Steve Reynaud

Justyna Ciolek

Michel Degueldre

Natalie J Saez

Ana Filipa Sequeira

Yoan Duhoo

Joana L A Brás

Herve Meudal

Miguel Cabo Díez

Victoria Fernández Pedrosa

Herve Meudal

Miguel Cabo Diez

Victoria Fernández Pedrosa

Marion Verdenaud

Julia Boeri

Oscar Pereira Ramos

Frédéric Ducancel

Margot Vanden Driessche

Rudy Fourmy

Aude Violette

Gregory Upert

Gilles Mourier

Annette G Beck-Sickinger

Karin Mörl

Céline Landon

Carlos M G A Fontes

Rebeca Miñambres Herráiz

Ricardo Canek Rodríguez De La Vega

Steve Peigneur

Jan Tytgat

Loïc Quinton

Edwin De Pauw

Renaud Vincentelli

Denis Servent

Nicolas Gilles

Miñambres Herráiz

Ana Filipa Sequeira

Hervé Meudal

Victoria Fernández

Oscar Pereira

Margot Vanden

Violette Aude

Grégory Upert

Annette G Beck- Sickinger

Rebeca Miñambres

Ricardo C Rodríguez De La Vega

A Venomics Approach Coupled to High-Throughput Toxin Production Strategies Identifies the First Venom-Derived Melanocortin Receptor Agonists

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Introduction

It is estimated that more than 200,000 venomous species exist on Earth, found in almost all animal subgroups across a range of diverse habitats [START_REF] Jenner | The Secrets of Nature's Deadliest Weapon, The Natura[END_REF] . Many are terrestrial, such as some vertebrates (e.g., snakes, platypus, lizards) or arthropods (e.g., scorpions, spiders, centipedes, ticks, ants), some marine, such as cnidarians (e.g., sea anemones and jellyfish) or some mollusks (e.g., cuttlefish, octopus, marine cone snails, terebrids, squids), and some aerial including insects (e.g., mosquitoes, bugs, wasps, bees) and vampire bats. Venoms are used for defense and/or predation and are composed, among other components, of peptide toxins. Toxins are small proteins up to one hundred residues in size, reticulated by disulfide bridges. Number of toxins present in venoms depends of the species and of the method used to identify them. In the 1990's, publications mentioned few tens of toxins in Conus venom.

Recently, almost 6,000 toxins were reported in the species Conus lividus 2 . All in all, we can estimate that at least 40 million toxins exist on the planet and of these, less than 5,000 have been pharmacologically characterized. Despite this very small number, a plethora of activities have been reported. Toxins interacting with membrane receptors like voltage-and ligand-gated ion channels [START_REF] Beraud | Therapeutic Potential of Peptide Toxins That Target Ion Channels[END_REF]4 and G-Protein Coupled receptors (GPCR) [START_REF] Servent | Muscarinic Toxins: Tools for the Study of the Pharmacological and Functional Properties of Muscarinic Receptors[END_REF][START_REF] Ciolek | Green Mamba Peptide Targets Type-2 Vasopressin Receptor against Polycystic Kidney Disease[END_REF][START_REF] Petrel | Identification, Structural and Pharmacological Characterization of τ-CnVA, a Conopeptide That Selectively Interacts with Somatostatin Sst3 Receptor[END_REF][START_REF] Rouget | Identification of a Novel Snake Peptide Toxin Displaying High Affinity and Antagonist Behaviour for the Α2-Adrenoceptors[END_REF][START_REF] Quinton | Isolation and Pharmacological Characterization of AdTx1, a Natural Peptide Displaying Specific Insurmountable Antagonism of the α 1A-Adrenoceptor[END_REF][START_REF] Jolkkonen | A Snake Toxin against Muscarinic Acetylcholine Receptors: Amino Acid Sequence, Subtype Specificity and Effect on Guinea-Pig Ileum[END_REF] have been frequently described whereas other toxins inhibit enzymes or bind membranes to exert hemolytic and/or antimicrobial actions [START_REF] Cologna | Peptidomic Comparison and Characterization of the Major Components of the Venom of the Giant Ant Dinoponera Quadriceps Collected in Four Different Areas of Brazil[END_REF] . Toxins are very often selective (with the exception of some lytic toxins) and make excellent pharmacological tools to better understand the physiological or pathophysiological roles of their targets. In some cases, the toxins themselves or their derivatives have applied clinical uses, such as exenatide from the beaded lizard), a treatment against type-1 diabetes, ziconotide from a cone snail, a treatment for chronic pain, captopril derived from a snake toxin and widely used to treat hypertension, but also some other snake toxins e.g. batroxobin with clotting properties used to stop bleeding [START_REF] Kumar | Nanofibrous Snake Venom Hemostat[END_REF] . In addition, more than 20 other toxins are currently investigated in clinical trials as biopharmaceutical and therapeutic tools.

Venoms are a very promising source of selective bioactive molecules and considerable effort is being undertaken to discover new toxins with exploitable activities [START_REF] Pennington | Peptide Therapeutics from Venom: Current Status and Potential[END_REF][START_REF] Saez | Versatile Spider Venom Peptides and Their Medical and Agricultural Applications[END_REF] . Classically, toxins are identified by bioguidage, i.e. successive cycles of venom deconvolution and screening tests against a given target.

After defining its sequence, the novel toxin of interest must be produced to allow in-depth characterization. This strategy is long, costly and risky and requires large amounts of venoms, unavailable for most venomous animals which are very small in size. This strategy is not adapted for the vast majority of this fantastic biodiversity.

New sequencing strategies that appeared in the 21 st century are revolutionizing the toxinological field.

These technologies have been commonly applied to whole genome sequencing, however, eukaryote genomes are typically large with tandem repeats and only few genomes from venomous species were reported (several snakes, two scorpions, a handful of spiders and dozens of Hymenopteras) but none from cone snails. By contrast, many studies have taken advantage of these same strategies for the sequencing of various venom gland transcriptomes. Toxins are produced as precursor proteins by venom-apparatus specific cells and undergo several maturation steps before being secreted [START_REF] Mebs | Toxicity in Animals. Trends in Evolution?[END_REF] . The formation of disulfide bonds is the most important post-translational modification (PTM) and their arrangement is commonly used to classify toxins [START_REF] Kaas | Conopeptide Characterization and Classifications: An Analysis Using ConoServer[END_REF] . Sequenced transcripts are translated into their cognate amino acid sequences allowing identification of the endoplasmic reticulum signal peptide, the propeptide and mature peptide regions. While tools like SignalP often accurately predict the endoplasmic reticulum signal peptide boundary, relying on transcriptomics alone, it is far more complicated to obtain robust information regarding further processing to the mature peptide due to the lack of information on most pro-protein convertase specific cleavage sites. Moreover, it is important to note that using a purely transcriptomic strategy, the physical presence in the venoms of most of the sequences described in databases is almost never demonstrated. Alternatively, proteomic strategies study only secreted toxins and provide the opportunity to characterize post-translational modifications (PTMs). Thus, only the combination of these complementary proteomic and transcriptomic strategies leads to a higher level of confidence for mature peptide sequences. Consequently, it is expected that the toxin sequences present in these types of databases will become more and more accurate.

The last and most complicated challenge remains the biological characterization of these natural peptides. This step cannot be done by a simple sequence analysis, especially for peptides with a low sequence similarity with previously characterized toxins. It is necessary to build a physical bank of peptides ready to be screened. The FP7 Venomics Project is part of the biotechnology program "Smallto-Medium Enterprise (SME)-targeted research for developing tools and technologies for highthroughput research". The total project budget was close to 10 million euros (equivalent to 11 million USD) shared by eight partners from five countries. The vision associated with the Venomics Project is not only to investigate the diversity of venom peptides through the development of "omics" technologies but also to replicate toxin diversity in vitro to generate novel peptide banks to be used in drug discovery programs [START_REF] Gilles | The European FP7 Venomics Project[END_REF] . This unique toxin bank was generated and screened on G-Protein Coupled Receptor (GPCR) families for which no toxin modulators have previously been described.

