**1. Acknowledgments**

We warmly acknowledge the contribution of all those people who work daily at ensuring the national and international shinning of the French Society of Toxinology (SFET) and those who made the 27th Meeting on Toxinology a success. We also address special thanks to our sponsors who, this year again, supported our meeting (Figure 1).

**Figure 1.** Sponsor logos.

**Citation:** Ladant, D.; Marchot, P.; Diochot, S.; Prévost, G.; Popoff, M.R.; Benoit, E. Report from the 27th (Virtual) Meeting on Toxinology, "Toxins: Mr Hyde or Dr Jekyll?", Organized by the French Society of Toxinology, 9–10 December 2021. *Toxins* **2022**, *14*, 110. https://doi.org/ 10.3390/toxins14020110

Received: 11 January 2022 Accepted: 25 January 2022 Published: 1 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **2. Preface**

Toxins are biologically active substances produced by most kinds of living organisms, bacteria, fungi, plants and animals. They present a vast diversity of molecular structures and target a wide variety of receptors involved in a range of physiological processes. As toxins were selected during evolution to acquire/improve their disabling/lethal effect, they display finely tuned functional properties often associated with high affinities and selectivity. Moreover, toxins are valuable tools to unravel cellular processes due to their extreme specificity for cell surface and/or intracellular targets. Therefore, toxins are very attractive compounds because of their Janus-like character, being both poisons and remedies, and as such, they have been primarily investigated not only for the light they can throw on fundamental physiological processes but also for their potential therapeutic applications.

This 27th Meeting on Toxinology of the SFET was held on 9–10 December 2021 as a virtual meeting (e-RT27). The central theme selected for this meeting, "Toxins: Mr Hyde or Dr Jekyll?", gave rise to three thematic sessions: the first one on plant toxins, algal toxins and mycotoxins; the second one on animal toxins; and the third one on bacterial toxins, all sessions were aimed at emphasizing the latest findings on this topic. Apart from this central theme, a "miscellaneous" session was dedicated to recent results obtained in Toxinology. Ten speakers from seven countries (Australia, Brazil, Burkina Faso, France, Germany, the Netherlands and the United States of America) were invited as international experts to present their work, and other researchers and students presented theirs through 15 shorter lectures and 20 posters. Of the ca. 80 participants who registered, 38% were foreigners, a value highlighting the international attractiveness of the SFET meetings.

Owing to a donation from MDPI-Toxins, two prizes of EUR 300 each were awarded to the best oral communication and the best poster (Figure 2), both selected from a vote by all the participants to the meeting.

**Figure 2.** Winners of the "Best Oral Communication" and "Best Poster" Awards at the 27th Meeting of the French Society of Toxinology (SFET).

Last but not least, we warmly thank the Editors of MDPI—Toxins for supporting the publication of a Special Issue also entitled "Toxins: Mr Hyde or Dr Jekyll?", and gathering this meeting report, along with peer-reviewed original articles and reviews. We believe that this Special Issue will be attractive to all, including those of our colleagues who could not attend the e-RT27 meeting, and that it will represent a comprehensive source of information for researchers and students in Toxinology.

#### **3. Scientific and Organizing Committees (SFET Board of Directors)**

Julien Barbier, CEA de Saclay, Gif-sur-Yvette, France Evelyne Benoit, CEA de Saclay, Gif-sur-Yvette, France Alexandre Chenal, Institut Pasteur, Paris, France Michel De Waard, L'institut du thorax, Nantes, France Sylvie Diochot, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France Sébastien Dutertre, Institut des Biomolécules Max Mousseron, Montpellier, France Daniel Ladant, Institut Pasteur, Paris, France Christian Legros, Université d'Angers, Angers, France Pascale Marchot, CNRS/Aix-Marseille Université, Marseille, France Gilles Prévost, Institut de Bactériologie, Université de Strasbourg, France Michel R. Popoff, Institut Pasteur, Paris, France Loïc Quinton, Université de Liège, Liège, Belgium

#### **4. Invited Lectures (When More Than One Author, the Underlined Name Is That of the Presenter)**

*4.1. The Plant with the Scorpion Sting: Novel Pain-Causing Toxins from the Australian Giant Stinging Tree*

**Irina Vetter 1,4,\*, Edward K. Gilding 1, Sina Jami 1, Jennifer R. Deuis 1, Mathilde R. Israel 1,2, Peta J. Harvey 1, Aaron G. Poth 1, Fabian B.H. Rehm 1, Jennifer L. Stow 1, Samuel D. Robinson 1, Kuok Yap 1, Darren L. Brown 1, Brett R. Hamilton 3, David Andersson 2, David J. Craik 1, Thomas Durek <sup>1</sup>**

<sup>1</sup> Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.

<sup>2</sup> Wolfson Centre for Age-Related Diseases, Institute of Psychiatry, Psychology & Neuroscience, King's College London, London, SE5 8AF, United Kingdom.

<sup>3</sup> Centre for Advanced Imaging, The University of Queensland, Brisbane, QLD 4072, Australia.

<sup>4</sup> School of Pharmacy, The University of Queensland, Brisbane, QLD 4102, Australia.

\* Correspondence: i.vetter@uq.edu.au

**Abstract:** Stinging Trees from Australasia produce remarkably persistent and painful stings upon contact of their stiff epidermal hairs, called trichomes, with mammalian skin. *Dendrocnide*-induced acute pain typically lasts for several hours, and intermittent painful flares can persist for days and weeks. Our recent work shows that the venoms of Australian *Dendrocnide* species contain heretofore-unknown pain-inducing peptides that potently activate sensory neurons and delay the inactivation of voltage-gated sodium channels. These neurotoxins localize specifically to the stinging hairs and are mini-proteins of 4 kDa whose 3D structure is stabilized in an inhibitory cystine knot motif, a characteristic shared with neurotoxins found in spider and cone snail venoms. Our results provide an intriguing example of inter-kingdom convergent evolution of animal and plant venoms with shared modes of delivery, molecular structure and pharmacology.

