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Conotoxins targeting voltage-gated sodium channels: harnessing nature’s analgesics

R.S. Norton,1 K.K. Khoo,1,2,3 Z. Kuang,2,3 B.R. Green,1,4 K. Gupta,5 M.J. Wilson,4 M. Zhang,4 J.E.F. Rivier,6 P. Balaram,5 D. Yoshikami,4 B.M. Olivera4 and G. Bulaj,7 1Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia, 2The Walter & Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia, 3The Department of Medical Biology, The University of Melbourne, Parkville. VIC 3010, Australia, 4Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA, 5Molecular Biophysics Unit, Indian Institute of Science, Bangalore, 560 012, India, 6Salk Institute for Biological Studies, La Jolla, CA 92037, USA and 7Department of Medicinal Chemistry, College of Pharmacy, University of Utah, Salt Lake City, Utah 84108, USA .

μ-Conotoxins are a family of peptides from predatory marine cone snails that target voltage-gated sodium channels (VGSCs), blocking the passage of sodium ions through the channel. Several neuronal VGSC subtypes have been implicated in the perception of pain; as such, modulators of these subtypes could have potential therapeutic use as analgesics. μ-KIIIA shows potent analgesic activity following its systemic administration in mice (Zhang et al., 2007). Structure-activity studies indicated that the key residues important for VGSC-blocking activity (K7, W8, R10, D11, H12, R14) mostly resided on an α-helical motif and that a disulfide bond could be removed without significant loss of activity (Khoo et al., 2009; Han et al., 2009). These findings suggested a route for structural minimization of μ-KIIIA by retaining the key residues on an α-helical scaffold.

In stabilizing α-helices, the use of (i, i+4) lactam bridges has proven to be a successful approach. For a mimetic of μ-KIIIA, the result that Cys9 can be replaced with no significant loss in activity identifies a position in the helix that can be substituted to form a helix stabilizing (i, i+4) lactam bridge to either residue 5 or 13, both of which are non-essential residues and therefore replaceable. We have designed and synthesized several analogues of μ-KIIIA; all of them are truncated at both the N- and C-termini, and the remaining sequence is stabilized by a lactam bridge at strategic locations. The helicity of six lactam analogues has been analysed using NMR spectroscopy and molecular modelling, and their activities have been tested against a range of VGSC subtypes (Khoo et al., 2011). Our findings highlight important structure-activity relationships and provide a basis for the design of new minimized peptides and helical mimetics as novel analgesics.

In the course of our studies on μ-KIIIA it became apparent that the major product from oxidative refolding adopts a {1-15,2-9,4-16} disulfide pattern rather than the {1-9,2-15,4-16} disulfide pattern predicted on the basis of closely-related μ-conotoxins (Khoo et al., 2009). Moreover, a minor product adopts a {1-16,2-9,4-15} pattern. Surprisingly, both products are capable of blocking the channel (Khoo et al., 2012), highlighting the fact that different structures can present the key functional groups. The difficulties in defining the disulfide connectivities of the products of oxidative refolding in vitro further emphasize the value of alternative strategies such as those pursued here for stabilising peptide structures.

Han TS, Zhang MM, Walewska A, Gruszczynski P, Robertson CR, Cheatham TE 3rd, Yoshikami D, Olivera BM, Bulaj G. (2009) ChemMedChem 4, 406-414.

Khoo KK, Feng ZP, Smith BJ, Zhang MM, Yoshikami D, Olivera BM, Bulaj G, Norton RS. (2009) Biochemistry 48, 1210-9.

Khoo KK, Wilson MJ, Smith BJ, Zhang MM, Gulyas J, Yoshikami D, Rivier JE, Bulaj G, Norton RS. (2011) Journal of Medicinal Chemistry 54, 7558-7566.

Khoo KK, Gupta K, Green BR, Zhang M-M, Watkins M, Olivera BM, Balaram P, Yoshikami D, Bulaj G & Norton RS (2012) Biochemistry, submitted.

Zhang MM, Green BR, Catlin P, Fiedler B, Azam L, Chadwick A, Terlau H, McArthur JR, French RJ, Gulyas J, Rivier JE, Smith BJ, Norton RS, Olivera BM, Yoshikami D, Bulaj G. (2007) Journal of Biological Chemistry 282, 30699-30706.