Results

A large venomous animal collection. In total, 323 animal samples from 191 different species were gathered (Table 1). Almost every terrestrial and marine venomous clade was represented by at least one species such as ray (Potamotrygon motoro) or venomous lizard (Heloderma horridum exasperatum). Small terrestrial arthropods represented 95 species i.e. half of the sourcing. 55 species (29% of the total sourcing) were studied for the first time, including 44 new species from the most infamous venomous clades (Araneae, Conus, Scorpiones and Serpentes). For instance, 12 previouslyunexplored species of Neogastropods were sourced including Conus canonicus from the Conidae family, the most studied group of Neogastropoda. The Turridae and Terebridae groups also belong to the Neogastropoda order but have been largely neglected compared to their counterpart [START_REF] Turner | Bioactive Compounds Isolated from Neglected Predatory Marine Gastropods[END_REF] . For instance, among over 4,000 described species of Turridae, venom secretions of only twelve species have been studied so far. One of them, Unedogemmula bisaya was included in the sourcing. Concerning the Terebridae family, only seven of around 350 described species has been subject of venom analysis. In Venomics sourcing, two of them, Terebra subulata and Terebra guttata, were chosen. A third species, Terebra maculata, never explored before, completed the Terebridae species of the sourcing. Sourcing also included totally unexplored groups such as Mitridae. Two species considered non-venomous, the Silurus glanis fish and the Trachycephalus resinifictrix frog, but known to produce toxic skin secretions, were also part of the collection. The diverse collection of source animals encompassed various habitats, morphologies and animal sizes ranging from millimeters (Manica rubida ant) to several meters (Ophiophagus hannah snake). A huge sequence toxin diversity in animal venoms. De novo transcriptomics opens the door to the study of Nature without any previous knowledge of it and with a very high resolution, allowing the unbiased identification and quantification of transcripts in a broad spectrum, even for those of low expression. Total RNA was extracted from venom glands. In order to include species diversity, sample requirements were decreased from 2 µg to 100 ng of RNA, being even as low as 60 ng in specific samples. Illumina specific 2x100 pair-end libraries were constructed from each RNA following manufacturer´s protocols. Over 60 million reads were obtained per sample using Hiseq 2000 sequencing platform with an average length of 500 nucleotides.

Two proteomic strategies were used according to the size of the toxins [START_REF] Morsa | Multi-Enzymatic Limited Digestion: The Next-Generation Sequencing for Proteomics?[END_REF] . The first one, for venoms presenting most of toxins with less than 25 amino acid residues (like Neogastropoda, Hymenoptera, Anura), analyzed the peptides post-disulfide-reduction by liquid chromatography coupled to tandem mass spectrometry. The second strategy, dedicated to the sequencing of the long venom toxins (> 25 amino acid residues (like Chilopoda, Scorpiones, Araneae, Squamata) required additional alkylation and trypsin digestion (short time and low enzyme concentration) steps after disulfide treatment leading to the generation of overlapping peptides (+/-15 amino acids) [START_REF] Morsa | Multi-Enzymatic Limited Digestion: The Next-Generation Sequencing for Proteomics?[END_REF] . These resulting peptides were then analyzed with the same mass spectrometry platform. The number of sequences varied between fifty to several hundred, depending on the venom. A high number of sequences are observed in Conidae venoms by proteomics. This diversity relies on the presence of post-translational modifications at different sites in a single toxin and has been discussed in previous publications 2, [START_REF] Dutertre | Deep Venomics Reveals the Mechanism for Expanded Peptide Diversity in Cone Snail Venom[END_REF] . A total of 21,300,000 sequence tags were obtained. Proteomic and transcriptomic data generated from the same animal were cross-validated to extract only sequences present in both the venom and venom gland transcripts, resulting in 20,206 validated toxin sequences (defined here as peptide up to 110 residues with at least one disulfide bridge). Figure 1 describes these sequences in term of origins, disulfide bridges (1 to 9), sizes (6 to 110) and PTMs.

The quasi-totality of Conus sequences (98%) bears at least one PTM, in addition to disulfide bridges.

Amidation is found in 2,889 sequences (14%) from all groups studied except Terebridae, Turridae, Mitridae and Teleostei. Hydroxylation on Pro, Met, Lys or Val residues was found in 2,630 sequences (13%) from various groups with a great majority from Conus (70%). In our study, 4-carboxyglutamic acid, pyroglutamic acid and bromo-tryptophan appeared to be Conus specific. Strangely, we did not find pyroglutamic acid in Arachnid venoms (Scorpiones and Araneae), although its presence has been described previously in many other studies [START_REF] Rong | Native Pyroglutamation of Huwentoxin-IV: A Post-Translational Modification That Increases the Trapping Ability to the Sodium Channel[END_REF][START_REF] Lucchesi | Analysis of the Blocking Activity of Charybdotoxin Homologs and Iodinated Derivatives against Ca2+-Activated K+ Channels[END_REF] . The 20,206 toxin sequences were divided into 137 different cysteine patterns, 97 with an even number of cysteines. Among these 97 patterns, 38 present new cysteine patterns never described in any venomous species. Regarding the 59 known patterns, C-C-CC-C-C framework is the most represented comprising 19% of the bank (Table S1). It is related to Inhibitory Cystine Knot Toxins (ICK), which is known to be particularly abundant in spider, snail and scorpion venoms [START_REF] Saez | Spider-Venom Peptides as Therapeutics[END_REF][START_REF] Gao | Functional Evolution of Scorpion Venom Peptides with an Inhibitor Cystine Knot Fold[END_REF][START_REF] Robinson | Conotoxin Gene Superfamilies[END_REF][START_REF] Imperial | The Augertoxins: Biochemical Characterization of Venom Components from the Toxoglossate Gastropod Terebra Subulata[END_REF] . For the first time, ICK peptides were identified at protein level from Mitridae, Turridae, Manica rubida ant, Chilopoda and the octopus Hapalochlaena lunulata.

Interestingly, 874 sequences covering 40 cysteine patterns display an odd number of cysteine residues (Table S2). Only thirteen were previously described. Two patterns (both lacking vicinal cysteine residues) are particularly abundant: C-C-C-C-C-C-C with 130 sequences (Scorpiones, Serpentes, Octopoda, Conus) and C-C-C-C-C specific to the Araneae taxon with 179 sequences. We did not identify any sequences from Conus sharing the latter pattern, despite it has been previously described [START_REF] Lavergne | Optimized Deep-Targeted Proteotranscriptomic Profiling Reveals Unexplored Conus Toxin Diversity and Novel Cysteine Frameworks[END_REF] .

Among these 20,206 sequences, 782 (4%) display a new cysteine pattern and 4,393 (22%) share less than 50% sequence identity with any described peptide or protein in the UniprotKB/SwissProt database using default settings (e-value max =1). It must be noted that the cysteine connectivity of the native toxins can not be explicitly determined using this pipeline.

From sequences to a high throughput screening compatible toxin bank. In order to allow screening and characterization of peptide activity, we developed two high-throughput production protocols.

5,923 sequences representing the broad diversity of the sequence database were selected and produced in the dedicated time of 18 months. Coming from 64 different species, they cover 91 different cysteine patterns, including 14 with an odd number of cysteine residues (Tables S1 andS2).

We selected 931 peptides shorter than 35 residues, with or without PTMs, to be produced by solidphase chemical synthesis [START_REF] Upert | High-Throughput Production of Two Disulphide-Bridge Toxins[END_REF] . The crude synthetic product was cleaved from the resin, desalted and subjected to an optimized oxidation process, whatever the number or pattern of cysteines [START_REF] Upert | High-Throughput Production of Two Disulphide-Bridge Toxins[END_REF] . After a final desalting step, liquid chromatography and mass analysis were performed to validate the presence of the expected oxidized mass (Figure S1). 893 toxins from 9 to 34 residues with 1 to 3 disulfide bridges were successfully produced, representing a success rate of 96%.

