New Analogs of Polyamine Toxins from Spiders and Wasps: Liquid Phase Fragment Synthesis and Evaluation of Antiproliferative Activity.

Polyamine toxins (PATs) are conjugates of polyamines (PAs) with lipophilic carboxylic acids, which have been recently shown to present antiproliferative activity. Ten analogs of the spider PATs Agel 416, HO-416b, and JSTX-3 and the wasp PAT PhTX-433 were synthesized with changes in the lipophilic head group and/or the PA chain, and their antiproliferative activity was evaluated on MCF-7 and MDA-MB-231 breast cancer cells, using Agel 416 and HO-416b as reference compounds. All five analogs of PhTX-433 were of very low activity on both cell lines, whereas the two analogs of JSTX-3 were highly active only on the MCF-7 cell line with IC50 values of 2.63-2.81 µΜ. Of the remaining three Agel 416 or HO-416b analogs, only the one with the spermidine chain was highly active on both cells with IC50 values of 3.15-12.6 µM. The two most potent compounds in this series, Agel 416 and HO-416b, with IC50 values of 0.09-3.98 µΜ for both cell lines, were found to have a very weak cytotoxic effect on the MCF-12A normal breast cells. The present study points out that the structure of both the head group and the PA chain determine the strength of the antiproliferative activity of PATs and their selectivity towards different cells.

Development of ProTx-II Analogues as Highly Selective Peptide Blockers of Nav1.7 for the Treatment of Pain.

Inhibitor cystine knot peptides, derived from venom, have evolved to block ion channel function but are often toxic when dosed at pharmacologically relevant levels in vivo. The article describes the design of analogues of ProTx-II that safely display systemic in vivo blocking of Nav1.7, resulting in a latency of response to thermal stimuli in rodents. The new designs achieve a better in vivo profile by improving ion channel selectivity and limiting the ability of the peptides to cause mast cell degranulation. The design rationale, structural modeling, in vitro profiles, and rat tail flick outcomes are disclosed and discussed.

Two for the Price of One: Heterobivalent Ligand Design Targeting Two Binding Sites on Voltage-Gated Sodium Channels Slows Ligand Dissociation and Enhances Potency.

Voltage-gated sodium (NaV) channels are pore-forming transmembrane proteins that play essential roles in excitable cells, and they are key targets for antiepileptic, antiarrhythmic, and analgesic drugs. We implemented a heterobivalent design strategy to modulate the potency, selectivity, and binding kinetics of NaV channel ligands. We conjugated µ-conotoxin KIIIA, which occludes the pore of the NaV channels, to an analogue of huwentoxin-IV, a spider-venom peptide that allosterically modulates channel gating. Bioorthogonal hydrazide and copper-assisted azide-alkyne cycloaddition conjugation chemistries were employed to generate heterobivalent ligands using polyethylene glycol linkers spanning 40-120 Å. The ligand with an 80 Å linker had the most pronounced bivalent effects, with a significantly slower dissociation rate and 4-24-fold higher potency compared to those of the monovalent peptides for the human NaV1.4 channel. This study highlights the power of heterobivalent ligand design and expands the repertoire of pharmacological probes for exploring the function of NaV channels.

Development of Photocrosslinking Probes Based on Huwentoxin-IV to Map the Site of Interaction on Nav1.7.

Voltage-gated sodium (Nav) channels respond to changes in the membrane potential of excitable cells through the concerted action of four voltage-sensor domains (VSDs). Subtype Nav1.7 plays an important role in the propagation of signals in pain-sensing neurons and is a target for the clinical development of novel analgesics. Certain inhibitory cystine knot (ICK) peptides produced by venomous animals potently modulate Nav1.7; however, the molecular mechanisms underlying their selective binding and activity remain elusive. This study reports on the design of a library of photoprobes based on the potent spider toxin Huwentoxin-IV and the determination of the toxin binding interface on VSD2 of Nav1.7 through a photocrosslinking and tandem mass spectrometry approach. Our Huwentoxin-IV probes selectively crosslink to extracellular loop S1-S2 and helix S3 of VSD2 in a chimeric channel system. Our results provide a strategy that will enable mapping of sites of interaction of other ICK peptides on Nav channels.

