Accepted Manuscript

Versatile 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.

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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 have been evolving complex and diverse repertoires of peptides in their 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 ) and predators 23 (other arthropods or ). 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 ; ; . 39 40 Abbreviations 41 AD = Alzheimer’s disease; AMPs = antimicrobial peptides; ASIC = Acid-sensing ; Ca V = 42 voltage-gated ; 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 ; 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 ; 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 = nigriventer whole 52 venom; RA = rheumatoid arthritis; SAR = structure/activity relationship; STAT3 = signal transducer 53 and activator of transcription 3; TNF-α = 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 (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' 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 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 -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 . 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 -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 higher potency 111 associated with peptide modulators means that lesser amounts are generally required for efficacy. 112 Perhaps a more challenging factor to negotiate is bioavailability. By their very nature spider venom 113 peptides are injected via the fangs, meaning from an evolutionary perspective that there is likely to 114 have been limited selection pressure to produce orally active peptides, however this does not 115 completely exclude spider peptides from possessing oral bioactivity. Oral administration is known to 116 produce better outcomes with regard to patient compliance (Lau and Dunn, 2018), however, in the 117 treatment of severe or chronic illness alternative more invasive methods of administration may be 118 considered acceptable. Alternatively, rational design or conjugation to other molecules may also be 119 employed in order to improve the aforementioned properties (Renukuntla et al., 2013). 120 121 The medical value of spider venom peptides is not limited to treating disease directly. Several spider 122 venom peptides have been invaluable as pharmacological tools in the elucidation of ion channel 123 function, and disease pathology. Guangxitoxin-1E has been instrumental in elucidating the role of 124 KV2 delayed-rectifier potassium channels in neuronal excitability (Liu and Bean, 2014; Bishop et al., 125 2015; Honigsperger et al., 2017), PcTx1 in understanding the roles of acid-sensing ion channels and 126 related pathologies (Escoubas et al., 2000a; Baron et al., 2013), and α- and ω-agatoxins for probing 127 mammalian glutamate receptor and calcium channel activity (Adams, 2004), just to name a few. 128 Phrixotoxin-3, first described in 2006 (Bosmans et al., 2006), is a selective blocker of voltage-gated 129 sodium channel Na V1.2. Given the observed in mice upon intracerebroventricular 130 injection, ultimately leading to death, in vivo use of the is not advisable, however, the peptide 131 has been successfully used ex vivo as a pharmacological tool, highlighting the role of Na V1.2 in 132 neuronal excitability and febrile seizure generation (Ye et al., 2018). An unfolding area of research 133 has begun to exploit labelled analogs of venom peptides furthering their use as pharmacological tools 134 for the specific labelling and visualisation of subpopulations of cell types/structures or in clinical 135 diagnostics (Kuzmenkov and Vassilevski, 2018). 136 137 Some interesting spider venom peptides with potential as future drugs will be discussed in the 138 following sections according to their respective therapeuticMANUSCRIPT target (ion channel or signalling pathway). 139 A summary of their potential therapeutic applications is presented in Tables 1 and 2. 140 141 2.1 Modulators of Acid-sensing ion channels 142 Acid-sensing ion channels (ASICs) are activated during acidosis. A recent review of ASICs can be 143 found in Cristofori-Armstrong and Rash, 2017. Due to their implication in a range of conditions 144 including ischemic stroke, neurodegenerative diseases and , ASIC modulators are considered to 145 have potential therapeutic applications. Furthermore, administration of selective ASIC inhibitors is 146 likely to be well tolerated from a therapeutic standpoint as knockout of individual ASIC subtypes 147 produces viable (discussed further in Lin et al., 2015). 148 149 At the time of our last review (Saez et al., 2010), PcTx1 (also known as -1), a peptide 150 from the venom of the Trinidad Chevron , Psalmopoeus cambridgei , had shown 151 encouraging results in a rodent model of acute pain (Mazzuca et al., 2007) and the 152 intracerebroventricular injection of the whole P. cambridgei venom was protective in models of 153 ischemic stroke (Xiong et al., 2004; Pignataro et al., 2007). Since that time, the pure PcTx1 peptide, 154 which selectively inhibits acid-sensing ion channel 1a (ASIC1a), has also been demonstrated to be 155 neuroprotective inACCEPTED both mouse (Duan et al., 2011) and piglet (Yang et al., 2011) models of neuronal 156 ischemic injury. More recently, PcTx1 has been tested in an arguably more clinically-relevant stroke 157 model using conscious hypertensive rats where the peptide was administered two hours post-ischemic 158 insult (McCarthy et al., 2015). Peptide administration was neuroprotective, both structurally and 159 functionally, resulting in a reduction in brain infarct size and increased neuronal survival as well as 160 improvements in post-stroke neurological scores and behavioural performance (McCarthy et al., 161 2015). More recently, PcTx1 was shown to inhibit rat articular chondrocyte autophagy, through its 162 action on ASIC1a, not only demonstrating a role for ASIC1a in rheumatoid arthritis (RA), but also 163 implicating the peptide as a potential treatment to prevent or diminish cartilage destruction for RA 164 patients (Dai et al., 2017). Over-expression of ASIC2a, in conjunction with PcTx1 administration

3 ACCEPTED MANUSCRIPT 165 (and inhibition of ASIC3 by sea anemone peptide APETx2), further promoted chondroprotection 166 (Zhou et al., 2018), suggesting that combining ASIC1a (+ASIC3) inhibition with ASIC2a potentiation 167 or agonism may be effective in combating acid-induced chondrocyte apoptosis and may provide an 168 additional avenue for treating rheumatoid arthritis. 169 170 Due to its obvious sequence similarity to PcTx1, another peptide named Hi1a was identified from the 171 transcriptome of the Australian funnel web spider, Hadronyche infensa (Chassagnon et al., 2017). 172 Curiously, Hi1a is composed of two PcTx1-like folds attached by a short structured linker totalling 75 173 residues long and containing six disulfide bonds in a double knot arrangement (Bohlen et al., 2010; 174 Chassagnon et al., 2017). The peptide also has a different mechanism of action and functional effect 175 to PcTx1, Hi1a delaying the activation of ASIC1a, probably by the stabilization of closed channels, 176 and resulting in an incomplete (~20% residual current) but more slowly reversible inhibition of the 177 channels than PcTx1. Furthermore, Hi1a is also a more potent inhibitor than PcTx1 on ASIC1a 178 channels (IC 50 values of 0.40 nM for rat ASIC1a and 0.52 nM for human ASIC1a) and is more 179 robustly selective having no effect on ASIC2a and ASIC3 channels, and only a very mild potentiating 180 effect on ASIC1b when compared to PcTx1 (Chassagnon et al., 2017; Cristofori-Armstrong and Rash, 181 2017). Hi1a was tested in the same clinically-relevant stroke model as PcTx1 (McCarthy et al., 2015; 182 Chassagnon et al., 2017), using conscious hypertensive rats and demonstrated remarkable 183 neuroprotection even when administered as a single dose up to 8 hours post-stroke (Chassagnon et al., 184 2017). Perhaps most striking is the ability of Hi1a to partially recover damaged tissue in the ischemic 185 core (previously thought to be unsalvageable), in addition to the penumbral zone of injury. Hi1a- 186 mediated neuroprotection also correlates with reduced neurological deficits and less impairment of 187 motor skills post-stroke. The slow dissociation and residual ASIC1a activity afforded by Hi1a, as well 188 as lack of adverse effects, have been proposed to make it an ideal therapeutic candidate for the 189 treatment of stroke and other conditions involving ischemic neuronal damage (Chassagnon et al., 190 2017). 191 192 Another PcTx1-like peptide, Hm3a, was discovered from the Togo starburst tarantula, Heteroscodra 193 maculata (Er et al., 2017). The peptide was demonstratedMANUSCRIPT to be pharmacologically similar to PcTx1 194 (completely inhibiting ASIC1a with high potency (IC 50 of 1–2 nM) and potentiating ASIC1b with 195 much lower potency), but with longer biological stability, making it a more desirable candidate for 196 drug development and in vivo use than its predecessor. Hm3a can also be reliably produced via the 197 same recombinant methods as for PcTx1 making it a strong candidate for future development and use 198 in ASIC1a-related pathophysiologies. Obvious therapeutic indications exist for Hm3a, overlapping 199 with those demonstrated previously for PcTx1, such as in neuroprotection or treatment of rheumatoid 200 arthritis, though they remain to be tested for Hm3a. 201 202 (insert Table 1) 203 204 2.2 Modulators of Voltage-gated sodium channels 205 Recent reviews of voltage-gated sodium (Na V) channels as therapeutic targets and their associated 206 pathophysiologies can be found in Bagal et al., 2015 and Kwong and Carr, 2015. Na V antagonists are 207 commonly found in spider venom (see Table 2). However, for therapeutic development it is necessary 208 to avoid targeting Na Vs involved in fundamental physiological processes such as 209 propagation in smooth muscle (Na V1.4), cardiac muscle (Na V1.5) and Nodes of Ranvier (Na V1.6) 210 (with the exceptionACCEPTED of treating channelopathies linked to those channels). 211 212 Originally published in 2002 for its activity on voltage-gated potassium channels (Escoubas et al., 213 2002), Hm1a, also from the Togo starburst tarantula has more recently been characterised for its 214 ability to selectively activate voltage-gated sodium channels, in particular the Na V1.1 subtype (Osteen 215 et al., 2016). Heterozygous loss-of-function mutations in the Na V1.1 gene ( SCN1A ) are responsible for 216 the development of the vast majority of Dravet syndrome cases, a severe early-onset pediatric 217 epilepsy coincident with cognitive impairment, deterioration of motor skills and ataxia (De Jonghe, 218 2011; Catterall, 2018). The disease is often resistant to current pharmacotherapies for seizure 219 management, and in some cases, can prove fatal. In the context of Dravet syndrome, the reduction in

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220 functional Na V1.1 channels in inhibitory interneurons leads to neuronal hyperexcitation and seizure 221 generation. The mechanism of action of Hm1a, which delays inactivation of Na V1.1, effectively 222 increasing activation of the channel to improve inhibitory interneuron function (Richards et al., 2018), 223 makes it a promising therapeutic candidate. Indeed, administration of Hm1a in an in vivo mouse 224 model of Dravet syndrome lead to improved seizure inhibition resulting in a drastic improvement in 225 number of seizures per day and overall survival outcome (Richards et al., 2018). Thus, Hm1a 226 provides real promise for the treatment of Dravet patients. 227 228 Originally described as an insecticidal peptide (Berkut et al., 2015), Hm-3 from the araneomorph 229 spider Heriaeus melloteei has since been demonstrated to modify gating pore currents of voltage-

230 gated sodium channel Na V1.4 in skeletal muscle. Specific mutations in skeletal Na V1.4 gene SCN4A 231 (particularly in the S4 helix of the voltage sensor domain I) contribute to hypokalemic periodic 232 paralysis (HypoPP) type 2 (Sternberg et al., 2001; Kim et al., 2011). The HypoPP mutations permit

233 leaky gating-pore currents leading to abnormal depolarization of Na V1.4 channels resulting in muscle 234 weakness and/or paralysis. Specifically, Hm-3 has been shown to modify channel gating, minimizing

235 the causative leaky gating-pore currents, however it also inhibits the main Na V1.4 current, therefore it 236 is not selective enough to be used therapeutically in its current form. Structure-activity relationship 237 (SAR) studies and rational drug design may help improve specificity for the problematic gating pore 238 current component.

239 The voltage-gated sodium channel Na V1.7 is known to play a critical role in sensing pain with loss of 240 function mutations to the SCN9A gene resulting in a congenital insensitivity to pain (Drenth and 241 Waxman, 2007). A recent review of Na V1.7 as a therapeutic target is given in Vetter et al., 2017. At 242 the time of our last review, Protoxin-II was the most potent blocker of Na V1.7 reported but was 243 neurotoxic at higher concentrations by intravenous or intrathecal injection in rats, likely through its 244 action on the cardiac voltage gated sodium channels (Schmalhofer et al., 2008), minimising its 245 potential for therapeutic use. Another peptide GsAF-I was known to inhibit -sensitive 246 voltage-gated sodium channels, but the subtype selectivityMANUSCRIPT remained to be determined. GsAF-I is a 247 less potent blocker of sodium channels than Protoxi n-II, retaining some selectivity for Na V1.7 with 248 IC 50 s across the subtypes in the low micromolar range, and additional activity at the cardiac hERG 249 channel (human Ether-a-go-go-Related-Gene, K V11.1) also in the micromolar range. In addition to 250 their previously described activities, Protoxin-II and GsAF-I were also found to modulate voltage- 251 sensitive calcium channels (Bladen et al., 2014; Salari et al., 2016). Their apparent promiscuity and 252 potential for cardiac toxicity make them unattractive for drug development. More recently a peptide 253 from the South American tarantula Pamphobeteus nigricolor , Pn3a was found to potently and very- 254 selectively inhibit Na V1.7 (Deuis et al., 2017), making it the leading candidate for drug development 255 of a Na V1.7 inhibitor. While the peptide produced analgesia in a Na V1.7-mediated (OD1-induced) 256 spontaneous pain model in vivo in various other rodent models of acute or inflammatory pain the 257 peptide, along with other known Na V1.7 inhibitors, surprisingly lacked efficacy, unless co- 258 administered with subtherapeutic doses of opioids leading to a synergistic analgesic effect (Deuis et 259 al., 2017). This suggests that Pn3a does not completely inhibit Na V1.7 in vivo or that Na V1.7 260 inhibition alone is not is not sufficient to induce analgesia and highlights the complex interplay 261 between pain sensing pathways. Other Na V1.7 inhibitors, Cd1a (Sousa et al., 2017) and Protoxin-III 262 (Cardoso et al., 2015) also showed promising results in the same OD1-induced model of pain, 263 however have notACCEPTED been tested in other pain models. Further research will be essential in determining 264 whether Na V1.7 inhibition alone or in combination with other will be useful in the clinical 265 treatment of pain in humans. 266 267 Many Na V1.7 inhibitors have been discovered (see Table 2), seemingly consistently in the venoms of 268 various species of theraphosid (tarantula) spiders. This may be due to facile high throughput 269 fluorescence screening for identifying Na V1.7 channel modulators (Klint et al., 2015b; Chernov- 270 Rogan et al., 2018) over more time-consuming methods for other targets. Secondly, being 271 large spiders provide more venom than smaller non-theraphosid spiders. This is highly advantageous 272 as it allows enough venom to be available for initial screening through to venom fractionation and