**Keywords:** pain; peptide toxin; sodium channel; stinging nettle

*4.2. Evidences That Pinnatoxin-G Crosses Intestinal, Hemato-Encephalic and Maternofetal Barriers to Reach Central and Peripheral Nicotinic Acetylcholine Receptors*

**Denis Servent 1,\*, Carole Malgorn 1, Sophie Gil 2, Christelle Simasotchi 2, Anne-Sophie Hérard 3, Thierry Delzescaux 3, Robert Thai 1, Peggy Barbe 1, Mathilde Keck 1, Fabrice Beau 1, Armen Zakarian 4, Vincent Dive 1, Jordi Molgó <sup>1</sup>**

<sup>1</sup> Université Paris-Saclay, CEA, Département Médicaments et Technologies pour la Santé (DMTS), Service d'Ingénierie Moléculaire pour la Santé (SIMoS), ERL CNRS 9004, F-91191 Gif-sur-Yvette, France.

<sup>2</sup> Université de Paris, UMR S1139, Faculté de Pharmacie de Paris, France.

<sup>3</sup> Université Paris-Saclay, UMR 9199, CNRS, CEA, MIRCen, Fontenay-aux-Roses, France. <sup>4</sup> University of California Santa Barbara, Department of Chemistry and Biochemistry,

California 93106, USA.

\* Correspondence: denis.servent@cea.fr

**Abstract:** The warming of ocean temperatures and the increase in nutrients has driven an intensification of harmful algal bloom events. During these active dinoflagellate proliferations, phycotoxins may accumulate in shellfish tissues and can be transferred into fish, marine mammals and ultimately to humans. Cyclic imines, produced by various species of marine dinoflagellates, constitute a widely distributed group of phycotoxins, including pinnatoxins. Pinnatoxin-G (PnTx-G) produced by the cosmopolitan dinoflagellate *Vulcanodinium rugosum* is considered the precursor of other pinnatoxins, and it is a fast-acting toxin in the mouse bioassay. PnTx-G is regularly detected in European coastal environments and in contaminated shellfish samples and may represent a human health risk. In this work, exploiting the ability of PnTx-G to be chemically synthesized and radiolabeled, we studied in vivo the toxicokinetics of [3H]-PnTx-G and its capacity to interact with neuronal and muscle-type nicotinic acetylcholine receptors (nAChRs). The biodistribution of [3H]-PnTx-G, using high-resolution digital radio-imaging after oral or intravenous administration to rats, revealed the presence of the radiolabeled toxin in various peripheral organs, as well as in specific regions of the central nervous system, highlighting its property to cross both the intestinal and blood–brain barriers. In addition, we demonstrate that PnTx-G crosses the materno–fetal barrier, first in rats by detecting the radiolabeled toxin in embryos after its injection to pregnant rats and secondly in humans, using a perfused ex vivo cotyledon model and mass spectrometry analysis. Furthermore, to assess the nAChR subtypes labeled by [3H]-PnTx-G, we performed competition experiments on brain and embryos sections in the presence of selective nAChR antagonists, revealing the major role of muscle-type and α7 subtype in the peripheral and central labeling, respectively. In conclusion, this work shows that PnTx-G efficiently crosses the intestinal, blood–brain and placental barriers to interact with central and peripheral nAChRs, supporting its in vivo effects. The mechanism used by pinnatoxins to cross these physiological barriers and the possible involvement of a receptor-mediated process are still under investigation.

**Keywords:** biodistribution; nicotinic acetylcholine receptor; pinnatoxin

*4.3. A Blue Mountains Funnel-Web Spider Toxin Expressed under Control of a Hemolymph-Specific Promoter Increases Fungal Lethality against Insecticide-Resistant Malaria-Vector Mosquitoes*

## **Étienne Bilgo 1,\*, Brian Lovett 2, Raymond St. Leger 2, Abdoulaye Diabate <sup>1</sup>**

<sup>1</sup> Institut de Recherche en Sciences de la Santé/Centre Muraz, Bobo-Dioulasso, Burkina Faso.

<sup>2</sup> Department of Entomology, University of Maryland, College Park, MD 20742, USA.

\* Correspondence: bilgo02@yahoo.fr

**Abstract:** The continued success of malaria control efforts requires the development, study and implementation of new technologies that circumvent insecticide resistance. We previously demonstrated that fungal pathogens could provide an effective delivery system

for mosquitocidal or malariacidal biomolecules. In this study, we firstly compared genes from arthropod predators encoding insect-specific sodium, potassium and calcium channel blockers for their ability to improve the efficacy of Metarhizium against wild-caught, insecticide-resistant anophelines. Toxins expressed under the control of a hemolymphspecific promoter increased fungal lethality to mosquitoes at spore dosages as low as one conidium per mosquito. One of the most potent, the EPA-approved Hybrid (Ca2+/K+ channel blocker), was studied for pre-lethal effects. These included reduced blood-feeding behavior, with almost 100% of insects infected with ca. six spores unable to transmit malaria within five days post-infection, surpassing the World Health Organization threshold for successful vector control agents. Furthermore, recombinant strains co-expressing Hybrid toxin and AaIT (Na<sup>+</sup> channel blocker) produced synergistic effects, requiring 45% fewer spores to kill half of the mosquitoes in five days as single toxin strains. Secondly, through a semifield trial in a MosquitoSphere (a contained, near-natural environment) in Soumousso, a region of Burkina Faso where malaria is endemic, we confirmed the proof of our concept in the field. The expression of Hybrid toxin increased fungal lethality and the likelihood that insecticide-resistant mosquitoes would be eliminated from this site. In summary, our results identified a repertoire of toxins with different modes of action that improve the utility of entomopathogens as a technology that is compatible with existing insecticide-based control methods.

**Keywords:** biotechnology; malaria; mosquito; mycology; toxin

*4.4. Bee Venom and Its Component Apamin Have Anti-Parkinsonian Properties in Animal Models of Parkinson's Disease*

## **Marianne Amalric 1,\*, Nathalie Turle-Lorenzo 1, Christophe Melon 2, Nicolas Maurice <sup>2</sup>**

<sup>1</sup> Laboratoire de Neurosciences Cognitives (LNC) UMR 7291, CNRS, Aix-Marseille Univ., Marseille, France.

