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BBSRC White Rose CASE studentship - Novel approaches for the identification of allosteric inhibitors of high value therapeutic targets

  • Full or part time
    Prof J P Waltho
  • Application Deadline
    Monday, January 06, 2020
  • Funded PhD Project (European/UK Students Only)
    Funded PhD Project (European/UK Students Only)

Project Description

This study will develop a novel approach to identify allosteric inhibitor sites on a wide variety of high-value therapeutic targets, including protein kinases, protein phosphatases and small G-proteins.

Protein kinases, protein phosphatases and small G-proteins are of enormous interest to the pharmaceutical industry. They are key controllers of intracellular signalling and cellular homeostasis, and inappropriate levels of their activity are fundamental to a wide range of diseases, including many cancers. To-date a range of inhibitors has been developed against protein kinases and, to a lesser extent, protein phosphatases and small G-proteins but numerous issues remain. One of the primary difficulties for protein kinases and small G-proteins is the dominant effect of the nucleotide binding sites on ligand affinity, which has skewed efforts towards nucleotide mimics. However, the lack of selectivity of nucleotide mimics is the Achilles heel of kinase drug discovery, because of the homology of the co-factor binding sites across the protein sub-families. Innovation requires a workflow that delivers selective kinase inhibitors with confidence. Correspondingly, there is great current interest in identifying allosteric sites, which inhibit these enzymes without binding in the nucleotide or substrate binding sites. Such sites have a far greater likelihood to be specific for individual enzymes and to be targetable by inhibitors with the required properties for clinical use.

We intend to exploit our newly developed, detailed understanding of the mechanisms of phosphoryl transfer enzymes to identify novel allosteric inhibitors. This should lead to the identification of novel modalities of functional modulation by kinase inhibitors. Allosteric sites are hard to identify without a clear knowledge of the conformational transitions that the enzymes undergo during their catalytic cycle. Our extensive work on ground and transition state analogue binding has mapped the conformational transitions of a number of archetypal phosphoryl transfer enzyme classes. Trapping these enzymes stably in these conformations should lead to novel cryptic pockets being exposed, which then become druggable. We introduce metal fluoride analogues of phosphate groups at different points within the catalytic cycle, which enables the use of 1D 19F NMR measurements to provide highly sensitive reporters of the environment in the immediate vicinity of the substrate(s). These NMR signals will also report on the perturbation of catalysis by inhibitors binding in allosteric sites, since the nucleotide and substrate binding sites are fully occupied by the species native to the catalysed reaction. We will exploit this novel technology and use NMR, X-ray crystallography and molecular enzymology approaches to discover new sites and new inhibitors that specifically target medically important enzymes. We will show proof of principle using a protein kinase, PKA or Abl, and a small G-protein, RhoA or Ras, which we have already characterised extensively in terms of their enzymology. We will then expand the programme to other readily tractable protein kinases, protein phosphatases and small G-proteins targets.

The appointee will develop specialist level expertise in experimental structural and molecular biology methods, and computational chemistry/biochemistry. Training in the experimental and computational components, including in enzymology, protein biochemistry and analytical methods, will be overseen by experienced researchers within the Academic Partner. Training in screening approaches will be led by the Industrial Partner, C4X Discover. C4X Discovery is a virtual drug discovery company, utilising cutting-edge technologies to design best-in-class drug candidates. C4X has multiple proprietary technologies which enable rapid discovery of novel drugs for diseases with high unmet medical need across therapeutic areas. The appointee will be seconded to the offices of C4X Discovery where they will access state-of-the-art computational equipment and software (including proprietary software of the company) for the modelling of protein:ligand interactions, which they will apply to the systems on which they are working. This may include simulations to aid in the characterisation of allosteric modulators identified in the project and/or in silico screening to identify additional chemical matter to further exploit the mechanisms identified through the project. The company runs training seminars and informal mentoring sessions with members of the discovery team: there will be opportunities covering broad aspects of drug discovery chemistry, biology and pharmacology as well as commercial analysis and intellectual property. This will enable the appointee to develop the applicability of their findings beyond the proof-of-principle systems already identified.

Funding Notes

- preferred start date October 2020
- a tax-free stipend at the standard Research Council rate (~£15,009, to be confirmed for 2020) for 4 years
- tuition fees at the UK/EU rate for 4 years.
- research costs
At least a 2:1 honours degree in a relevant subject or equivalent is required.
Studentships are available to UK and EU students who meet the UK residency requirements.

References

Prioritization of charge over geometry in transition state analogues of a dual specificity protein kinase. Xiaoxia L, Marston JP, Baxter NJ, Hounslow AM, Yufen Z, Blackburn GM, Cliff MJ, Waltho JP. J Am Chem Soc. (2011) 133 3989-94.

Near attack conformers dominate beta-phosphoglucomutase complexes where geometry and charge distribution reflect those of substrate. Griffin JL, Bowler MW, Baxter NJ, Leigh KN, Dannatt HRW, Hounslow AM, Blackburn GM, Webster CE, Cliff MJ, Waltho JP. Proc. Nat. Acad. Sci. USA (2012) 109 6910-5.

Charge-balanced metal fluoride complexes for protein kinase A with adenoside diphosphate and substrate peptide SP20. Jin Y, Cliff MJ, Baxter NJ, Dannatt HRW, Hounslow AM, Bowler MW, Blackburn GM, Waltho JP. Angew. Chemie (2012) 51 12242-5.

α-Fluorophosphonates reveal how a phosphomutase conserves transition state conformation over hexose recognition in its two step reaction. Jin Y, Bhattasali D, Pellegrini E, Forget SM, Baxter NJ, Cliff MJ, Bowler MW, Jakeman DL, Blackburn GM, Waltho JP. Proc. Nat. Acad. Sci. USA (2014) 111 12384-12389.

Assessing the Influence of Mutation on GTPase Transition States using X-ray, 19F NMR, and DFT Approaches. Jin Y, Molt RW Jr, Pellegrini E, Cliff MJ, Bowler MW, Richards NG, Blackburn GM, Waltho JP. Angew Chem Int Ed Engl. (2017) 56 9732-5.

van der Waals Contact between Nucleophile and Transferring Phosphorus Is Insufficient To Achieve Enzyme Transition-State Architecture. Johnson LA, Robertson AJ, Baxter NJ, Trevitt CR, Bisson C, Jin Y, Wood HP, Hounslow AM, Cliff MJ, Blackburn GM, Bowler MW, Waltho JP. ACS Catalysis (2018) 8 8140-8153.

How good is research at University of Sheffield in Biological Sciences?

FTE Category A staff submitted: 44.90

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