biomedicines Article Characterisation of a Novel A-Superfamily Conotoxin David T. Wilson 1, Paramjit S. Bansal 1, David A. Carter 2, Irina Vetter 2,3, Annette Nicke 4, Sébastien Dutertre 5 and Norelle L. Daly 1,* 1 Centre for Molecular Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Smithfield, QLD 4878, Australia; [email protected] (D.T.W.); [email protected] (P.S.B.) 2 Centre for Pain Research, Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD 4072, Australia; [email protected] (D.A.C.); [email protected] (I.V.) 3 School of Pharmacy, The University of Queensland, Woolloongabba, QLD 4102, Australia 4 Walther Straub Institute of Pharmacology and Toxicology, Faculty of Medicine, LMU Munich, Nußbaumstraße 26, 80336 Munich, Germany; [email protected] 5 Institut des Biomolécules Max Mousseron, UMR 5247, Université de Montpellier, CNRS, 34095 Montpellier, France; [email protected] * Correspondence: [email protected]; Tel.: +61-7-4232-1815 Received: 3 May 2020; Accepted: 18 May 2020; Published: 20 May 2020 Abstract: Conopeptides belonging to the A-superfamily from the venomous molluscs, Conus, are typically α-conotoxins. The α-conotoxins are of interest as therapeutic leads and pharmacological tools due to their selectivity and potency at nicotinic acetylcholine receptor (nAChR) subtypes. Structurally, the α-conotoxins have a consensus fold containing two conserved disulfide bonds that define the two-loop framework and brace a helical region. Here we report on a novel α-conotoxin Pl168, identified from the transcriptome of Conus planorbis, which has an unusual 4/8 loop framework. Unexpectedly, NMR determination of its three-dimensional structure reveals a new structural type of A-superfamily conotoxins with a different disulfide-stabilized fold, despite containing the conserved cysteine framework and disulfide connectivity of classical α-conotoxins. The peptide did not demonstrate activity on a range of nAChRs, or Ca2+ and Na+ channels suggesting that it might represent a new pharmacological class of conotoxins. Keywords: conopeptide; NMR spectroscopy; disulfide framework 1. Introduction Cone snail venoms comprise mainly small peptides, termed conotoxins, and represent one of the most extensive libraries of bioactive compounds from marine creatures. Conotoxins generally have selectivity and potency for a range of ion channels and G-protein coupled receptors and consequently have been useful as pharmacological tools and therapeutic leads [1]. Several conotoxins have been tested in clinical trials, with an N-type calcium channel blocker from Conus magus (!-MVIIA) approved by the Federal Drug Administration (FDA) as Prialt® for the treatment of chronic pain [2–4]. While the majority of studies aimed at developing conotoxins as drug leads focussed on treatment of pain (e.g., !-CVID and χ-MrIA [5]) other studies have expanded the potential applications of conotoxins. Conantokin G, a N-methyl-D-aspartate (NMDA) antagonist from Conus geographus, has been of interest for development as an anticonvulsant [6], while more recent studies have shown antimycobacterial activity (O1_cal29b from Californiconus californicus)[7] and inhibitory effects against the growth of lung cancer cells (TxID from Conus textile)[8]. The venom of a single species can contain hundreds of different peptides and with at least 750 different species of cone snails [9,10], it is estimated that Biomedicines 2020, 8, 128; doi:10.3390/biomedicines8050128 www.mdpi.com/journal/biomedicines Biomedicines 2020, 8, x FOR PEER REVIEW 2 of 10 Conotoxins have been classified into various gene superfamilies based on signal sequence conservation, in addition to classification into families based on cysteine framework and receptor targets [11]. The number of disulfide bonds is typically 2-4, but the connectivity can vary even amongst conotoxins containing the same number of cysteine residues, as can the bioactivity. The known cone snail venom peptide sequences and their known functions have been collated in the database Conoserver [12,13]. Twenty-nine superfamilies and thirty different cysteine frameworks have been identified in Conoserver to date, highlighting the diversity in the sequences of conotoxins. The A-superfamily is one of the most well characterised, with the majority containing cysteine framework I (CC-C-C). This framework is primarily associated with the α-conotoxin family, members of which specifically antagonise the nicotinic acetylcholine receptors (nAChRs) [14,15]. nAChRs are ligand-gated ion channels involved in a range of physiological and pathophysiological processes, including muscle contraction, pain sensation and nicotine addiction. They are classified as muscle- type and neuronal, and have been implicated in neurological disorders such as Parkinson’s and Biomedicines 2020, 8, 128 2 of 10 Alzheimer’s diseases [16] making them potential drug targets. nAChRs exist as homopentamers or heteropentamers comprising a range of different subunits [17]. α-Conotoxins are one of the most moremedically than 1relevant million families unique peptidesof conotoxins, exist [10 highlighted]. However, by despite a cyclic extensive version study of Vc1.1 in the displaying field, we haveoral currentlyactivity in sampled an animal less model than 1%of neuropathic of this diversity pain [ 10[3,18].]