous chemokine peptides (including CXCL8 / interleukin 8 (IL8)) at the orthosteric binding site is a complex, multi-step process which presents significant challenges when designing small molecule competitive orthosteric antagonists. This has led to the development of CXCR2 allosteric modulators targeting an intracellular pocket near the C-terminal of the receptor, formed by the transmembrane domains overlapping the G-protein binding site. Navarixin (2) is an intracellular negative allosteric modulator (NAM) belonging to the 3,4-diamino-3-cyclobutene-1,2-dione class and it exhibits sub-nanomolar potency and selectivity for CXCR2 over the related CXCR1 subtype. Our understanding of the binding and mode of action of navarixin and other CXCR2 NAMs has been limited by the availability of suitable tracer ligands for competition studies, that allow convenient direct measurements of ligand binding. Here we report the rational design, synthesis and pharmacological evaluation of a series of fluorescent NAMs, based on navarixin (2), which are the first examples of ligands able to bind to intracellular binding sites of CXCR1 and CXCR2. These novel fluorescent ligands display high affinity and selectivity for CXCR2 over CXCR1, and, notably, they can be applied in both NanoBRET assay formats in whole cells or membranes and in fluorescence imaging. We also demonstrate that the NanoBRET binding assay using these fluorescence probes is capable of direct measurement of the affinity of unlabelled NAMs acting at the intracellular site, thus providing a platform to screen for alternative chemophores targeting these receptors.
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