<sup>2</sup> Institut de Biologie du Développement de Marseille (IBDM) UMR 7288, NeuroMarseille, Aix-Marseille Univ., CNRS, Marseille, France.

\* Correspondence: marianne.amalric@univ-amu.fr

**Abstract:** Parkinson's disease (PD) is an age-related neurological disorder that affects more than 6.3 million people worldwide. PD is characterized by progressive loss of dopaminergic (DA) neurons in the substantia nigra (SN), which leads to a wide range of debilitating motor, cognitive and neuropsychiatric symptoms. L-DOPA therapy is effective on motor symptoms and motor fluctuations but leads over the years to dyskinesia. Calcium-activated potassium channels (SK channels) have recently emerged as alternative therapeutic targets because they regulate the neuronal firing of midbrain dopamine neurons. SK channels (in particular SK2/SK3 subtypes) are highly expressed in the basal ganglia (BG). These brain structures are dysregulated by DA neuronal degeneration. Potassium channels are privileged targets because they regulate neuronal excitability in order to maintain balance in the input-output circuits of the BG. Our study aimed to assess the behavioral effects of apamin, a selective SK2/SK3 channel blocker peptide extracted from bee venom, and compare them with those produced by bee venom, in pharmacological and 6-hydroxydopamine (6-OHDA) lesional models of PD in rats. Both bee venom and apamin reversed haloperidol-induced catalepsy, a model of akinetic symptoms, while a co-treatment with the SK opener CYPPA prevented this antiakinetic effect. After extensive unilateral 6-OHDA nigrostriatal lesions, acute and subchronic administration of bee venom and apamin reduced forelimb asymmetry in the cylinder test and apomorphine-induced rotations revealing an antiparkinsonian action on motor symptoms. In another rat model of partial bilateral DA nigrostriatal lesions, apamin also reduced deficits revealed on anxiety, social interaction and visuospatial memory. The neural substrates of these effects were investigated by in vivo electrophysiological recordings of neuronal activity of the BG output structure substantia nigra pars reticulata (SNr). Bee venom restored the balance between the inhibitory and excitatory influence exerted by the trans-striatal direct and indirect

pathways that were disrupted by the pharmacological blockade of DA receptors. These results suggest that bee venom and apamin restore the functional properties of the basal ganglia circuitry in PD conditions and emphasize the crucial role of potassium channels (Ca2-dependent) in these anti-parkinsonian effects. Supported by CNRS, AMU, Fondation de France, Association France Parkinson.

**Keywords:** bee venom; dopamine; Parkinson's disease

*4.5. Slithering Stem Cells—Understanding Snake Venom Production Using Organoids*

**Jens Puschhof 1,2,\*, Yorick Post 1, Joep Beumer 1, Harald M. Kerkkamp 3, Julien Slagboom 4, Buys De Barbanson 1,2, Nienke R. Wevers 5, Xandor Spijkers 5,6, Thomas Olivier 5, Taline D. Kazandijan 7, Stuart Ainsworth 7, Carmen Lopez Iglesias 8, Willine van de Wetering 1,8, Maria C. Heinz 2,9, Ravian L. Van Ineveld 2,10, Regina G.D.M. Van Kleef 11, Harry Begthel 1, Jeroen Korving 1, Yotam E. Bar-Ephraim 1,2, Walter Getreuer 12, Anne C. Rios 2,10, Remco H.S. Westerink 11, Hugo J.G. Snippert 2,9, Alexander Van Oudenaarden 1,2, Peter J. Peters 8, Freek J. Vonk 3, Jeroen Kool 4, Michael K. Richardson 3, Nicholas R. Casewell 7, Hans Clevers 1,2,10**

<sup>1</sup> Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, 3584 CT Utrecht, The Netherlands.

<sup>2</sup> Oncode Institute, Hubrecht Institute, 3584 CT Utrecht, The Netherlands.

<sup>3</sup> Institute of Biology Leiden, Department of Animal Science and Health, 2333 BE Leiden, The Netherlands.

<sup>4</sup> Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit Amsterdam, 1081 LA Amsterdam, The Netherlands.

<sup>5</sup> Mimetas BV, Organ-on-a-chip Company, 2333 CH Leiden, The Netherlands.

<sup>6</sup> Department of Translational Neuroscience, University Medical Center, 3584 CG, Utrecht, The Netherlands.

<sup>7</sup> Centre for Snakebite Research & Interventions, Parasitology Department, Liverpool School of Tropical Medicine, Liverpool, L3 5QA, UK.

<sup>8</sup> The Maastricht Multimodal Molecular Imaging institute, Maastricht University, 6229 ER Maastricht, The Netherlands.

<sup>9</sup> Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, 3584 CX Utrecht, The Netherlands.

<sup>10</sup> The Princess Maxima Center for Pediatric Oncology, 3584 CS Utrecht, The Netherlands. <sup>11</sup> Neurotoxicology Research Group, Division of Toxicology, Institute for Risk Assessment Sciences (IRAS), Utrecht University, 3584 CL Utrecht, The Netherlands.

<sup>12</sup> Serpo, 2288 ED Rijswijk, The Netherlands.

\* Correspondence: jens.puschhof@dkfz-heidelberg.de

**Abstract:** Recent advances in organoid technology have proven this system to be a valuable tool in understanding human organ development and pathologies. These adult stem cellderived cultures closely recapitulate the structural and functional properties of their organ of origin. Here, we expand the organoid technology toolbox by describing a protocol to culture non-mammalian organoids derived from a snake venom gland. The complexity of venom production, composition and function remain largely unknown for many species. Organoids derived from an *Aspidelaps lubricus* venom gland can be long-term expanded and histologically resemble the gland. Expression of typical venom-related transcripts (threefinger toxins and Kunitz-type protease inhibitors) can be detected in proliferating organoids with RNA sequencing. Single-cell RNA sequencing reveals distinct venom-expressing cell types, as well as proliferating cells with features of mammalian stem cells. By using mass spectrometry, we identified peptides in the culture medium supernatant that match the composition of the crude venom of the same species. Venom gland organoids furthermore consist of specialized secretory cells visible by transmission electron microscopy. The system enables the investigation of venom production and function on a cellular level in controlled conditions and without the need for experimental animals. This study describes

the adaption of organoid technology to a non-mammalian species, providing a model to understand the complexity of the snake venom gland.