Racemic crystal structures of peptide toxins, GsMTx4 prepared by protein total synthesis.

The Piezo channel is a versatile mechanosensitive cation channel that mediates tactile, vascular development, and proprioception. GsMTx4 is the only reported inhibitor specifically targeting Piezo channels. Although the sequence of GsMTx4 is reported, the crystal structure of GsMTx4 is still unknown. Here, we achieved the two-segment synthesis of GsMTx4 and its enantiomer, enGsMTx4, through hydrazide based Native Chemical Ligation, and analyzed the crystal structure of GsMTx4 through the racemic crystallization technology. By analyzing the structure, we found that there is a hydrophobic patch surrounded by aromatic residues and charged residues.

Spermaurin, an La1-like peptide from the venom of the scorpion Scorpio maurus palmatus, improves sperm motility and fertilization in different mammalian species.

STUDY QUESTION: Is it possible to identify original compounds that are able to enhance sperm motility from the venom of the scorpion Scorpio maurus palmatus? SUMMARY ANSWER: We identified a potent disulfide-rich peptide (DRP) of 73 amino acids that significantly improved the motility of fresh and frozen-thawed sperm in different mammalian species, including human, and improved fertilization outcome in mouse IVF experiments. WHAT IS KNOWN ALREADY: Any disturbance of sperm motility has a strong impact on fertilization and can lead to subfertility or infertility. Significant efforts have, therefore,  been made to identify pharmacological drugs that might improve sperm motility. Such compounds are particularly useful in azoospermia to improve testicular sperm extraction and in the domain of cryopreservation because the motility of frozen-thawed sperm is reduced. STUDY DESIGN, SIZE, DURATION: This was a basic science/medical research study aimed at identifying original compounds from a library of venoms able to enhance mammalian sperm motility, including human. We first identified in the venom of a scorpion S. m. palmatus a fraction able to potently activate sperm motility. We next purified and characterized the compound by liquid chromatography, mass spectrometry and peptide synthesis. Finally, the potency and toxicity of both purified and synthetic versions of the identified compound on sperm motility were assessed using different in vitro tests in different mammalian species. PARTICIPANTS/MATERIALS, SETTING, METHODS: For human sperm, biological samples were collected from normozoospermic donors and subfertile patients attending a reproduction department for diagnostic semen analysis. Testicular sperm was collected from cynomolgus monkeys (Macaca fascicularis) euthanized for the needs of specific authorized research projects. The peptide was also tested on bovine and mouse epidydimal sperm. We measured different sperm motility parameters with a computer-assisted sperm analysis system in the presence or absence of the peptide. MAIN RESULTS AND THE ROLE OF CHANCE: Size exclusion chromatography enabled us to isolate a fraction of the venom of S. m. palmatus able to increase sperm motility. By liquid chromatography and mass spectrometry, a peptide comprising 73 amino acids with 4 disulfide bridges was identified as responsible for the biological activity and called 'spermaurin'. The identity of spermaurin was confirmed by chemical synthesis. We showed that the peptide increased the motility of fresh and frozen-thawed human sperm. We observed that the potency of the peptide was higher on fresh ejaculated spermatozoa with a low motility, achieving a 100% increase of curvilinear velocity in poorly performing sperm. We also demonstrated that peptide is effective on bovine and mouse fresh epididymal, bovine frozen-thawed ejaculated and fresh non-human primate testicular sperm. Finally, in mouse IVF, the production of 2-cell embryos was increased by 24% when sperm were treated with the peptide. LIMITATIONS, REASONS FOR CAUTION: This work is an in vitro evaluation of the ability of spermaurin to improve sperm motility parameters. Another limitation of this study is the small number of human sperm samples tested with the natural (n = 36) and synthetic (n = 12) peptides. Moreover, the effect of the peptide on IVF outcome was only tested in mouse and further tests with human and bovine gametes are required to confirm and extend this result in other mammalian species. WIDER IMPLICATIONS OF THE FINDINGS: This work confirms our initial study showing that venoms represent an interesting source of molecules that are able to modify sperm physiology. Moreover, this work presents the first demonstrated biological action of a venom peptide from the scorpion S. m. palmatus with sequence similarities to La1 peptide from Liocheles australasiae (Wood scorpion), a widespread family of DRPs. LARGE SCALE DATA: Not applicable. STUDY FUNDING/COMPETING INTEREST(S): This work is part of the project 'LAB COM-14 LAB7 0004 01-LIPAV', funded by the program LabCom 2014 from the French Research Agency (ANR). Dr Arnoult reports grants from IMV Technologies during the conduct of the study. In addition, Drs Arnoult, Martinez, Ray and Schmitt have a patent EP16305642.7 pending containing some of the information presented in this manuscript.

