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Antibody-assisted investigations into allosteric regulation of Wnt signalling: building the sclerostin franchise

Project Supervisor: Prof Mark Carr

Co-supervisor: Dr Lorna Waters

Non-academic partner: Dr Gill Holdsworth, UCB

PhD project title: Antibody-assisted investigations into allosteric regulation of Wnt signalling: building the sclerostin franchise

Project outline:

The bone disorder osteoporosis affects hundreds of millions of people worldwide and occurs following a loss of bone strength and integrity. Despite a growing number of people affected by osteoporosis current therapeutic options offer only limited benefit, with the majority inhibiting the resorption of bone but having little effect on the restoration of bone that has already been lost. The canonical Wnt signalling pathway has emerged as a key regulator of bone homeostasis and activation of this pathway is tightly regulated by a number of secreted inhibitory proteins, including sclerostin and members of the Dkk (Dkk1-4) family. These soluble inhibitors of Wnt signalling have been shown to interact with specific sites on the closely related Wnt co-receptors LRP4, LRP5 and LRP6. The central role of Wnt signalling in bone growth and turnover, together with the existence of natural regulatory systems, highlights modulation of Wnt signalling in bone as an attractive target for the development of effective therapeutics to treat osteoporosis.

In pioneering studies of a range of camelid VHH antibody fragments raised to a cytokine, we have identified hitherto unknown allosteric sites, where binding of specific VHHs results in modulation of distant functional sites. Determination of structures for VHHs bound to the cytokine has identified specific interactions responsible for the allosteric regulation and highlighted the potential to identify synthetic small molecules that would similarly modulate activity. Structural plasticity of the cytokine is key to its allosteric regulation, which is a property shared by many secreted regulators of Wnt signalling, including sclerostin and Dkk family proteins.

NMR-based structural studies have demonstrated that both sclerostin and Dkk proteins are characterised by regions that exist in multiple interconverting conformations and show significant flexibility, which often correspond to sites involved in interactions with functional partners such as LRP6. Antibody-assisted structural approaches similar to those pursued for the cytokine are likely to identify unknown allosteric sites on sclerostin and/or stabilise the conformations of key regions required for specific binding to LRP co-receptors. This will facilitate the identification of new ways to modulate the function of sclerostin through structure-based design of small molecules, which has the potential to yield novel therapeutics to treat a variety of low bone mass disorders. The proposed Ph.D. project will initially focus on the identification of VHHs raised to sclerostin that modulate the interactions with LRP4, LRP5 or LRP6 via allosteric sites and/or stabilise the conformations of key functional regions of the protein. A range of biophysical techniques will be employed alongside existing cell-based assays to identify and characterise function-modifying VHHs, with structures then determined for selected VHH-sclerostin complexes. Gaining an understanding of the allosteric potential of sclerostin and how this translates into regulation of protein function will give valuable insights into the mechanism of action of this highly dynamic and flexible protein. In addition, further development of antibody-assisted drug discovery technology, together with enhanced understanding of the potential to regulate sclerostin, will unlock future identification of small molecules that inhibit sclerostin.

The proposed project will provide the Ph.D. student with a thorough research training in structural biology, protein biochemistry and molecular cell biology. In addition, there is strong synergy with the research programmes of other experienced postdoctoral scientists and research students in the Leicester group. The proposed project will also further validate the innovative use of antibodies as tools in structure-based drug discovery, which has the potential to revolutionise the therapeutic targeting of protein-protein interactions with synthetic small molecules.

References:

  1. Wnt signaling in bone homeostasis and disease: from human mutations to treatments. Baron R., Kneissel M (2013) Nat. Med. 19(2), 179-92.
  2. Romosozumab in postmenopausal women with low bone mineral density. McClung MR1, Grauer A, Boonen S, Bolognese MA, Brown JP, Diez-Perez A, Langdahl BL, Reginster JY, Zanchetta JR, Wasserman SM, Katz L, Maddox J, Yang YC, Libanati C, Bone HG (2014) N. Engl. J. Med. 370(5), 412-20.
  3. Characterization of the structural features and interactions of sclerostin: molecular insight into a key regulator of Wnt-mediated bone formation. Veverka V, Henry AJ, Slocombe PM, Ventom A, Mulloy B, Muskett FW, Muzylak M, Greenslade K, Moore A, Zhang L, Gong J, Qian X, Paszty C, Taylor RJ, Robinson MK, Carr MD (2009) J. Biol. Chem. 284(16), 10890-900.
  4. Characterization of the interaction of sclerostin with the low density lipoprotein receptor-related protein (LRP) family of Wnt co-receptors. Holdsworth G, Slocombe P, Doyle C, Sweeney B, Veverka V, Le Riche K, Franklin RJ, Compson J, Brookings D, Turner J, Kennedy J, Garlish R, Shi J, Newnham L, McMillan D, Muzylak M, Carr MD, Henry AJ, Ceska T, Robinson MK (2012) J. Biol. Chem. 287(32), 26464-77.
  5. Antibody-enabled small-molecule drug discovery. Lawson AD (2012) Nat Rev Drug Discov. 11(7), 519-25.

Techniques that will be undertaken during the project:

The project will involve a range of cell biology, protein biochemistry and structural biology techniques including the following:

  • NMR spectroscopy
  • X-ray crystallography
  • Surface plasmon resonance (Biacore)
  • Protein expression in mammalian and bacterial cells
  • Protein refolding
  • Protein purification
  • Cell-based Wnt reporter assays

Interview date: TBA