**Keywords:** in vitro; organoid; snake; venom gland

*4.6. Prospection of Animal Toxins in Drug Discovery—Challenges and Perspectives*

#### **Karla de Castro Figueiredo Bordon \***

University of São Paulo, School of Pharmaceutical Sciences of Ribeirão Preto, Ribeirão Preto, São Paulo, Brazil.

\* Correspondence: karlabordon@yahoo.com.br

**Abstract:** Venomous animals may cause severe medical complications and untimely death, but their venoms are also sources of molecules acting on the nervous, cardiovascular, immune and other systems. Animal toxins are often used as pharmacological tools for the validation of therapeutic targets, but they are also used as cosmeceuticals and diagnostic tools in the design of new therapeutic agents and to improve drug libraries. Some drugs used in the therapy of many disorders, such as diabetes and cardiovascular diseases, were developed based on the molecular structures of animal toxins. Captopril was the first animal toxin-based drug approved for human use. Scorpion neurotoxins are known to be responsible for the pathological manifestations of scorpionism. They are a threat to human health but may serve as leads for the development of new therapies against current and emerging diseases. The scorpion toxin chlorotoxin is undergoing clinical phase trials as a fluorescent molecular probe that paints tumors, while the CPP-Ts peptide is a potential intranuclear delivery tool targeting cancer cells. The bioprospection of neurotoxins that block KV1.3 potassium channels may lead to the development of drugs to treat autoimmune diseases since these channels are found in macrophages, platelets and T cells. The overexpression of KV1.3 in lymphocytes is related to diseases such as atherosclerosis, some types of cancers, obesity and autoimmune diseases such as Crohn's disease, dermatitis, psoriasis and rheumatoid arthritis, among others. Related to snake toxins, a heterologous fibrin sealant, comprising a cryoprecipitate rich in fibrinogen extracted from the blood of buffaloes in association with a serine protease from rattlesnake, is a biodegradable biological product that reduces surgical time, promotes faster postoperative recovery, is highly adhesive and can also be used as an adjuvant in suture procedures and as a carrier for drug delivery. The use of biotechnological tools, such as the heterologous expression of peptides and proteins, enables the production of biologically active molecules in sufficient quantity for the evaluation and development of new medicines. Recently, a serine protease was recombinantly expressed with functional and structural integrity and showed fibrinogenolytic activity and inhibition against the hEAG1 channel, highly expressed in tumor cells, in a mechanism independent of its catalytic activity. Non-PEGylated and PEGylated forms of this enzyme share similar kinetic and functional characteristics, and the latter showed no evidence of immunogenicity or cytotoxicity, even at high concentrations. Given the examples, approaches to improve the druggability of animal toxins are fruitful fields for future research.

**Keywords:** biological dressing; heterologous expression; immunosuppression; PEGylation; poison; toxin

#### *4.7. Redirecting the Target of Muscarinic Toxin*

**Shoji Maeda 1,7,\*, Jun Xu 1, Francois Marie N. Kadji 2, Mary J. Clark 3, Jiawei Zhao 4, Naotaka Tsutsumi 5, Junken Aoki 2, Roger K. Sunahara 3, Asuka Inoue 2, K. Christopher Garcia 1,5,6, Brian K. Kobilka <sup>1</sup>**

<sup>1</sup> Stanford University School of Medicine, Stanford, CA 94305, USA;

<sup>2</sup> Tohoku University, Sendai, Japan;

<sup>3</sup> University of California San Diego School of Medicine, La Jolla, CA 92093, USA;

<sup>5</sup> Stanford University School of Medicine, Stanford, CA 94305, USA;

<sup>6</sup> Howard Hughes Medical Institute; <sup>7</sup> University of Michigan, MI 48109, USA.

\* Correspondence: shojim@umich.edu

**Abstract:** Muscarinic acetylcholine receptors are prototypical class-A GPCRs that are distributed throughout the human body and play critical roles in the maintenance of fundamental human physiology by responding to the neurotransmitter acetylcholine. Five muscarinic receptor subtypes (M1R–M5R) have been identified in humans with distinct G protein coupling preferences, distribution profile and physiological roles. With their conserved orthosteric ligand-binding site and the presence of an allosteric ligand-binding site at the extracellular region, muscarinic receptors were extensively studied as a model system for the subtype-selective targeting at a GPCR through small molecule allosteric modulators. Animals have evolved venoms to hunt prey and/or run from predators. These venoms have been a rich source to isolate natural products that modulate numerous protein functions, including GPCRs. Peptide and small protein toxins are emerging modalities because of their superior selectivity and stability. Muscarinic toxin belongs to the three-finger toxin family and is one of the best-characterized peptide toxins produced by venomous snakes. A member of muscarinic toxin, MT7, shows extremely high selectivity to M1R over other muscarinic receptors and elicits an inhibitory effect. We solved the crystal structure of the M1R–MT7 complex and revealed the exquisite design of this toxin to exclusively fit into M1R by selectively engaging at the allosteric site with residues unique to M1R. By using the structural information and the surface display platform, we succeeded in converting the subtype selectivity of MT7 to another muscarinic receptor, M2R. Furthermore, we obtained conformational selective three-finger proteins exclusively targeting the active conformation of muscarinic receptors. Our data indicate a promise of repurposing this natural toxin scaffold to a broader range of target systems.

**Keywords:** muscarinic acetylcholine receptor; protein engineering; three-finger toxin

*4.8. Synthetic AB-Toxins for Targeted Pharmacological Modulation of Cancer and Immune Cells* **Holger Barth \***

University of Ulm Medical Center, Institute of Pharmacology and Toxicology, Albert-Einstein-Allee 11, 89081 Ulm, Germany.

