Accepted Manuscript Versatile spider venom peptides and their medical and agricultural applications Natalie J. Saez, Volker Herzig PII: S0041-0101(18)31019-5 DOI: https://doi.org/10.1016/j.toxicon.2018.11.298 Reference: TOXCON 6024 To appear in: Toxicon Received Date: 2 May 2018 Revised Date: 12 November 2018 Accepted Date: 14 November 2018 Please cite this article as: Saez, N.J., Herzig, V., Versatile spider venom peptides and their medical and agricultural applications, Toxicon (2019), doi: https://doi.org/10.1016/j.toxicon.2018.11.298. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 1 Versatile spider venom peptides and their medical and agricultural applications 2 3 Natalie J. Saez 1, #, *, Volker Herzig 1, #, * 4 5 1 Institute for Molecular Bioscience, The University of Queensland, St. Lucia QLD 4072, Australia 6 7 # joint first author 8 9 *Address correspondence to: 10 Dr Natalie Saez, Institute for Molecular Bioscience, The University of Queensland, St. Lucia QLD 11 4072, Australia; Phone: +61 7 3346 2011, Fax: +61 7 3346 2101, Email: [email protected] 12 Dr Volker Herzig, Institute for Molecular Bioscience, The University of Queensland, St. Lucia QLD 13 4072, Australia; Phone: +61 7 3346 2018, Fax: +61 7 3346 2101, Email: [email protected] 14 15 Running head: Versatile spider venom peptides and their applications 16 17 18 19 Abstract 20 Spiders have been evolving complex and diverse repertoires of peptides in their venoms with vast 21 pharmacological activities for more than 300 million years. Spiders use their venoms for prey capture 22 and defense, hence they contain peptides that target both prey (mainly arthropods) and predators 23 (other arthropods or vertebrates). This includes peptides that potently and selectively modulate a 24 range of targets such as ion channels, receptors and signaling pathways involved in physiological 25 processes. The contribution of these targets in particular disease pathophysiologies makes spider 26 venoms a valuable source of peptides with potential therapeutic use. In addition, peptides with 27 insecticidal activities, used for prey capture, can be exploited for the development of novel 28 bioinsecticides for agricultural use. Although we have already reviewed potential applications of 29 spider venom peptides as therapeutics (in 2010) andMANUSCRIPT as bioinsecticides (in 2012), a considerable 30 number of research articles on both topics have been published since, warranting an updated review. 31 Here we explore the most recent research on the use of spider venom peptides for both medical and 32 agricultural applications. 33 34 35 36 Keywords 37 Spider; venom-based drug discovery; therapeutics; insecticidal spider venom peptide; bioinsecticide; 38 arachnid; arthropod; toxins. 39 40 Abbreviations 41 AD = Alzheimer’s disease; AMPs = antimicrobial peptides; ASIC = Acid-sensing ion channel; Ca V = 42 voltage-gated calcium channel; cGMP = cyclic guanosine monophosphate; DMD = Duchenne 43 Muscular Dystrophy; EAE = experimental autoimmune encephalomyelitis; EPA = Environmental 44 Protection Agency; hERG = human Ether-a-go-go-Related Gene; HypoPP = hypokalemic periodic 45 paralysis; IC 50 = concentrationACCEPTED resulting in 50% inhibition; ISP = Insecticidal spider venom protein or 46 peptide; KV = voltage-gated potassium channel; LD50 = dose resulting in 50% lethality; MPTP = 1- 47 methyl-4-phenyl-1,2,3,6-tetra hydropyridine; MS = Multiple sclerosis; MSC = mechanosensitive 48 channel; Na V = voltage-gated sodium channel; NCX1 = sodium-calcium exchanger 1; NF-κB = 49 nuclear factor kappa-light-chain-enhancer of activated B cells; NMDA = N-Methyl-d-Aspartate; NO 50 = nitric oxide; PD 50 = dose resulting in 50% paralysis; OAIP = orally active insecticidal peptide; PD = 51 Parkinson’s disease; PDE5i = phosphodiesterase-5 inhibitor; PnV = Phoneutria nigriventer whole 52 venom; RA = rheumatoid arthritis; SAR = structure/activity relationship; STAT3 = signal transducer 53 and activator of transcription 3; TNF-α = tumor necrosis factor alpha; TRPA1 = transient receptor 54 potential ankyrin 1 channel; TRPV1 = transient receptor potential vanilloid channel 1. 