**Brigitte Girard 1, Aurélie Couesnon 2, Émmanuelle Girard 3, Jordi Molgó 2,4,\***

<sup>1</sup> Service d'Ophtalmologie—Hôpital Tenon, 4 rue de la Chine 75020 Paris, Sorbonne Université, UPMC, France.

<sup>2</sup> Institut des Neurosciences Paris-Saclay, UMR 9197, CNRS/Université Paris-Sud, 91198 Gif-sur-Yvette cedex, France.

<sup>3</sup> Institut NeuroMyoGene, CNRS UMR 5310, INSERM U1217, Université Lyon1, 8 avenue Rockefeller, 69008 Lyon, France.

<sup>4</sup> Université Paris-Saclay, CEA, INRAE, Institut des sciences du vivant Frédéric Joliot, Département Médicaments et Technologies pour la Santé, Service d'Ingénierie Moléculaire pour la Santé, ERL CNRS 9004, F-91191 Gif-sur-Yvette, France.

\* Correspondence: jordi.molgo@cea.fr

**Abstract:** Currently, a number of pathological conditions, including movement disorders, are treated with botulinum type-A neurotoxin (BoNT/A). This neurotoxin is known to block quantal acetylcholine (ACh) release from motor nerve terminals of the skeletal neuromuscular junction (NMJ), causing temporary muscle paralysis. The treatment with BoNT/A has shown its clinical efficacy in the management of benign blepharospasm, a form of focal dystonia. Due to the slow functional recovery of neuromuscular transmission after BoNT/A-treatment, patients with blepharospasm receive recurrent (every 2–3 months) intramuscular toxin injections in the affected muscles. Regardless of this limitation, the toxin securely relieves patients from their dystonia symptoms and considerably enriches their quality of life. Nevertheless, in less than 4% of the blepharospasm patients treated at Tenon Hospital, BoNT/A-treatment was no longer effective in relieving the patient's symptoms, and it was compulsory to perform the upper myectomy of the *Orbicularis*

*oculi* muscle. In this study, we used surgical waste muscle specimens from 14 patients treated with repeated injections of either Dysport®, abobotulinumtoxinA Ipsen or Xeomin® incobotulinumtoxinA, Merz. These muscle specimens were compared to others, obtained in normal subjects (naïve of BoNT/A) during blepharoplasty. The morphological study was performed blinded to the BoNT/A sample treatment. Neuromuscular specimens analyzed by confocal laser scanning microscopy, using fluorescent staining and immune-labeling of presynaptic proteins, revealed that the pattern of innervation, the muscle nicotinic ACh receptors (nAChR) and the NMJs exhibited marked differences in BoNT/A-treated muscles (regardless of the toxin clinically used), with respect to controls. The control muscles were constantly innervated by a single motor axon (mono-innervated), and NMJs were relatively simple with the typical nAChR array. In contrast, most of the BoNT/A-treated muscles exhibited profuse and stable poly-neuronal innervation, with two unambiguous features: one in which multiple axons innervated a single muscle fiber, the other in which distinct motor axons converged to a unique endplate (convergent innervation). The ability to increase the proportion of poly-innervated muscle fibers may be related to either the stimulation of nerve sprouting (due to muscle inactivity caused by BoNT/A) or the absence of synapse elimination. During development, the structures composing the NMJ undergo rapid formation and elimination. In rodents, unessential synapses are eliminated. We previously reported synapse elimination in mature rodent NMJs following a single BoNT/A injection. The new findings reported here raise a number of questions about the origin and factors contributing to the plasticity changes observed and the expected detrimental functioning of NMJs and muscle fibers.

**Keywords:** botulinum neurotoxin type A; innervation pattern; sprouting

*6.16. Synthetic Analogues of Huwentoxin-IV Spider Peptide with Altered Human NaV1.7/NaV1.6 Selectivity Ratios*

**Ludivine Lopez 1, Jérôme Montnach 1,\*, Barbara Oliveira-Mendes 1, Baptiste Thomas 2, Cécile Caumes 2, Sébastien Nicolas 1, Denis Servent 3, Charles Cohen 4, Rémy Béroud 2, Evelyne Benoit 3, Michel De Waard 1,2,5**

<sup>1</sup> L'institut du thorax, INSERM, CNRS, UNIV NANTES, Nantes, France.