\* Correspondence: holger.barth@uni-ulm.de

**Abstract:** Bacterial AB-toxins are highly toxic proteins because of their unique modular structure: these toxins bind to mammalian cells by a specific receptor binding (B) subunit and deliver their enzymatically active (A) subunit into the host cell cytosol via their intrinsic translocation (T) subunit. In the cytosol, the A subunit modifies its specific cellular substrate, which alters cellular structures and/or functions and is the reason for the clinical symptoms of the particular toxin-associated disease. Because of their intracellular, highly specific and extremely potent mode of action, some AB-toxins serve as valuable molecular tools in pharmacology and cell biology for the targeted modulation of cell functions and are used as drugs. In past years, we and others used the non-toxic B and T subunits of various toxins to deliver pharmacologically active proteins (e.g., enzymes) and peptides into the cytosol of cells and developed cell type-selective transport systems to modulate functions of immune and cancer cells. However, for some cell types, there are no selective AB toxins available by nature, and therefore, we aimed to develop "synthetic" AB-toxins. In one approach, supramolecular transporter systems were generated, where avidin with four biotin-binding sites serves as a central binding platform for three biotinylated binding peptides that selectively bind to receptors on target cells and trigger internalization of the transporter molecule, and biotinylated cargo molecules that act as "A subunit" in the cytosol. Proofof-concept was provided by using clostridial C3 Rho-inhibitor as cargo enzyme and the peptide somatostatin as a ligand to selectively target human lung cancer cells in vitro and in

<sup>4</sup> Tsinghua University, Beijing, China;

the HET/CAM model for a human xenograft lung tumor that overexpresses somatostatin receptor. This strategy also served for the generation of a novel cell type-selective Rhoinhibitor for human neutrophils. This universal "LEGO-like" modular approach represents a promising platform for the generation of cell-type selective synthetic AB-toxins for the targeted pharmacological modulation of cell functions.

**Keywords:** bacterial AB-toxin; cellular uptake; targeted drug delivery; receptor binding; membrane translocation; avidin-biotin technology; (semi)synthetic AB-type molecule

*4.9. Phenotypic Screening for Anti-Toxin Molecules Selects Inhibitors of Intracellular Trafficking with Broad-Spectrum Anti-Infectious Properties*

#### **Daniel Gillet \***

Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SIMoS, 91191 Gif-sur-Yvette, France. \* Correspondence: daniel.gillet@cea.fr

**Abstract:** In the aftermath of the anthrax letter attacks in the fall of 2001, the French authorities mandated the French health products sanitary safety agency (AFSSAPS) to determine the needs for medical countermeasures (MCM) against biothreat agents. A working group under the lead of Prof. Dominique Dormont established the cruel lack of medications against bacterial and plant toxins prone to be used as bioweapons, among which the plant toxin ricin. Then, the growing concerns about antimicrobial resistance led to suggest that antitoxin drugs should be developed to treat common public health bacterial infections as an alternative to antibiotics. In these respects, we developed two phenotypic screenings of small chemical molecules in 2005 and 2015; the first was against ricin toxin, and the second (in collaboration with Emmanuel Lemichez, Institut Pasteur) was against the cytotoxic necrotizing factor CNF1, produced by uropathogenic and extra-intestinal strains of *Escherichia coli*. The first screening was designed to select molecules capable of rescuing cells from ricin-induced toxicity. The second screening was designed to select molecules inhibiting the degradation of CNF1-induced activated Rac1. Overall, the anti-ricin screening identified three original inhibitors of intracellular trafficking. Retro-1 and Retro-2 act on the retrograde trafficking pathway (collaboration with Ludger Johannes, Institut Curie). In particular, Retro-2 interferes with the ERES protein Sec16A and the circulation of syntaxin-5 along the anterograde and retrograde routes. ABMA acts on the endolysosomal pathway by the accumulation of late endosomes and alteration of the autophagic flux. The CNF1 screening identified C910 as an original inhibitor of early endosome sorting function. Most interestingly, all four inhibitors displayed a broad spectrum of inhibition against a series of bacterial toxins, but also viruses, bacteria and parasites exploiting these pathways for intoxication or infection of cells. There are at least eight examples published or under the submission of in vivo protection of mice by these compounds against intoxications and/or infections. Compound optimization by medicinal chemistry was performed. The challenges of turning these molecules into drugs are discussed. Nevertheless, 16 years of research by us and others showed that intracellular trafficking pathways might be interesting druggable targets to fight intracellular toxins and pathogens.

**Keywords:** bacterial toxin; broad-spectrum; intracellular trafficking; ricin toxin; small chemical inhibitor

*4.10. NLRP3 Inflammasome Sensing of RhoGTPase-Activating Toxins during Bacteremia*

#### **Laurent Boyer \***

C3M, INSERM U1065, Université Cote d'Azur, Nice, France. \* Correspondence: boyerl@unice.fr

**Abstract:** The detection of the activities of pathogen-encoded virulence factors by the innate immune system has emerged as a new paradigm of pathogen recognition. Much remains to be determined regarding the molecular components contributing to this defense mechanism in mammals and its importance during infection. Our team showed the central role of the IL-1β signaling axis in controlling the Escherichia coli burden in the blood in response to the sensing of the RhoGTPase-activating toxin CNF1. Using the CNF1 toxin, we provided evidence of the role of the NLRP3 inflammasome in sensing the activity of bacterial toxins and virulence factors that activates host RhoGTPases. We demonstrated that this activation relies on monitoring of the toxin activity on the RhoGTPase Rac2. We also showed that the NLRP3 inflammasome is activated by a signaling cascade involving the P21-activated kinases (Pak)-1/2. The Pak1-mediated phosphorylation of threonine-659 of NLRP3 was necessary for NLRP3-Nek7 interaction, inflammasome activation and IL-1ß cytokine maturation. Furthermore, inhibition of the Pak1-NLRP3 axis diminished the bacterial clearance of CNF1-expressing E. coli during bacteremia. Altogether, our results established Pak1/2 as critical regulators of the NLRP3 inflammasome and revealed the role of the Pak1-NLRP3 signaling axis in vivo during bacteremia.

