Development of intracelullar CXCR1 modulators

The aim of our full triangle project is to design, synthesise and characterise novel fluorescent ligands for the intracellular allosteric binding site of the chemokine receptor CXCR1. The student will then use these to probe structure activity relationships, and the nature of allostery, at this novel target site for modulating G protein-coupled receptor (GPCR) signalling.

CXCR1 belongs to the G protein-coupled receptor (GPCR) superfamily and is found on the surface of a range of inflammatory cells, with a key role in pulmonary function and a therapeutic target of interest in asthma and chronic obstructive pulmonary disease. The orthosteric agonists for CXCR1 are large peptide chemokines (CXCL1 and CXCL8), but this receptor was one of the first GPCRs for which a topographically distinct allosteric intracellular binding site for small molecules was described (by a group now at Nottingham; 1. Salchow et al 2010). Ligands at this site act as negative allosteric modulators, inhibiting the chemokine response. Since this discovery, similar intracellular allosteric binding sites have been identified structurally in other receptors (e.g. Beta2-adrenoceptor, CCR2, CCR9- refs 2 - 4).

There is limited information about how this class of allosteric ligands interacts with GPCRs and influences signalling to functionally different effectors (G proteins or arrestins). For example we now know that binding of G proteins and arrestins to the GPCR intracellular domain exploits different molecular footprints, with the intracellular modulator binding sites positioned at this GPCR-effector interface. Better structure activity relationships (SARs) at this position thus have potential to reveal new ligand classes - for example "biased" modulators that selectively regulate G protein or arrestin interaction to tune the functional chemokine response. However the development of good allosteric SAR is often limited by the poor range of compound analogues and the lack of suitable probe ligands to monitor binding at the target site directly.

To explore allosteric SAR at the CXCR1 receptor, the student will therefore develop fluorescent high affinity ligands, and use them to monitor ligand binding and kinetics by our established methods (Sykes et al 2017). In conjunction with receptor modelling studies this will advance our understanding of the nature of GPCR allostery at this site, and how allosteric ligands influence both chemokine messenger and effector protein binding in the three partner activated GPCR complex. In due course, these data will also inform future drug discovery projects focused on CXCR1 allosteric ligands.

Specific aims of the project will be:
1. To design, synthesis and characterise novel fluorescent allosteric ligands for the CXCR1 receptor (related to rotation 1, Mistry).
2. To develop TR-FRET based competition binding kinetic assays using these fluorescent modulators to probe CXCR1 modulator SAR directly (Sykes et al 2017).
3. To develop models of modulator- CXCR1 interaction, and use these to understand the allosteric regulation of chemokine and effector binding (rotation 2, Laughton).
4. To pharmacologically characterise novel compound pharmacology on CXCR1 signalling, and the effects of receptor mutants as informed by modelling studies (rotation 3, Holliday)

This chemical biology-focused project will span the disciplines of synthetic chemistry, modelling and pharmacology to increase our understanding of CXCR1 biology. With increasing numbers of allosteric ligands being discovered for the wider GPCR family, the results will be of direct relevance to many other important receptors.