The remaining 4,992 sequences containing more than 34 residues were selected for heterologous expression in E. coli [START_REF] Duhoo | High-Throughput Production of Oxidized Animal Toxins in Escherichia Coli[END_REF][START_REF] Sequeira | Gene Design, Fusion Technology and TEV Cleavage Conditions Influence the Purification of Oxidized Disulphide-Rich Venom Peptides in Escherichia Coli[END_REF][START_REF] Turchetto | High-Throughput Expression of Animal Venom Toxins in Escherichia Coli to Generate a Large Library of Oxidized Disulphide-Reticulated Peptides for Drug Discovery[END_REF] . All synthetic genes encoding toxin sequences were subcloned into an optimized periplasmic expression plasmid containing an N-terminal E. coli Disulfide bond Isomerase C (DsbC) chaperone fusion tag, a 6xHIS tag for purification and a tobacco etch virus (TEV) protease cleavage site to release the native peptide from the fusion protein. Expressed fusion proteins are purified on a Nickel resin, subjected to overnight TEV cleavage and finally desalted. The correct mass was confirmed by mass analysis (Figure S2). 2,704 toxins, from 35 to 110 residues with 1 to 9 disulfide bridges were successfully produced representing a success rate of 54%.

Determining disulfide connectivity in native toxins is a challenge and the Venomics project did not have the resources to carry out this task. In the absence of this information, cysteine residues were randomly oxidized and the vast majority of toxins presented a unique peak in LC, suggesting that they adopted only one conformation, hopefully the natural one. In the case where LC profiles presented two or three peaks with the good expected oxidized mass, we assumed that it could be due to the generation of peptide isomers [START_REF] Massonnet | Disulfide Connectivity Analysis of Peptides Bearing Two Intramolecular Disulfide Bonds Using MALDI In-Source Decay[END_REF] (Figures S1 andS2).

Together, these two strategies resulted in a bank of 3,597 peptides composed of 71 different cysteine frameworks (Tables S1 andS2). In addition, 158 toxins (4%) display a novel cysteine pattern and 1,026 (29%) share less than 50% sequence identity with any previously described peptide or protein in the UniprotKB/SwissProt database. All together, we have successfully translated the high complexity of the omics bank into a physical toxin bank via high throughput methods.

First animal toxins on melanocortin receptors. Animal toxins are mostly known for their bioactivity on ion channels. Few but very interesting toxins active on GPCRs have also been identified [START_REF] Muratspahić | Nature-Derived Peptides: A Growing Niche for GPCR Ligand Discovery[END_REF] 125 I-NDP-α-MSH, 97% and 86% respectively (Figure 2). Further pharmacological investigations were then done with newly synthetically produced toxins of more than 95% purity (Figures 3 andS3). A B 13 control hMCR-free HEK293. Both toxins tested at 100 µM induced cAMP production on hMC1R, similar to 1 nM of α-MSH, while no effect was observed on hMC4R (Figure 4B). In the presence of 1 nM of α-MSH, the toxins slightly enhance hMC1R related cAMP production but did not reduce the cAMP production related to hMC4R (Figure 4B). Both toxins were also tested at 100 µM on several

human sodium channels (Na V 1.1, Na V 1.2, Na V 1.3, Na V 1.4, Na V 1.5, Na V 1.6, Na V 1.7 and Na V 1.8), potassium channels (K V 1.1, K V 1.2, K V 1.3, K V 1.4, K V 1.5, K V 1.6, K V 2.1, K V 3.1, K V 4.3, K V 7.1, K V 7.3, K V 10.1, K V 11.1(hERG), GIRK1
), a voltage-gated calcium channel (Ca V 3.3) and ligand-gated ion channels nAChR α1 2 β1γδ and nAChR α7. No activity could be detected (Figure S4 and S5).

N-TRTX-Preg1a

N-BUTX-Ptr1a A set of 546 NOEs for N-TRTX-Preg1a were used in the final ARIA run, which includes 454 unambiguous restraints (Table S3). Additional constraints were introduced during the iterative ARIA procedure: 38 dihedral angle restraints, 10 H-bond constraints observed within the β-strands and 3 covalent bonds between sulphur atoms (C2-C19, C9-C22, C18-C27). Indeed, when enough experimental data had been introduced into the first runs of the iterative ARIA process, the C18-C27 disulfide bond is formed in two-thirds of the N-TRTX-Preg1a structures. The fine analysis of NOE constraints and the evaluation of distance between sulfur atoms allowed discrimination between the two remaining possibilities: C2-C19, C9-C22, C18-C27 (favorable) or C2-C22, C9-C19, C18-C27 (excluded). Therefore, the corresponding covalent bonds were added between sulfur atoms to refine the N-TRTX-Preg1a structure in the last runs of the calculations. The 10 models representative of the solution structure (Figure 5A) display a very low RMSD, small potential energy values, respect all the experimental data and are in perfect agreement with standard covalent geometry with 64.8% of the residues in the most favored regions of the Ramachandran plot, and 35.2% in additional allowed regions (Table S3). The N-TRTX-Preg1a structure is very well defined, organized into the ICK motif without ambiguities regarding cysteine pairing. It is composed of a short antiparallel β-sheet made up of two short strands (22-23 and 26-27). The very short turn between these two strands, containing two arginine residues (R24 and R25), is highly constrained. Note the presence of an extended region (7-9), which faces the two-stranded β-sheet, resembling a third quasi-β-strand. Proline residue 12 is in a majority trans configuration (about 80%). N-TRTX-Preg1a sequence produced only a handful of significant hits in public databases and shares only 61% identity with the closest toxin with a known activity, Hs1a from Cyriopagopus schmidti (Figure 5C), a calcium channel blocker [START_REF] Liu | Function and Solution Structure of Huwentoxin-X, a Specific Blocker of N-Type Calcium Channels, from the Chinese Bird Spider Ornithoctonus Huwena[END_REF] . Regarding structural determination of N-BUTX-Ptr1a, 839 NOEs (including 558 unambiguous distance restraints) and 4 hydrogen bonds present within the β-sheet were used in the final ARIA run (Table S4). 92% of the 250 final structures display a C1-C4, C2-C5, C3-C6 disulfide bridge array, without adding any constraint on the bonds. The 10 models representative of the solution structure (Figure 5B) display low root mean square deviations (RMSD), small potential energy values, and respect all the experimental data, as attested by the absence of residual violations (Table S4). The Ramachandran plot exhibits 53% of the (,) angles in the most favored regions, and 40% in the additional allowed regions. The N-BUTX-Ptr1a structure scaffold is a well-known motif within scorpion toxins [START_REF] Kuzmenkov | Diversity of Potassium Channel Ligands: Focus on Scorpion Toxins[END_REF] , plant defensins [START_REF] Carvalho | Plant Defensins--Prospects for the Biological Functions and Biotechnological Properties[END_REF] and insect defensins [START_REF] Koehbach | Structure-Activity Relationships of Insect Defensins[END_REF] , and was designated as a cysteine-stabilized α-helix β-sheet motif (CSαβ motif) [START_REF] Cornet | Refined Three-Dimensional Solution Structure of Insect Defensin A[END_REF] . The structure is composed of an α-helix (11-19) and a 2-stranded short β-sheet (26-28; 31-33), stabilized by three disulfide bridges (C6-C27, C12-C32, C16-C34) unambiguously assigned. N-BUTX-Ptr1a shares the same sequence pattern as the majority of α-KTx toxins known to block potassium channels: C-x(4,5