**Keywords:** bacterial toxin; inflammasome; NLPR3 RhoGTPase

#### **5. Oral Presentations (When More Than One Author, the Underlined Name Is That of the Presenter)**

## *5.1. Screening an In-House Library of Isoquinoline Alkaloids with Fluorescent Probes for Discovering New Ligands of Voltage-Gated Na<sup>+</sup> and Ca2+ Channels*

**Claire Legendre 1,\*, Jacinthe Frangieh 1,2, Léa Réthoré 1, Quentin Coquerel 1, Daniel Henrion 1, César Mattei 1, Ziad Fajloun 2,4, Anne-Marie Le Ray 3, Christian Legros <sup>1</sup>**

<sup>1</sup> Laboratory Mitochondrial and Cardiovascular Pathophysiology (MITOVASC), CNRS UMR 6015—INSERM U1083, CHU Angers, Université d'Angers, Angers, France.

<sup>2</sup> Laboratory of Applied Biotechnology (LBA3B), Azm Center for Research in Biotechnology and its Applications, EDST, Lebanese University, 1300 Tripoli, Lebanon.

<sup>3</sup> SONAS Laboratory, SFR QUASAV, Université d'Angers, Angers, France.

<sup>4</sup> Department of Biology, American University of Beirut, 11-0236 Beirut, Lebanon.

\* Correspondence: claire.legendre@univ-angers.fr

**Abstract:** The isoquinoline alkaloids (IA) is a large chemical group of natural compounds with various structures and acting on multiple pharmacological targets of therapeutical interest, including ion channels, such as voltage-gated Na+ (Nav) and Ca2+ (Cav) channels. These ion channels are established molecular targets for the development of therapeutical agents in cardiovascular diseases and particularly arrhythmia. Here, we screened 62 IA from an in-house vegetal chemical library, using a model of excitable cells, the rat pituitary GH3b6 cells, which endogenously express both Nav and Cav channels, to find new blockers for these channels. Moreover, we demonstrated that Nav activation by selective neurotoxins induced an intracellular Ca2+ concentration [Ca2+]i elevation mediated by Cav channels, highlighting crosstalk between Nav and Cav channels that we used for pharmacological studies. We first tested these IA for their abilities to inhibit batrachotoxin (BTX)-induced depolarization using fluorescent voltage-sensor probes (VSP) and identified two oxoaporphines, namely liriodenine and oxostephanine, which abolished BTX-induced VSP signal with IC50 values in the micromolar range. While the blocking activity of liriodenine was already reported, the activity of oxostephanine on Nav channels has never been described yet. Interestingly, oxostephanine differs from liriodenine only by a methoxy group on the benzyl part of the skeleton. This subclass of IA might constitute a new group of ligands of Nav channels. We confirmed the blocking effect of both molecules in a Na+ influx assay using the Na+ fluorescent probe ANG-2. Since liriodenine is also known to block Cav channels, we hypothesized that oxostephanine probably targets Cav channels. Thus, we investigated the effects of both IA on L-type Cav channels (LTCC) expressed in GH3b6 cells. In order to activate LTCC, we used a chemical depolarization with KCl or the agonist Bay K8644 and monitored [Ca2+]i change with the fura-2 probe. Our results showed that liriodenine and oxostephanine induced a concentration-dependent inhibition

of KCl- and Bay K8644-evoked Ca2+ responses, with similar IC50 values in the micromolar range. In addition, this interaction of liriodenine and oxostephanine on LTCC was also highlighted by their ability to inhibit veratridine (VTD)- and BTX-induced [Ca2+]i elevation. In conclusion, our data showed that liriodenine and oxostephanine, two oxoaporphine alkaloids, inhibit Nav and Cav channels with similar potency. The oxoaporphine skeleton might bring an interesting pharmacophore for structure-function relationship studies for designing more selective ligands toward Cav and Nav channels and for developing new antiarrhythmic therapeutical leads.

**Keywords:** Cav channel; isoquinoline alkaloid; Nav channel

*5.2. The Venom of the Lebanese Viper, Montivipera bornmuelleri, Contains Vasoactive Compounds* **Jacinthe Frangieh 1,2,\*, Joohee Park 1, Ziad Fajloun 2, Loïc Quinton 3, Riyad Sadek 4, Daniel Henrion 1, César Mattei 1, Christian Legros <sup>1</sup>**

<sup>1</sup> Laboratory Mitochondrial and Cardiovascular Pathophysiology—MITOVASC, CNRS UMR 6015, INSERM U1083, CHU Angers, Université d'Angers, Angers, France.

<sup>2</sup> Laboratory of Applied Biotechnology (LBA3B), Azm Center for Research in Biotechnology and its Applications, EDST, Lebanese University, 1300 Tripoli, Lebanon.

<sup>3</sup> Laboratory of Mass Spectrometry, Department of Chemistry, MolSys Research Unit, University of Liège, 4000 Liège, Belgium.

<sup>4</sup> Department of Biology, American University of Beirut, 11-0236 Beirut, Lebanon.

\* Correspondence: jacynthefrangieh@gmail.com

**Abstract:** Snake venoms are rich mixtures of polypeptides that target, among other physiological networks, the cardiovascular system. Snake toxins such as phospholipase A2, natriuretic peptides, and bradykinin-potentiating peptides exert various effects on the cardiovascular system (hypotension, vasorelaxation, etc.). Some of these toxins were developed as drugs with antihypertensive properties, such as Captopril™. We previously showed that the venom of the viper *Montivipera bornmuelleri* induces relaxation of rat aortic rings. Here, we aim to identify and characterize the vasoactive compounds of this venom. We fractionated the venom by HPLC. The fractions were assayed on the endothelial MS1 cell line and CHO cells lines expressing muscarinic receptors M1, M3 or M5. We screened for compounds able to inhibit acetylcholine-induced intracellular Ca2+ rise, using the fluorescence probe FURA-2. In addition, wire myography on isolated mesenteric arteries was used. Proteomic analysis was performed to characterize selected fractions. The crude venom exhibited a vasorelaxant effect on mesenteric arteries. Among 23 fractions of *Mb* venom, one fraction, namely P14, was selected for its ability to reduce acetylcholine-induced Ca2+ rise in MS1 cells. The proteomic analysis allowed us to identify a novel peptidyl toxin (P14) as a homolog of the vascular endothelial growth factor (VEGF). However, P14 was unable to antagonize acetylcholine-induced Ca2+ rise in cells expressing muscarinic receptors, suggesting that this peptide interferes with other target(s) of the Ca2+ signaling pathway in endothelial cells. Further studies will be carried out to characterize the molecular targets of P14 and its effects on vascular function.

