DiMeN Doctoral Training Partnership: Mapping the CDK interactome using Fraglites – a set chemical probes that probe protein interaction sites

This PhD offers an interdisciplinary opportunity to develop and apply novel technology that will increase our understanding of cell division and gene expression, and may initiate the development of novel anti-cancer therapeutics.

Cyclin dependent kinases (CDKs) mediate vital biological functions through a combination of kinase activity and scaffolding protein complexes. Cyclins activate CDKs and enhance substrate and regulator recognition by providing additional docking sites. CDKs 1/2/4 and 6 regulate the eukaryotic cell cycle, are closely related and bind specifically to cognate cyclin partners cyclins A/B/D and E. In many cases, the mechanism of action of CDKs and cyclins, executed through specific interactions with protein partners, remains unknown. Identification of sites of CDK-protein and cyclin-protein interaction would significantly advance our understanding of CDK biology and would provide potential opportunities for new therapies.

We have recently developed a set of very small (≤12 heavy atoms) fragment molecules (termed Fraglites), that display an array of two-point hydrogen bonding groups in combination with lipophilic ring structures, designed to recapitulate the interactions that underlie most protein-protein interactions. Our set of Fraglites all also contain an halogen atom that can be exploited in structural and biophysical studies to enable sensitive detection of binding. We have demonstrated that Fraglites correctly identify both orthosteric and allosteric small molecule binding sites (known and previously-unknown) on CDK2. We have subsequently generated an extended set of Fraglites that include molecules chemically related to amino acids. We propose that this set, designed to mimic individual backbone and side chain protein interactions, might allow for an extensive mapping of potential sites of protein-protein interaction.

The successful applicant will use this technology to identify potential protein binding sites on CDKs and cyclins that regulate the eukaryotic cell cycle. Potential protein binding sites will be verified by mutagenesis coupled with in vitro biochemical and biophysical methods (for example solution NMR to be carried out in the laboratory of Prof. A. Breeze at Leeds University) and in cellulo functional studies. An unbiased method to identify which residues drive CDK-cyclin protein interactions will provide the rationale to design mutant tool proteins to interrogate CDK1,2,4 and 6 function. More generally, this project will provide a vital proof of principle of technology that should be generally applicable to mapping of the protein interactome of crystallisable proteins.

The project is aligned to ongoing research in the laboratories of Prof. Waring, Prof.Noble at Newcastle University and Prof. Breeze at Leeds University. We have access to world-leading facilities for chemistry, molecular and cellular biological studies. Our laboratories are working together to explore novel routes to identifying potential drug targets and to develop new approaches to treat cancer.