5 ACCEPTED MANUSCRIPT 273 ultimately, peptide characterisation. Therefore it is not too surprising that tarantulas are a popular 274 choice for researchers over smaller spiders that produce significantly smaller amounts of venom. 275 Thus, the observation that spider-derived Na V1.7 inhibitors have only been found in tarantulas is 276 likely a skewed representation of the real phyletic distribution of Na V1.7 antagonists among spiders 277 and global tarantula venom bioactivity. Evidently, the challenge isn’t in identifying Na V1.7 inhibitors, 278 but in finding or engineering inhibitors with improved Na V subtype selectivity for therapeutic use. 279 Researchers have demonstrated their ability to rationally design analogues of spider-venom derived 280 peptides with improved potency and selectivity against Na V1.7, which are likely to represent a new 281 generation of venom-derived Na V1.7 blockers for clinical development (also see Table 2, mutated 282 peptides in italics). The tarantula peptide CcoTx1 was rationally engineered using four substitutions 283 plus amidation to improve potency and sub-type selectivity (Shcherbatko et al., 2016), similar 284 improvements in potency and sub-type selectivity also emerged when tarantula peptide GpTx1 was 285 re-engineered using five single substitutions (Murray et al., 2015b). Furthermore, 286 homodimerisation of GpTx1 improved potency and duration of Na V1.7 inhibition by reducing its off- 287 rate at the channel (Murray et al., 2015a). The native GpTx1 peptide was shown to be analgesic when 288 administered locally, but not systemically, in an OD1-induced spontaneous pain model in mice (Deuis 289 et al., 2016). The synthetically produced (Murray et al., 2015a; Chen et al., 2018) panel of GpTx1-like 290 analogues with variable pharmacokinetic properties will be ideal in establishing the essential 291 requirements for effecting Na V1.7-mediated analgesia in vivo . Regardless of therapeutic target or 292 indication, rational re-engineering of spider venom peptides is a likely strategy key to improving their 293 success as therapeutics. 294 295 (insert Table 2) 296 297 2.3 Modulators of Voltage-gated calcium channels 298 There are many types of voltage-gated calcium channels with various physiological roles. 299 Consequently, their function or dysfunction have been observed in a range of therapeutically relevant 300 conditions, most notably neurological, neuropsychiatric and chronic pain disorders (for 301 comprehensive reviews please see Zamponi et al., 2015MANUSCRIPT and Nanou and Catterall, 2018). 302 303 Ph α1β (also known as PnTx3-6, PhTx3-6) is a peptide from spider Phoneutria nigriventer with 304 analgesic potential. The peptide selectively inhibits voltage-gated calcium channels (particularly Ca V2 305 channels) (Vieira et al., 2005) and transient receptor potential ankyrin 1 (TRPA1) channels (Tonello 306 et al., 2017). Delivered intrathecally in vivo , it has been shown to relieve pain in a range of animal 307 models including: acute pain (Souza et al., 2008; Tonello et al., 2017), neuropathic pain (Souza et al., 308 2008; Rosa et al., 2014; Rigo et al., 2017b), inflammatory pain (Souza et al., 2008; Rigo et al., 309 2017b), thermal pain (Souza et al., 2008), post-operative pain (de Souza et al., 2011), mechanical and 310 cold hyperalgesia (Tonello et al., 2017) and cancer pain (Rigo et al., 2013; Rigo et al., 2017b). When 311 compared to contemporary drugs morphine and Ziconitide ( ω- MVIIA – delivered through 312 an intrathecal pump), Ph α1β is more effective, with less side-effects and is even able to control 313 cancer-related pain in morphine-tolerant mice (Rigo et al., 2013). In fact Ph α1β displays extraordinary 314 versatility. The peptide reduced scratching behaviour in a dry skin model suggesting it may be utilized 315 to control chronic refractory pruritus (itching) resulting from a range of clinical disorders (Maciel et 316 al., 2014). In a model of cyclophosphamide-induced hemorrhagic cystitis reductions in neutrophil 317 migration, levels of proinflammatory cytokines and oxidative stress markers were reported upon 318 treatment with Ph ACCEPTEDα1β, in line with an observed decrease in pain, inflammation and hemorrhage (Silva 319 et al., 2015b). The peptide is antiproliferative against glioma cell lines in vitro and in vivo without 320 affecting non-tumor glial cell proliferation (Nicoletti et al., 2017) (see also ‘Anti-cancer peptides’). In 321 an experimental autoimmune encephalomyelitis (EAE) model of Multiple sclerosis (MS) the peptide 322 improved motor deficits, pain, neurological and clinical scores, as well as decreased infiltration of 323 inflammatory mediators and the characteristic demyelination associated with MS, often with more 324 efficacy than fingolimod (an immunomodulator currently in use for treatment of MS), whether 325 administered intrathecally or intravenously (Silva et al., 2018). There is also evidence of synergistic 326 analgesic action of Ph α1β with a TRPV1 blocker increasing analgesic potency beyond that achievable

6 ACCEPTED MANUSCRIPT 327 through either component alone (Palhares et al., 2017; Silva et al., 2017). One limitation of the 328 peptide is that it is not likely to be orally available, however given the severe impacts on quality of 329 life caused by some of the aforementioned conditions, more invasive methods of delivery such as 330 intrathecal or intravenous administration may be acceptable to patients. Alternatively, novel delivery 331 technologies may be employed to improve bioavailability via other routes. Despite delivery 332 constraints, with its remarkable catalogue of actions and ability to be made recombinantly (Tonello et 333 al., 2014), combined with lack of observed toxicity (also see ‘Modulators of TRP Channels’section), 334 Ph α1β is likely to be an excellent drug candidate.

335 Venom from the spider Phoneutria nigriventer has, in fact, been a valuable source of peptides with 336 therapeutic potential (also see Peigneur et al., 2018 in this special issue). Several isoforms of Ph α1β 337 (PnTx3-6) have also been shown to have analgesic potential, including PnTx3-3 (Dalmolin et al., 338 2011; Dalmolin et al., 2017), PnTx3-4 (da Silva et al., 2015) and PnTx3-5 (Oliveira et al., 2016). 339 While PnTx3-5 is less studied than Ph α1β, it may prove to be a better drug candidate, its improved 340 potency and selectivity resulting in a more desirable therapeutic index compared with Ph α1β (Oliveira 341 et al., 2016). The therapeutic potential of analgesic Ca V2 blocker PnTx3-4 (PhTx3-4 in Binda et al., 342 2016) has also been examined in neurodegenerative retinopathy (Binda et al., 2016). In a rat model of 343 NMDA-induced retinal injury, intravitreal co-injection of PnTx3-4 with NMDA significantly 344 improved retinal damage (as measured by electroretinogram) and reduced pathophysiological 345 processes associated with retinal injury, such as glutamate excitotoxicity, oxidative stress and cell 346 death, demonstrating its potential for therapeutic use in certain ophthalmic conditions (including 347 diabetic neuropathy and glaucoma) that could otherwise lead to blindness. Given these promising 348 results, it is still rather disappointing that the complete sequence of PnTx3-4 matching the published 349 experimental mass spectrometry data has not yet been fully determined (Herzig, 2016; Gomez, 2016). 350 351 Originally discovered in 1993, -I an inhibitor of the voltage-gated calcium channel 352 Ca V2.2 and tetrodotoxin-sensitive voltage-gated sodium channels was recently shown to be 353 neuroprotective in a mouse model of chronic cerebral ischemic injury (Mao et al., 2017). In ischemic 354 conditions, Ca 2+ overload can trigger cell death.MANUSCRIPT The authors report that administration of 355 Huwentoxin-I minimises Ca 2+ overload by activating sodium-calcium exchanger 1 (NCX1) and 356 recruiting the Notch1 pathway, reducing neuronal damage. Combining an aerobic exercise regimen 357 with Huwentoxin-I treatment further promoted neuroprotection. However, the promiscuous action of 358 Huwentoxin-I and observed lethality in mice at higher dosages by irreversible block of neuromuscular 359 transmission (Liang et al., 1993) means that it is likely to have a poor therapeutic index, and holds 360 limited promise as a therapeutic. 361 362 The recently discovered Huwentoxin-XVI is likely to have greater therapeutic potential. Through its 363 potent and selective, yet reversible action on Ca V2.2, the peptide reduced pain in a rat formalin model, 364 postincision and thermal pain models without affecting general motor coordination (Deng et 365 al., 2014). The peptide was also more effective over a longer time course in the postincision model 366 when compared to morphine. In addition Huwentoxin-XVI lacked or toxicity. 367 Synthetic Huwentoxin-XVI is available from several companies therefore production of the peptide 368 should not be a limitation for further preclinical testing and drug development. 369 370 2.4 Modulators of Voltage-gated potassium channels 371 A thorough overviewACCEPTED of Voltage-gated potassium channels, their physiological roles, contribution to 372 disease and peptide modulators can be found in Norton and Chandy, 2017. 373 374 The Phoneutria nigriventer peptide PhK V (previously Tx3-1) has shown a remarkable ability to 375 rescue long-term memory in a mouse model of Alzheimer’s disease (AD), due to its selective + 376 modulation of A-type transient outward K currents ( IA) (Kv4.1–4.3 in CA1 pyramidal ) 377 (Gomes et al., 2013). Furthermore, no observable adverse side-effects were reported in contrast to the 378 non-selective voltage-gated potassium channel blocker, 4-aminopyridine, which showed no 379 statistically significant improvement in long-term memory but induced toxic side-effects at all doses 380 tested. The toxin’s potency in A β25-35 -treated AD-like mice makes it a promising molecule for

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381 exploration in therapeutics to treat cognitive decline. PhK V has also shown an antinociceptive effect in 382 a mouse model of chronic constriction injury (Rigo et al., 2017a) and a reduction in aberrant cardiac 383 activity in arrhythmic rat (Almeida et al., 2011), however these effects are likely to be mediated 384 by an acetylcholine-dependent mechanism (Almeida et al., 2011; Rigo et al., 2017a). 385 386 As noted in the introduction, several spider venom peptides have been utilized as pharmacological 387 tools to elucidate the role of particular channels in disease. Guangxitoxin-1E (Liu and Bean, 2014; 388 Bishop et al., 2015) was recently used to implicate voltage-gated potassium channel K V2.1 in a 389 subchronic MPTP-induced mouse model of Parkinson’s disease (PD) (Chao et al., 2018). In this 390 model, expression of the channel is increased leading to deleterious effects on dopaminergic neuronal 391 function and motor coordination. Guangxitoxin-1E was almost able to completely restore motor 392 deficits in the PD-like mice, evidently by reversing the MPTP-induced increase in expression of 393 KV2.1, however the exact mechanisms remain to be elucidated. Similarly, K V2.1 was implicated in the 394 regulation of glucose-stimulated insulin secretion from pancreatic beta cells using Guangxitoxin-1E 395 and genetic knockouts (Li et al., 2013). While further research is required on both fronts, it may 396 suggest that selective spider venom inhibitors of KV2.1 may have therapeutic relevance, and could be 397 an avenue for future drug development. 398 399 2.5 Modulators of TRP channels 400 Transient receptor potential (TRP) channels are a large family of ion channels with a broad spectrum 401 of involvement in sensory perception (Emir, 2017). Several TRP channels have been implicated in 402 nociception and inhibitors may be useful for the treatment of chronic pain (Moran and Szallasi, 2018). 403 404 The prototypical TRP channel modulators from spider venom are the vanillotoxins (Siemens et al., 405 2006) that activate TRPV1 channels, however, being agonists of channel function, they are pain 406 promoting therefore are not therapeutically useful. The analgesic peptide Ph α1β (also known as 407 PnTx3-6, PhTx3-6) was recently shown to inhibit transient receptor potential ankyrin 1 (TRPA1) 408 channels (Tonello et al., 2017) in addition to acting on voltage-gated calcium channels (Vieira et al., 409 2005)(discussed above). A recombinant form of Ph α1βMANUSCRIPT specifically inhibited TRPA1 channels in vitro 410 without effect on other TRP channels and in a TRPA1 -dependent form of chemotherapy-induced 411 peripheral neuropathy the peptide proved antihyperalgesic in mice (Tonello et al., 2017). In this case, 412 the dual pro-analgesic functions of the peptide on voltage-gated calcium channels in addition to 413 TRPA1 channels could be beneficial therapeutically, however, extra precautions must be taken to 414 ensure that the additional functionality of the peptide does not lead to off-target adverse reactions, 415 though none have been reported thus far. 416 417 2.6 Modulators of P2X receptors 418 Purinergic receptors have been implicated in a broad range of conditions (Burnstock, 2017), therefore, 419 specific modulators of purinergic receptor function could be highly valuable therapeutically. At the 420 time of our last review, only one P2X 3 purinergic receptor antagonist from spider venom had been 421 reported, known as Purotoxin-1. The peptide had demonstrated potential as an analgesic in rat pain 422 models (Grishin et al., 2010). While P2X 3 receptors themselves have continued to receive a lot of 423 attention as therapeutic targets, the peptide itself as a receptor antagonist has lost favour to bioactive 424 small molecule inhibitors as pharmacological tools or in clinical trials (Ford and Undem, 2013; 425 Abdulqawi et al., 2015; Burnstock, 2017; Ginnetti et al., 2018). Shortly after the discovery of 426 Purotoxin-1, anotherACCEPTED selective P2X 3 receptor inhibitor, named Purotoxin-2, was discovered and 427 characterised (Kabanova et al., 2012) and the structure was recently reported (Oparin et al., 2016), 428 however no further evidence of therapeutic or clinical development can be found. 429 430 2.7 Modulators of Mechanosensitive ion channels 431 The peptide GsMTx4, from the venom of the tarantula Grammostola rosea , is a mechanosensitive ion 432 channel (MSC) inhibitor, with potential therapeutic activity in a range of conditions including cardiac 433 arrhythmias, spinal cord injuries, muscular dystrophies, and gliomas, or even as antibiotics (Bowman 434 et al., 2007) (further details are discussed in our earlier review (Saez et al., 2010)). However, in recent 435 years, the most promising and advanced therapeutic applications lie in treating Duchenne muscular

8 ACCEPTED MANUSCRIPT 436 dystrophy (DMD), a severe and lethal X-linked muscle-wasting disease affecting boys and young 437 men, as well as in treating cardiac ischemic injury. GsMTx4 is a good therapeutic candidate for 438 several reasons: it is relatively specific in that it does not affect mechanosensitive ion channels 439 involved in hearing and touch (which are transmitted by a different class of MSCs stimulated by stress 440 in the cytoskeleton, rather than stress to the surrounding bilayer) (Bowman et al., 2007) and it has 441 good therapeutic properties in that it is non-toxic, non-immunogenic and biologically stable (Sachs, 442 2015). The peptide is thought to deposit itself in membranes, expanding the membrane area and 443 acting like a shock absorber, in effect reducing the transfer of stimulating mechanical forces from the 444 membrane to the channels (Gnanasambandam et al., 2017). Both L- and D-enantiomers of GsMTx4 445 inhibit mechano-gated Piezo channels (Bae et al., 2011; Copp et al., 2016; Alcaino et al., 2017). Early 446 studies of GsMTx4 on cultured muscle cells from DMD-like mice showed promising results (Yeung 447 et al., 2005). After modest success of GsMTx4 in DMD preclinical trials in 2014, the peptide 448 (renamed AT-300) was picked up by a therapeutics company (Blaustein, 2014) for further 449 development and clinical testing (DiMilia, 2017). GsMTx4 has also been shown to reduce myocardial 450 infarction size in a mouse model of ischemic reperfusion injury (Wang et al., 2016a). A recent review 451 of GsMTx4 and its action on mechanosensitive ion channels can be found in Suchyna, 2017. Should 452 GsMTx4 behave well in clinical testing it is likely to be a promising treatment for Piezo-related 453 diseases and channelopathies, of which there are many (Wu et al., 2017). 454 455 2.8 Anti-cancer peptides 456 Since the first description of the peptide and report of its anti-cancer activity in 2012, research into 457 Lycosin-I from the venom of Lycosa singoriensis has rapidly progressed. The peptide is able to 458 suppress tumor growth in vivo , by a two-fold mechanism consisting of activation of the mitochondrial 459 death pathway leading to apoptosis and inhibition of cell proliferation of cancer cells (Liu et al., 460 2012b). However, due to its somewhat limited penetration ability with regard to solid tumors, recent 461 research has involved increasing selectivity for tumor cells and improving internalization efficiency of 462 the peptide. A simple substitution of the seven lysines in Lycosin-I to arginine increased the stability, 463 membrane binding and cell penetration (by improving cellular uptake and endocytic release 464 intracellularly) into cancer cells in an ex vivo modelMANUSCRIPT of human tumors (Zhang et al., 2017). 465 Remarkably, arginine substitution did not drastically alter the peptides cell penetrating ability into 466 non-cancer cells, effectively improving its selectivity. Recently the anti-cancer effect of Lycosin-I 467 was investigated in prostate cancer, and its mechanisms further explored. By inactivation of the signal 468 transducer and activator of transcription 3 (STAT3) pathway, a known oncogenic target (Siveen et al., 469 2014), the peptide concentration-dependently induced apoptosis (at concentrations of 10 M and 470 above) or inhibited cell invasion, migration and metastasis, without inducing apoptosis, at a lower 471 peptide concentration (5 M) (Shen et al., 2018). This has implications for cancer treatment in 472 general, but particularly with regard to prostate cancer, which can have poor diagnosis and survival 473 rates and high risk of metastasis (Gartrell et al., 2015; Pascale et al., 2017). Lycosin-I is an extremely 474 promising candidate for anti-cancer therapeutic development both to induce apoptosis of cancer cells 475 and reduce likelihood of further metastases. The arginine-substituted form of the peptide has the more 476 desirable properties in terms of stability, size and penetrability (Zhang et al., 2017) and its ability to 477 be chemically synthesized makes it highly suitable for further preclinical development. Several more 478 recent activities and potential therapeutic applications for the peptide have also been discovered 479 (discussed below and in ‘Modulators of Nitric Oxide signaling’, ‘Modulators of NF-κB signaling’ and 480 ‘Anti-pathogenic peptides’). 481 ACCEPTED 482 Gold nanoparticle technologies are emerging for the diagnosis and treatment of cancers (Huang and 483 El-Sayed, 2010). Lycosin-I-conjugated gold nanoparticles have been shown to target and accumulate 484 in tumors in an in vivo xenograft model, demonstrating their potential for use as a contrast agent in the 485 imaging and localisation of tumor tissue (Tan et al., 2017). Due to their different spectroscopic 486 properties gold nanorods show promise in photothermal therapy owing to their ability to convert light 487 into heat that destroys tissue (Huang and El-Sayed, 2010). Consequently, Lycosin-I-conjugated gold 488 nanorods have also been developed that show promise in vivo by selectively accumulating within 489 cancer cells allowing efficient and selective delivery of photothermal therapy, without damage to 490 surrounding non-tumor tissue (Tan et al., 2017).

