Molecular approaches to understanding intracellular allosteric modulation (rotation three)

 

Lab rotation project description

In this mini-project of our triangle, you will predict how novel allosteric modulators interact with the intracellular CXCR2 allosteric binding site. CXCR2 is a membrane-bound protein residing at the cell surface which belongs to family A of the G protein-coupled receptors (GPCRs). Allosteric ligands are those that bind to topographically distinct regions of the receptor, relative to the endogenous (orthosteric) ligand binding site. Until now, the development of allosteric ligands for family A GPCRs has focused on extracellular allosteric binding sites that tend to overlap with the orthosteric ligand entry channel at the top of the GPCR. Recently, x-ray crystal structures have been published for a small number of GPCRs (2-adrenoceptor, CCR2 and CCR9 chemokine receptors), which reveal an intracellular binding pocket, targetable by small molecules.

Whilst currently, there is no x-ray crystal structure for the CXCR2, mutagenesis studies have identified a series of residues on the intracellular face of this receptor which affect the binding of three structurally distinct ligands, believed to share a common intracellular binding site.

The aim of this training project will be to use state-of-the-art computational modelling methods to predict the detailed molecular structure of CXCR2 in complex with these ligands. This data will then guide the rational design and synthesis of improved ligands, including fluorescent probes (chemistry rotation). This training project will provide you with experience in:

• computational methods for predicting protein structure (homology modelling)

• computational methods for predicting ligand binding sites (docking, and potentially molecular dynamics)

• use of molecular graphics and modelling methods for ligand design.

This arm of the triangle will be supervised by Professor Charlie Laughton (BSMC, School of Pharmacy). Initially you will build models for the CXCR2 receptor using data from structurally-related proteins whose X-ray structure is known (the technique of homology modelling). The ways in which known and potential new allosteric modulators (from the chemistry arm of the training triangle) might bind to the intracellular allosteric site in these protein models will then be predicted (docking studies and, potentially, molecular dynamics simulations). 

Fact file

Research theme

MCO

Location

tbc

Rotation

tbc

Contact

2nd supervisor

Shailesh Mistry

BBSRC Doctoral Training Partnerships
 

Linked PhD Project Outline

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 CXCR2. You 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.

CXCR2 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 CXCR2 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. 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 (for example, 2-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 CXCR2 receptor, you will therefore develop fluorescent high affinity ligands, and use them to monitor ligand binding and kinetics by our established methods. 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 CXCR2 allosteric ligands.

The specific aims of this project will be:

  • to design, synthesis and characterise novel fluorescent allosteric ligands for the CXCR2 receptor
  • to develop TR-FRET based competition binding kinetic assays using these fluorescent modulators to probe CXCR2 modulator SAR directly
  • to develop models of modulator- CXCR2 interaction, and use these to understand the allosteric regulation of chemokine and effector binding
  • to pharmacologically characterise novel compound pharmacology on CXCR2 signalling, and the effects of receptor mutants as informed by modelling studies

This chemical biology-focused project will span the disciplines of synthetic chemistry, modelling and pharmacology to increase our understanding of CXCR2 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.

 

Biotechnology and Biological Sciences Doctoral Training Programme

The University of Nottingham
University Park
Nottingham, NG7 2RD

Tel: +44 (0) 115 8466946
Email: bbdtp@nottingham.ac.uk