1 ACCEPTED MANUSCRIPT 55 56 1. Introduction 57 Spiders have evolved > 300 million years ago (Wang et al., 2018) and almost all extant species (with 58 the exemption of the Uloboridae family, comprising 0.6% of all species) are actually venomous 59 (World Spider Catalog, 2018). However, being venomous does not necessarily imply that they are 60 also dangerous to humans, as we've recently classified only 0.5% of all extant spider species as 61 potentially dangerous to humans (Hauke and Herzig, 2017). Nevertheless, the abundant usage of 62 venom in spiders reflects the evolutionary advantage that spiders have gained by employing venom 63 for prey capture and defense, despite the significant energetic costs required for venom synthesis 64 (Morgenstern and King, 2013). While venom of the few spider species that are potentially dangerous 65 to humans has been used for > 100 years to develop antivenoms for treatment of spider bites (for 66 details see Maretic and Stanic, 1954), only the recent decade has seen a boom into research on other 67 potential applications. Based on the fact that spiders mainly use their venoms to overcome insect prey, 68 an obvious application of spider venom components such as venom peptides includes the 69 development of novel bioinsecticides. And given that the great majority of spider venom peptides 70 target ion channels or receptors (Klint et al., 2012), another avenue of potential applications are as 71 specific modulators of these targets in basic research or in medicine to treat diseases, which are 72 caused by altered activity of these channels or receptors (Saez et al., 2010). In the present review, we 73 present an update on recent research on spider venom peptides and their applications in medicine and 74 as bioinsecticides. 75 76 2. Spider venoms as a source of therapeutics 77 Venomous organisms are ubiquitous and many animal lineages have independently evolved complex 78 compositions of bioactive molecules in their venoms for the purposes of prey capture and/or self- 79 defence (Holford et al., 2018). Spider venom is a highly complex cocktail comprising inorganic salts, 80 organic small molecules, small polypeptides as well as higher mass proteins and enzymes. Detailed 81 reviews of spider venom composition and pharmacology can be found in Escoubas et al., 2000b; 82 Vassilevski et al., 2009 and Kuhn-Nentwig et al., 2011a. Typically, the small polypeptides are 83 disulfide-rich molecules below 10 kDa in mass and cMANUSCRIPTomprise the majority of the venom, except for 84 some membrane active antimicrobial peptides found in spiders from the sub-order Araneomorphae. 85 Venom peptides are known for their potency and selectivity as compared with traditional small 86 molecule drugs, making them desirable for therapeutic development, and spider venom peptides are 87 no exception (Pennington et al., 2018). 88 89 Currently, most drugs used in clinical settings are small molecules. However, their limited 90 effectiveness combined with off-target activity leading to potentially severe side-effects necessitates 91 the development of a new generation of targeted, potent and efficacious therapeutics. Because of their 92 robust selectivity, spider-venom peptides provide a source for true pharmaceutical innovation in this 93 regard. Many spider venom peptides possess stringent biological activities (often with sub-type 94 selectivity at their targets, minimizing off-target effects) at low nanomolar or even picomolar 95 concentrations. However, it takes more than potency and selectivity to achieve eventual clinical 96 success. In addition, drug candidates should have a reliable production method, physiological 97 stability, desirable pharmacology, an applicable route of administration promoting bioavailability and 98 suitable pharmacokinetics, as well as be safe for in vivo use with no observed toxic effects (Hefti, 99 2008). 100 ACCEPTED 101 A broad discussion of peptides as therapeutics is outside the scope of this review but is given in Lau 102 and Dunn, 2018 and Fosgerau and Hoffmann, 2015. Venom peptides are largely cysteine-rich, 103 disulfide-reticulated molecules (Vassilevski et al., 2009). From a structural standpoint, these disulfide 104 bonds can protect the peptides, promoting chemical and biological stability; in particular spider 105 venom peptides often contain an inhibitor cystine knot (ICK) fold, which can provide them with 106 extraordinary stability (Saez et al., 2010; King and Hardy, 2013; Kikuchi et al., 2015). This complex 107 folding can be challenging for production, but is not unfeasible, with several methods for recombinant 108 expression or chemical synthesis available to toxinologists (Bae et al., 2012; Upert et al., 2014; 109 Clement et al., 2015; Saez et al., 2017; Turchetto et al., 2017). While production can be more 2 ACCEPTED MANUSCRIPT 110 challenging and costly than for some small molecules (Lau and Dunn, 2018), the