<sup>2</sup> Smartox Biotechnology, Saint-Egrève, France.

<sup>3</sup> Université Paris Saclay, CEA, Institut des sciences du vivant Frédéric Joliot, Département Médicaments et Technologies pour la Santé (DMTS), Service d'Ingénierie Moléculaire pour la Santé (SIMoS), ERL CNRS/CEA 9004, Gif-sur-Yvette, France.

<sup>4</sup> Xenon Pharmaceuticals, Burnaby, British Columbia, Canad. <sup>5</sup> LabEx "Ion Channels, Science and Therapeutics", Valbonne, France.

\* Correspondence: jerome.montnach@univ-nantes.fr

**Abstract:** Huwentoxin-IV (HwTx-IV), a peptide discovered in the venom of the Chinese bird spider *Cyriopagopus schmidti*, has been reported to be a potent antinociceptive compound due to its action on the genetically validated NaV1.7 pain target. Using this peptide for antinociceptive applications in vivo suffers from one major drawback, namely its negative impact on the neuromuscular system. Although it was studied only recently, this effect appears to be due to an interaction between the peptide and the NaV1.6 channel subtype located at the presynaptic level. The aim of this work was to investigate how HwTx-IV could be modified in order to alter the original human (h) NaV1.7/NaV1.6 selectivity ratio of 23. Nineteen HwTx-IV analogs were chemically synthesized and tested for their blocking effects on the Na<sup>+</sup> currents flowing through these two channel subtypes stably expressed in cell lines. The dose–response curves for these analogs were generated, thanks to the use of an automated patch-clamp system. Several key amino acid positions were targeted owing to the information provided by earlier structure–activity relationship (SAR) studies. Among the analogs tested, the potency of HwTx-IV E4K was significantly improved for hNaV1.6, leading to a decreased hNaV1.7/hNaV1.6 selectivity ratio (close to 1). Similar decreased selectivity ratios, but with increased potency for both subtypes, were observed

for HwTx-IV analogs that combine a substitution at position 4 with a modification of amino acid 1 or 26 (HwTx-IV E1G/E4G and HwTx-IV E4K/R26Q). In contrast, increased selectivity ratios (>46) were obtained if the E4K mutation was combined to an additional double substitution (R26A/Y33W) or simply by further substituting the C-terminal amidation of the peptide by a carboxylated motif, linked to a marked loss of potency on hNaV1.6 in this latter case. These results demonstrate that it is possible to significantly modulate the selectivity ratio for these two channel subtypes in order to improve the potency of a given analog for hNaV1.6 and/or hNaV1.7 subtypes. In addition, selective analogs for hNaV1.7, possessing better safety profiles, were produced to limit neuromuscular impairments.

**Keywords:** huwentoxin-IV; selectivity; sodium channel

*6.17. Characterization of a Highly Neutralizing Single Monoclonal Antibody to Botulinum Neurotoxin Type A*

**Christine Rasetti-Escargueil 2,\*, Sébastien Brier 1, Anne Wijkhuisen 3, Stéphanie Simon 3, Maud Marechal 2, Émmanuel Lemichez 2, Michel R. Popoff <sup>2</sup>**

<sup>1</sup> Biological NMR Technological Plateform, Institut Pasteur, CNRS UMR 3528, 75015 Paris, France.

<sup>2</sup> Institut Pasteur, Toxines Bactériennes, CNRS ERL6002, 75015 Paris, France.

<sup>3</sup> Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé, 91191 Gif-sur-Yvette, France.

