The identification of allosteric inhibitors of high value therapeutic targets

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.

https://www.sheffield.ac.uk/biosciences/people/academic-staff/jon-waltho