A

EVDMRCKSSKECLVKCKQATGRPNGKCMNRKCKCYPR 59% K v 1.1, K v 1.2, K v 1.3 37 Hongotoxin-2 HFIDVKCTTSKECWPPCKAATGKAAGKCMNKKCKCQ 53% Unknown Rj1m1 IVIDVPCTVSKECWSACKKAVGTDRGKCMGKKCKCYP 53% Unknown Tst26 VFINAKCRGSPECLPKCKQAIGKAAGKCMNGKCKCYP 53% K v 1.2, K v 1.3 38 Ts7 VFINAKCRGSPECLPKCKEAIGKAAGKCMNGKCKCYP 53% K v 1.1, K v 1.2, K v 1.3, K v 1.6, Shaker K v 39- 41 Tco30 WCSTCLDLECGASRECYDPCFKAFGRAHGKCMNNKCRCYT 50% Shaker B KCh 42 TtBut WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCRCYT 50% Unknown Charybdotoxin ZFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS 41% K v 1.2, K v 1.3, K v 1.5, K v 1.6, KCa1.1, Shaker K v 22,43 Agitoxin-2 GVPINVSCTGSPQCIKPCKDA-GMRFGKCMNRKCHCTPK 38% K v 1.1, K v 1.2, K v 1.3, K v 1.6 43,44
)-C-x(3)-C-x(6,9)-[GAS]-K-C-[IMQT]-x(3)-C-x- C (Prosite PS01138
). Yet, N-BUTX-Ptr1a shares only 59% identity with the closest toxin with a known activity, Parabutoxin-3 from the same species Parabuthus transvaalicus (Figure 5D), a potassium channel blocker [START_REF] Huys | Characterization and Biosynthesis of Parabutoxin 3, a Component of Parabuthus Transvaalicus Venom[END_REF] . The coordinates of structures have been deposited in the Protein Data Bank (http://www.pdb.org) with the entry codes 6SAA and 6SAB for N-TRTX-Preg1a and N-BUTX-Ptr1a, respectively.

While N-TRTX-Preg1a and N-BUTX-Ptr1a display the canonical ICK and CSαβ structure, respectively, their sequences diverged so much from most described toxins that homology searches or phylogenetic reconstruction failed to assign them into known toxin groups. On one hand, N-TRTX-Preg1a matched only a handful of spider toxins that could not be linked to the main group of spider ICK toxins. On the other hand, phylogenetic reconstruction with all known alpha-KTx (UniProt)

placed N-BUTX-Ptr1a as sister to a clade consisting of several monophyletic alpha-KTx subfamilies (alpha-KTx 2, 4 and 12).

Discussion and conclusions

To our knowledge, this is the first time that a thorough '-omic' analysis of venoms has been carried out on such a wide number and diversity of venomous species from various habitats (128 terrestrial, new structural organizations. This is an opportunity to discover peptides with attractive biophysical properties in terms of engineering and thermodynamic stability for optimal drugability. Another level of complexity, not investigated here, is the dimerization (homo-and/or hetero-) of toxins containing an odd number of cysteines. A specific proteomic analysis should be designed to address this point.

Converting a sequence database into physical peptides is challenging, in particular for the oxidation of disulfide bridges. Our recombinant expression strategy was able to directly produce oxidized peptide and has proven to be effective in terms of high throughput production. The two-step chemical synthesis strategy was also extremely efficient. This is mainly thanks to the use of a universal folding strategy applied on all toxins regardless of size or number of cysteines. These production strategies provided peptides with impurities, as no high-efficiency purification step was performed. While the presence of these contaminations could have caused numerous false positive results during the screening tests [START_REF] Feng | Synergy and Antagonism of Promiscuous Inhibition in Multiple-Compound Mixtures[END_REF] we shown this was not the case as 90% of the tested hits were confirmed.

GPCRs constitute one of the largest and most versatile families of receptors. They are involved in a wide spectrum of physiological functions. Since the late 20 th century, pharmaceutical companies have significantly diminished their natural product programs, partly because of the advances in both HTS and combinatorial synthesis, thereby the creation of large synthetic libraries of small but artificial molecules. Natural product libraries consist of extracts and partially purified fractions and are perceived to be incompatible with HTS. Unfortunately, this loss of interest in natural products correlated with the overall reduction in new leads in the drug development pipeline and the substantial decline in new drug approval [START_REF] Li | Drug Discovery and Natural Products: End of an Era or an Endless Frontier?[END_REF] . The Venomics bank reconciles venoms, a particularly difficult exploitable resource of natural peptides, with HTS. The screening of the Venomics bank provided an extraordinary hit rate of 3%, much higher than the one reported in literature concerning artificial molecules and estimated at around 0.05% [START_REF] Bender | Which Aspects of HTS Are Empirically Correlated with Downstream Success?[END_REF][START_REF] Oprea | Current Trends in Lead Discovery: Are We Looking for the Appropriate Properties?[END_REF] . To our knowledge, this is the first large-scale demonstration of the power of natural peptide toxins to provide GPCR ligands. This strategy opens up an unexplored avenue for the discovery of new ligands on receptors linked to diseases with unmet medical needs such as obesity.

20

MC4R is mainly expressed in the paraventricular nucleus of the hypothalamus. Its activity is finely regulated by both anorexigenic agonists, the melanocyte stimulating hormones (MSH), and the orexigenic antagonist, Agouti-related protein (AgRP) [START_REF] Andermann | Toward a Wiring Diagram Understanding of Appetite Control[END_REF] . Loss-of-function mutations on hMC4R are the primary cause of monogenic obesity (http://www.orpha.net/) making it a therapeutic target in eating disorders. Currently, only one MC4R ligand is in clinical trials (Setmelanotide, https://clinicaltrials.gov/). Subtype 1 (MC1R) is also of therapeutic interest with its protective role against melanoma. It elicits eumelanin production, a dark, UV radiation-absorbing, photoprotective pigment [START_REF] Kobayashi | Supranuclear Melanin Caps Reduce Ultraviolet Induced DNA Photoproducts in Human Epidermis[END_REF] and promotes nucleotide excision repair of DNA damaged by UV radiation [START_REF] Kadekaro | Melanocortin 1 Receptor Genotype: An Important Determinant of the Damage Response of Melanocytes to Ultraviolet Radiation[END_REF] . There is an urgent need for development of innovative ligands to better understand MCR functions and to generate new medicines. N-TRTX-Preg1a and N-BUTX-Ptr1a are the first MCR venom-peptide ligands, structurally unrelated to natural endogenous MCR agonists, while similarities and differences exist with other natural ligands such as human Agouti-related protein (hAgRP) or human β-defensin 3 (HBD3). As N-TRTX-Preg1a, hAgRP displays an ICK fold in its C-terminal part (hAgRP 83-132) representing its hypothesized mature form [START_REF] Creemers | Agouti-Related Protein Is Posttranslationally Cleaved by Proprotein Convertase 1 to Generate Agouti-Related Protein (AGRP)83-132: Interaction between AGRP83-132 and Melanocortin Receptors Cannot Be Influenced by Syndecan-3[END_REF] with two additional disulfide bridges. As presented in Figure 6A, cysteine residues involved in ICK fold in N-TRTX-Preg1a and hAgRP 83-132 are well aligned with similarly β-sheets involved the two last cysteine residues of the ICK pattern. Composition and loop sizes are quite divergent between the two peptides especially for the third loop which is almost two times longer in hAgRP 83-132 (C3-C9). This loop contains the Arg-Phe-Phe pharmacophore of AgRP [START_REF] Creemers | Agouti-Related Protein Is Posttranslationally Cleaved by Proprotein Convertase 1 to Generate Agouti-Related Protein (AGRP)83-132: Interaction between AGRP83-132 and Melanocortin Receptors Cannot Be Influenced by Syndecan-3[END_REF][START_REF] Tota | Molecular Interaction of Agouti Protein and Agouti-Related Protein with Human Melanocortin Receptors[END_REF] that is lacking in N-TRTX-Ptr1a. This difference suggests that in spite of sharing the same structural ICK family, N-TRTX-Preg1a and hAgRP 83-132 have a different mode of action on MCRs. Regarding N-BUTX-Ptr1a, we noticed that it shared with HBD3 interesting features.