. Additional engineering studies have further highlightedConotoxins the potential have been of this classified peptide into in drug various design gene [19,20]. superfamilies based on signal sequence conservation,α-Conotoxins in addition are generally to classification less than into20 residues families in based length, on cysteinehave a CysI-CysIII, framework andCysII-CysIV receptor targetsdisulfide [11 connectivity]. The number and of disulfidethe majority bonds have is typicallya 4/7 loop 2-4, spacing, but the connectivitywhich represents can vary 4 residues even amongst in the conotoxinsfirst inter-cysteine containing loop the and same 7 in number the second of cysteine loop (Figure residues, 1a). as A can well-studied the bioactivity. example The known containing cone snail this venomloop spacing peptide issequences Vc1.1 [21]. and Several their knownother loop functions spacings have have been been collated identified in the database in the α-conotoxin Conoserver family,[12,13]. Twenty-nineand the size of superfamilies the loops correlates and thirty to different some extent cysteine with frameworks specificity for have different been identified nAChR insubtypes. Conoserver For toexample, date, highlighting 3/4 α-conotoxins the diversity target homome in the sequencesric neuronal of nAChRs, conotoxins. 3/5 Theα-conotoxins A-superfamily target ismuscle-type one of the mostnAChRs well and characterised, 4/4, 4/6 and with 4/7 theα-conotoxins majority containing target different cysteine hetero frameworkmeric and/or I (CC-C-C). homomeric This framework neuronal isnAChRs primarily [14]. associated with the α-conotoxin family, members of which specifically antagonise the nicotinicThe acetylcholinestructures of receptorsseveral α-conotoxins (nAChRs) [14 have,15]. been nAChRs determined are ligand-gated using nuclear ion channels magnetic involved resonance in a range(NMR) of spectroscopy. physiological andThis pathophysiological technique is well processes,suited to includingthe determination muscle contraction, of the structures pain sensation of α- andconotoxins nicotine because addiction. of their They small are size, classified high aqueous as muscle-type solubility and and neuronal, relatively and well-defined have been implicatedstructures α in[22]. neurological In addition, disorders some studies such asused Parkinson’s X-ray crystallography and Alzheimer’s to study diseases -conotoxins [16] making either them in potentialisolation drugor in targets.complex nAChRs with binding exist aspartners. homopentamers Despite the or heteropentamersvariation in inter-cystei comprisingne loop a range sizes of across different the α subunitsfamily, the [17 majority]. α-Conotoxins of -conotoxins are one of are the characterised most medically by relevant a small familieshelical structure, of conotoxins, which highlighted is braced by athe cyclic CysI-CysIII version of disulfide Vc1.1 displaying bond. Th orale activityadditional in an disulfide animal model bond of connecting neuropathic CysII-CysIV pain [3,18]. Additionalgenerally engineeringtethers the C-terminal studies have region further to the highlighted N-terminus. the potential of this peptide in drug design [19,20]. αHere-Conotoxins we show arethat generally a minor change less than in the 20 inter-cysteine residues in length, loop spacing have a in CysI-CysIII, framework CysII-CysIVI can have a disulfidesignificant connectivity impact on andthe thestructure majority and have bioactivity. a 4/7 loop Pl168, spacing, a which22-residue represents framework 4 residues I peptide in the that first α inter-cysteinecontains an unusual loop and 4/8 7 spacing, in the second was identified loop (Figure in 1thea). transcriptome A well-studied of example Conus planorbis containing as thisa new loop - α spacingconotoxin is Vc1.1[23]. [21The]. Severalsequence other of loopPl168, spacings along havewith been the identifiedsequences in theof twoα-conotoxin well characterised family, and the- sizeconotoxins, of the loops Vc1.1 correlates and MII, to are some given extent in Figure with specificity 1b. The three-dimensional for