**Keywords:** calcium signaling; *Montivipera bornmuelleri* venom; vascular function

*5.3. Lipids and Cholesterol Mediate the Cytotoxicity of Spider Peptides*

**Javier Moral-Sanz 1, Sergey Kurdyukov 2, Ana Vela-Sebastián 1, Zoltan Dekan 3, Thomas Kremsmayr 4, Markus Muttenthaler 3,4, Paul F. Alewood 3, Gregory G. Neely 2, Évelyne Deplazes 3, Maria P. Ikonomopoulou 1,3,\***

<sup>1</sup> Madrid Institute for Advanced Studies in Food, Madrid, E28049, Spain.


<sup>4</sup> University of Vienna, Vienna 1090, Austria.

\* Correspondence: maria.ikonomopoulou@imdea.org

**Abstract:** Spider gomesin peptides target melanoma cells of BRAF-mutation with minimum effect on non-transformed neonatal foreskin fibroblasts. We hypothesized that the selectivity of gomesin peptides towards melanoma of BRAF mutation could be influenced by interactions with lipids or cholesterol present in cell membranes. In order to elucidate upon this, we employed a multidisciplinary approach, including CRISPR/Cas9 genomewide screening, to identify key players underlying the antiproliferative mechanisms of gomesins. We also used a combination of fluorescence spectroscopy to measure membrane dipole changes, electrical impedance spectroscopy (EIS) with tethered bilayer membranes (tBLMs) to quantify alterations in membrane conductance and membrane thickness, and cell viability assays. Gomesin and variants showed weak binding to POPC membranes alone. However, the presence of POPS and cholesterol significantly improved the binding of the peptides and lessened membrane disruption. In addition, the cytotoxicity of gomesin was blunted by increasing concentrations of cholesterol in both melanoma cells and fibroblasts. Conversely, cholesterol depletion potentiated the cytotoxicity of peptides in fibroblasts to almost the levels originally observed in melanoma cells. In conclusion, we postulate a specific role of cholesterol in the selective cytotoxic profile of gomesin in melanoma of BRAF mutation.

**Keywords:** cytotoxicity; melanoma; spider peptide

#### *5.4. ExoU and ExoY, Two Toxins from Pseudomonas aeruginosa with Different Mechanisms of Action and Biological Effects*

## **Vincent Deruelle 1,\*, Dorothée Raoux-Barbot 1, Undine Mechold 1, Philippe Huber <sup>2</sup>**

<sup>1</sup> Unité de Biochimie des Interactions Macromoléculaires, Institut Pasteur, Département de Biologie Structurale et Chimie, CNRS UMR 3528, Paris, France.

<sup>2</sup> Center for Immunology of Viral, Auto-immune, Hematological and Bacterial diseases (IMVA-HB/IDMIT), Université Paris-Saclay, INSERM, CEA, Fontenay-aux-Roses, France. \* Correspondence: vincent.deruelle@pasteur.fr

**Abstract:** *Pseudomonas aeruginosa* is a ubiquitous and opportunistic Gram-negative bacterium. It is a leading cause of nosocomial infections and is responsible for acute and chronic infections thanks to a broad panel of virulence factors. Among them, the Type 3 Secretion System (T3SS) plays an essential role in the pathogenicity of the bacterium. This system pierces the cell membrane and allows the delivery of toxins into the host cytoplasm. Four T3SS effectors have been identified—ExoS, ExoU, ExoT and ExoY. Most strains inject two or three of these effectors simultaneously, with ExoU and ExoS being mutually exclusive. Each T3SS effector is inactive when injected and requires a host cell factor to be activated. Strains injecting ExoU toxin induce the most severe pathologies because the damage caused by the toxin is rapid and irreversible, leaving no time for proper clinical management. Its mechanism of action is well described. Inside host cells, ExoU is a phospholipase that interacts with a specific lipid at the cytosolic side of the plasma membrane, allowing its activation and leading to cell necrosis. However, several aspects of ExoU activation and its trafficking in human cells upon injection remain elusive. Thanks to genetic approaches and analyses by microscopy, we identified a favored anterograde transport, led by the co-chaperone DNAJC5, which targets ExoU to the plasma membrane. Inactivation of DNAJC5 gene disrupts ExoU-dependent toxicity in vitro and in vivo. Unlike ExoU, the role of ExoY in the pathogenicity of *P. aeruginosa* is still controversial. This toxin is a nucleotidyl cyclase that interacts with the eukaryotic filamentous actin (F-actin) to be activated and thereby to massively convert nucleotide triphosphates into their cyclic form. It was reported that ExoS toxicity to the epithelial cells is enhanced in the absence of ExoY and that in many ExoU+ strains, ExoY is not active. Thus, ExoY seems to limit the action of other toxins. We will discuss our advances in exploring the role of ExoY by studying its effects in infected cells.

**Keywords:** CRISPR-Cas9; DNAJC5; ExoU; ExoY; virulence

*5.5. Moving Forward Tetanus Therapy: Two Exceptionally Potent humAbs Are Effective for Prophylaxis and Treatment in Mice*

**Marco Pirazzini 1,\*, Alessandro Grinzato 1, Davide Corti 2, Sonia Barbieri 2, Oneda Leka 1, Francesca Vallese 1, Marika Tonellato 1, Chiara Silacci-Fregni 3, Luca Piccoli 3, Eaazhisai Kandiah 4, Giampietro Schiavo 5,6, Giuseppe Zanotti 1, Antonio Lanzavecchia 3,7, Cesare Montecucco 1,8**

<sup>1</sup> Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58/B, Padova, 35131, Italy.