They both display similar secondary structures composed of a short α-helix and several antiparallel βsheets reticulated by three disulfide bridges (Figure 6B). In addition, they are both rich in cationic residues with high basic pIs of 10 for HBD3 and 9. . Without testing HBD3 in our conditions, we cannot be sure that HBD3 and N-BUTX-Ptr1a share or not similar affinities on melanocortin receptors. Nevertheless, the micromolar affinity of HBD3 on hMC1R was supported by functional results who described the peptide as a micromolar agonist of the Gs pathway on hMC1R (EC 50 = 0.40 μM) [START_REF] Ericson | Human β-Defensin 1 and β-Defensin 3 (Mouse Ortholog MBD14) Function as Full Endogenous Agonists at Select Melanocortin Receptors[END_REF] , a feature shared with N-BUTX-Ptr1a. Despite these similarities, important differences between the two peptides have also to be noticed. Firstly, they do not share global three-dimensional structures, surely linked to the difference in cysteine pattern (Figure 6B). Secondly, Nix et al. showed that the absence of disulfide bridges, by cysteine replacement in serine in HBD3 sequence, decreased HBD3 affinities by less than 10 times [START_REF] Nix | Molecular and Functional Analysis of Human β-Defensin 3 Action at Melanocortin Receptors[END_REF] contrary to the linear N-BUTX-Ptr1, which lost at least 100 times in affinity. Therefore, the structural constraints induced by disulfide bridges may be more important in N-BUTX-Ptr1a binding than in HBD3. These two structural differences may be linked in distinct interaction profile of the two peptides with melanocortin receptors.

N-TRTX-Preg1a and N-BUTX-Ptr1a also lack the HFRW pharmacophore, described in endogenous agonists as the minimal active sequence [START_REF] Hruby | Alpha-Melanotropin: The Minimal Active Sequence in the Frog Skin Bioassay[END_REF][START_REF] Singh | Structure-Activity Relationships (SAR) of Melanocortin and Agouti-Related (AGRP) Peptides[END_REF] , suggesting that they activate hMC1R in a different way, that would be interesting to deeply study. In addition, both toxins promote a synergistic effect with endogenous agonist α-MSH. This activity deserves to be finely studied as both ligands may activate hMC1R by a new molecular mechanism. N-TRTX-Preg1a and N-BUTX-Ptr1a inhibit 125 I-NDP-α-MSH binding on hMC4R but are not able to modulate activate or antagonize the hMC4R/Gs pathway, under our experimental conditions. This discrepancy can come from our experimental procedure. We perhaps did not incubate enough time as the toxins display low affinities and may need longer time to bind the receptor. Indeed, functional tests are designed according to the pharmacological characteristic of the agonist, which is nM. Another possibility is that these toxins display biased functions, with activities on other pathways than the Gs one. A deeper exploration of the functions of the two toxins on various MC4R signaling pathways (Gq, β-arrestin, Kinases, etc.) will be performed to better understand their modes of action. N-TRTX-Preg1a exhibits a common ICK fold only described so far for activity on ion channels. Its sequence is close to calcium channel toxins but does not possess the tyrosine residue in position 16

(N-TRTX-Preg1a numbering), described to be essential for blocking Ca V 2.2 channel conductivity [START_REF] Sousa | Venom Peptides as a Rich Source of Cav2.2 Channel Blockers[END_REF] .

N-BUTX-Ptr1a, close in sequence to potassium channel toxins, possesses a dyad composed of a basic and an aromatic residue [START_REF] Gasparini | Delineation of the Functional Site of Alpha-Dendrotoxin. The Functional Topographies of Dendrotoxins Are Different but Share a Conserved Core with Those of Other Kv1 Potassium Channel-Blocking Toxins[END_REF][START_REF] Dauplais | On the Convergent Evolution of Animal Toxins. Conservation of a Diad of Functional Residues in Potassium Channel-Blocking Toxins with Unrelated Structures[END_REF] (F24 and K26, N-BUTX-Ptr1a numbering). This dyad, stated as essential for potassium activity [START_REF] Gasparini | Delineation of the Functional Site of Alpha-Dendrotoxin. The Functional Topographies of Dendrotoxins Are Different but Share a Conserved Core with Those of Other Kv1 Potassium Channel-Blocking Toxins[END_REF][START_REF] Dauplais | On the Convergent Evolution of Animal Toxins. Conservation of a Diad of Functional Residues in Potassium Channel-Blocking Toxins with Unrelated Structures[END_REF] but we showed that is clearly not sufficient for N-BUTX-Ptr1a to bind potassium channels. N-TRTX-Preg1a and N-BUTX-Ptr1a sequences diverge so much within their own group that it was even impossible to phylogenetically anchor them into known toxin families. Thus, they constitute new clades for ICK and CSαβ families. Several other toxins of the produced bank sharing more than 80% of sequence identity with these two toxins are present in the Venomics database, suggesting that N-TRTX-Preg1a and N-BUTX-Ptr1a are not the only members of these new families. Production and pharmacological characterization of these natural variants should provide interesting insights with regard to the mode of action of these toxins on hMCRs. This may aid the generation of ligands with improved affinity and selectivity for development of new hMC1R agonists with applications in the treatment and/or prevention of melanoma.

To our knowledge, the Venomics bank is the largest one ever built. We demonstrated that it represents a real opportunity to identify ligands with unusual structures and pharmacological properties. Its high hit rate shows that quality can beat quantity. Two toxins, with classical toxin structures but original pharmacological activities highlight the diversity of toxin properties and open the door to the development of new hMCR modulators.

Experimental Section

Sourcing. We paid important caution to respect the natural resources to which we had access. Thus, we limited the number of collected specimens to the strict number necessary to ensure the amounts and volumes of biological samples were compatible with the experiments to be carried out within the Venomics consortium. For instance, during the Mayotte and Polynesia expeditions, supernumerary cone snails collected during the day, were systematically dropped back to water. In the case of the venomous lizard specimen (Heloderma horridum exasperatum), the lizard was anaesthetized while a veterinary surgeon performed surgery to remove only one of the venomous salivary glands of the animal. The animal fully recovered. This demonstrates the applicability of such a strategy of tissue extraction that could certainly be applied with success to rare snakes too.

Transcriptomic analysis. The Illumina paired-end NGS platform was selected to assure sequence and data quality. 203 RNA samples from different venomous species were sequenced through a low-input method. Quality and quantity of total RNA was determined using a Bio-analyzer 2100 (Agilent, California), and Qubit 2.0 (ThermoFisher, Massachussetts). The poly(A)+ mRNA fraction was isolated from total RNA and cDNA libraries were obtained following Illumina´s recommendations.

Briefly, poly(A)+ RNA was isolated on poly-T oligo-attached magnetic beads and chemically fragmented prior to reverse transcription and cDNA generation. cDNA fragments then went through an end repair process, the addition of a single 'A' base to the 3' end and then ligation of the adapters.

Finally, the products were purified and enriched with PCR to create the indexed final double stranded cDNA library. Library quality was analyzed via a Bioanalyzer 2100 High Sensitivity assay and quantity was determined by real-time PCR using a LightCycler 480 (Roche, Basilea, Suiza). The pool of libraries was sequenced by paired-end sequencing (100 x 2) on an Illumina HiSeq 2000 sequencer (Illumina, California). Bioinformatic analysis was a crucial step of the process to identify peptide sequences. This analysis started with NGS raw data quality control, followed by exhaustive cleaning of primers, adapters, indexes and low-quality bases. The next step consisted of joining the paired-end reads and dereplicating all reads before entering the assembly step itself. To assure the quality of the assembly two different assemblers were used; Velvet (v1.2.10) [START_REF] Zerbino | Velvet: Algorithms for de Novo Short Read Assembly Using de Bruijn Graphs[END_REF] and Trinity (v2.1.1) [START_REF] Grabherr | Full-Length Transcriptome Assembly from RNA-Seq Data without a Reference Genome[END_REF] , creating a robust and comprehensive de novo assembly pipeline.

Proteomic analysis. Venoms predominantly containing toxins with less than 25 amino acid residues such as cone snails and wasps, were treated with dithiothreitol (10 mM final concentration, 1 hour at 56 °C) and alkylated with iodoacetamide (20 mM final concentration, 1 hour at room temperature).