9 ACCEPTED MANUSCRIPT 491 492 Similarly, a membrane-targeting, cell-penetrating peptide from spider venom, latarcin-1, was 493 produced as part of a multifunctional cancer-targeting fusion protein (in combination with anticancer 494 peptide Tachyplesin 1 and ribosome inactivating protein MAP30). The fusion protein selectively 495 targeted cancer cells, and when used in combination with a pre-existing chemotherapy promoted 496 enhanced delivery and anti-cancer effects (Rothan et al., 2015). The latarcins are discussed further in 497 ‘Anti-pathogenic peptides’. 498 499 Phoneutria nigriventer whole venom (PnV) also shows potential in the treatment of gliomas (tumors 500 of the brain and central nervous system). Although preliminary, application of the whole venom has 501 been shown to reduce glioma cell viability in vitro (Raposo, 2017) by either inducing cell death or 502 arresting the cell cycle, in addition to reducing migration of tumor cells (Barreto Dos Santos et al., 503 2018). Experiments are in progress to identify the responsible anti-cancer venom component/s within 504 PnV and their specific mode/s of action. Ph α1β, a voltage-gated calcium-channel and TRPA1 505 inhibitor (see ‘Modulators of Voltage-gated calcium channels’ and ‘Modulators of TRP channels’), is 506 one component of the venom that may be responsible (solely or partially) for the anticancer activity of 507 PnV. The peptide has antigliomal activity in vitro and in vivo without affecting non-tumor glial cell 508 proliferation (Nicoletti et al., 2017). 509 510 The peptide gomesin was originally identified in hemocytes from the tarantula Acanthoscurria 511 gomesiana and characterized as an antimicrobial peptide (Lorenzini et al., 2003). Recently, the 512 peptide and a homolog identified from the venom-gland transcriptome of Australian funnel-web 513 spider Hadronyche infensa , were shown to be antiproliferative and cytotoxic to tumor cells from 514 Tasmanian devils, native Australian marsupials, suffering from devil facial tumour disease, a highly 515 transmittable disease threatening wild devil populations (Fernandez-Rojo et al., 2018; Ikonomopoulou 516 and Fernandez-Rojo, 2018). Furthermore, the peptides were also effective in a xenograft model of 517 melanoma, selectively disrupting the proliferation of cancer cells in contrast to healthy cells 518 (Ikonomopoulou et al., 2018). The gomesins hold extreme promise as candidates for further 519 development of treatments against skin cancers. MANUSCRIPT 520 521 2.9 Modulators of the Nitric Oxide signalling 522 A curious symptom that has long been observed in cases of envenomation by Phoneutria nigriventer 523 is the induction of priapism (long-lasting, painful erections) in males (Lopesmartins et al., 1994). The 524 ion-channel toxin PnTx2-6 was finally identified as the causative molecule in 2008 (Andrade et al., 525 2008). Consequently, PnTx2-6 was investigated as a potential treatment for erectile dysfunction 526 (research prior to 2010 is discussed in our previous review (Saez et al., 2010)). Erections are caused 527 by downstream effects of nitrous oxide (NO) release upon sexual stimulation and impaired NO 528 signalling can hinder penile erection (Burnett, 2006). Due to extremely unfavourable dose-dependent 529 side-effects of the full length PnTx2-6 peptide in vivo (Leite et al., 2012), a smaller 19-amino acid 530 synthetic peptide called PnPP-19 was engineered, to mimic the spider peptide’s pharmacophore (Silva 531 et al., 2015a). In contrast to PnTx2-6, PnPP-19 showed no apparent toxicity in mice, having lost 532 activity against sodium channels, however retained the ability to induce erection, through NO and 533 downstream cGMP signalling pathways (Silva et al., 2015a). Of particular importance is the novel 534 mode of action when compared with current treatments for erectile dysfunction. This novel activity 535 may be beneficial for patients who are refractory to presently available phosphodiesterase-5 inhibitor 536 (PDE5i) drugs likeACCEPTED Viagra. Recently, topical administration of PnPP-19 using cationic transfersomes 537 was reported to allow permeation of the peptide across human skin and shows promise as a topical 538 therapeutic for erectile dysfunction (Almeida et al., 2018). 539 540 PnPP-19 has also been reported to elicit central and peripheral antinociceptive effects in vivo (da 541 Fonseca Pacheco et al., 2016; Freitas et al., 2016; Freitas et al., 2017). The peptide has been shown to 542 selectively and directly activate -opioid receptors (Freitas et al., 2018), but its mechanism of action 543 is also likely to include indirect activation of δ-opioid receptors, CB 1 cannabinoid receptors (da 544 Fonseca Pacheco et al., 2016; Freitas et al., 2016) and inhibition of downstream calcium signalling 545 (Freitas et al., 2018), in addition to activation of the nitric oxide signalling system (Freitas et al.,

10 ACCEPTED MANUSCRIPT 546 2017). In its current state, however, without additional modification, PnPP-19 prescribed as an 547 analgesic is likely to also have the potentially undesirable side-effect of potentiating erectile function. 548 549 In addition to its anti-cancer activity, Lycosin-I (discussed above in ‘Anti-cancer peptides’, and below 550 in ‘Modulators of NF-κB signaling’ and ‘Anti-pathogenic peptides’) has also been implicated as a 551 potential treatment for hypertension due to its recently-characterised effects on the endothelial nitric 552 oxide signaling pathway (Ma et al., 2018). 553 554 2.10 Modulators of NF-κB signaling 555 New research highlights further therapeutic applications of the Lycosin-I peptide in inflammatory 556 vascular diseases. Lycosin-I has been demonstrated to reduce TNF-α-enhanced expression of pro- 557 inflammatory mediators and suppresses NF-κB activation in human umbilical vein endothelial cells, 558 inhibiting vascular inflammation. Furthermore, significant reductions in inflammatory mortality were 559 observed in a mouse model of septic shock when animals were pre-treated with Lycosin-I (Li et al., 560 2018). 561 562 2.11 Anti-pathogenic peptides 563 With antibiotic resistance posing an ever-increasing threat, there has never been a more urgent need 564 for the development of novel antimicrobial drugs. Antimicrobial activity is a key feature observed in a 565 range of spider venoms (for a thorough review see Santos et al., 2016). Because different organisms 566 have different membrane lipid compositions and many antimicrobial peptides (AMPs) interact 567 directly with membranes, there is an opportunity for selective interaction. To be clinically useful 568 AMPs must be targeted towards the pathogen, rather than host cells, in order to avoid complications 569 such as hemolysis. Some AMPs from mygalomorph spiders have been reported since our last review 570 (Zhao et al., 2011; Ayroza et al., 2012; Ferreira et al., 2016), demonstrating their presence in the 571 venom of both lineages of spiders, not just in araneomorphs, however their structure and mechanism 572 of action may differ, those from mygalomorphs being disulfide-rich knottin structures rather than 573 being dominated by linear amphipathic helices, as in araneomorphs. 574 MANUSCRIPT 575 Two novel mygalomorph AMPs have been characterised, both with inhibitor cystine knot (ICK) 576 folds. Oh-defensin, identified in venom from Cyriopagopus hainanus (previously Ornithoctonus 577 hainana ) displays broad-spectrum activity, inhibiting the growth of all six microbial strains tested 578 including Gram-positive bacteria, Gram-negative bacteria and fungus, while only inducing very little 579 hemolysis at much higher concentrations (Zhao et al., 2011). Juruin from the venom of Avicularia 580 juruensis was not antibacterial but rather highly fungicidal to yeast, including clinical isolates of the 581 yeast Candida glabrata , which displays resistance to conventional azole antifungals. Despite the 582 potent antifungal activity, Juruin still lacked hemolytic activity on human erythrocytes at the highest 583 concentration tested (Ayroza et al., 2012). While the exact mode of antimicrobial action for each 584 peptide remains to be determined, it suggests a more specific mechanism than general membrane 585 disruption, and thus, while not as well studied at the current time, these AMPs may prove to be more 586 therapeutically-relevant in the future than some of the less selective, better-characterized linear AMPs 587 described below. For a full overview of AMPs discussed in this section see Table 3. 588 589 Unfortunately general cytolytic activity belied most of the early reports of antimicrobial peptides from 590 spider venom, such as the lycotoxins (Yan and Adams, 1998), oxyopinins (Corzo et al., 2002) and 591 cupiennins (Kuhn-NentwigACCEPTED et al., 2011b), with peptides often exhibiting cytotoxicity due to 592 undesirable lysis of non-target host cells, like blood or muscle cells. 593 594 In the case of latarcins, a group of AMPs from the venom of Lachesana tarabaevi (Kozlov et al., 595 2006), not all exhibit hemolysis to the same degree, and they have been used as tools to investigate 596 mechanisms of AMP penetration and lysis at different membranes (Polyansky et al., 2009; Dubovskii 597 et al., 2015). Latarcin-3a and latarcin-1 have also shown to be antiviral in vitro in the context of 598 human immunodeficiency virus type 1 (HIV) (Wang et al., 2010) and Dengue virus (Rothan et al., 599 2014), respectively. Further exploiting their inherent membrane permeability, latarcin-derived cell 600 penetrating peptides have even been proposed as a novel vector for intracellular drug delivery

11 ACCEPTED MANUSCRIPT 601 (Ponnappan and Chugh, 2017; Budagavi and Chugh, 2018). Similarly, a multifunctional latarcin-1- 602 fusion protein has also been used alone or in combination with pre-existing chemotherapies for 603 enhanced delivery and anti-cancer effects (Rothan et al., 2015) (see ‘Anti-cancer peptides’). 604 605 Lycosin-I has also displayed potent bactericidal activity in an in vivo model of S. aureus infection 606 (Tan et al., 2013). In vitro , antimicrobial activity of Lycosin-I was demonstrated in 19 species of 607 medically-relevant microbe tested, including gram-negative and gram-positive bacteria and fungi 608 (Tan et al., 2013; Wang et al., 2014). It also showed synergistic activity, improving efficacy, when 609 used in conjunction with traditional antibiotics (Tan et al., 2013). In fact, research has demonstrated 610 that for several arachnid AMPs combining treatment with commercially available antibiotics can 611 reduce the doses required, production costs, side-effects and lower the risk of further antimicrobial 612 resistance (Garcia et al., 2013; Arenas et al., 2016), making it a practical and feasible approach for 613 future clinical treatments. A related AMP from the same spider was discovered in 2016 known as 614 Lycosin-II (Wang et al., 2016b), showing similarly potent and broad-spectrum antibacterial activity 615 on clinical isolates of multi-drug-resistant bacteria, with limited hemolysis. 616 617 Another Lycosid venom peptide with promising antimicrobial potential is LyeTx1 (Santos et al., 618 2010). LyeTx1 is antibacterial ( S. aureus and E. coli ) and antifungal ( C. krusei and C. neoformans ), 619 only weakly hemolytic at high concentrations and preferentially targets microbial membranes. A 620 formulation containing LyeTx1 and β-cyclodextrin further improved microbial susceptibility in 621 Aggregatibacter actinomycetemcomitans , a periodontal bacterium, lowering amounts of peptide 622 required for effective dosing, however, some cytotoxicity to host cells was still observed (Consuegra 623 et al., 2013). The β-cyclodextrin formulation also doubled potency on microbial biofilms in the early 624 stages of growth, despite biofilms being more resistant to antimicrobial treatment than their 625 planktonic counterparts (Cruz Olivo et al., 2017). A LyeTx1 derivative, LyeTx1-b (with a single 626 histidine deletion and acetylated N-terminus) was recently shown to be 10-fold more potent against 627 planktonic E. coli than the native peptide in vitro , and was effective in an S. aureus -induced model of 628 septic arthritis in mice when administered by intra-articular injection (Reis et al., 2018). The peptide 629 reduced bacterial accumulation within the joint, asMANUSCRIPT well as reducing the number of infiltrating 630 neutrophils and mononuclear cells, minimising infla mmation and preventing damage to articular 631 cartilage thereby protecting joint integrity. LyeTx1-b is a promising lead molecule for development of 632 future antibiotics. Modifications to enhance bioavailability or delivery as well as efficacy of the 633 peptide with commercial antibiotics as an adjunct therapy should also be explored. 634 635 Another strategy for delivery of spider venom AMPs is by heterologous recombinant expression 636 through a plasmid vector for localised, inducible, precisely regulated antimicrobial action inside the 637 host cells. This gene technology has been used to express several AMPs intracellularly, at the site of 638 infection (Lazarev et al., 2011). Cyto-insectotoxin 1a (CIT1a) is a cytolytic and insecticidal peptide 639 from Lachesana tarabaevi that also bears anti-chlamydial activity (Lazarev et al., 2011). When CIT1a 640 expression was induced inside human embryonic kidney (HEK293) cells challenged by Chlamydia 641 trachomatis infection, peptide expression dramatically reduced the development of infection, but not 642 host cell viability (Polina et al., 2012), with inhibition being most effective in the early stages of the 643 Chlamydial life cycle (Lazarev et al., 2013). Exactly how CIT1a selectively exerts its activity inside 644 the infected cells remains to be determined. 645 646 At the time of ourACCEPTED last review, the Psalmopeotoxins from Psalmopeous cambridgei (PcFK1 and 647 PcFKII) were being investigated as antiplasmodials, specifically, in the treatment of malaria (Choi et 648 al., 2004; Pimentel et al., 2006; Combes et al., 2009; Kamolkijkarn et al., 2010; Bastianelli et al., 649 2011; Gleeson et al., 2011). Incidentally, no research indicating their therapeutic development has 650 been published on this topic lately. Recent efforts have focussed on using computational modelling to 651 better understand the mechanism of their antiplasmodial actions (Bastianelli et al., 2011; Lhouvum et 652 al., 2015). 653 654 (insert table 3) 655