t-boc synthesis of huwentoxin-i through native chemical ligation incorporating a trifluoromethanesulfonic acid cleavage strategy.

Tert-butyloxycarbonyl (t-Boc)-based native chemical ligation (NCL) techniques commonly employ hydrogen fluoride (HF) to create the thioester fragment required for the ligation process. Our research aimed to assess the replacement of HF with Trifluoromethanesulfonic acid (TFMSA). Here we examined a 33 amino acid test peptide, Huwentoxin-I (HwTx-I) as a novel candidate for our TFMSA cleavage protocol. Structurally HwTx-I has an X-Cys(16) -Cys(17) -X sequence mid-region, which makes it an ideal candidate for NCL. Experiments determined that the best yields (16.8%) obtained for 50 mg of a thioester support resin were achieved with a TFMSA volume of 100 µL with a 0.5-h incubation on ice, followed by 2.0 h at room temperature. RP-HPLC/UV and mass spectra indicated the appropriate parent mass and retention of the cleaved HwTx-I N-terminal thioester fragment (Ala(1) -Cys(16) ), which was used in preparation for NCL. The resulting chemically ligated HwTx-I was oxidized/folded, purified, and then assessed for pharmacological target selectivity. Native-like HwTx-I produced by this method yielded an EC50 value of 340.5 ± 26.8 nM for Nav 1.2 and an EC50 value of 504.1 ± 81.3 nM for Nav 1.3, this being similar to previous literature results using native material. This article represents the first NCL based synthesis of this potent sodium channel blocker. Our illustrated approach removes potential restrictions in the advancement of NCL as a common peptide laboratory technique with minimal investment, and removes the hazards associated with HF usage. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 737-745, 2016.

Tarantula toxins use common surfaces for interacting with Kv and ASIC ion channels.

Tarantula toxins that bind to voltage-sensing domains of voltage-activated ion channels are thought to partition into the membrane and bind to the channel within the bilayer. While no structures of a voltage-sensor toxin bound to a channel have been solved, a structural homolog, psalmotoxin (PcTx1), was recently crystalized in complex with the extracellular domain of an acid sensing ion channel (ASIC). In the present study we use spectroscopic, biophysical and computational approaches to compare membrane interaction properties and channel binding surfaces of PcTx1 with the voltage-sensor toxin guangxitoxin (GxTx-1E). Our results show that both types of tarantula toxins interact with membranes, but that voltage-sensor toxins partition deeper into the bilayer. In addition, our results suggest that tarantula toxins have evolved a similar concave surface for clamping onto α-helices that is effective in aqueous or lipidic physical environments.

Regioselective solid-phase synthesis of N-mono-hydroxylated and N-mono-methylated acylpolyamine spider toxins using an 2-(ortho-nitrophenyl)ethanal-modified resin.

A recently introduced new SPS resin, possessing a 2-(ortho-nitrophenyl)ethanal linker, was used for the regioselective on-resin synthesis of N-mono-hydroxylated and N-mono-methylated polyamine spider toxins of Agelenopsis aperta and Larinioides folium. The polyamine backbones of the target compounds were efficiently constructed from the center by reductive amination of the aldehyde linker, followed by stepwise alkylation and acylation on solid support. Depending on the cleavage conditions, employing either oxidation/Cope elimination or methylation/Hofmann elimination, regioselectively the respective N-hydroxyl or N-methyl products were obtained. Employing this methodology, a number of acylpolyamine spider toxins were synthesized and identified as venom components by UHPLC and ESI-MS/MS.