Reduced and alkylated peptidic toxins were separated by reverse phase chromatography (nanoLC-UPLC Acquity, Waters, Milford, Massachusetts, United Stated) and analyzed by tandem mass spectrometry (Q-Exactive, ThermoFisher, Waltham, Massachusetts ,United States). This "top-down" proteomics approach has the advantage of measuring the mass of the intact full-size toxins but also allows for sequence determination using collision-based fragmentation mass spectrometry experiments. Venoms containing large toxins, such as snake, spider and scorpion venoms, were, after reduction and alkylation, enzymatically cleaved (by using a ratio of 1/50 protein/trypsin, with incubation at 37°C over 1 hour). These cleavage conditions yield truncated peptides cleaved at different positions, with several overlapping peptides covering the full toxin sequence. The peptides were then analyzed with the same nanoUPLC-MS/MS platform, in the same way as the smaller toxins permitting the full toxin sequences to be determined. As large toxins have been transformed into a collection of smaller peptides, the fragmentation yield is then much better and MS/MS spectra can provide useful sequence information.

Sequence validation.

For the transcriptomics data, different databases were generated: (1) the first one including genes coding for peptidic toxins by using predictive bioinformatics tools and (2) the rest of the venom gland transcriptome translated into the six possible open reading frames. The bioinformatics data analysis has been supported by Peaks software (Bioinformatics Solutions Inc., Waterloo, Canada). In addition to classical data analysis, Peaks was also used to de novo sequence peptides not identified from the databases, and to predict mutations with Spider algorithms. All the peptides discovered was classified and only those with a false discovery rate of less than 0.1% were considered for inclusion in the Venomics database.

High-throughput peptide production by solid-phase synthesis. The high-throughput chemical production of toxins up to 34 residues with or without post-translational modifications (PTMs) was undertaken by solid-phase peptide synthesis on an automated Prelude synthesizer (Protein Technologies, USA) equipped with six reactions vessels using the Fmoc strategy. Amino acids were coupled twice for 10 minutes using five or twenty equivalents for toxins up to 25 residues or between 26 and 34 residues, respectively, on a ChemMatrix ® or pre-loaded Wang resins using HCTU as a coupling reagent. Both strategies lead to toxins with excellent purities higher than 50%. After a desalting step on homemade C18 columns and a drying step, the refolding of the different toxins was performed directly on the crude material. A universal folding protocol was applied for each toxin as already described 28 using 50 mM HEPES buffer pH 7.5, 20% acetronitrile, 1 mM cysteine, 0.1 mM cystine, over 48 hours at 4 °C. After a desalting step and analytical characterizations by analytical HPLC on a Waters e2695 separation module equipped with a XBridge BEH300 C18 column ( 5µm , 4.6x250mm, Waters) and by mass spectrometry on a MALDI-TOF spectrometer (4800 MALDI TOF-TOF Analyser from Applied Biosystems, USA), the toxins were distributed in 96 well plates at a concentration between 2-120 µM.

High-throughput peptide production by recombinant expression. Optimization of gene synthesis and expression clone constructs for venom-peptide production have been published previously [START_REF] Turchetto | High-Throughput Expression of Animal Venom Toxins in Escherichia Coli to Generate a Large Library of Oxidized Disulphide-Reticulated Peptides for Drug Discovery[END_REF] , in addition to the final protocols used in this project [START_REF] Duhoo | High-Throughput Production of Oxidized Animal Toxins in Escherichia Coli[END_REF] . Briefly, genes encoding animal venom peptides were designed with codon optimization for expression in E. coli using the ATGenium codon optimization algorithm (NZYTech genes & enzymes, Portugal). Synthetic genes were produced in a high-throughput pipeline and purified PCR products were subsequently cloned into the pHTP4 expression vector using the NZYEasy Cloning kit (NZYTech genes & enzymes, Portugal), according to ligation independent cloning (LIC) technology protocols. In pHTP4, peptide genes are placed under the control of a T7 promoter, and are produced as fusion proteins in the E. coli periplasmic space.

Fusions contain an N-terminal Disulphide bond isomerase C (DsbC) fusion, an internal hexa-histidine tag for purification and a TEV protease recognition sequence to allow cleavage and isolation of the native peptides. Optimization of expression and purification conditions [START_REF] Sequeira | Gene Design, Fusion Technology and TEV Cleavage Conditions Influence the Purification of Oxidized Disulphide-Rich Venom Peptides in Escherichia Coli[END_REF][START_REF] Turchetto | High-Throughput Expression of Animal Venom Toxins in Escherichia Coli to Generate a Large Library of Oxidized Disulphide-Reticulated Peptides for Drug Discovery[END_REF] and the final recombinant production protocols used herein [START_REF] Duhoo | High-Throughput Production of Oxidized Animal Toxins in Escherichia Coli[END_REF] were carried out following the classical procedure, distance constraints were deduced from the volume integration of NOE correlations and introduced into the standard iterative calculation process of the Aria (ambiguous restraints for iterative assignment) software. For each Aria run, 500 structure calculations were performed per iteration, and 250 structures were calculated in the final iteration in water. For both samples, the first runs were performed allowing all combinations of disulfide pairing (ambiguous intersulfur constraints option). This option was kept for the N-BUTX-Ptr1a, for which the calculations converged towards a unique array of disulfide bridges, and the only additional constraints were the H-bonds observed in the β-sheet already formed in the first runs. For the N-TRTX-Preg1a sample, (φ,ψ) angular restraints deduced by the TALOS software 72 from [START_REF] Pennington | Peptide Therapeutics from Venom: Current Status and Potential[END_REF] C and 1 H chemical shifts were added to NOE constraints. Covalent bonds between sulphur atoms involved in each defined bridge were introduced in the last runs of the calculation. Finally, for each peptide, ten of the structures refined in water in the final iteration were selected according to their agreement with experimental data. The final sets of structures were displayed and analyzed using PyMol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC), and their quality were evaluated using Promotif [START_REF] Hutchinson | PROMOTIF--a Program to Identify and Analyze Structural Motifs in Proteins[END_REF] and PROCHECK3.5 74 software.

Screening. The 125 I-NDP-α-MSH and membranes expressing receptors were purchased from Perkin Elmer (Courtaboeuf, France). Screening conditions were optimised in order to get a Z'-factor value superior to 0.5. Z'-factor is defined as: , where MAD equals Median 1 -3𝑀𝐴𝐷 𝑚𝑎𝑥 + 3𝑀𝐴𝐷 𝑚𝑖𝑛 𝑀𝑒𝑑𝑖𝑎𝑛 𝑚𝑎𝑥 -𝑀𝑒𝑑𝑖𝑎𝑛 𝑚𝑖𝑛 Absolute Deviation. On each 96-well plate, eight wells were dedicated to negative controls and correspond to total binding of the radioligand in absence of any other ligand. Nonspecific binding was measured in the presence of 1 µM NDP-α-MSH (Tocris Bioscience) in eight other wells per plate.

Following screening, peptides with an associated binding value bigger or lower than Median -3xMAD median [START_REF] Zhang | A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays[END_REF] were classified as hits. Peptides were screened at concentrations between 0.1 to 100 µM according to their production yields.

Radioligand binding assays. The 125 I-NDP-α-MSH and melanocortin receptors were purchased from Perkin Elmer (Courtaboeuf, France). Binding experiments were performed using radioligand at 50 pM in a 100-µL reaction mixture at room temperature in buffer composed of 25 mM Hepes pH 7.4, 1 mM MgCl 2 , 1 mM CaCl 2 , 0.2 g/L BSA with various concentrations of competitors. Controls were the same as for the primary screening on hMC4R. After 3 hours of incubation time, samples were filtered through 96 well GF/C filter plates preincubated with 0.5% polyethylenimine. 25 µL of MicroScint 0 was added onto each dry filter and the radioactivity was quantified on a TopCount beta counter (Perkin Elmer, Courtaboeuf, France) with 45% yield. Competition binding data were fitted to a one-site/state inhibition mass action curve using Prism software, v5.0.0 (GraphPad, La Jolla,CA, USA). IC 50 values were converted to K i , using the Cheng-Prusoff equation [START_REF] Cheng | Relationship between the Inhibition Constant (K1) and the Concentration of Inhibitor Which Causes 50 per Cent Inhibition (I50) of an Enzymatic Reaction[END_REF] . Kd values were 0.34 nM, 0.47 nM, 0.25 nM and 0.88 nM, respectively, for hMC1R, hMC3R, hMC4R and hMC5R. Data represent at least three independent experiments performed in duplicate and are presented as mean ± SEM. or 100 µM N-BUTX-Ptr1a) for 3 h at 37 °C, 5% CO 2 . As a positive control 1 µM forskolin (Sigma-Aldrich) was used, while serum-free medium was used as the negative control (baseline). For Luciferase detection 15 µl OneGlo Substrate (Promega) was added at RT to each well. The 384-well plate was transferred directly to the plate reader (Tecan Infinite 200 PRO) and luminescence was measured after shaking for 5 sec (double orbital, amplitude: 3.5 mm) and 5 min incubation with an integration time of 1000 ms at room temperature. Data analysis was performed with GraphPad Prism.