12 ACCEPTED MANUSCRIPT 656 2.12 Summary 657 In the last decade there has been an obvious shift from using venom-derived peptides as 658 pharmacological tools for studying ion channel function to studying their activity in vivo with the aim 659 of determining their therapeutic potential. Even so, venom-based drug discovery is still in its relative 660 infancy, as evidenced by the limited range of spider species from which the peptides summarised in 661 this review were sourced (see Tables 1–3). The history of venom-based drug discovery has relied on 662 the study of very few extremely-well characterized venoms (like P. nigriventer venom) or 663 prototypical peptides (such as PcTx1). However, with the development of increasingly-cheaper 664 technologies for venomic exploration through transcriptomic and proteomic analyses, it is likely that 665 the vast diversity seen in nature will be better represented in future venomics peptide libraries for drug 666 discovery. Even in the present case of Hi1a, PcTx1 and Hm3a it is evident that identification of 667 similar peptides from different species with slightly differing activities or properties can be beneficial 668 for improving therapeutic outcomes, even when targeting the same ion channel. There are several 669 other lessons that can also be learned from the examples presented in this review. In certain instances 670 the native peptide may not be an ideal therapeutic candidate, but through a range of potential 671 modifications, improvements in potency, selectivity, toxicity and stability are attainable, as in the case 672 of PnTx2-6 and PnPP-19. In conditions that are extremely detrimental to the patients’ quality of life, 673 oral availability is not essential, particularly when alternative treatments that are currently available 674 are inadequate or produce severe side-effects (compare morphine and Ziconitide with Ph α1β in the 675 treatment of refractory chronic pain, such as cancer pain). New medical technologies offer growing 676 opportunities for applications using venom peptides, exemplified by the conjugation of Lycosin-I to 677 gold nanoparticles and nanorods for the identification, localization and ultimately, treatment of 678 tumors, or in the use of cationic transfersomes for the topical application of PnPP-19 in erectile 679 dysfunction. Other options that are yet to be explored therapeutically are the synergistic use of 680 peptides with specific activities to treat multiple aspects of complex diseases. With all of these options 681 available to researchers, venom-based drug discovery will have a bright and buzzing future ahead. 682 683 3. Spider venoms as a source of bioinsecticides 684 Arthropods and in particular insects cause an estimatedMANUSCRIPT annual damage of $470 billion to agriculture 685 worldwide, destroying about 18–26% of the crop production (Culliney, 2014). In addition, many 686 insect species are also vectors for a wide range of human diseases (for details see Windley et al., 687 2012), with malaria alone accounting for 216 million cases and a resulting 445,000 deaths in 2016 688 (WHO, 2017). Unfortunately, many commercial that are used for insect control suffer 689 from problems of resistance development and unwanted environmental side-effects (Hardy, 2014). 690 Therefore, the development of environmentally-friendly alternatives, for example the so-called 691 "bioinsecticides" is urgently needed to sustain a growing human population. Venomous animals in 692 general (Smith et al., 2013) and in particular spiders, as the world's most successful venomous insect 693 predators, have come into the focus of research, as their venoms contain a plethora of insecticidal 694 toxins that could potentially be developed into bioinsecticides (King and Hardy, 2013). We have 695 already reviewed the potential of spider venom peptides as bioinsecticides previously (Herzig and 696 King, 2010; Windley et al., 2012), but due to exciting new developments in this field an update on the 697 more recent findings is warranted. 698 699 The major breakthrough in the field has undoubtedly been the commercial registration by the 700 Vestaron Corporation (USA) of a spider venom-derived peptide as a bioinsecticide with the US 701 Environmental ProtectionACCEPTED Agency in 2014. The active ingredient in their commercial product Spear ® 702 T is GS ‐ω/κ‐HXTX ‐Hv1a, a targeting insect Bk Ca and Ca V channels that was originally 703 discovered in the laboratory of Prof. Glenn F. King (Bomgardner, 2017). Spear T ® is currently aimed 704 at greenhouse pests, targeting a wide range of insects from the orders lepidoptera, coleoptera, diptera, 705 orthoptera, thysanoptera and hemiptera, while being safe for mammals and honeybees (see 706 www.vestaron.com for more information). The example of GS ‐ω/κ‐HXTX ‐Hv1a provides proof-of- 707 concept that spider venom peptides can actually go all the way to become commercial products such 708 as bioinsecticides. Furthermore, it also highlights that these peptides can be economically produced in 709 large scale to be competitive with other commercial insecticides on the market. However, in order to 710 achieve this, several criteria need to be fulfilled as indicated in table 4.

13 ACCEPTED MANUSCRIPT 711 712 Table 4: Desirable criteria (in alphabetical order) for the development of novel bioinsecticides 713 (adapted from Windley et al., 2012). Criteria Reason Costs need to be competitive with chemical Economical production insecticides currently on the market No long-term persistence in environment Prevent resistance development Large scale application is usually by Oral or topical bioavailability spraying or direct expression within High potency and bioavailability Saves material and thereby production costs Selectivity towards target organism Protects beneficial insects (e.g. pollinators) and or non-lethal mode of action vertebrates (e.g. livestock and humans) Should be easy to apply to achieve compliance by staff Solubility/formulation in the field Extremes in temperature, pH, UV radiation could lead Stability under field conditions to inactivation 714 715 3.1 Novel insecticidal spider peptides 716 In table 5 we provide an overview of those insecticidal spider peptides and proteins (ISPs), which 717 have been described since our previous review (Windley et al., 2012). Interestingly, not all of these 718 new toxins have been sourced from spider venoms, with one exception being solely found in spider 719 eggs (Lei et al., 2015). Latroeggtoxin-III is a cockroach-specific toxin isolated from the eggs of 720 Latrodectus tredecimguttatus (Lei et al., 2015). Despite the low potency of Latroeggtoxin-III, it is 721 rather exciting that spider eggs could be considered as an additional source of ISPs, which further 722 increases the diversity of spider-derived ISPs. Phospholipases D such as LaSicTox-αIB2bi have 723 recently been shown as extremely potent insecticides with a low PD 50 of 71 pmol/g in crickets (Zobel- 724 Thropp et al., 2012). In addition, some Phospholipases D were also found to exhibit phyletic 725 specificity, which can be explained by different headgroup alcohols between mammalian and insect 726 lipid substrates (Lajoie et al., 2015). However, the largeMANUSCRIPT size of Phospholipases D of around 30 kDa 727 might have a negative impact on their stability as well as on their economical production in large 728 scale in comparison to the shorter venom peptides. At the current stage, the Phospholipases D would 729 therefore not be considered as useful candidates for bioinsecticide development. 730 731 (insert table 5) 732 733 With the exception of the Latroeggtoxin-III (36 kDa), Phopholipases D (30 kDa), CpTx1-4 (15 kDa) 734 and OtTx1 (12 kDa), all other novel ISPs ranged from 3.9 to 8.4 kDa. While the ISPs in table 5 were 735 sourced from ten different spider families, a large percentage (40.6%) was isolated from spiders of the 736 Theraphosidae family, reflecting their relatively large venom yields and the accessibility of these 737 species and their venoms for research. Overall, they exhibited activity against 6 different insect 738 orders, with their potency (based on the respective PD 50 and LD 50 values) showing considerable 739 variations. Furthermore, the insect target species for these novel ISPs as listed in table 5 are usually 740 more a reflection of those insect species available to the researchers for testing rather than an exact 741 representation of their phyletic specificity. In most cases, we would presume that the ISPs have a 742 much broader phyletic activity than indicated in table 5. 743 ACCEPTED 744 Due to their larger size and reduced membrane permeability, peptides usually have a slower mode of 745 action compared to conventional small molecule insecticides. Among the key advantages of spider 746 venom peptides are their remarkable potency as well as selectivity towards their molecular target. One 747 example of such a potent and selective ISP is -diguetoxin-Dc1a (Dc1a), which blocks insect voltage- 748 gated sodium (Na V) channels (Bende et al., 2014). Interestingly, Dc1a was found to potently kill 749 German cockroaches, whereas American cockroaches were unaffected by the toxin. A closer 750 examination revealed two residues at the Dc1a binding site on the Na V channel that differed between 751 both cockroach species and were therefore presumed to be responsible for the differences in toxin 752 potency (Bende et al., 2014). In another study with the unrelated ISP -theraphotoxin-Ae1a (Ae1a),

14 ACCEPTED MANUSCRIPT

753 which shares the same binding site on insect Na V channels as Dc1a, the mutation responsible for the 754 phyletic specificity was narrowed down to a single residue mutation in the S1-S2 loop of domain II of 755 the Na V channel (Herzig et al., 2016). These examples of potent yet specific ISPs such as Dc1a and 756 Ae1a highlight the possibility that peptides exist that selectively target certain pest species, without 757 affecting beneficial insects or vertebrates (including humans), thereby minimizing unwanted 758 environmental side-effects. And being peptides, they have the added advantage of being fully 759 biodegradable into amino acids, the basic building blocks of life on earth. 760 761 In regards to the potency of ISPs, the most potent candidates from our previous review had PD 50 or 762 LD 50 values ranging from 10-380 pmol/g on at least one of the target insects (Windley et al., 2012). A 763 number of new ISPs showed LD 50 values in the sub nmol/g (e.g. -NPTX-Nc1a, CsTx-1, β/δ-PrIT1, 764 CpTx1, OxyTx1) or in the low nmol/g range (for details, see table 5). While a high potency does not 765 necessarily make a these ISPs potential bioinsecticide candidates, ISPs with rather poor potency such 766 as Latroeggtoxin III (LD 50 = 278 nmol/g in cockroaches, Lei et al., 2015), CsTx-2b (LD 50 = 66.5 767 nmol/g in Drosophila, (Kuhn-Nentwig et al., 2012) or Magi3 (LD 50 = 22.8 nmol/g in crickets, 768 (Titaux-Delgado et al., 2018) can be excluded. Besides a high potency, other criteria such as potential 769 unwanted side-effects should be considered to judge the suitability of ISPs as bioinsecticide 770 candidates. For example, ISPs with unspecific cytolytic activity such as OtTx1 ((Vassilevski et al., 771 2013)) or CsTx-1 (Kuhn-Nentwig et al., 2012) are most likely unsuitable. Also, some sub-types of 772 mammalian cardiac voltage-gated sodium (Na V) and calcium (Ca V) channels (i.e. Na V1.5, Ca V1.3 and 773 Ca V3.1), as well as Na V channels in the skeletal muscle (Na V1.4) and nodes of Ranvier (Na V1.6) are 774 important off-targets which should not be targeted by any bioinsecticide in order to avoid potentially 775 serious side-effects. Interestingly, the recently discovered ISP β-theraphotoxin-Cd1a (Cd1a) was 776 active in the low nanomolar range against dipterans and also inhibited important peripheral 777 nociceptive targets (i.e. Na V1.7, Na V1.8 and Ca V2.2), while being inactive against the above 778 mentioned off-target channels (Sousa et al., 2017). Thus, besides analgesia (which might even be 779 considered beneficial), no serious side-effects would be expected from Cd1a when used as a 780 bioinsecticide. The example of Cd1a therefore shows that ISPs with the desired sub-type selectivity 781 on a range of on- and off-target voltage-gated ion channelsMANUSCRIPT actually exist, leaving the major challenge 782 for future research to find the most suitable ISPs for each specific application or target species. 783 784 Phyletic specificity towards the target species would be another important requirement for a good 785 bioinsecticide candidate, aiming to spare arthropods that are beneficial to agriculture such as 786 honeybees or spiders. The type of activity induced by the ISP might also need to be considered for 787 each application. For ISPs that are expressed in transgenic plants or applied by spraying, a reversibly 788 paralytic activity would be sufficient to protect those plants from being devastated by pest insects 789 such as caterpillars or locusts that constantly consume material. Every time sufficient amounts 790 of a non-lethal ISP have been consumed, these pests would then become paralysed, impairing their 791 growth and making them vulnerable to predators such as ants, reptiles or birds, thereby significantly 792 affecting their long-term survival. The advantage of ISPs with a non-lethal mode of action would be 793 the lack of long-lasting effects on beneficial species that have received an accidental exposure for 794 example when spraying the crops with the ISP. Such considerations could help in minimising the use 795 of lethal toxins and thereby protect beneficial species. As indicated in table 5, not all novel ISPs 796 immediately induced lethality in the target insects. Some ISPs caused completely reversible paralysis 797 (Herzig et al., 2016; Smith et al., 2017; Sousa et al., 2017), while others caused a long-lasting 798 paralysis that eventuallyACCEPTED resulted in death by starvation (Ikonomopoulou et al., 2016; Matsubara et al., 799 2017). And these ISP-induced effects did not only vary between ISPs, but also within ISPs tested 800 across a range of target insect species. For example Ae1a was lethal to fruit flies, but induced only a 801 reversible paralysis in sheep blowflies and was completely inactive when tested against an assassin 802 bug species (Herzig et al., 2016). 803 804 3.2 Oral toxicity 805 A significant challenge for the development of spider venom peptides into bioinsecticides will be the 806 requirement for oral toxicity when used on large-scale agricultural applications, as common ways of 807 distributing insecticides to large acre crops is either by spraying or by using genetically-modified

15 ACCEPTED MANUSCRIPT 808 crops that produce insecticidal compounds. In these scenarios, the insecticide becomes attached to the 809 plant surface or is produced inside the plant and then subsequently ingested by the pest insect 810 consuming the plant. Thus, in order to be active, these insecticides need to have some degree of oral 811 toxicity. Topical activity would be another possible mechanism for spider venom peptides, although 812 there is currently very little evidence in the literature to support it. Khan et al. described topical 813 activity against caterpillars of a recombinant fusion protein containing the spider venom peptide ω- 814 hexatoxin-Hv1a (Hv1a) (Khan et al., 2006). However, their buffer also contained imidazole, which is 815 known to be insecticidal itself and therefore casts some doubt on whether the observed topical activity 816 was indeed caused by Hv1a. The most likely routes for peptides to gain access through the insect 817 cuticle is either through spiracles or by using formulations that enable a direct penetration of the 818 cuticle. An advantage of topically-active peptides is that they can be applied by spraying to large acre 819 crops. However, only pest insects that are present at the time of spraying would be treated by 820 topically-active insecticides, whereas orally-active insecticides (depending on their stability in the 821 field) would persist on the leaves for longer and thereby also treat pests that appear after the spraying. 822 As spiders usually inject the venoms into their prey, there was no evolutionary pressure to develop 823 either oral or topical toxicity. For that reason, spider venoms have been deemed as being not suitable 824 as insecticides (Nentwig, 1993). The discovery of an orally-active insecticidal peptide termed OAIP-1 825 from an Australian theraphosid species was therefore rather surprising (Hardy et al., 2013). On the 826 other hand, Hv1a was already known to be orally-active in ticks (which are not insects but ) 827 (Mukherjee et al., 2006) and against caterpillars when recombinantly expressed in plants (Khan et al., 828 2006). Therefore, despite the lack of an evolutionary selection pressure towards oral toxicity, at least 829 some spider venom peptides such as Hv1a or OAIP-1 exist with the inherent characteristic of oral 830 toxicity. Furthermore, in a recent study we were able to demonstrate that oral insecticidal activity of 831 arachnid venoms and toxins is far more common than previously anticipated (Guo et al., 2018). We 832 then employed chemical modifications aiming to further improve the oral toxicity of Hv1a, but 833 neither selective replacement of disulfide by diselenide bonds nor N- to C-terminal cyclisation were 834 successful (Herzig et al., 2018). Cyclised Hv1a was even found to have a dramatically reduced oral 835 toxicity, which was due to a dramatically reduced translocation rate of the peptide across midgut 836 (Herzig et al., 2018). Thus, besides potency, bioavailabilityMANUSCRIPT is another feature that would be beneficial 837 for any bioinsecticide candidate. 838 839 A complete lack of oral activity on the other hand does not necessarily preclude promising ISPs from 840 being developed into bioinsecticides, as there are other routes of transport available to get these 841 peptides into the target pest insects. We have previously reviewed the strategies available to increase 842 oral peptide bioavailability in insects in more detail (Herzig et al., 2014). One strategy involves the 843 binding of a peptide to a carrier protein such as plant lectins or viral coat proteins (Herzig et al., 844 2014). In a series of experiments, Elaine Fitches, John Gatehouse and co-workers have pioneered this 845 research area and demonstrated the feasibility of conjugating spider venom peptides and lectins to 846 improve oral toxicity (Fitches et al., 2004; Down et al., 2006; Yang et al., 2014). Furthermore, by 847 using a carrier protein in combination with a suitable spider venom peptide, toxicity to beneficial 848 insects such as honeybees can be avoided (Nakasu et al., 2014a). And this method can even be used to 849 construct transgenic plants expressing both the venom peptide and the lectin (Nakasu et al., 2014b). 850 Another benefit of conjugating spider venom peptides and lectins is that the lectins can be 851 fluorescently labeled and might therefore help in determining the mode of action and in localizing the 852 targets of venom peptides within insects (Fitches et al., 2012). 853 ACCEPTED 854 3.3 Application strategies 855 As detailed in fig. 1, a range of strategies is available to transport ISPs into target insects by 856 employing different entomopathogens, including baculoviruses (for review see Kroemer et al., 2015), 857 entomopathogenic fungi (e.g. Metarhizium and Beauveria ) and the soil bacterium Bacillus 858 thuringiensis (Cao et al., 2010). A major disadvantage of baculoviruses and entomopathogenic fungi 859 is their slow speed in killing insects, which has already been improved by combining them with ISPs 860 (Ardisson-Araujo et al., 2013; Bilgo et al., 2017). In case of the fungus Metarhizium , another 861 beneficial outcome of the combination treatment was that the spore dose required for insecticidal 862 activity was also decreased (Bilgo et al., 2017). The combination of insecticidal B. thuringiensis Cry