Structure-activity relationship study of spider polyamine toxins as inhibitors of ionotropic glutamate receptors.

The spider polyamine toxins Joro spider toxin-3 (JSTX-3) and Nephila polyamine toxins-1 and -8 (NPTX-1 and NPTX-8) are isolated from the venom of the orb-weaver spider Nephila clavata (Joro spider). They share a high degree of structural resemblance, their aromatic head groups being the only difference, and were recently found to be very potent open-channel blockers of ionotropic glutamate (iGlu) receptors. In this study we designed and synthesized a collection of 24 analogues of these toxins using a recently developed solid-phase synthetic methodology. Systematic variation in two regions of the toxins and subsequent evaluation of biological activity at AMPA and NMDA subtypes of iGlu receptors provided succinct information on structure-activity relationships. In particular, one set of analogues were found to display exquisite selectivity and potency for AMPA receptors relative to the natural products. Thus, this systematic SAR study has provided new pharmacological tools for studies of iGlu receptors.

Synthesis and analgesic effects of µ-TRTX-Hhn1b on models of inflammatory and neuropathic pain.

µ-TRTX-Hhn1b (HNTX-IV) is a 35-amino acid peptide isolated from the venom of the spider, Ornithoctonus hainana. It inhibits voltage-gated sodium channel Nav1.7, which has been considered as a therapeutic target for pain. The goal of the present study is to elucidate the analgesic effects of synthetic µ-TRTX-Hhn1b on animal models of pain. The peptide was first synthesized and then successfully refolded/oxidized. The synthetic peptide had the same inhibitory effect on human Nav1.7 current transiently expressed in HEK 293 cells as the native toxin. Furthermore, the analgesic potentials of the synthetic peptide were examined on models of inflammatory pain and neuropathic pain. µ-TRTX-Hhn1b produced an efficient reversal of acute nociceptive pain in the abdominal constriction model, and significantly reduced the pain scores over the 40-min period in the formalin model. The efficiency of µ-TRTX-Hhn1b on both models was equivalent to that of morphine. In the spinal nerve model, the reversal effect of µ-TRTX-Hhn1b on allodynia was longer and higher than mexiletine. These results demonstrated that µ-TRTX-Hhn1b efficiently alleviated acute inflammatory pain and chronic neuropathic pain in animals and provided an attractive template for further clinical analgesic drug design.

Studies examining the relationship between the chemical structure of protoxin II and its activity on voltage gated sodium channels.

The aqueous solution structure of protoxin II (ProTx II) indicated that the toxin comprises a well-defined inhibitor cystine knot (ICK) backbone region and a flexible C-terminal tail region, similar to previously described NaSpTx III tarantula toxins. In the present study we sought to explore the structure-activity relationship of the two regions of the ProTx II molecule. As a first step, chimeric toxins of ProTx II and PaTx I were synthesized and their biological activities on Nav1.7 and Nav1.2 channels were investigated. Other tail region modifications to this chimera explored the effects of tail length and tertiary structure on sodium channel activity. In addition, the activity of various C-terminal modifications of the native ProTx II was assayed and resulted in the identification of protoxin II-NHCH3, a molecule with greater potency against Nav1.7 channels (IC50=42 pM) than the original ProTx II.

Isolation, synthesis and characterization of ω-TRTX-Cc1a, a novel tarantula venom peptide that selectively targets L-type Cav channels.

