An in silico structure-based approach to map the druggable allosteric space of membrane receptors

Membrane receptors enable cells to respond to chemical and physical signals, activate a complex chain of biological events inside the cell and ultimately lead to a change in cell biology. The G protein-coupled receptors (GPCRs), ligand- and voltage-gated ion channel receptors and tyrosine kinase receptors are effective targets of nearly 60 % of the medicines currently used in clinics. The receptor biological state can be modulated by small molecule ligands acting on spatially distinct allosteric binding sites that result in more selective and safer medicines than those targeting orthosteric sites. Recent studies point to common mechanisms of allosteric regulation and the discovery of synthetic allosteric modulators for these receptor families. However, the discovery of allosteric sites and modulators has been largely serendipitous, achieved through high-throughput screening. A recent breakthrough in structural biology disclosed the crystal structures of several GPCRs bounded to allosteric modulators providing opportunities to develop predictive computational methodologies for allosteric medicine discovery in the membrane receptors.

We aim to develop a general structure-based computational methodology to search for allosteric binding sites in the membrane receptors. Since the newly available crystal structures of GPCR-allosteric modulator complexes indicate a diverse location of allosteric sites, we aim to develop computational procedures to map allosteric sites inside of the receptors; and outside of the receptor at the interface with the lipid bilayer. Our methodology will rely on the study of receptor dynamics in realistic cellular environment with the presence of carefully selected organic solvents. The organic solvents will be used as probes to search for binding sites. We will initially develop the methodology, where we predict allosteric sites for the receptors with the available crystal structures of a receptor-allosteric ligand complex (the training set) and then explore developed computational procedures predicting allosteric sites for new receptors (the test set) with further validation of the results of prediction in mutagenesis and compound screening. The bioamine, peptide and nucleotide receptors will be used to develop the methodology. We will use our receptor case studies together with sequence approaches to predict a general molecular basis of allosteric site location in GPCRs.

Our computational methodology will facilitate the development of new therapies for the treatment of GPCR-related diseases such as inflammation, infertility, metabolic and neurological disorders, viral infections and cancer. The computational strategy developed for one membrane receptor family will enable its applications to other receptor families.

We intend to develop a computational methodology for the search of allosteric binding sites in the membrane receptors based on the cutting-edge enhanced sampling molecular dynamics combined with smart cosolvent mapping. We will address several current limitations in cosolvent mapping for the membrane proteins: probe non-specific binding, protein denaturation, limited probe sampling, and membrane distortion. We will initially develop the computational methodology of allosteric site mapping using the M2, PAR2, CCR9 and P2Y1 receptors of Class A, and the CRF1 and GCGR receptors of Class B GPCRs with the available crystal structures of a receptor-allosteric modulator complex. We will develop approaches of assessing the functional relevance of the sites that include construction of allosteric networks, druggability assessment and sequence analysis. Next, we will apply the developed protocols to predict allosteric sites of the D3, CXCR4 and PAR1 receptors. The predicted allosteric sites will be further tested in site-directed mutagenesis. In the case of PAR1, virtual screening of compound libraries followed by experimental test will be performed to further prove the computational methodology. Using the results of cosolvent mapping together with the phylogenetic analysis we will start rationalization of allosteric site location across the GPCR family.

Understanding of allosteric regulation in biomolecules is essential for in-depth comprehension of a broad range of complex biological systems under physiological conditions and in diseases, and will greatly benefit the development of more selective, potent and effective allosteric drugs. Our study of allosteric modulation in GPCRs through mapping of putative binding sites and understanding their functional relevance and location across the GPCR family will facilitate identification novel pharmacological tools to further decipher the signalling complexities of GPCRs. In addition, our work will provide a conceptual framework to study allostery in other receptor families, such as ligand- and voltage-gated ion channels, tyrosine kinases and nuclear hormone receptors.

Our computational methodology will aim to overcome the limitations of currently available protocols for MD-cosolvent mapping and to develop a complex analysis of simulation data involving application of bio/chemoinformatics tools. This will be beneficial in atomistic computer simulations of other biosystems as well as materials, nanostructures and synthetic molecules.

The findings of this research will be disseminated through publications in leading international journals and presentations at international conferences. Research staff in this project may move on to careers in the industry where they could bring in the new multidisciplinary knowledge. The protocols to carry out research will be made widely available to academic and industrial scientists via various popular science and general online resources.

Allosteric modulators of GPCRs have emerged as a novel and highly desirable class of compounds. The direct outcome of the project will be an innovative, world leading, computational methodology for the search of allosteric sites that will facilitate allosteric drug discovery. There is a clear opportunity for the future exploitation of the results by engaging pharm and biotech companies. Therefore, in the long term our work will have a potential to improve the health and productivity of UK citizens and give a positive impact on the competitiveness of the UK pharmaceutical industry.
This project is strongly aligned with the BBSRC responsive mode priorities: Healthy ageing across the life course' 'Data driven biology' 'Systems approaches to the biosciences' and 'Technology development for the biosciences'. This research will therefore contribute towards achieving BBSRC's pathways to impact.

Pupils from primary and secondary schools, undergraduate students will benefit from our research development through public lectures within Science week in local schools, Researcher's night at Ulster Museum, Northern Ireland Science Festival in Belfast. We regularly host several high school students, who were introduced to on-going research in our labs sponsored by the Nuffield Research Foundation. General public and policy makers will learn about our research on Open Days at QUB and UoS, popular social networking sites (LinkedIn, Twitter and Facebook).