<sup>2</sup> Humabs BioMed SA, 6500 Bellinzona, Switzerland.

<sup>3</sup> Institute for Research in Biomedicine, Università della Svizzera Italiana, 6500 Bellinzona, Switzerland.

<sup>4</sup> European Synchrotron Radiation Facility, 71 avenue des Martyrs, F-38000 Grenoble, France.

<sup>5</sup> Department of Neuromuscular Diseases, Queen Square Institute of Neurology, University College London, London, WC1N 3BG, UK.

<sup>6</sup> UK Dementia Research Institute, University College London, London, WC1E 6BT UK.

<sup>7</sup> Fondazione Istituto Nazionale Genetica Molecolare c/o Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico di Milano, Via Francesco Sforza 35, 20122 Milano, Italy.

<sup>8</sup> Institute of Neuroscience, National Research Council, Via Ugo Bassi 58/B, Padova, 35131, Italy.

\* Correspondence: marco.pirazzini@unipd.it

**Abstract:** Tetanus neurotoxin (TeNT) is the causative agent of tetanus, a life-threatening disease of vertebrates, including humans, characterized by neurogenic muscle rigidity and spasticity. Although tetanus can be prevented by a very effective vaccine, a worldwide clinical practice in the emergency rooms is the administration of anti-TeNT immunoglobulins (TIG), which are used both for prophylaxis, to avoid tetanus development in wounded patients, and for therapy, to treat patients already carrying tetanus symptoms. TIG is produced from the blood of hyperimmune individuals, either humans or horses (in developing countries). As such, it exposes patients to several possible side effects, including infections by still-unknown pathogens as well as dangerous anaphylactic reactions. Human monoclonal antibodies (humAbs), which are emerging as superior therapeutics against several diseases, could overcome the drawbacks of TIG. Here, we screened the immortalized memory B cells pooled from the blood of immunized human donors and isolated two humAbs, dubbed TT104 and TT110, which display an unprecedented neutralization ability against TeNT. We determined the epitopes recognized by TT104 and TT110 via cryo-EM and defined how they interfere with the mechanism of neuron intoxication. These analyses pinpointed two novel mechanistic aspects of TeNT activity in neurons and unraveled at the same time the molecular bases of TT104 and TT110 exceptional neutralization ability. Crucially, the combination of TT104 and TT110 display a prophylactic activity in mice when injected long before TeNT, and the two Fab derivatives (TT104-Fab and TT110-Fab) neutralize TeNT in post-exposure experiments. Of note, in both these two paradigms of experimental tetanus, the humAbs and the Fabs show an activity fully comparable to TIG. Therefore, TT104 and TT110 humAbs and their Fab derivatives meet all requirements for being considered for prophylaxis and therapy of human tetanus and are ready for clinical trials.

**Keywords:** monoclonal antibody; spastic paralysis; tetanus neurotoxin; tetanus prophylaxis

*5.6. CyaA Toxin Cell Invasion Involves Membrane-Active and Calmodulin-Binding Properties of Its Translocation Domain*

**Alexis Voegele 1, Mirko Sadi 1, Darragh P. O'Brien 1, Pauline Gehan 2, Dorothée Raoux-Barbot 1, Maryline Davi 1, Sylviane Hoos 1, Sébastien Brulé 1, Bertrand Raynal 1, Patrick Weber 1, Ariel Mechaly 1, Ahmed Haouz 1, Nicolas Rodriguez 2, Patrice Vachette 3, Dominique Durand 3, Sébastien Brier 1, Daniel Ladant 1, Alexandre Chenal 1,\***

<sup>1</sup> Institut Pasteur, CNRS UMR 3528, 75015 Paris, France.

<sup>2</sup> Sorbonne Université, École Normale Supérieure, PSL University, CNRS, Laboratoire des biomolécules, LBM, 75005 Paris, France.

<sup>3</sup> Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France.

\* Correspondence: chenal@pasteur.fr

**Abstract:** The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells are still a matter of intense research. *Bordetella pertussis*, the causative agent of whooping cough, produces an adenylate cyclase (CyaA) toxin that plays an essential role in the early stages of respiratory tract colonization. CyaA displays a unique intoxication pathway of human cells via a direct translocation of its catalytic domain (AC) across the plasma membrane. Once in the cytosol, AC impairs the physiology of immune cells, leading to cell death. We showed that the P454 peptide (CyaA residues 454–484) is able to translocate across membranes and interact with calmodulin. The key residues involved in membrane-active and calmodulin-binding properties were identified. The mutational analysis demonstrates that these residues play a crucial role in CyaA translocation into target cells. We propose that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin (Figure 3). Trapping of CyaA by the calmodulin:P454 interaction in the cytosol may assist the entry of AC by converting the stochastic motion of the polypeptide chain through the membrane into an efficient vectorial chain translocation into host cells.

**Figure 3.** The membrane-active segment (yellow) of the CyaA toxin translocates across the plasma membrane and binds calmodulin (red), which assists the entry and refolding of the catalytic domain (blue) into host cells while the hydrophobic and acylation domains (green) interact with the membrane, and the C-terminal Repeat-in-Toxin domain (grey) remains in the extra-cellular milieu. The cAMP production ultimately leads to cell death.

**Keywords:** CyaA; adenylate cyclase toxin; membrane-active peptide; membrane translocation; entropic pulling; calmodulin-binding peptide

#### **References**

1. Voegele, A., Sadi, M., O'Brien, D. P., Gehan, P., Raoux-Barbot, D., Davi, M., Hoos, S., Brûlé, S., Raynal, B., Weber, P., Mechaly, A., et.al. A High-Affinity Calmodulin-Binding Site in the CyaA Toxin Translocation Domain is Essential for Invasion of Eukaryotic Cells. *Adv. Sci.* **2021**, 8, 2003630.


*5.7. Is the Regulation of Toxinogenesis the Same in Clostridium botulinum and Clostridium tetani?*