Spider venoms are replete with peptidic ion channel modulators, often with novel subtype selectivity, making them a rich source of pharmacological tools and drug leads. In a search for subtype-selective blockers of voltage-gated calcium (CaV) channels, we isolated and characterized a novel 39-residue peptide, ω-TRTX-Cc1a (Cc1a), from the venom of the tarantula Citharischius crawshayi (now Pelinobius muticus). Cc1a is 67% identical to the spider toxin ω-TRTX-Hg1a, an inhibitor of CaV2.3 channels. We assembled Cc1a using a combination of Boc solid-phase peptide synthesis and native chemical ligation. Oxidative folding yielded two stable, slowly interconverting isomers. Cc1a preferentially inhibited Ba(2+) currents (IBa) mediated by L-type (CaV1.2 and CaV1.3) CaV channels heterologously expressed in Xenopus oocytes, with half-maximal inhibitory concentration (IC50) values of 825nM and 2.24µM, respectively. In rat dorsal root ganglion neurons, Cc1a inhibited IBa mediated by high voltage-activated CaV channels but did not affect low voltage-activated T-type CaV channels. Cc1a exhibited weak activity at NaV1.5 and NaV1.7 voltage-gated sodium (NaV) channels stably expressed in mammalian HEK or CHO cells, respectively. Experiments with modified Cc1a peptides, truncated at the N-terminus (ΔG1-E5) or C-terminus (ΔW35-V39), demonstrated that the N- and C-termini are important for voltage-gated ion channel modulation. We conclude that Cc1a represents a novel pharmacological tool for probing the structure and function of L-type CaV channels.

Synthesis and biological characterization of synthetic analogs of Huwentoxin-IV (Mu-theraphotoxin-Hh2a), a neuronal tetrodotoxin-sensitive sodium channel inhibitor.

Huwentoxin-IV (HWTX-IV, also named Mu-theraphotoxin-Hh2a) is a typical inhibitor cystine knot peptide isolated from the venom of Chinese tarantula Ornithoctonus huwena and is found to inhibit tetrodotoxin-sensitive (TTX-S) sodium channels from mammalian sensory neurons. This peptide binds to neurotoxin receptor site 4 located at the extracellular S3-S4 linker of domain II in neuronal sodium channels. However, the molecular surface of HWTX-IV interaction with sodium channels remains unknown. In this study, we synthesized HWTX-IV and three mutants (T28D, R29A and Q34D) and characterized their functions on TTX-S sodium channels from adult rat dorsal root ganglion (DRG) neurons. Analysis of liquid chromatography, mass spectrometry and circular dichroism spectrum indicated that all four synthetic peptides are properly folded. Synthetic HWTX-IV exhibited the same activity as native HWTX-IV, while three mutations reduced toxin binding affinities by 10-200 fold, indicating that the basic or vicinal polar residues Thr²8, Arg²9, and Gln³4 in C-terminus might play critical roles in the interaction of HWTX-IV with TTX-S sodium channels.

Potency optimization of Huwentoxin-IV on hNav1.7: a neurotoxin TTX-S sodium-channel antagonist from the venom of the Chinese bird-eating spider Selenocosmia huwena.

The spider venom peptide Huwentoxin-IV (HwTx-IV) 1 is a potent antagonist of hNav1.7 (IC50 determined herein as 17 ± 2 nM). Nav1.7 is a voltage-gated sodium channel involved in the generation and conduction of neuropathic and nociceptive pain signals. We prepared a number of HwTx-IV analogs as part of a structure-function study into Nav1.7 antagonism. The inhibitory potency of these analogs was determined by automated electrophysiology and is reported herein. In particular, the native residues Glu(1), Glu(4), Phe(6) and Tyr(33) were revealed as important activity modulators and several peptides bearing mutations in these positions showed significantly increased potency on hNav1.7 while maintaining the original selectivity profile of the wild-type peptide 1 on hNav1.5. Peptide 47 (Gly(1), Gly(4), Trp(33)-HwTx) demonstrated the largest potency increase on hNav1.7 (IC50 0.4 ± 0.1 nM).

Cysteine racemization during the Fmoc solid phase peptide synthesis of the Nav1.7-selective peptide--protoxin II.