16 ACCEPTED MANUSCRIPT 863 toxins ("also known as Bt toxin") with ISPs has recently been suggested as another strategy to 864 facilitate the transport of ISPs across the insect midgut (Cao et al., 2010). Cry toxins are pore-forming 865 toxins that are secreted as water-soluble proteins and then undergo a range of conformational changes 866 (for details see Bravo et al., 2011) before forming stable pores in the plasma-membrane of their hosts. 867 The idea behind the combination of Cry toxins and ISPs is that the pores formed by the Cry toxins 868 might allow the passage of smaller ISPs from the midgut into the epithelial cells and then further by 869 transcytosis into the haemocoel. This strategy is already being employed by the Vestaron Corporation 870 in their commercial products Spear C ® and Spear P ®, which combine GS ‐ω/κ‐HXTX ‐Hv1a with 871 different strains of Bt to improve the control of lepidopteran pests or the Colorado potato beetle, 872 respectively. 873 874 (insert figure 1) 875 876 Besides a potential synergistic effect in terms of mortality and required toxin amounts, there are some 877 additional benefits resulting from combining entomopathogens with ISPs. The mode of action by 878 which the entomopathogens exert their insecticidal effects is usually quite different from those of 879 ISPs. For example, Cry toxins form pores in the plasma-membrane, whereas most ISPs target existing 880 ion channels (Bravo et al., 2011; King and Hardy, 2013). As it is less likely for insects to 881 simultaneously develop resistance against toxins with two different modes of action, pyramiding (= 882 combining) entomopathogens with ISPs would therefore prevent or at least substantially delay the 883 development of resistance. Furthermore, in the case of baculoviruses and entomopathogenic fungi, 884 which express the ISPs inside the target insect, the rather narrow host range of some of these 885 entomopathogens could help in limiting unwanted side-effects caused by the ISP on non-target 886 species and would even allow for less specific ISPs to be employed. 887 888 3.4 Summary 889 Overall, substantial progress has been made in recent years for discovering novel ISPs with the aim of 890 developing them as potential bioinsecticides. The EPA-registration of the first ISP-derived peptide as 891 bioinsecticide as well as the discovery of orally-activeMANUSCRIPT ISPs have been major breakthroughs in this 892 field. Other research revealed that oral toxicity of ISPs can be improved by either using carrier 893 molecules or by combination with entomopathogens such as baculoviruses, entomopathogenic fungi 894 or bacterial toxins. The criteria for selecting the most suitable ISP candidates for bioinsecticide 895 development include their potency, selectivity, oral activity, bioavailability, stability and an 896 economical means of large-scale production. Depending on the application at hand, some of these 897 criteria might be more important than others, but Vestaron's EPA-approved ISP is a good example 898 that all of these criteria can be met by a single peptide. Other successful examples will hopefully 899 follow in future to meet the urgent need of novel bioinsecticides in order to help sustain our growing 900 world population. 901 902 4. Conclusions 903 The last decade has seen considerable progress on spider venom exploration and peptide 904 characterization. The superior potency and selectivity of spider venom peptides over small molecule 905 drugs or insecticides is one key advantage, minimizing the risks of side effects and development of 906 resistance. Furthermore, spider venom peptides are active on a huge range of targets, including insects 907 as their main prey, which has already been utilised in a commercially registered bioinsectide (GS ‐ 908 ω/κ‐HXTX ‐Hv1a)ACCEPTED developed by Vestaron Corporation in the USA. Beyond their insecticidal activity, 909 applications of spider venom peptides further extend into therapeutics to treat a plethora of medical 910 conditions. GsMTx4 for example is entering clinical trials for the treatment of Duchenne muscular 911 dystrophy, demonstrating the true potential of these peptides. Other peptides that have already shown 912 promise in disease models and are likely to enter future clinical trials include Hi1a in the treatment of 913 stroke, Ph α1β in the treatment of chronic refractory pain and Lycosin-I-conjugates in the detection 914 and treatment of tumors. Surprisingly, of the 116 extant venomous spider families, all peptides 915 presented in this review originate from only fourteen families. Thus, there are still enormous 916 opportunities for discovery of novel peptides with exploitable bioactivities from unexplored spider 917 venoms. The increasing availability of high throughput transcriptomic and proteomic data is likely to

17 ACCEPTED MANUSCRIPT 918 aid further discovery and characterization. In some cases, a peptide’s properties and pharmacokinetics 919 may fall short of ideal, however emerging technologies for improving delivery, bioavailability or 920 enhancing activity or selectivity of the parenteral peptides will also broaden their commercial 921 applications. 922 923 5. Acknowledgements 924 For the images used for the graphical abstract, the authors like to thank M. Lilly (Wikimedia 925 Commons, under Creative Commons 3.0 licence) for the agriculture picture and E.A.B. Undheim 926 (Australia) for the spider picture. 927 928 6. References 929 Abdulqawi, R., Dockry, R., Holt, K., Layton, G., McCarthy, B.G., Ford, A.P., and Smith, J.A., 2015. 930 P2X3 receptor antagonist (AF-219) in refractory chronic cough: a randomised, double-blind, 931 placebo-controlled phase 2 study. Lancet 385, 1198-1205. 932 Adams, M.E., 2004. Agatoxins: ion channel specific toxins from the American funnel web spider, 933 Agelenopsis aperta . Toxicon 43, 509-525. 934 Agwa, A.J., Lawrence, N., Deplazes, E., Cheneval, O., Chen, R.M., Craik, D.J., Schroeder, C.I., 935 Henriques, S.T., 2017. Spider peptide toxin HwTx-IV engineered to bind to lipid membranes has 936 an increased inhibitory potency at human voltage-gated sodium channel hNa V1.7. Biochimica et 937 Biophysica Acta Biomembranes 1859, 835-844. 938 Alcaino, C., Knutson, K., Gottlieb, P.A., Farrugia, G., and Beyder, A., 2017. Mechanosensitive ion 939 channel Piezo2 is inhibited by D-GsMTx4. Channels (Austin) 11, 245-253. 940 Almeida, F.M., Silva, C.N., de Araujo Lopes, S.C., Santos, D.M., Torres, F.S., Cardoso, F.L., 941 Martinelli, P.M., da Silva, E.R., de Lima, M.E., Miranda, L.A.F., and Oliveira, M.C., 2018. 942 Physicochemical characterization and skin permeation of cationic transfersomes containing the 943 synthetic peptide PnPP-19. Curr. Drug. Deliv. 944 Andrade, E., Villanova, F., Borra, P., Leite, K., Troncone, L., Cortez, I., Messina, L., Paranhos, M., 945 Claro, J., and Srougi, M., 2008. Penile erection induced in vivo by a purified toxin from the 946 Brazilian spider Phoneutria nigriventer . BJU InternationalMANUSCRIPT 102, 835-837. 947 Ardisson-Araujo, D.M., Morgado Fda, S., Schwartz, E.F., Corzo, G., and Ribeiro, B.M., 2013. A new 948 theraphosid spider toxin causes early insect cell death by necrosis when expressed in vitro during 949 recombinant baculovirus infection. PLoS One 8, e84404. 950 Arenas, I., Villegas, E., Walls, O., Barrios, H., Rodriguez, R., and Corzo, G., 2016. Antimicrobial 951 activity and stability of short and long based arachnid synthetic peptides in the presence of 952 commercial antibiotics. Molecules 21. 953 Ayroza, G., Ferreira, I.L., Sayegh, R.S., Tashima, A.K., and da Silva Junior, P.I., 2012. Juruin: an 954 antifungal peptide from the venom of the Amazonian Pink Toe spider, Avicularia juruensis , 955 which contains the inhibitory cystine knot motif. Front. Microbiol. 3, 324. 956 Bae, C., Sachs, F., and Gottlieb, P.A., 2011. The mechanosensitive ion channel Piezo1 is inhibited by 957 the peptide GsMTx4. Biochemistry 50, 6295-6300. 958 Bae, C., Kalia, J., Song, I., Yu, J., Kim, H.H., Swartz, K.J., and Kim, J.I., 2012. High yield production 959 and refolding of the double-knot toxin, an activator of TRPV1 channels. PLoS One 7, e51516. 960 Bagal, S.K., Marron, B.E., Owen, R.M., Storer, R.I., and Swain, N.A., 2015. Voltage gated sodium 961 channels as drug discovery targets. Channels (Austin) 9, 360-366. 962 Baron, A., Diochot, S., Salinas, M., Deval, E., Noel, J., and Lingueglia, E., 2013. Venom toxins in the 963 exploration ACCEPTED of molecular, physiological and pathophysiological functions of acid-sensing ion 964 channels. Toxicon 75, 187-204. 965 Barreto Dos Santos, N., Bonfanti, A.P., Rocha, E.S.T., da Silva, P.I., Jr., da Cruz-Hofling, M.A., 966 Verinaud, L., and Raposo, C., 2018. Venom of the Phoneutria nigriventer spider alters the cell 967 cycle, viability, and migration of cancer cells. J. Cell. Physiol. 968 Bastianelli, G., Bouillon, A., Nguyen, C., Crublet, E., Petres, S., Gorgette, O., Le-Nguyen, D., Barale, 969 J.C., and Nilges, M., 2011. Computational reverse-engineering of a spider-venom derived 970 peptide active against Plasmodium falciparum SUB1. PLoS One 6, e21812.

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22 ACCEPTED MANUSCRIPT 1191 Guo, S., Herzig, V., and King, G.F., 2018. Dipteran toxicity assays for determining the oral 1192 insecticidal activity of venoms and toxins. Toxicon 150, 297-303. 1193 Hardy, M.C., Daly, N.L., Mobli, M., Morales, R.A., and King, G.F., 2013. Isolation of an orally 1194 active insecticidal toxin from the venom of an Australian tarantula. PLoS One 8, e73136. 1195 Hardy, M.C., 2014. Resistance is not futile: it shapes insecticide discovery. Insects 5, 227-242. 1196 Hauke, T.J., and Herzig, V., 2017. Dangerous arachnids-Fake news or reality? Toxicon 138, 173-183. 1197 Hefti, F.F., 2008. Requirements for a lead compound to become a clinical candidate. BMC Neurosci. 1198 9 Suppl 3, S7. 1199 Herzig, V., and King, G.F., 2010. Spider toxins and their potential for insect control. In: Gilbert, L.I. 1200 and Gill, S.S. (Eds), Insect Pharmacology, Elsevier, Oxford, pp. 119-123. 1201 Herzig, V., Bende, N.S., Alam, M.S., Tedford, H.W., Kennedy, R.M., and King, G.F., 2014. Methods 1202 for deployment of spider-venom peptides as bioinsecticides. In: Dhadialla, T.S. and Gill, S.S. 1203 (Eds), Advances in insect physiology: Insect midgut and insecticidal proteins for insect control 1204 Vol. 7, Academic Press, London, pp. 389–411. 1205 Herzig, V., 2016. Create guidelines for characterization of venom peptides. Toxins 8, 252, 1-4. 1206 Herzig, V., Ikonomopoulou, M., Smith, J.J., Dziemborowicz, S., Gilchrist, J., Kuhn-Nentwig, L., 1207 Rezende, F.O., Moreira, L.A., Nicholson, G.M., Bosmans, F., and King, G.F., 2016. Molecular 1208 basis of the remarkable species selectivity of an insecticidal sodium channel toxin from the 1209 African spider Augacephalus ezendami . Sci. Rep. 6, 29538. 1210 Herzig, V., de Araujo, A.D., Greenwood, K.P., Chin, Y.K., Windley, M.J., Chong, Y., Muttenthaler, 1211 M., Mobli, M., Audsley, N., Nicholson, G.M., Alewood, P.F., and King, G.F., 2018. Evaluation 1212 of chemical strategies for improving the stability and oral toxicity of insecticidal peptides. 1213 Biomedicines 6, 90, 1-16. 1214 Holford, M., Daly, M., King, G.F., and Norton, R.S., 2018. Venoms to the rescue. Science 361, 842- 1215 844. 1216 Honigsperger, C., Nigro, M.J., and Storm, J.F., 2017. Physiological roles of KV2 channels in 1217 entorhinal cortex layer II stellate cells revealed by Guangxitoxin-1E. J. Physiol. 595, 739-757. 1218 Huang, X., and El-Sayed, M.A., 2010. Gold nanoparticles: Optical properties and implementations in 1219 cancer diagnosis and photothermal therapy. J. Adv.MANUSCRIPT Res. 1, 13-28. 1220 Ikonomopoulou, M.P., Smith, J.J., Herzig, V., Pined a, S.S., Dziemborowicz, S., Er, S.Y., Durek, T., 1221 Gilchrist, J., Alewood, P.F., Nicholson, G.M., Bosmans, F., and King, G.F., 2016. Isolation of 1222 two insecticidal toxins from venom of the Australian theraphosid spider Coremiocnemis tropix . 1223 Toxicon 123, 62-70. 1224 Ikonomopoulou, M.P., Fernandez-Rojo, M.A., 2018. The antiproliferative and apoptotic profile of 1225 gomesin against DFTD. Cell Death Dis. 9, 833. 1226 Ikonomopoulou, M.P., Fernandez-Rojo, M.A., Pineda, S.S., Cabezas-Sainz, P., Winnen, B., Morales, 1227 R.A.V., Brust, A., Sanchez, L., Alewood, P.F., Ramm, G.A., Miles, J.J., King, G.F., 2018. 1228 Gomesin inhibits melanoma growth by manipulating key signaling cascades that control cell 1229 death and proliferation. Sci. Rep. 8, 11519. 1230 Jin, L., Fang, M., Chen, M., Zhou, C., Ombati, R., Hakim, M.A., Mo, G., Lai, R., Yan, X., Wang, Y., 1231 and Yang, S., 2017. An insecticidal toxin from Nephila clavata spider venom. Amino Acids 49, 1232 1237-1245. 1233 Kabanova, N.V., Vassilevski, A.A., Rogachevskaja, O.A., Bystrova, M.F., Korolkova, Y.V., 1234 Pluzhnikov, K.A., Romanov, R.A., Grishin, E.V., and Kolesnikov, S.S., 2012. Modulation of 1235 P2X3 receptors by spider toxins. Biochim. Biophys. Acta 1818, 2868-2875. 1236 Kamolkijkarn, P.,ACCEPTED Prasertdee, T., Netirojjanakul, C., Sarnpitak, P., Ruchirawat, S., and Deechongkit, 1237 S., 2010. Synthesis, biophysical, and biological studies of wild-type and mutant 1238 psalmopeotoxins--anti-malarial cysteine knot peptides from Psalmopoeus cambridgei . Peptides 1239 31, 533-540. 1240 Khan, S.A., Zafar, Y., Briddon, R.W., Malik, K.A., and Mukhtar, Z., 2006. Spider venom toxin 1241 protects plants from insect attack. Transgen. Res. 15, 349-357. 1242 Kikuchi, K., Sugiura, M., Kimura, T., 2015. High proteolytic resistance of spider-derived inhibitor 1243 cystine knots. Int. J. Pept. 2015, 537508. 1244 Kim, H., Hwang, H., Cheong, H.I., and Park, H.W., 2011. Hypokalemic periodic paralysis; two 1245 different genes responsible for similar clinical manifestations. Korean J. Pediatr. 54, 473-476.

