Structural and functional characterisation of peptide inhibitors of voltage-gated sodium channels from piscivorous cone snails
thesisposted on 02.03.2017, 02:23 by Green, Brad Reed
The venoms of piscivorous (fish-hunting) snails of the genus Conus contain a vast array of neuroactive peptides used to immobilize prey and for defence. The intrinsic specificity and potency of these components for certain molecular targets has attracted the interest of researchers as novel drug leads for a wide range of neurological disorders. Beyond their therapeutic potential, these compounds have been invaluable as pharmacological probes to better understand the structure and function of their respective targets. Members of the M- and O1-conotoxin superfamilies are disulfide-rich neurotoxins characterised by the ability to potently and specifically inhibit voltage-gated sodium channels (VGSCs). As with other peptide-based therapeutics, issues of poor bioavailability, low metabolic stability and a general lack of VGSC subtype-selectivity have hindered the development of these toxins as viable therapeutic options. Structure-activity relationship (SAR) studies, such as those described here, have served as a basis from which important structural features of the peptide could be identified and peptide analogues with improved pharmacological profiles could be designed. The overall goal of this thesis is to identify the key structural components of two potent conotoxins, µ-conotoxin BuIIIB (M-superfamily) and µO§-conotoxin GVIIJ (O1-superfamily), that contribute to VGSC blockade. Chapter 3 reviews the SAR studies of µ-conotoxins and summarizes efforts to identify the structural features that contribute to their biological activity. To date, over 20 members of this family have been described, many of which possess an innate preference for certain VGSC subtypes. This chapter discusses what is known about the interactions of µ-conotoxins with the channel and highlights their therapeutic potential for the treatment of neurological disorders and severe pain. One member of the µ-conotoxin family, µ-BuIIIB, is of interest for its purported role in inhibiting the sodium channel subtype NaV1.3. Chapter 4 describes efforts to produce this peptide by recombinant expression and explores the importance of disulfide-connectivity for inhibition of NaV1.3. Chapter 5 details SAR studies of µ-BuIIIB against NaV1.3, using a disulfide-deficient, selenocysteine-containing analogue of µ-BuIIIB. This analogue simplified the oxidative folding of µ-BuIIIB and facilitated an alanine-scan of non-cysteine residues to identify amino acid residues critical for biological activity. Chapter 6 explores a different class of VGSC-inhibiting peptides that exert their block by covalently linking to the channel at a recently identified site on the channel that is close to, but physically distinct from, the site of interaction of the µ-conotoxins. In this chapter we solved the structure of the O1-superfamily toxin µO§-GVIIJ by NMR and performed complimentary SAR studies to identify structural features that contribute to VGSC blockade. Members of the M- and O1-superfamilies hold promise as therapeutic leads due to their intrinsic potency and specificity for VGSCs. SAR studies were performed to identify the structural features that are important for the biological activity of these two peptides. These studies identified structural components of µ-BuIIIB and µO§-GVIIJ that could be replaced to alter potency against pain-relevant VGSC subtypes. Studies such as those described here are an important step in designing peptide analogues that improve upon the existing pharmacological properties of venom-derived peptides.