After baseline subtraction, data were normalized to stimulation with 1 µM Forskolin. Data represent the mean ± SEM from three independent experiments performed in triplicates. Statistical analysis was performed with GraphPad Prism 5.03 (GraphPad Software, Inc., San Diego, California, USA).

Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. *** p < 0.001.

Electrophysiology.

For the expression of Na V channels (human (h)Na V 1.1, rat (r)Na V 1.2, rNa V 1.3, rNa V 1.4, hNa V 1.5, mouse (m)Na V 1.6, hNa V 1.7, rNa V 1.8 channels and the auxiliary subunits rβ1, hβ1), K V channels (rK V 1.1, rK V 1.2, hK V 1.3, rK V 1.4, rK V 1.5, rK V 1.6, rK V 2.1, hK V 3.1, rK V 4.3, hK V 7.2, hK V 7.3, hK V 10.1, hK V 11.1), Ca V channels (rCa V 3.3), hGIRK1 and nAChRs (hα1β1γδ and hα7) in Xenopus laevis oocytes, the linearized plasmids were transcribed using the T7 or SP6 mMESSAGE-mMACHINE transcription kit (Ambion, Carlsbad, CA). The harvesting of stage V-VI oocytes from anesthetized female Xenopus laevis frogs was previously described [START_REF] Peigneur | Where Cone Snails and Spiders Meet: Design of Small Cyclic Sodium-Channel Inhibitors[END_REF] . Oocytes were injected with 50 nl of cRNA at a concentration of 1 ng/nl using a microinjector (Drummond Scientific, Broomall, PA).

The oocytes were incubated in a solution containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 2 mM MgCl 2 , and 5 mM HEPES pH 7.4, supplemented with 50 mg/L gentamycin sulfate. Two-electrode voltage-clamp recordings were performed at room temperature (18-22 °C) using a Geneclamp 500 amplifier (Molecular Devices, Downingtown, PA) controlled by a pClamp data acquisition system (Axon Instruments, Union City, CA). Whole cell currents from oocytes were recorded 1-4 days after injection. Bath solution composition was 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 2 mM MgCl 2 , and 5 mM HEPES pH 7.4. Toxins were applied directly to the bath. Resistances of both electrodes were kept between 0.8 and 1.5 MΩ. The elicited currents were filtered at 2 kHz (Na V ), 0.5 kHz (K V & Ca V ), 0.05 kHz (GIRK & nAChR) and sampled at 20 kHz (Na V ), 2 kHz (K V & Ca V ) and 0.1 kHz (GIRK & nAChR) using a four-pole low-pass Bessel filter. Leak subtraction was performed using a -P/4 protocol. For the electrophysiological analysis, a number of protocols were applied from a holding potential of -90 mV. K V 1.1-K V 1.6 currents were evoked by 250 ms depolarizations to 0 mV followed by a 250 ms pulse to -50 mV. K V 2.1, K V 3.1, and K V 4.3 currents were elicited by 250 ms pulses to +20 mV from a holding potential of -90 mV. K V 7 and K V 10.1 currents were evoked by 2 s depolarizing pulses to 0 mV from a holding potential of -90 mV. Current traces of K V 11.1 channels were elicited by applying a +40 mV prepulse for 2 s followed by a step to -120 mV for 2 s. Sodium current traces were evoked by 100 ms depolarization to the voltage corresponding to maximal sodium current in control conditions. For GIRK measurements, the oocytes were clamped at -70 mV and superfused with a high K+ solution (96 mM KCl, 2 mM NaCl, 1 mM MgCl 2 , 1.8 mM CaCl 2 , 5 mM HEPES pH 7.5). Ca V channel currents, carried by 10 mM Ba 2+ , were elicited by 200 ms test depolarizations applied from -90 mV to voltages ranging from -50 mV or +30 mV. For nAChRs, oocytes were clamped at a holding potential of -70 mV throughout all recordings.

To minimize interference of endogenous Ca 2+ -activated Cl -channels during the experiments, a Ca 2+free bath perfusion solution was used, containing 82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl 2 , 2.5 mM BaCl 2 , and 5 mM HEPES pH 7.4. Current responses were evoked by 10 s applications of increasing concentrations of acetylcholine (starting at 200 μM). All data were analyzed using pClamp Clampfit 10.1 (Molecular Devices) and Origin 7.5 software (OriginLab, Northampton, MA).

Toxins nomenclature. Newly characterized toxins were named according to the rational nomenclature proposed by King et al. [START_REF] King | A Rational Nomenclature for Naming Peptide Toxins from Spiders and Other Venomous Animals[END_REF] with a greek letter associated with a biological function followed by the generic name indicating from which taxon the toxin is derived, genus and species indicated

Figure 1 .

 1 Figure 1. Richness of animal origin and structural features (number of disulfide bridges, length and

  . For example, the commercially available type 2 diabetes treatment, exenatide derived from a venomous lizard, targets a class A GPCR. Belonging to the same class, human melanocortin 4 receptor (hMC4R) regulates food intake and its genetic invalidation is responsible of morbid obesity. Screening of the Venomics bank on hMC4R gave an amazing high hit rate of 8%, 163 coming from chemical synthesis and 123 from recombinant expression. We decided to further investigate two hits displaying cysteine patterns related to two of the largest structural families in animal toxins, ICK-related pattern (-C-C-CC-C-C-) and the CSαβ-related cysteine patterns (-C-C-C-C-C-K-C-), and primary renowned for their activities on ion channels. The ICK-related N-TRTX-Preg1a comes from the spider Poecilotheria regalis while the CSαβ-related N-BUTX-Ptr1a from the scorpion Parabuthus transvaalicus. N letter (uppercase nu greek letter) has been chosen as the activity descriptor for modulators of hMCRs. In primary screening on hMC4R, N-TRTX-Preg1a and N-BUTX-Ptr1a both showed strong inhibition of 12

Figure 2 .

 2 Figure 2. Screening results on hMC4R from the two plates containing N-TRTX-Preg1a (A) and N-

Figure 3 .

 3 Figure 3. Analytical HPLC chromatogram of the final purified products of N-TRTX-Preg1a and N-

Figure 4 .

 4 Figure 4. Pharmacological characterization of N-TRTX-Preg1a and N-BUTX-Ptr1a. (A)

Figure 5 . 4 from

 54 Figure 5. Structural characterization of N-TRTX-Preg1a and N-BUTX-Ptr1a. Primary structure and

55 aquatic and 8

 8 aerial) including two European species (Manica rubida ant and Buthus occitanus scorpion). The Manica rubida ant was the smallest species of the project, studied for the first time and 35 sequences were validated. The number of validated sequences ranges from 0, due to experimental failures (some collecting conditions were not compatible with proper mRNA extraction), to 1,163, in the venom of the Mediterranean scorpion Buthus occitanus. Finally, no validated toxin sequences could be detected in the skin secretions of Silurus glanis fish and the Trachycephalus resinifictrix frog.An average of 106 toxin sequences per venom were validated, much less than what could be expected according to the assumed richness of venoms. This difference highlights the difficulties in obtaining full and unambiguous toxin sequences. The gotten sequence diversity also highlights our very limited state of knowledge of the toxin world. Indeed, almost one quarter of the toxins share less than 50% sequence identity with known toxin sequences, suggesting unpredictable and original biological activities. Furthermore, 65 new naturally occurring cysteine patterns have been found, suggesting 65

Figure 6 .