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26 ACCEPTED MANUSCRIPT 1409 Palhares, M.R., Silva, J.F., Rezende, M.J.S., Santos, D.C., Silva-Junior, C.A., Borges, M.H., Ferreira, 1410 J., Gomez, M.V., and Castro-Junior, C.J., 2017. Synergistic antinociceptive effect of a calcium 1411 channel blocker and a TRPV1 blocker in an acute pain model in mice. Life Sci. 182, 122-128. 1412 Pascale, M., Azinwi, C.N., Marongiu, B., Pesce, G., Stoffel, F., and Roggero, E., 2017. The outcome 1413 of prostate cancer patients treated with curative intent strongly depends on survival after 1414 metastatic progression. BMC Cancer 17, 651. 1415 Peigneur, S., de Lima, M.E., and Tytgat, J., 2018. Phoneutria nigriventer venom: a pharmacological 1416 treasure. Toxicon 151, 96-110. 1417 Peng, K., Shu, Q., Liu, Z., Liang, S., 2002. Function and solution structure of huwentoxin-IV, a potent 1418 neuronal tetrodotoxin (TTX)-sensitive sodium channel antagonist from Chinese bird spider 1419 Selenocosmia huwena . J. Biol. Chem. 277, 47564-47571. 1420 Pennington, M.W., Czerwinski, A., and Norton, R.S., 2018. Peptide therapeutics from venom: current 1421 status and potential. Bioorg. Med. Chem. 26, 2738-2758. 1422 Pignataro, G., Simon, R.P., and Xiong, Z.G., 2007. Prolonged activation of ASIC1a and the time 1423 window for neuroprotection in cerebral ischaemia. Brain 130, 151-158. 1424 Pimentel, C., Choi, S.J., Chagot, B., Guette, C., Camadro, J.M., and Darbon, H., 2006. Solution 1425 structure of PcFK1, a spider peptide active against Plasmodium falciparum . Protein Sci. 15, 628- 1426 634. 1427 Polina, N.F., Shkarupeta, M.M., Popenko, A.S., Vassilevski, A.A., Kozlov, S.A., Grishin, E.V., 1428 Lazarev, V.N., and Govorun, V.M., 2012. Cyto-Insectotoxin 1a from Lachesana tarabaevi spider 1429 venom inhibits Chlamydia trachomatis infection. Probiotics Antimicrob. Proteins 4, 208-216. 1430 Polyansky, A.A., Vassilevski, A.A., Volynsky, P.E., Vorontsova, O.V., Samsonova, O.V., Egorova, 1431 N.S., Krylov, N.A., Feofanov, A.V., Arseniev, A.S., Grishin, E.V., and Efremov, R.G., 2009. N- 1432 terminal amphipathic helix as a trigger of hemolytic activity in antimicrobial peptides: a case 1433 study in latarcins. FEBS Lett. 583, 2425-2428. 1434 Ponnappan, N., and Chugh, A., 2017. Cell-penetrating and cargo-delivery ability of a spider toxin- 1435 derived peptide in mammalian cells. Eur. J. Pharm. Biopharm. 114, 145-153. 1436 Priest, B.T., Blumenthal, K.M., Smith, J.J., Warren, V.A., Smith, M.M., 2007. ProTx-I and ProTx-II: 1437 gating modifiers of voltage-gated sodium channels.MANUSCRIPT Toxicon 49, 194-201. 1438 Raposo, C., 2017. and spider venoms in can cer treatment: state of the art, challenges, and 1439 perspectives. J. Clin. Transl. Res. 3, 233-249. 1440 Redaelli, E., Cassulini, R.R., Silva, D.F., Clement, H., Schiavon, E., Zamudio, F.Z., Odell, G., 1441 Arcangeli, A., Clare, J.J., Alagon, A., de la Vega, R.C., Possani, L.D., Wanke, E., 2010. Target 1442 promiscuity and heterogeneous effects of tarantula venom peptides affecting Na + and K + ion 1443 channels. J. Biol. Chem. 285, 4130-4142. 1444 Reis, P.V.M., Boff, D., Verly, R.M., Melo-Braga, M.N., Cortes, M.E., Santos, D.M., Pimenta, A.M.C., 1445 Amaral, F.A., Resende, J.M., and de Lima, M.E., 2018. LyeTxI-b, a synthetic peptide derived 1446 from Lycosa erythrognatha spider venom, shows potent antibiotic activity in vitro and in vivo . 1447 Front. Microbiol. 9, 667. 1448 Renukuntla, J., Vadlapudi, A.D., Patel, A., Boddu, S.H., and Mitra, A.K., 2013. Approaches for 1449 enhancing oral bioavailability of peptides and proteins. Int. J. Pharm. 447, 75-93. 1450 Revell, J.D., Lund, P.E., Linley, J.E., Metcalfe, J., Burmeister, N., Sridharan, S., Jones, C., Jermutus, 1451 L., Bednarek, M.A., 2013. Potency optimization of Huwentoxin-IV on hNa V1.7: a neurotoxin 1452 TTX-S sodium-channel antagonist from the venom of the Chinese bird-eating spider 1453 Selenocosmia huwena . Peptides 44, 40-46. 1454 Richards, K.L., ACCEPTED Milligan, C.J., Richardson, R.J., Jancovski, N., Grunnet, M., Jacobson, L.H., 1455 Undheim, E.A.B., Mobli, M., Chow, C.Y., Herzig, V., Csoti, A., Panyi, G., Reid, C.A., King, 1456 G.F., and Petrou, S., 2018. Selective Na V1.1 activation rescues Dravet syndrome mice from 1457 seizures and premature death. Proc. Natl. Acad. Sci. USA 115, E8077-E8085. 1458 Rigo, F.K., Trevisan, G., Rosa, F., Dalmolin, G.D., Otuki, M.F., Cueto, A.P., de Castro Junior, C.J., 1459 Romano-Silva, M.A., Cordeiro Mdo, N., Richardson, M., Ferreira, J., and Gomez, M.V., 2013. 1460 Spider peptide Ph α1β induces analgesic effect in a model of cancer pain. Cancer Sci. 104, 1226- 1461 1230. 1462 Rigo, F.K., Rossato, M.F., Trevisan, G., De Pra, S.D., Ineu, R.P., Duarte, M.B., de Castro Junior, C.J., 1463 Ferreira, J., Gomez, M.V., 2017a. PhKv a toxin isolated from the spider venom induces

27 ACCEPTED MANUSCRIPT 1464 antinociception by inhibition of cholinesterase activating cholinergic system. Scand. J. Pain 17, 1465 203-210. 1466 Rigo, F.K., Trevisan, G., De Pra, S.D., Cordeiro, M.N., Borges, M.H., Silva, J.F., Santa Cecilia, F.V., 1467 de Souza, A.H., de Oliveira Adamante, G., Milioli, A.M., de Castro Junior, C.J., Ferreira, J., and 1468 Gomez, M.V., 2017b. The spider toxin Ph α1β recombinant possesses strong analgesic activity. 1469 Toxicon 133, 145-152. 1470 Robinson, S.D., Undheim, E.A.B., Ueberheide, B., and King, G.F., 2017. Venom peptides as 1471 therapeutics: advances, challenges and the future of venom-peptide discovery. Expert Rev. 1472 Proteomics 14, 931-939. 1473 Rosa, F., Trevisan, G., Rigo, F.K., Tonello, R., Andrade, E.L., Cordeiro M.N., Calixto, J.B., Gomez, 1474 M.V., and Ferreira, J., 2014. Ph α1β, a peptide from the venom of the spider Phoneutria 1475 nigriventer shows antinociceptive effects after continuous infusion in a neuropathic pain model 1476 in rats. Anesth. Analg. 119, 196-202. 1477 Rothan, H.A., Bahrani, H., Rahman, N.A., and Yusof, R., 2014. Identification of natural antimicrobial 1478 agents to treat dengue infection: in vitro analysis of latarcin peptide activity against dengue virus. 1479 BMC Microbiol. 14, 140. 1480 Rothan, H.A., Ambikabothy, J., Abdulrahman, A.Y., Bahrani, H., Golpich, M., Amini, E., N, A.R., 1481 Teoh, T.C., Mohamed, Z., and Yusof, R., 2015. Scalable production of recombinant membrane 1482 active peptides and its potential as a complementary adjunct to conventional chemotherapeutics. 1483 PLoS One 10, e0139248. 1484 Sachkova, M.Y., Slavokhotova, A.A., Grishin, E.V., and Vassilevski, A.A., 2014. Structure of the 1485 yellow sac spider Cheiracanthium punctorium genes provides clues to evolution of insecticidal 1486 two-domain knottin toxins. Insect Mol. Biol. 23, 527-538. 1487 Sachs, F., 2015. Mechanical transduction by ion channels: a cautionary tale. World J. Neurol. 5, 74-87. 1488 Saez, N.J., Senff, S., Jensen, J.E., Er, S.Y., Herzig, V., Rash, L.D., and King, G.F., 2010. Spider- 1489 venom peptides as therapeutics. Toxins 2, 2851-2871. 1490 Saez, N.J., Cristofori-Armstrong, B., Anangi, R., and King, G.F., 2017. A strategy for production of 1491 correctly folded disulfide-rich peptides in the periplasm of E. coli . Methods Mol. Biol. 1586, 1492 155-180. MANUSCRIPT 1493 Salari, A., Vega, B.S., Milescu, L.S., and Milescu, M., 2016. Molecular interactions between tarantula 1494 toxins and low-voltage-activated calcium channels. Sci. Rep. 6, 23894. 1495 Santos, D.M., Verly, R.M., Pilo-Veloso, D., de Maria, M., de Carvalho, M.A., Cisalpino, P.S., Soares, 1496 B.M., Diniz, C.G., Farias, L.M., Moreira, D.F., Frezard, F., Bemquerer, M.P., Pimenta, A.M., 1497 and de Lima, M.E., 2010. LyeTx I, a potent antimicrobial peptide from the venom of the spider 1498 Lycosa erythrognatha . Amino Acids 39, 135-144. 1499 Santos, D.M., Reis, P.V., and Pimenta, A.M.C., 2016. Antimicrobial peptides in spider venoms. In: 1500 Gopalakrishnakone, P., Corzo, G.A., de Lima, M.E. and Diego-García, E. (Eds), Spider venoms, 1501 Springer Netherlands, Dordrecht, pp. 361-377. 1502 Schmalhofer, W.A., Calhoun, J., Burrows, R., Bailey, T., Kohler, M.G., Weinglass, A.B., 1503 Kaczorowski, G.J., Garcia, M.L., Koltzenburg, M., and Priest, B.T., 2008. ProTx-II, a selective 1504 inhibitor of Na V1.7 sodium channels, blocks action potential propagation in nociceptors. Mol. 1505 Pharmacol. 74, 1476-1484. 1506 Shcherbatko, A., Rossi, A., Foletti, D., Zhu, G., Bogin, O., Galindo Casas, M., Rickert, M., Hasa- 1507 Moreno, A., Bartsevich, V., Crameri, A., Steiner, A.R., Henningsen, R., Gill, A., Pons, J., 1508 Shelton, D.L., Rajpal, A., and Strop, P., 2016. Engineering highly potent and selective 1509 microproteinsACCEPTED against Na V1.7 sodium channel for treatment of pain. J. Biol. Chem. 291, 13974- 1510 13986. 1511 Shen, H., Xie, Y., Ye, S., He, K., Yi, L., and Cui, R., 2018. Spider peptide toxin lycosin-I induces 1512 apoptosis and inhibits migration of prostate cancer cells. Exp. Biol. Med. 243, 725-735. 1513 Siemens, J., Zhou, S., Piskorowski, R., Nikai, T., Lumpkin, E.A., Basbaum, A.I., King, D., and Julius, 1514 D., 2006. Spider toxins activate the capsaicin receptor to produce inflammatory pain. Nature 444, 1515 208-212. 1516 Sievers, F., Wilm, A., Dineen, D., Gibson, T.J., Karplus, K., Li, W., Lopez, R., McWilliam, H., 1517 Remmert, M., Soding, J., Thompson, J.D., Higgins, D.G., 2011. Fast, scalable generation of 1518 high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539.

