MT9, a natural peptide from black mamba venom antagonizes the muscarinic type 2 receptor and reverses the M2R-agonist-induced relaxation in rat and human arteries.

All five muscarinic receptors have important physiological roles. The endothelial M2 and M3 subtypes regulate arterial tone through direct coupling to Gq or Gi/o proteins. Yet, we lack selective pharmacological drugs to assess the respective contribution of muscarinic receptors to a given function. We used mamba snake venoms to identify a selective M2R ligand to investigate its contribution to arterial contractions. Using a bio-guided screening binding assay, we isolated MT9 from the black mamba venom, a three-finger toxin active on the M2R subtype. After sequencing and chemical synthesis of MT9, we characterized its structure by X-ray diffraction and determined its pharmacological characteristics by binding assays, functional tests, and ex vivo experiments on rat and human arteries. Although MT9 belongs to the three-finger fold toxins family, it is phylogenetically apart from the previously discovered muscarinic toxins, suggesting that two groups of peptides evolved independently and in a convergent way to target muscarinic receptors. The affinity of MT9 for the M2R is 100 times stronger than that for the four other muscarinic receptors. It also antagonizes the M2R/Gi pathways in cell-based assays. MT9 acts as a non-competitive antagonist against acetylcholine or arecaine, with low nM potency, for the activation of isolated rat mesenteric arteries. These results were confirmed on human internal mammary arteries. In conclusion, MT9 is the first fully characterized M2R-specific natural toxin. It should provide a tool for further understanding of the effect of M2R in various arteries and may position itself as a new drug candidate in cardio-vascular diseases.

2-Hydroxy-4-methoxybenzyl as a Thiol-Protecting Group for Directed-Disulfide Bond Formation.

The chemical synthesis of disulfide-rich peptides such as toxins can be accomplished by using numerous orthogonal cysteine-protecting groups. Herein we report the use of the Hmboff/on protecting group for directed disulfide bond formation. Its combination with classical Trt, Acm, and Mob groups was studied for the synthesis of NMB-1 and phlotoxin-1 toxins highlighting new orthogonal strategies for directed disulfide bond formation.

Chemical Synthesis of a Functional Fluorescent-Tagged α-Bungarotoxin.

α-bungarotoxin is a large, 74 amino acid toxin containing five disulphide bridges, initially identified in the venom of Bungarus multicinctus snake. Like most large toxins, chemical synthesis of α-bungarotoxin is challenging, explaining why all previous reports use purified or recombinant α-bungarotoxin. However, only chemical synthesis allows easy insertion of non-natural amino acids or new chemical functionalities. Herein, we describe a procedure for the chemical synthesis of a fluorescent-tagged α-bungarotoxin. The full-length peptide was designed to include an alkyne function at the amino-terminus through the addition of a pentynoic acid linker. Chemical synthesis of α-bungarotoxin requires hydrazide-based coupling of three peptide fragments in successive steps. After completion of the oxidative folding, an azide-modified Cy5 fluorophore was coupled by click chemistry onto the toxin. Next, we determined the efficacy of the fluorescent-tagged α-bungarotoxin to block acetylcholine (ACh)-mediated currents in response to muscle nicotinic receptor activation in TE671 cells. Using automated patch-clamp recordings, we demonstrate that fluorescent synthetic α-bungarotoxin has the expected nanomolar affinity for the nicotinic receptor. The blocking effect of fluorescent α-bungarotoxin could be displaced by incubation with a 20-mer peptide mimicking the α-bungarotoxin binding site. In addition, TE671 cells could be labelled with fluorescent toxin, as witnessed by confocal microscopy, and this labelling was partially displaced by the 20-mer competitive peptide. We thus demonstrate that synthetic fluorescent-tagged α-bungarotoxin preserves excellent properties for binding onto muscle nicotinic receptors.

Fluorescent- and tagged-protoxin II peptides: potent markers of the Nav 1.7 channel pain target.