\* Correspondence: christine.rasetti-escargueil@pasteur.fr

**Abstract:** Monoclonal antibodies (mAbs) represent an alternative and safer way to treat botulism, a fatal flaccid paralysis due to botulinum neurotoxins (BoNTs). In addition, mAbs offer the advantage of being produced in a reproducible manner. We previously identified a unique and potent mouse mAb (TA12) targeting BoNT/A1 with high affinity and neutralizing activity. **Methods**—We characterized the molecular basis of TA12 neutralization by combining Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) with site-directed mutagenesis and neutralization studies. **Results**—The TA12 recognized a conformational epitope located at the interface between the HCN and HCC subdomains of the BoNT/A1 receptor-binding domain (HC). This interface shares common structural features with the ciA-C2 VHH epitope and lies on the face opposite recognized by ciA-C2 and the CR1/CR2 neutralizing mAbs. The single substitution of N1006 was sufficient to affect TA12 binding to HC, confirming the position of the epitope. We further uncovered that the TA12 epitope overlaps with the BoNT/A1 binding site for both the neuronal receptor synaptic vesicle glycoprotein 2 isoform C (SV2C) and the GT1b ganglioside. In vivo neutralization studies confirmed that TA12 is highly neutralizing the most potent subtype BoNT/A1 with medium neutralizing activity against BoNT/A2 and A3 subtypes and low neutralizing activity against BoNT/A5. **Conclusion**—Hence, TA12 potently blocks the entry of BoNT/A1 into neurons by interfering simultaneously with the binding of SV2C and, to a lower extent, GT1b. Our study reveals the unique neutralization mechanism of TA12 and emphasizes the benefit of single mAbs to treat botulism type A.

**Keywords:** botulinum neurotoxin; epitope; monoclonal antibody

*6.18. Steatoda nobilis: A Comparative Study including Full-Body MALDI Imaging and Application to the Venomics Field*

**Damien Redureau 1, Mathieu Tiquet 1, John P. Dunbar 2, Fernanda Gobbi Amorim 1, Michel M. Dugon 2, Loïc Quinton 1,\***

<sup>1</sup> Mass Spectrometry Laboratory, MolSys RU, University of Liège, 4000 Liège, Belgium.

<sup>2</sup> Venom Systems & Proteomics Lab, School of Natural Sciences, Ryan Institute, National University of Ireland Galway, H91 TK33 Galway, Ireland.

\* Correspondence: loic.quinton@uliege.be

**Abstract:** The Noble false widow spider *Steatoda nobilis* is a member of the Theridiidae family, akin to the "true" Black widows of the genus Latrodectus. *S. nobilis* is rapidly expanding its geographic range throughout Europe and parts of the Americas, particularly in and around human dwellings. *S. nobilis* has also been shown to be of medical significance in the UK and in Ireland, where a growing number of severe cases of envenomation have occurred over the past five years [Dunbar, 2021]. The comparison of venom composition of male and female *S. nobilis*, using a proteo-transcriptomic approach, demonstrates that male venom contained a lower quantity and diversity of latrotoxins. In order to illustrate the comparison of both male and female *S. nobilis* profiles, we additionally present the first whole-body imaging of a spider using MALDI mass spectrometry. This proof-of-concept allows one to compare the anatomy of females and males based on molecular markers (specific m/z distribution on spider slices). This is the first time this approach has been performed on a spider and could allow identifying regionalization on toxin production in venom glands when applied to other venomous animal groups. In the next year, this technology could also drive the identification of the spatial activity of toxin in tissue slice after envenomation by comparing the distribution of toxin in the tissue and the spatial distribution of substrate of some toxin/enzyme or again markers of inflammatory response and cellular death.

**Keywords:** MALDI imaging; *Steatoda nobilis*; venomics

#### *6.19. Characterization of Innovative Animal Toxins for the Functional Study of Angiotensin Receptors*

**Anne-Cécile Van Baelen 1,\*, Denis Servent 1, Nicolas Gilles 1, Philippe Robin <sup>1</sup>** Université Paris-Saclay, CEA, Département Médicaments et Technologies pour la Santé (DMTS), SIMoS, F-91191 Gif-sur-Yvette, France.