28 ACCEPTED MANUSCRIPT 1519 Silva, C.N., Nunes, K.P., Torres, F.S., Cassoli, J.S., Santos, D.M., Almeida Fde, M., Matavel, A., 1520 Cruz, J.S., Santos-Miranda, A., Nunes, A.D., Castro, C.H., Machado de Avila, R.A., Chavez- 1521 Olortegui, C., Lauar, S.S., Felicori, L., Resende, J.M., Camargos, E.R., Borges, M.H., Cordeiro, 1522 M.N., Peigneur, S., Tytgat, J., and de Lima, M.E., 2015a. PnPP-19, a synthetic and nontoxic 1523 peptide designed from a Phoneutria nigriventer toxin, potentiates erectile function via NO/cGMP. 1524 J. Urol. 194, 1481-1490. 1525 Silva, J.F., Palhares, M.R., Santos, D.C., Silva-Junior, C.A., Ferreira, J., Gomez, M.V., and Castro 1526 Junior, C.J., 2017. Data and calculus on isobolographic analysis to determine the antinociceptive 1527 interaction between calcium channel blocker and a TRPV1 blocker in acute pain model in mice. 1528 Data Brief 14, 440-452. 1529 Silva, R.B., Sperotto, N.D., Andrade, E.L., Pereira, T.C., Leite, C.E., de Souza, A.H., Bogo, M.R., 1530 Morrone, F.B., Gomez, M.V., and Campos, M.M., 2015b. Spinal blockage of P/Q- or N-type 1531 voltage-gated calcium channels modulates functional and symptomatic changes related to 1532 haemorrhagic cystitis in mice. Br. J. Pharmacol. 172, 924-939. 1533 Silva, R.B.M., Greggio, S., Venturin, G.T., da Costa, J.C., Gomez, M.V., and Campos, M.M., 2018. 1534 Beneficial effects of the calcium channel blocker CTK 01512-2 in a mouse model of multiple 1535 sclerosis. Mol. Neurobiol. 1536 Siveen, K.S., Sikka, S., Surana, R., Dai, X., Zhang, J., Kumar, A.P., Tan, B.K., Sethi, G., and 1537 Bishayee, A., 2014. Targeting the STAT3 signaling pathway in cancer: role of synthetic and 1538 natural inhibitors. Biochim. Biophys. Acta 1845, 136-154. 1539 Smith, J.J., Herzig, V., King, G.F., and Alewood, P.F., 2013. The insecticidal potential of venom 1540 peptides. Cell. Mol. Life Sci. 70, 3665–3693. 1541 Smith, J.J., Herzig, V., Ikonomopoulou, M.P., Dziemborowicz, S., Bosmans, F., Nicholson, G.M., 1542 and King, G.F., 2017. Insect-active toxins with promiscuous pharmacology from the African 1543 theraphosid spider Monocentropus balfouri . Toxins 9, 155, 1-18. 1544 Sousa, S.R., Wingerd, J.S., Brust, A., Bladen, C., Ragnarsson, L., Herzig, V., Deuis, J.R., Dutertre, S., 1545 Vetter, I., Zamponi, G.W., King, G.F., Alewood, P.F., and Lewis, R.J., 2017. Discovery and 1546 mode of action of a novel analgesic β-toxin from the African spider darlingi . PLoS 1547 One 12, e0182848. MANUSCRIPT 1548 Souza, A.H., Ferreira, J., Cordeiro M.N., Vieira, L.B., De Castro, C.J., Trevisan, G., Reis, H., Souza, 1549 I.A., Richardson, M., Prado, M.A., Prado, V.F., and Gomez, M.V., 2008. Analgesic effect in 1550 rodents of native and recombinant Ph α1β toxin, a high-voltage-activated calcium channel blocker 1551 isolated from armed spider venom. Pain 140, 115-126. 1552 Sternberg, D., Maisonobe, T., Jurkat-Rott, K., Nicole, S., Launay, E., Chauveau, D., Tabti, N., 1553 Lehmann-Horn, F., Hainque, B., and Fontaine, B., 2001. Hypokalaemic periodic paralysis type 2 1554 caused by mutations at codon 672 in the muscle sodium channel gene SCN4A. Brain 124, 1091- 1555 1099. 1556 Tao, H., Chen, X., Lu, M., Wu, Y., Deng, M., Zeng, X., Liu, Z., Liang, S., 2016. Molecular 1557 determinant for the tarantula toxin Jingzhaotoxin-I slowing the fast inactivation of voltage-gated 1558 sodium channels. Toxicon 111, 13-21. 1559 Tan, H., Ding, X., Meng, S., Liu, C., Wang, H., Xia, L., Liu, Z., and Liang, S., 2013. Antimicrobial 1560 potential of lycosin-I, a cationic and amphiphilic peptide from the venom of the spider Lycosa 1561 singorensis . Curr. Mol. Med. 13, 900-910. 1562 Tan, H., Huang, Y., Xu, J., Chen, B., Zhang, P., Ye, Z., Liang, S., Xiao, L., and Liu, Z., 2017. Spider 1563 toxin peptide Lycosin-I functionalized gold nanoparticles for in vivo tumor targeting and therapy. 1564 TheranosticsACCEPTED 7, 3168-3178. 1565 Titaux-Delgado, G., Carrillo, E., Mendoza, A., Mayorga-Flores, M., Escobedo-Gonzalez, F.C., Cano- 1566 Sanchez, P., Lopez-Vera, E., Corzo, G., and Del Rio-Portilla, F., 2018. Successful refolding and 1567 NMR structure of rMagi3: a disulfide-rich insecticidal spider toxin. Protein Sci. 27, 692-701. 1568 Tonello, R., Rigo, F., Gewehr, C., Trevisan, G., Pereira, E.M., Gomez, M.V., and Ferreira, J., 2014. 1569 Action of Ph α1β, a peptide from the venom of the spider Phoneutria nigriventer , on the analgesic 1570 and adverse effects caused by morphine in mice. J. Pain 15, 619-631. 1571 Tonello, R., Fusi, C., Materazzi, S., Marone, I.M., De Logu, F., Benemei, S., Goncalves, M.C., Coppi, 1572 E., Castro-Junior, C.J., Gomez, M.V., Geppetti, P., Ferreira, J., and Nassini, R., 2017. The

29 ACCEPTED MANUSCRIPT 1573 peptide Ph α1β, from spider venom, acts as a TRPA1 channel antagonist with antinociceptive 1574 effects in mice. Br. J. Pharmacol. 174, 57-69. 1575 Turchetto, J., Sequeira, A.F., Ramond, L., Peysson, F., Bras, J.L., Saez, N.J., Duhoo, Y., Blemont, M., 1576 Guerreiro, C.I., Quinton, L., De Pauw, E., Gilles, N., Darbon, H., Fontes, C.M., and Vincentelli, 1577 R., 2017. High-throughput expression of animal venom toxins in Escherichia coli to generate a 1578 large library of oxidized disulphide-reticulated peptides for drug discovery. Microb. Cell. Fact. 1579 16, 6. 1580 Upert, G., Mourier, G., Pastor, A., Verdenaud, M., Alili, D., Servent, D., and Gilles, N., 2014. High- 1581 throughput production of two disulphide-bridge toxins. Chem. Commun. 50, 8408-8411. 1582 Vassilevski, A.A., Kozlov, S.A., Samsonova, O.V., Egorova, N.S., Karpunin, D.V., Pluzhnikov, K.A., 1583 Feofanov, A.V., and Grishin, E.V., 2008. Cyto-insectotoxins, a novel class of cytolytic and 1584 insecticidal peptides from spider venom. Biochem. J. 411, 687-696. 1585 Vassilevski, A.A., Kozlov, S.A., and Grishin, E.V., 2009. Molecular diversity of spider venom. 1586 Biochemistry 74, 1505-1534. 1587 Vassilevski, A.A., Fedorova, I.M., Maleeva, E.E., Korolkova, Y.V., Efimova, S.S., Samsonova, O.V., 1588 Schagina, L.V., Feofanov, A.V., Magazanik, L.G., and Grishin, E.V., 2010. Novel class of spider 1589 toxin: active principle from the yellow sac spider Cheiracanthium punctorium venom is a unique 1590 two-domain polypeptide. J. Biol. Chem. 285, 32293-32302. 1591 Vassilevski, A.A., Sachkova, M.Y., Ignatova, A.A., Kozlov, S.A., Feofanov, A.V., and Grishin, E.V., 1592 2013. Spider toxins comprising disulfide-rich and linear amphipathic domains: a new class of 1593 molecules identified in the Oxyopes takobius . FEBS J. 280, 6247-6261. 1594 Vetter, I., Deuis, J.R., Mueller, A., Israel, M.R., Starobova, H., Zhang, A., Rash, L.D., and Mobli, M., 1595 2017. Na V1.7 as a pain target - from gene to pharmacology. Pharmacol. Ther. 172, 73-100. 1596 Vieira, L.B., Kushmerick, C., Hildebrand, M.E., Garcia, E., Stea, A., Cordeiro, M.N., Richardson, M., 1597 Gomez, M.V., and Snutch, T.P., 2005. Inhibition of high voltage-activated calcium channels by 1598 spider toxin PnTx3-6. J. Pharmacol. Exp. Ther. 314, 1370-1377. 1599 Wang, B., Dunlop, J.A., Selden, P.A., Garwood, R.J., Shear, W.A., Muller, P., and Lei, X., 2018. 1600 Cretaceous arachnid Chimerarachne yingi gen. et sp. nov. illuminates spider origins. Nat. Ecol. 1601 Evol. MANUSCRIPT 1602 Wang, G., Watson, K.M., Peterkofsky, A., and Buckhe it, R.W., Jr., 2010. Identification of novel 1603 human immunodeficiency virus type 1-inhibitory peptides based on the antimicrobial peptide 1604 database. Antimicrob. Agents Chemother. 54, 1343-1346. 1605 Wang, J., Ma, Y., Sachs, F., Li, J., and Suchyna, T.M., 2016a. GsMTx4-D is a cardioprotectant 1606 against myocardial infarction during ischemia and reperfusion. J. Mol. Cell. Cardiol. 98, 83-94. 1607 Wang, L., Wang, Y.J., Liu, Y.Y., Li, H., Guo, L.X., Liu, Z.H., Shi, X.L., and Hu, M., 2014. In vitro 1608 potential of Lycosin-I as an alternative antimicrobial drug for treatment of multidrug-resistant 1609 Acinetobacter baumannii infections. Antimicrob. Agents Chemother. 58, 6999-7002. 1610 Wang, Y., Wang, L., Yang, H., Xiao, H., Farooq, A., Liu, Z., Hu, M., and Shi, X., 2016b. The spider 1611 venom peptide Lycosin-II has potent antimicrobial activity against clinically isolated bacteria. 1612 Toxins 8, 119, 1-9. 1613 WHO, 2017. World Malaria Report. World Health Organisation. 1614 Windley, M.J., Herzig, V., Dziemborowicz, S.A., Hardy, M.C., King, G.F., and Nicholson, G.M., 1615 2012. Spider-venom peptides as bioinsecticides. Toxins 4, 191–227. 1616 Wingerd, J.S., Mozar, C.A., Ussing, C.A., Murali, S.S., Chin, Y.K., Cristofori-Armstrong, B., Durek, 1617 T., Gilchrist, J., Vaughan, C.W., Bosmans, F., Adams, D.J., Lewis, R.J., Alewood, P.F., Mobli, 1618 M., Christie,ACCEPTED M.J., Rash, L.D., 2017. The tarantula toxin β/δ-TRTX-Pre1a highlights the 1619 importance of the S1-S2 voltage-sensor region for sodium channel subtype selectivity. Sci. Rep. 1620 7, 974. 1621 World Spider Catalog, 2018. World spider catalog (http://wsc.nmbe.ch ), Vol. 2018. Natural History 1622 Museum Bern. 1623 Wu, J., Lewis, A.H., and Grandl, J., 2017. Touch, tension, and transduction - the function and 1624 regulation of Piezo ion channels. Trends Biochem. Sci. 42, 57-71. 1625 Xiao, Y., Bingham, J.P., Zhu, W., Moczydlowski, E., Liang, S., Cummins, T.R., 2008. Tarantula 1626 huwentoxin-IV inhibits neuronal sodium channels by binding to receptor site 4 and trapping the 1627 domain ii voltage sensor in the closed configuration. J. Biol. Chem. 283, 27300-27313.

30 ACCEPTED MANUSCRIPT 1628 Xiao, Y., Liang, S., 2003. Inhibition of neuronal tetrodotoxin-sensitive Na + channels by two spider 1629 toxins: hainantoxin-III and hainantoxin-IV. Eur. J. Pharmacol. 477, 1-7. 1630 Xiao, Z., Zhang, Y., Zeng, J., Liang, S., Tang, C., and Liu, Z., 2018. Purification and characterization 1631 of a novel insecticidal toxin, -sparatoxin-Hv2, from the venom of the spider Heteropoda 1632 venatoria . Toxins 10, 233, 1-12. 1633 Xiong, Z.G., Zhu, X.M., Chu, X.P., Minami, M., Hey, J., Wei, W.L., MacDonald, J.F., Wemmie, J.A., 1634 Price, M.P., Welsh, M.J., and Simon, R.P., 2004. Neuroprotection in ischemia: blocking calcium- 1635 permeable acid-sensing ion channels. Cell 118, 687-698. 1636 Yan, L., and Adams, M.E., 1998. Lycotoxins, antimicrobial peptides from venom of the wolf spider 1637 Lycosa carolinensis . J. Biol. Chem. 273, 2059-2066. 1638 Yang, S., Pyati, P., Fitches, E., and Gatehouse, J.A., 2014. A recombinant fusion protein containing a 1639 spider toxin specific for the insect voltage-gated sodium ion channel shows oral toxicity towards 1640 insects of different orders. Insect Biochem. Mol. Biol. 47, 1-11. 1641 Yang, Z.J., Ni, X., Carter, E.L., Kibler, K., Martin, L.J., and Koehler, R.C., 2011. Neuroprotective 1642 effect of acid-sensing ion channel inhibitor psalmotoxin-1 after hypoxia-ischemia in newborn 1643 piglet striatum. Neurobiol. Dis. 43, 446-454. 1644 Ye, M., Yang, J., Tian, C., Zhu, Q., Yin, L., Jiang, S., Yang, M., and Shu, Y., 2018. Differential roles 1645 of Na V1.2 and Na V1.6 in regulating neuronal excitability at febrile temperature and distinct 1646 contributions to febrile seizures. Sci. Rep. 8, 753. 1647 Yeung, E.W., Whitehead, N.P., Suchyna, T.M., Gottlieb, P.A., Sachs, F., and Allen, D.G., 2005. 1648 Effects of stretch-activated channel blockers on [Ca 2+ ]i and muscle damage in the mdx mouse. J. 1649 Physiol. 562, 367-380. 1650 Yuan, C., Yang, S., Liao, Z., Liang, S., 2007. Effects and mechanism of Chinese tarantula toxins on 1651 the K V2.1 potassium channels. Biochem. Biophys. Res. Commun. 352, 799-804. 1652 Zamponi, G.W., Striessnig, J., Koschak, A., and Dolphin, A.C., 2015. The physiology, pathology, and 1653 pharmacology of voltage-gated calcium channels and their future therapeutic potential. 1654 Pharmacol. Rev. 67, 821-870. 1655 Zeng, X., Deng, M., Lin, Y., Yuan, C., Pi, J., Liang, S., 2007. Isolation and characterization of 1656 Jingzhaotoxin-V, a novel neurotoxin from the MANUSCRIPT venom of the spider Chilobrachys jingzhao . 1657 Toxicon 49, 388-399. 1658 Zhang, F., Liu, Y., Zhang, C., Li, J., Yang, Z., Gong, X., Gan, Y., Chen, P., Liu, Z., Liang, S., 2015. 1659 Natural mutations change the affinity of µ-theraphotoxin-Hhn2a to voltage-gated sodium 1660 channels. Toxicon 93, 24-30. 1661 Zhang, J., Tang, D., Liu, S., Hu, H., Liang, S., Tang, C., Liu, Z., 2018. Purification and 1662 characterization of JZTx-14, a potent antagonist of mammalian and prokaryotic voltage-gated 1663 sodium channels. Toxins 10, 408, 1-15. 1664 Zhang, P., Ma, J., Yan, Y., Chen, B., Liu, B., Jian, C., Zhu, B., Liang, S., Zeng, Y., and Liu, Z., 2017. 1665 Arginine modification of lycosin-I to improve inhibitory activity against cancer cells. Org. 1666 Biomol. Chem. 15, 9379-9388. 1667 Zhao, H., Kong, Y., Wang, H., Yan, T., Feng, F., Bian, J., Yang, Y., and Yu, H., 2011. A defensin- 1668 like antimicrobial peptide from the venoms of spider, Ornithoctonus hainana . J. Pept. Sci. 17, 1669 540-544. 1670 Zhong, Y., Song, B., Mo, G., Yuan, M., Li, H., Wang, P., Yuan, M., and Lu, Q., 2014. A novel 1671 neurotoxin from venom of the spider Brachypelma albopilosum . PLoS One 9, e110221. 1672 Zhou, R.P., Ni, W.L., Dai, B.B., Wu, X.S., Wang, Z.S., Xie, Y.Y., Wang, Z.Q., Yang, W.J., Ge, J.F., 1673 Hu, W., andACCEPTED Chen, F.H., 2018. ASIC2a overexpression enhances the protective effect of PcTx1 1674 and APETx2 against acidosis-induced articular chondrocyte apoptosis and cytotoxicity. Gene 1675 642, 230-240. 1676 Zobel-Thropp, P.A., Kerins, A.E., and Binford, G.J., 2012. Sphingomyelinase D in sicariid spider 1677 venom is a potent insecticidal toxin. Toxicon 60, 265-271.