BACKGROUND AND PURPOSE: Protoxin II (ProTx II) is a high affinity gating modifier that is thought to selectively block the Nav 1.7 voltage-dependent Na+ channel, a major therapeutic target for the control of pain. We aimed at producing ProTx II analogues entitled with novel functionalities for cell distribution studies and biochemical characterization of its Nav channel targets. EXPERIMENTAL APPROACH: We took advantage of the high affinity properties of the peptide, combined to its slow off rate, to design a number of new tagged analogues useful for imaging and biochemistry purposes. We used high-throughput automated patch-clamp to identify the analogues best matching the native properties of ProTx II and validated them on various Nav -expressing cells in pull-down and cell distribution studies. KEY RESULTS: Two of the produced ProTx II analogues, Biot-ProTx II and ATTO488-ProTx II, best emulate the pharmacological properties of unlabelled ProTx II, whereas other analogues remain high affinity blockers of Nav 1.7. The biotinylated version of ProTx II efficiently works for the pull-down of several Nav isoforms tested in a concentration-dependent manner, whereas the fluorescent ATTO488-ProTx II specifically labels the Nav 1.7 channel over other Nav isoforms tested in various experimental conditions. CONCLUSIONS AND IMPLICATIONS: The properties of these ProTx II analogues as tools for Nav channel purification and cell distribution studies pave the way for a better understanding of ProTx II channel receptors in pain and their pathophysiological implications in sensory neuronal processing. The new fluorescent ProTx II should also be useful in the design of new drug screening strategies.

Identification, Characterization and Synthesis of Walterospermin, a Sperm Motility Activator from the Egyptian Black Snake Walterinnesia aegyptia Venom.

Animal venoms are small natural mixtures highly enriched in bioactive components. They are known to target at least two important pharmacological classes of cell surface receptors: ion channels and G protein coupled receptors. Since sperm cells express a wide variety of ion channels and membrane receptors, required for the control of cell motility and acrosome reaction, two functions that are defective in infertility issues, animal venoms should contain interesting compounds capable of modulating these two essential physiological functions. Herein, we screened for bioactive compounds from the venom of the Egyptian black snake Walterinnesia aegyptia (Wa) that possess the property to activate sperm motility in vitro from male mice OF1. Using RP-HPLC and cation exchange chromatography, we identified a new toxin of 6389.89 Da (termed walterospermin) that activates sperm motility. Walterospermin was de novo sequenced using a combination of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF MS/MS) and liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF MS/MS) following reduction, alkylation, and enzymatic proteolytic digestion with trypsin, chymotrypsin or V8 protease. The peptide is 57 amino acid residues long and contains three disulfide bridges and was found to be identical to the previously cloned Wa Kunitz-type protease inhibitor II (Wa Kln-II) sequence. Moreover, it has strong homology with several other hitherto cloned Elapidae and Viperidae snake toxins suggesting that it belongs to a family of compounds able to regulate sperm function. The synthetic peptide shows promising activation of sperm motility from a variety of species, including humans. Its fluorescently-labelled analog predominantly marks the flagellum, a localization in agreement with a receptor that controls motility function.

Chemical Synthesis, Proper Folding, Nav Channel Selectivity Profile and Analgesic Properties of the Spider Peptide Phlotoxin 1.

Phlotoxin-1 (PhlTx1) is a peptide previously identified in tarantula venom (Phlogius species) that belongs to the inhibitory cysteine-knot (ICK) toxin family. Like many ICK-based spider toxins, the synthesis of PhlTx1 appears particularly challenging, mostly for obtaining appropriate folding and concomitant suitable disulfide bridge formation. Herein, we describe a procedure for the chemical synthesis and the directed sequential disulfide bridge formation of PhlTx1 that allows for a straightforward production of this challenging peptide. We also performed extensive functional testing of PhlTx1 on 31 ion channel types and identified the voltage-gated sodium (Nav) channel Nav1.7 as the main target of this toxin. Moreover, we compared PhlTx1 activity to 10 other spider toxin activities on an automated patch-clamp system with Chinese Hamster Ovary (CHO) cells expressing human Nav1.7. Performing these analyses in reproducible conditions allowed for classification according to the potency of the best natural Nav1.7 peptide blockers. Finally, subsequent in vivo testing revealed that intrathecal injection of PhlTx1 reduces the response of mice to formalin in both the acute pain and inflammation phase without signs of neurotoxicity. PhlTx1 is thus an interesting toxin to investigate Nav1.7 involvement in cellular excitability and pain.

Synthesis by native chemical ligation and characterization of the scorpion toxin AmmTx3.

The scorpion toxin AmmTx3 is a specific blocker of Kv4 channels. It was shown to have interesting potential for neurological disorders. In this study, we report the first chemical synthesis of AmmTx3 by using the native chemical ligation strategy and validate its biological activity. We determined its 3D structure by nuclear magnetic resonance spectroscopy, and pointed out that AmmTx3 possesses the well-known CSαß structural motif, which is found in a large number of scorpion toxins. Overall, this study establishes an easy synthetic access to biologically active AmmTx3 toxin.