Protoxin II is biologically active peptide containing the inhibitory cystine knot motif. A synthetic version of the toxin was generated with standard Fmoc solid phase peptide synthesis. If N-methylmorpholine was used as a base during synthesis of the linear protoxin II, it was found that a significant amount of racemization (approximately 50%) was observed during the process of cysteine residue coupling. This racemization could be suppressed by substituting N-methylmorpholine with 2,4,6-collidine. The crude linear toxin was then air oxidized and purified. Electrophysiological assessment of the synthesized protoxin II confirmed its previously described interactions with voltage-gated sodium channels. Eight other naturally occurring inhibitory knot peptides were also synthesized using this same methodology. The inhibitory potencies of these synthesized toxins on Nav1.7 and Nav1.2 channels are summarized.

The effects of huwentoxin-I on the voltage-gated sodium channels of rat hippocampal and cockroach dorsal unpaired median neurons.

Huwentoxin-I (HWTX-I) is a 33-residue peptide isolated from the venom of Ornithoctonus huwena and could inhibit TTX-sensitive voltage-gated sodium channels and N-type calcium channels in mammalian dorsal root ganglion (DRG) neurons. However, the effects of HWTX-I on mammalian central neuronal and insect sodium channel subtypes remain unknown. In this study, we found that HWTX-I potently inhibited sodium channels in rat hippocampal and cockroach dorsal unpaired median (DUM) neurons with the IC(50) values of 66.1±5.2 and 4.80±0.58nM, respectively. Taken together with our previous work on DRG neurons (IC(50)≈55nM), the order of sodium channel sensitivity to HWTX-I inhibition was insect central DUM≫mammalian peripheral>mammalian central neurons. HWTX-I exhibited no effect on the steady-state activation and inactivation of sodium channels in rat hippocampal and cockroach DUM neurons.

Loxosceles gaucho Venom-Induced Acute Kidney Injury In Vivo and In Vitro Studies

Accidents caused by Loxosceles spider may cause severe systemic reactions, including acute kidney injury(AKI). There are few experimental studies assessing Loxosceles venom effects on kidney function in vivo.In order to test Loxosceles gaucho venom (LV) nephrotoxicity and to assess some of the possible mechanisms of renal injury, rats were studied up to 60 minutes after LV 0.24 mg/kg or saline IV injection (control). LV caused a sharp and significant drop in glomerular filtration rate, renal blood flow and urinary output and increased renal vascular resistance, without changing blood pressure. Venom infusion increased significantly serum creatine kinase and aspartate aminotransferase. In the LV group renal histology analysis found acute epithelial tubular cells degenerative changes, presence of cell debris and detached epithelial cells in tubular lumen without glomerular or vascular changes.Immunohistochemistry disclosed renal deposition of myoglobin and hemoglobin. LV did not cause injury to a suspension of fresh proximal tubules isolated from rats.

Syntheses and biological activities of fluorescent-labeled analogs of acylpolyamine toxin NPTX-594 isolated from the venom of Madagascar Joro spider.

Acylpolyamine-type spider toxins are known to be potent and specific blockers against glutamate receptors (GluRs). The present study describes the syntheses and biological activities of several fluorescent-labeled analogs related to a Madagascar Joro spider toxin NPTX-594 to analyze visually the unknown interaction between spider toxins and GluRs.

Membrane structure and interactions of a short Lycotoxin I analogue.

Lycotoxin I and Lycotoxin II are natural anti-microbial peptides that were identified in the venom of the Wolf Spider Lycosa carolinensis. These peptides were found to be potent growth inhibitors for bacteria (Escherichia coli) and yeast (Candida glabrata) at micromolar concentrations. Recently, shortened analogues of LycoI and LycoII have been reported to have decreased haemolytic effects. A shorter Lyco-I analogue studied, LycoI 1-15 (H-IWLTALKFLGKHAAK-NH2), was active only above 10 microM, but was also the least haemolytic. On the basis of these findings, we became interested in obtaining a deeper insight into the membrane activity of LycoI 1-15, as this peptide may represent the first major step for the future development of selective, i.e. non-haemolytic, Lycotoxin-based antibiotics. The interaction of this peptide with liposomes of different composition was studied by microcalorimetry [differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC)] and CD. The results obtained from the calorimetric and spectroscopic techniques were jointly discussed in an attempt to further understand the interaction of this peptide with model membranes.