Award details

Structural and Fragment approaches to the modulation of O-GlcNAc in cells

Reference	BB/K003836/1
Principal Investigator / Supervisor	Professor Gideon Davies
Co-Investigators / Co-Supervisors	Professor Roderick Eliot Hubbard
Institution	University of York
Department	Chemistry
Funding type	Research
Value (£)	730,226
Status	Completed
Type	Research Grant
Start date	29/04/2013
End date	31/10/2016
Duration	42 months

Abstract

The dynamic post-translational modification of serine and threonine by O-GlcNAc is of developing importance in cellular biology by virtue both of its reciprocity with phosphorylation and the distinct phenotypes of an O-GlcNAc modified protein. The modification of transcription factors, cytoskeletal network components and proteasome subunits as well as its emerging role as an epigenetic modifier involved in the temporal regulation of gene expression are all of major cellular importance. Furthermore, the aberrant under and over O-GlcNAcaylation of proteins is linked to the stress response, to Alzheimer's and cancer. There is a major impetus to obtain better small molecule modulators of O-GlcNAc levels in cells in order both to probe the cellular biology of O-GlcNAc and to establish a basis for therapeutic intervention. We will build upon past success to determine the structure of the mammalian O-GlcNAc hydrolase OGA, to probe the structure and roles of its unusual active centre pocket, defining its peptide binding sites and its tandem putative histone acetyl transferase domain. 3-D structure will inform further cycles of inhibitor design and these compounds will be applied in cellular assays of O-GlcNAc level and its cellular consequences. In preliminary studies, we have used fragment-based discovery methods to identify compounds which bind to bacterial OGA and elicit different effects - either inhibit, enhance inhibition of a known inhibitor, or (uniquely) enhance the activity by decreasing KM. Crystal structures show the enhancers stabilize a loop conformation, presumably thereby increasing activity. We seek to capitalize on these preliminary results to discover inhibitors and/or enhancers of activity of the human OGA, and the O-GlcNAc transferase OGT, and to optimize the affinity of the fragments. This will provide chemical tools with which to probe the effects on cell biology of modulating the O-GlcNAc state.

Summary

The function of many of the large macromolecules that control biological processes in a cell can be changed by chemical modification with a sugar called O-GlcNAc. Two enzymes control this chemical modification: a transferase (OGT) that adds the sugar to the protein and a hydrolase (OGA) which removes it. This addition of a sugar is analogous to other mechanisms of control in the cell - such as phosphorylation where a phosphate is added (with a kinase) or removed (with a phosphatase). A great deal is known about how phosphorylation occurs and its consequences on the cell - indeed, many of the more recent drugs that have been discovered are kinase inhibitors that have an impact in conditions such as cancer. However, the O-GlcNAc modification has only recently been discovered and we are only now beginning to discover what happens to cells when the pattern of modification is changed. One recent discovery is that the O-GlcNAc modification has a role in maintaining healthy neurones, and disruption has some impact on neurodegenerative diseases such as Alzheimer's. The aim of our research is to understand more about what these enzymes, OGA and OGT, look like and how they work. This knowledge will be of interest in its own right, but it also provides information which can be used to design small molecule compounds that can bind to the enzymes and change their activity - interestingly, we can both increase and decrease how quickly the enzymes work. It is difficult to find such small molecules by trial and error. One new technique that has been developed recently is the method of fragment-based discovery. Instead of having to find the complete molecule that fits the binding site, this approach begins by identifying smaller pieces of molecule that bind. If the way in which these small fragments bind can be understood, then the chemist can design changes that merge or grow these fragments into the larger compound with the correct properties. We will use these methods to discover small molecules that change the activity of OGA and OGT. These can then be added to cells to see what effect changing the O-GlcNAc has on the way the cells behave. The principle aim is to understand the effect on cell biology, though the compounds may provide a starting place for drug design.

Impact Summary

Who will benefit from this research as well as the benefit of increased understanding of fundamental cell and molecular biology (see academic beneficiaries), there are two major ways in which this research could have significant impact in the pharmaceutical sector. The first is exploring the potential therapeutic effects of modulating O-GlcNAc status in cells. A currently known impact would be possible translation to strategies for drug discovery for agents to affect neurodegeneration; the chemical tools we develop may also lead to discovery of other health and disease related pathways where the O-GlcNAc tone is important. The second major area of impact could be in demonstrating that enzyme enhancers can be used to modulate cell biological processes. In addition to these major impacts, the work could provide additional demonstrations of the power of a structural approach to understanding mechanisms of disease and, also, the use of fragment and structure based design methods for identifying novel starting points that could be optimised to drug candidates. Timescale to impact This three-year project should result in chemical tools that establish the principles and benefits of modulating O-GlcNAc status in cells. As discussed in the pathway to impact, it takes some 2-3 years to develop from such hit start points to a clinical candidate and at least a further 5 years of clinical trials to demonstrate clinical efficacy and safety. The understanding and projected output of our research therefore has the potential to significantly enhance quality of life and health. Research skills for project staff This is a multi-disciplinary project, developing a full range of biomolecular, biophysical and cell biology skills in the post-doctoral workers. They will also gain an increased appreciation of the issues and methods in modern structure-based ligand discovery, equipping them with the skills needed for a continuing career in chemical biology in academia or in drug discovery in the pharmaceutical sector.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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