31 ACCEPTED MANUSCRIPT 1678 Figure legends 1679 1680 Fig.1: Summary of methods for delivering insecticidal spider peptides (ISPs) to the insect haemocoel: 1681 (1) Some ISPs such as OAIP-1 access the haemocoel via paracellular uptake possibly through leaky 1682 septate junctions. (2) ISPs can be fused to carrier proteins such as snowdrop lectin (GNA) or the coat 1683 protein of pea enation mosaic virus, which are recognised by specific receptors on gut epithelial cells. 1684 These fusion proteins can be taken up via receptor-mediated endocytosis and delivered to the 1685 haemocoel by transcytosis. (3) Insecticidal Cry proteins produced by the soil bacterium Bacillus 1686 thuringiensis oligomerize to form stable transmembrane pores. ISPs might enter epithelial cells 1687 through these pores and then be delivered to the haemocoel by transcytosis (4) Baculovirus can be 1688 engineered to encode an ISP transgene. Upon recognition by specific gut receptors, budded virions 1689 infect gut epithelial cells and other body tissues and release ISPs. (5) Entomopathogenic fungi such as 1690 Metarhizium and Beauveria can also be engineered to encode an ISVP transgene. Fungal spores 1691 (conidia) adhere to the insect cuticle, germinate and then invade the integument. The fungus then 1692 proliferates in the haemocoel in the form of wall-less blastopores, which produce and release ISPs. 1693 The figure and legend have been adapted from Herzig et al., 2014.

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Table 1: Spider-venom peptides and their potential therapeutic applications (excluding Antimicrobials, see Table 3) Indication Specific indication Peptide Source (Family 1: Species ) Expected target Reference Neurodegenerative Stroke PcTx1 T: Psalmopoeus cambridgei ASIC1a (McCarthy et al., 2015) diseases Hi1a At: Hadronyche infensa ASIC1a (Chassagnon et al., 2017) Hm3a T: Heteroscodra maculata ASIC1a (Er et al., 2017) Dravet Syndrome Hm1a T: Heteroscodra maculata Na V1.1 (Richards et al., 2018) Multiple sclerosis Ph α1β C: Phoneutria nigriventer Ca V2 (Silva et al., 2018) Retinopathy PnTx3-4 C: Phoneutria nigriventer Ca V2 (Binda et al., 2016) Chronic cerebral ischemia Huwentoxin-I T: Cyriopagopus schmidti Ca V2.2 (Mao et al., 2017) Alzheimer's disease PhK V C: Phoneutria nigriventer KV4.1, 4.2, 4.3 (Gomes et al., 2013) Parkinson's disease Guangxitoxin-1E T: Chilobrachys guangxiensis KV2.1 (Chao et al., 2018) Pain Na V1.7-mediated pain Pn3a T: Pamphobeteus nigricolor Na V1.7 (Deuis et al., 2017) Cd1a T: Ceratogyrus darlingi Na V1.7 (Sousa et al., 2017) Protoxin-III T: Thrixopelma pruriens Na V1.7 (Cardoso et al., 2015) CcoTx1 T: Ceratogyrus marshalli Na V1.7 (Shcherbatko et al., 2016) GpTx1 T: Grammostola porteri Na V1.7 (Deuis et al., 2016) Ca V2-mediated pain Ph α1β C: Phoneutria nigriventer Ca V2 (So uza et al., 2008; de Souza et al., 2011; Rigo et al., 2013; Rosa et al., 2014; Rigo et al., 2017b; Tonello et al., 2017) PnTx3-3 C: Phoneutria nigriventer Ca V2 (Dalmolin et al., 2011; Dalmolin et al., 2017) PnTx3-4 C: Phoneutria nigriventer Ca V2 (da Silva et al., 2015) PnTx3-5 C: Phoneutria nigriventer Ca V2 (Oliveira et al., 2016) Huwentoxin-XVI T: Cyriopagopus schmidti Ca V2.2 (Deng et al., 2014) TRPA1-mediated pain Ph α1β C: Phoneutria nigriventer TRPA1 (Tonello et al., 2017) P2X 3-mediated pain Purotoxin-1 L: Geolycosa sp. MANUSCRIPT P2X 3 receptors (Grishin et al., 2010) Skeletal muscle Hypokalemic periodic paralysis Hm-3 Tm: Heriaeus melloteei Na V1.4 (Mannikko et al., 2018) diseases Duchenne muscular dystrophy GsMTx4 T: Grammostola rosea Piezo MSCs (Yeung et al., 2005) Skin disorders Chronic refractory pruritus Ph α1β C: Phoneutria nigriventer Ca V2 (Maciel et al., 2014) Urinary system Hemorrhagic cystitis Ph α1β C: Phoneutria nigriventer Ca V2 (Silva et al., 2015b) disorders Metabolic disorders Diabetes type 2 Guangxitoxin-1E T: Chilobrachys guangxiensis KV2.1 (Li et al., 2013) Anti-cancer Various cancers Lycosin-I L: Lycosa singoriensis tumor cell membranes (Liu et al., 2012b; Tan et al., 2017; Zhang et al., 2017; Shen et al., 2018) (+derivatives) Latarcin-I Z: Lachesana tarabaevi cell membranes (Rothan et al., 2015) Ph α1β C: Phoneutria nigriventer Ca V2 (Nicoletti et al., 2017) Melanoma and skin cancers HiGomesin At: Hadronyche infensa antiproliferative (Ikonomopoulou,and Fernandez-Rojo, 2018; Ikonomopoulou et al., proapoptotic pathways 2018; Fernandez-Rojo, et al., 2018) Vascular disorders Cardiac arrhythmias PhK V C: Phoneutria nigriventer KV4.1, 4.2, 4.3 and/or (Almeida et al., 2011) cholinergic system Myocardial infarction GsMTx4ACCEPTED T: Grammostola rosea Piezo MSCs (Wang et al., 2016a) Erectile dysfunction PnPP-19 (derived C: Phoneutria nigriventer NO signaling (Silva et al., 2015a; Almeida et al., 2018) from PnTx2-6) Hypertension Lycosin-I L: Lycosa singoriensis NO signaling (Ma et al., 2018) Vascular inflammation Lycosin-I L: Lycosa singoriensis NF κB signaling (Li et al., 2018) 1Family of source spiders: At = Atracidae; C = Ctenidae; L = Lycosidae; T = Theraphosidae; Tm = Thomisidae; Z = Zodariidae.

33 ACCEPTED MANUSCRIPT (Table 2 is in a separate excel document)

Table 3: Potential medical applications and activity spectrum of antimicrobial spider venom peptides

Peptide Source (Family 1: Potential medical Gram- Gram- Fungus Virus Plasmodium Cytotoxic to Reference Spider species ) application positive negative host at bacteria bacteria therapeutic concentration Oh-defensin T: Cyriopagopus Broad spectrum Y Y Y N.D. N.D. N (Zhao et al., 2011) hainanus antibiotic Juruin T: Avicularia Antifungal N N Y N.D. N.D. N (Ayroza et al., 2012) juruensis Latarcin-3a Z: Lachesana Antiviral (HIV), Y Y Y Y N.D. N (Kozlov et al., 2006; Wang tarabaevi broad spectrum et al., 2010) antibiotic Latarcin-1 Z: Lachesana Antiviral (Dengue), Y Y Y Y N.D. mildly (Kozlov et al., 2006; Rothan tarabaevi broad spectrum et al., 2014) antibiotic Lycosin-I L: Lycosa Broad spectrum Y Y Y N.D. N.D. mildly (Tan et al., 2013) singoriensis antibiotic Lycosin-II L: Lycosa Broad spectrum Y Y Y N.D. N.D. N (Wang et al., 2016b) singoriensis antibiotic, active against multi-drug resistant bacteria MANUSCRIPT LyeTx1/1b L: Lycosa Broad spectrum Y Y Y N.D. N.D. N (Santos et al., 2010) erythrognatha antibiotic, useful for biofilms Cyto- Z: Lachesana Antichlamydial (by Y Y N.D. N.D. N.D. Y (not observed (Vassilevski et al., 2008; insectotoxin 1a tarabaevi gene therapy) using gene Lazarev et al., 2011; Polina therapy) et al., 2012) PcFK1 T: Psalmopoeus Antimalarial N N N N.D. Y N (Choi et al., 2004) cambridgei PcFK2 T: Psalmopoeus Antimalarial N N N N.D. Y N (Choi et al., 2004) cambridgei 1Family of source spiders: L = Lycosidae; T = Theraphosidae; Z = Zodariidae. N.D. = not determined ACCEPTED

34 ACCEPTED MANUSCRIPT Table 5: Recently discovered insecticidal spider peptides/proteins (ISPs)

Toxin name Source MW (kDa) Insect target PD50 (nmol/g) LD50 (nmol/g) Activity Reference -NPTX-Nc1a A: Nephila clavata 4.2 B: Periplaneta americana 0.14 (@24h) lethal (Jin et al., 2017) CsTx-1 C: Cupiennius salei 8.4 D: Drosophila melanogaster 0.35 (@24h) lethal (Kuhn-Nentwig et al., 2012) CsTx-2a C: Cupiennius salei 6.9 D: Drosophila melanogaster 2.58 (@24h) lethal (Kuhn-Nentwig et al., 2012) CsTx-2b C: Cupiennius salei 6.7 D: Drosophila melanogaster 66.51 (@24h) lethal (Kuhn-Nentwig et al., 2012) β/δ-PrIT1 C: Phoneutria reidyi 5.6 D: Musca domestica 0.004 (@24h) lethal (de Oliveira et al., 2015) B: Periplaneta americana 0.23 (@24h) CpTx1 E: Cheiracanthium punctorium 15.1 D: Sarcophaga carnaria 0.2 (@24h) lethal (Vassilevski et al., 2010) CpTx2a E: Cheiracanthium punctorium 15.0 D: Sarcophaga carnaria 2.2-3.3 (@24h) paralytic (Sachkova et al., 2014) CpTx3a E: Cheiracanthium punctorium 15.1 D: Sarcophaga carnaria 2.2-3.3 (@24h) lethal (Sachkova et al., 2014) CpTx4a E: Cheiracanthium punctorium 15.1 D: Sarcophaga carnaria 2.2-3.3 (@24h) lethal (Sachkova et al., 2014) Magi3 M: Macrothele gigas 5.2 O: Gryllus bimaculatus 0.18 (@2h) 3 22.8 (@2h) 3 lethal (Titaux-Delgado et al., 2018) OxyTx1 O: Oxyopes lineatus 8.1 L: Spodoptera frugiperda 0.97 (@24h) lethal (Estrada et al., 2016) OxyTx2 O: Oxyopes lineatus 6.2 L: Spodoptera frugiperda 1.88 (@24h) lethal (Estrada et al., 2016) OtTx1 O: Oxyopes takobius 12.0 D: Sarcophaga carnaria 6.2 (@24h) lethal (Vassilevski et al., 2013) ω-Tbo-IT1 P: Tibellus oblongus 4.3 D: Musca domestica 4.40 (@48h) lethal (Mikov et al., 2015) B: Gromphadorhina portentosa lethal 2 LaSicTox-αIB2bi S: Loxosceles arizonica 30.1 O: Acheta domesticus 0.07 (@1h) lethal (Zobel-Thropp et al., 2012) U2-SCTX-Li1b S: Loxosceles intermedia 5.6 D: Lucilia cuprina 0.83 (@1h) irreversibly paralytic (Matsubara et al., 2017) -SPRTX-Hv2 Sp: Heteropoda venatoria 4.2 B: Periplaneta americana 2.8 (@24h) lethal (Xiao et al., 2018) -TRTX-Ae1a T: Augacephalus ezendami 4.3 D: Lucilia cuprina 0.96 (@1h) reversibly paralytic (Herzig et al., 2016) D: Drosophila melanogaster 0.29 (@3h) 4.3(@24h) lethal H: Rhodnius prolixus inactive @500nmol/g Ba1 T: Brachypelma albiceps 4.4 O: Acheta domesticus 2.45 (@24h) lethal (Clement et al., 2015) brachyin T: Brachypelma albopilosum 4.9 B: Periplaneta americana 1.2 (@48h) 1 lethal (Zhong et al., 2014) C: Tenebrio molitor 1.55 (@48h) 1 lethal β-TRTX-Cd1a T: Ceratogyrus darlingi 4.0 D: Lucilia cuprina 0.13 (@1h) reversibly paralytic (Sousa et al., 2017) U1-TRTX-Ct1a T: Coremiocnemis tropix 4.3 D: Lucilia cuprina 1.34 (@24h)MANUSCRIPT 1.69 (@24h) irreversibly paralytic (Ikonomopoulou et al., 2016) U1-TRTX-Ct1b T: Coremiocnemis tropix 4.2 D: Lucilia cuprina irreversibly paralytic 2 (Ikonomopoulou et al., 2016) /ω-TRTX-Mb1a T: Monocentropus balfouri 4.2 D: Lucilia cuprina 5.9 (@1h) not lethal reversibly paralytic (Smith et al., 2017) /ω-TRTX-Mb1b T: Monocentropus balfouri 4.2 D: Lucilia cuprina 6.0 (@1h) not lethal reversibly paralytic (Smith et al., 2017) OAIP-1 T: Selenotypus plumipes 3.9 C: Tenebrio molitor 1.85 (@24h) 4 lethal (Hardy et al., 2013) L: Helicoverpa armigera lethal 5 Latroeggtoxin-III Th: Latrodectus tredecimguttatus 36.0 B: Periplaneta americana 278 (@30h) 3 lethal (Lei et al., 2015)

Family of source spiders: A = Araneidae; C = Ctenidae; E = Eutichuridae; M = Macrothelidae; O = Oxyopidae; P = Philodromidae; S = Sicariidae; Sp = Sparassidae; T = Theraphosidae; Th = Theridiidae. Orders of insect targets: B = Blattaria, C = Coleoptera; D = Diptera; H= Hemiptera; L = Lepidoptera; O = Orthoptera In the columns for PD 50 and LD 50 , the time point (after injection of the ISP) at which the respective behavior was measured is indicated in brackets 1 LD 50 conversion to nmol/g based on updated information provided by authors of original publication (Zhong et al., 2014) 2 No PD 50 or LD 50 data available ACCEPTED 3 Activity based on a single dose, no PD 50 or LD 50 data available 4 Orally active at 170.5 nmol/g 5 Orally active at 104.2 pmol/g

35 ACCEPTED MANUSCRIPT Figure 1:

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36 ACCEPTED MANUSCRIPT Highlights

− Spider venoms contain a huge diversity of potent and target-selective peptides with a correspondingly vast array of pharmacological activities.

− Their potential medical applications have focused on the treatment of diseases such as stroke, cancer, pain and erectile dysfunction.

− They also have great potential for application in agriculture as environmentally benign bioinsecticides.

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ACCEPTED ACCEPTED MANUSCRIPT Ethical Statement This work reported in this manuscript complied with all institutional, state, and national policies governing the ethical treatment of the experimental subjects. Moreover, we wish to acknowledge that:

(i) the paper is not currently being considered for publication elsewhere;

(ii) all authors have been personally and actively involved in substantive work leading to the review, and will hold themselves jointly and individually responsible for its content.

Natalie Saez Volker Herzig

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