#### \* Correspondence: anne-cecile.vanbaelen@cea.fr

**Abstract:** An overactivation of the renin–angiotensin system (RAS) with an increase in angiotensin II level is the main cause of arterial hypertension. AT1 receptor antagonists are the goal standard for the treatment of this pathology. Nevertheless, other RAS-related GPCRs (AT2, Mas and MrgD) are also involved in the regulation of blood pressure but are under-exploited due to the lack of knowledge of their physiological properties. Furthermore, the development of biased ligand activating only certain intracellular signaling pathways is in full swing. The discovery of new ligands with original functions would be of great interest to better understand these receptors. Toxins from venomous animals are reticulated peptides that display antagonist, agonist or allosteric functions on GPCRs, and because of their high target specificity and selectivity, they constitute a large reservoir of unexploited promising ligands. The screening of an 800 toxin library was performed on the AT2 receptor by the mean of a radioligand binding assay, and it highlighted three original toxins which originate from different organisms with different structural profiles. These toxins display a micromolar affinity on AT2 and AT1 receptors, and so far, pharmacological trials are more advanced on this receptor. A-CTX-cMila is a small cyclic toxin of seven residues from the cone snail *Conus miliaris*, demonstrating a competitive antagonism on AT1 receptor with micromolar affinity, blocking both G-protein and β-arrestin pathways. A-CTX-MilVa, from the same cone snail, is composed of 20 residues and seems to be an AT1 agonist activating Gq protein pathway. Finally, A-TRTX-Por1a from the spider *Poecilotheria ornata*, is a three disulfide bridges peptide of 29 residues, which also appears to display an agonist activity on the AT1 receptor. These preliminary results describe the first animal toxins active on the RAS and highlight animal venoms as a prolific source of angiotensin receptor binders. A-CTX-cMila, A-CTX-MilVa and A-TRTX-Por1a, with their interesting structural and pharmacological profiles, may represent promising tools for the study of angiotensin receptors. More investigations are needed to characterize these toxins fully, especially on AT2. This work highlights a new family of GPCRs targeted by toxins.

**Keywords:** angiotensin; GPCR; toxin

*6.20. Mast Cells Modulate the Immune Response and Redox Status of Intestinal Tissue in Induced Venom Pathogenesis*

#### **Nehla Zerarka-Chabane \*, Fatima Laraba-Djebari, Djelila Hammoudi-Triki**

University of Science and Technology Houari Boumediene (USTHB), Faculty of Biological Sciences, Laboratory of Cellular and Molecular Biology, Bab-Ezzouar, 16111 Algiers, Algeria. \* Correspondence: chabane.nehla88@gmail.com

**Abstract:** The inflammatory response caused by scorpion venoms components is a key event in the pathogenesis of scorpion envenomation. The binding of neurotoxins on their targets leads to a massive release of inflammatory mediators, such as cytokines, eicosanoids and bioactive amines from activated immune cells. Moreover, mast cells undergo maturation and polarization in gut mucosal surfaces and can alter intestinal permeability, an important factor in many inflammatory mucosal disorders. The c-Kit ligand, stem cell factor (SCF), is a major regulator of mast cell migration, development, growth, survival and activation. This study was conducted to investigate the effect of mast cells in modulating intestinal inflammation induced by scorpion venom. NMRI mice were injected by a sublethal dose of *Androctonus australis hector* (Aah) venom (0.5 mg/kg, subcutaneously) and sacrificed 24 h after envenomation; blood and organs were then collected for further analysis. The assessment of inflammatory response and the evaluation of redox status biomarkers were performed in the small intestine and colonic homogenates. Hematoxylin– eosin staining was used for general histological assessment, and toluidine blue staining was performed for identification and quantification of mast cells. The results revealed that Aah venom induced a significant increase in inflammatory cell infiltration, nitrite oxide levels and malondialdehyde (MDA) concomitantly with a significant decrease in catalase and glutathione S-transferase activities companied by intestinal tissue alteration. Furthermore, the percentage of mast cells was increased in the peritoneal cavity. Pharmacological inhibition of tyrosine kinase receptors alleviated these alterations. These findings could help to better understand the mechanisms involved in scorpion envenoming syndrome and develop potential drugs targeting mast cells for the management of inflammatory disorders of the gut.

**Keywords:** mast cell; redox status; scorpion venom

**Author Contributions:** All authors equally contributed to the conceptualization, the writing original draft preparation and the writing—review and editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** Not applicable.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
