NMR and protein structure in drug design - David Craik

Professor David Craik

Our group uses peptide chemistry and NMR spectroscopy to determine the structures of proteins that are important in drug-design programs and in agriculture. By elucidating the structures of biologically active proteins we are able to identify regions crucial for activity and can use this information to design new drugs or crop protection agents. The proteins we study come from a range of animal and plant sources. Examples include conotoxins (venom components from marine snails) and the cyclotides (ultra-stable circular proteins from plants).

We have a particular interest in the discovery and structural characterisation of novel protein topologies. We aim to determine the mechanisms of biosynthesis and evolutionary origin of circular proteins and to apply protein-engineering principles to explore applications of circular proteins in drug design and agriculture. Circular proteins are particularly stable and thus have advantages over conventional proteins.

We undertake protein-engineering studies in which we modify protein frameworks either by “grafting” new biologically active epitopes onto them, or by stabilising them by cyclisation. We currently have molecules under development for the treatment of multiple sclerosis, cardiovascular disease, cancer and chronic pain. We also undertake fieldwork in Australia and overseas for the collection of plant species so that we can explore the diversity and evolution of the cyclotide family of plant proteins. We study the structures of a range of toxins from cone snails, spiders and snakes and use this information to understand their mode of action against ion channels and other receptors. We also study the protein-folding problem, i.e., how do proteins fold into the complex shapes that determine their functions?

Highlights of last year included the development of an orally active peptide for the treatment of pain. This molecule, IMB007, is an engineered form of a native conotoxin that we had earlier structurally characterised. Our structural studies were used to design a cyclic analogue that proved to be more potent and more stable than the natural conotoxin, and is orally active in an animal model of neuropathic pain. We also developed cyclotides with improved potency against parasites of sheep and cattle and have advanced our understanding of structure-activity relationships in this class of cyclic proteins.

Research Projects

• Discovery, biosynthesis and applications of circular proteins

• Development of new drugs for pain, cancer and cardiovascular disease

• Studying the structure-activity relationship of toxins


• Investigating plants as production factories to make peptide-based drugs

Key Publications

Clark, R.J., Jensen, J., Nevin, S.T., Callaghan, B.P., Adams, D.J., and Craik, D.J. (2010). The engineering of an orally active conotoxin for the treatment of neuropathic pain. Angewandte Chemie 49: 6545-6548.

Huang, Y.-H., Colgrave, M.L., Clark, R.J., Kotze, A.C., Craik, D.J. (2010). Lysine scanning mutagenesis reveals an amendable face of the cyclotide kalata B1 for the optimisation of nematocidal activity. Journal of Biological Chemistry 285: 10797-10805.

Gunasekera, S., Foley, F.M., Clark, R.J., Sando, L., Fabri, L.J., Craik, D.J., and Daly, N.L. (2009). Engineering stabilized VEGF-A antagonists: Synthesis, structural characterization and bioactivity of grafted analogues of cyclotides. Journal of Medicinal Chemistry 51: 7697-7704.

Halai, R., Clark, R.J., Nevin, S., Jensen, J.E., Adams, D.J., and Craik, D.J. (2009). Scanning mutagenesis of alpha-conotoxin Vc1.1 reveals residues crucial for activity at the alpha9alpha10 nicotinic acetylcholine receptor. Journal of Biological Chemistry 284: 20275-20284.

Huang, Y.H., Colgrave, M.L., Daly, N.L., Keleshian, A., Martinac, B., and Craik, D.J. (2009). The biological activity of the cyclotides is modulated by the formation of a multimeric pore. Journal of Biological Chemistry 284: 20699-20707.

Wang, C.K.L., Hu, S.-H., Martin, J.L., Sjogren, T., Hajdu, J., Bohlin, L., Claeson, P., Goransson, U., Rosengren, K.J., Tang, J., Tan, N.-H., and Craik, D.J. (2009). Combined X-ray and NMR analysis of the stability of the cyclotide cystine knot fold that underpins its insecticidal activity and potential use as a drug scaffold. Journal of Biological Chemistry 284: 10672-10683.

Gruber, C.W., Elliot, A., Ireland, D.C., Trabi, M., Göransson, U., Delprete, P.G., Dessein, S., Robbrecht, E.F., and Craik, D.J. (2008). Distribution and evolution of circular mini-proteins in flowering plants. The Plant Cell 20: 2471-2483.

Simonsen, S.M., Sando, L., Rosengren, K.J., Colgrave, M.L., Daly, N.L., and Craik, D.J. (2008). Alanine scanning mutagenesis of the prototypic cyclotide reveals a cluster of residues essential for bioactivity. Journal of Biological Chemistry 283: 9805-9813.

Gillon, A.D., Saska, I., Jennings, C.V., Renda, R.F., Craik, D.J., and Anderson, M.A. (2007). Biosynthesis of circular proteins in plants. The Plant Journal 53: 505-515.

Saska, I., Gillon, A.D., Hatsugai, N., Dietzgen, R., Hara-Nishimura, I., Anderson, M.A., and Craik, D.J. (2007). An asparaginyl endopeptidase mediates in vivo protein backbone cyclization. Journal of Biological Chemistry 282: 29721-29728.

Craik, D.J. (2006). Seamless proteins tie up their loose ends. Science 311: 1563-1564.

Lovelace, E.S., Armishaw, C.J., Colgrave, M.L., Wahlstrom, M.E., Alewood, P.F., Daly, N.L., and Craik, D.J. (2006). Cyclizing the backbone of conotoxin MrIA improves enzymatic stability while maintaining structure and activity. Journal of Medicinal Chemistry 49: 6561-6568.

Mulvenna, J.P., Bharathi, R., Mylne, J.S., Burton, R.A., Shirley, N.J., Fincher, G.B., Anderson, M.A., and Craik, D.J. (2006). Discovery of cyclotide-like protein sequences in graminaceous crop plants: Ancestral precursors of circular proteins? The Plant Cell 18: 2134-2144.

Clark, R.J., Fischer, H., Dempster, L., Daly, N.L., Rosengren, K.J., Nevin, S.T., Meunier, F.A., Adams, D.J., and Craik, D.J. (2005). Engineering stable peptide toxins by means of backbone cyclisation: Stabilization of the conotoxin MII. Proceedings of the National Academy of Sciences USA 102: 13767-13772.
 

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