 6 Figure 6. Structure comparison of the spider and scorpion toxins with melanocortin receptor

  have been described previously. All steps were carried out in 24deep-well plates or in 96-deep-well plates on a Tecan Freedom EVO 200 robot (Switzerland)[START_REF] Saez | High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-Rich Venom Proteins Produced in E. Coli[END_REF] . pHTP4 expression plasmids were transformed into BL21 (DE3) pLysS E. coli and cultured overnight at 25 °C before harvesting. Cell pellets were lysed enzymatically and by sonication. DsbC-His-peptide fusions were isolated from the soluble cell lysates by nickel affinity purification. Concentrations of the 96 purified DsbC-His-fusion proteins were estimated by UV absorbance at 280 nm in a microtiter plate reader (GENios Plus, TECAN, Switzerland). TEVsh protease was added (1/10 w/w) directly to the eluted fusion proteins. The mixture was supplemented with a final concentration of 0.1 mM DTT then incubated overnight at room temperature in a Microtron shaking incubator (INFORS-HT, Switzerland) (200 rpm). Finally, samples were acidified for 1 h with a final concentration of 5% acetonitrile and 0.1% formic acid (FA) at room temperature with mild agitation. TEV protease were precipitated during this acidification process and the 96 recombinant toxins per plate were subjected to a final purification step on C18 chromatographic resin by Solid-Phase Extraction (SPE). The target peptides were eluted from the C18 resin, and analyzed by UHPLC and MS to confirm the identity and the quantity of the toxin (FigureS2). The toxins were distributed in 96 well plates at a concentration between 1-10 µM.Production of N-TRTX-Preg1a and N-BUTX-Ptr1a toxins. Linear peptide was obtained by solidphase chemical synthesis following the same protocol described above using Fmoc-Thr(tBu)-Wang resin LL (Novabiochem ® ) for N-TRTX-Preg1a and H-Cys(Trt)-Trityl NovaPEG resin (Novabiochem ® ) for N-BUTX-Ptr1a. After resin cleavage and ether precipitation, linear peptide was then purified on Waters Preparative HPLC 600 with an XBridge BEH 300Å Prep C18 column (5 µm, 19 x 250 mm, Waters). If disulfide bridges were to be prevented from forming, linear peptide was treated in Guanidinium chloride 6M with iodoacetamide (ten times in excess) for 2h under agitation at room temperature. The oxidation step was performed using Venomics oxidation conditions[START_REF] Upert | High-Throughput Production of Two Disulphide-Bridge Toxins[END_REF] .Resulting oxidized peptide was finally purified on a Waters 600 controller using an XBridge BEH 300Å Prep C18 column (5 µm, 10 x 250 mm, Waters). Purity of the final product was above 95% and was assessed separately by MALDI-TOF spectrometry (4800 MALDI TOF-TOF Analyser (Applied Biosystems, USA)) and analytical HPLC on Waters e2695 separation module with an XBridge BEH 300Å C18 column (5 µm, 4.6 x 250 mm, Waters). Final quantity was measured by amino-acid quantification using the PicoTag kit (Waters Associates, Milford, MA).Sequence analysis and phylogeneticreconstruction. Sequences were queried against UniProt (release 2019_02) and refseq_protein (release-96) with blastp 2.29.1. Searching parameters were adjusted for short-length sequences. Spider and scorpion venom proteins in UniProt were annotated as in VenomZone (https://venomzone.expasy.org/ release May, 2019). Multiple sequence alignments were performed with mafft 7.310 --auto option. Phylogenetic trees were obtained with RAxML 8.2.11 --PROTGAMMAAUTO --# 100 options, and MrBayes v3.2.0 ngen=1000000 nchains=4 nruns=2. NMR structures. Solutions of 0.5 mM N-TRTX-Preg1a, and 1.2 mM N-BUTX-Ptr1a were prepared in 3 mm tubes (respectively, 315 μg of N-TRTX-Preg1a and about 1 mg of N-BUTX-Ptr1a were dissolved in 220 μL of H 2 0/D 2 0 (95/5), at pH 4.5). All NMR spectra were acquired on a BRUKER 700 MHz NMR spectrometer equipped with a 5 mm TCI cryoprobe, at 298K. The standard set of 2D 1 H-NMR experiments (COSY, 80 ms TOCSY, and 160 ms NOESY) were completed with natural abundance 15 N-and 13 C-HSQC spectra. Processing, analyses, and calculations were successively performed with Bruker's TopSpin3.2, CcpNMR 70 and Aria2.3 71 software. Chemical shift assignments

Functional assays .

 assays Human embryonic kidney cells (HEK293, DSMZ, ACC305, authentication: fullmatching STR reference profile of 293 in the database, authentic) and HEK293-MC1R (human embryonic kidney cells expressing the human melanocortin 1 receptor with an N-terminal HA-tag and C-terminal FLAG-tag due to stable transfection, selection marker: hygromycin, AG Beck-Sickinger, University Leipzig, Leipzig, Germany) were grown in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/L glucose and L-glutamine and Ham's F12 (1:1, v/v; Lonza) supplied with 15% (v/v) heatinactivated fetal calf serum (FCS; Lonza) in a humidified atmosphere at 37 °C and 5% CO 2 . HEK293 or stably transfected HEK293-HA_MC1R_FLAG cells were transfected in solution with 0.15 µl Lipofectamine 2000 (Invitrogen) per well and 30 ng Plasmid pGL4.29[luc2P/CRE/Hygro] (Promega) per well and additionally 30 ng pcDNA3-hMC4R per well when needed. Lipofectamine and plasmid solutions were prepared in 6 µl Opti-MEM medium (Gibco) per well. After 5 min incubation at RT the Lipofectamine solution was slowly added to the DNA solution and incubated for 30 min at RT. Transfection of 20,000 cells/well in 30 µl medium (DMEM/Ham's F12/ 15% FCS) without antibiotics was performed in solution by slowly adding the DNA/Lipofectamine solution to the cells. After careful mixing, 42 µl per well was plated on a 384-well microplate (Greiner bio-one, F-Bottom, white) and incubated overnight. The day after transfection, medium was removed and cells were stimulated with 15 µl peptide solution in serum free medium (1 nM α-MSH (AG Beck-Sickinger, University Leipzig, Leipzig, Germany) corresponding to ~EC 85 at hMC1R or ~EC 75 at hMC4R, 100 µM N-TRTX-Preg1a

ABBREVIATIONS

  ABBREVIATIONS

Table 1 . Selected taxa for sourcing.

 1

	Taxa	Main families	Total	Never studied
			N° of species	species
		Conidae	38	10
	Neogastropoda
		Other (Turridae, Terebridae & Mitridae)	5	2
	Octopoda	Octopodidae	3	0
	Chilopoda	Scolopendridae	6	2
		Buthidae, Scorpionidae, Vaejovidae, Scorpionidae,
	Scorpiones		40	7
		Hemiscorpiidae & Euscorpiidae
		Theraphosidae, Ctenidae, Agelenidae, Araneidae,
	Araneae	Dipluridae, Lycosidae, Nephilidae, Sparassidae &	41	23
		Theridiidae
	Hymenoptera	Apidae, Vespida, Formicidae & Myrmicinae	8	4
	Chondrichthyes	Potamotrygonidae	1	0
	Teleostei	Synanceiidae, Plotosidae & Siluridae	8	2
		Serpentes (Elapidae, Viperidae & Atractaspididae)	39	4
	Squamata
		Anguimorpha (Helodermatidae)	1	0
	Anura	Hylidae	1	1

Table 2 .

 2 Inhibition constant (K i ) values of N-TRTX-Preg1a, N-BUTX-Ptr1a on melanocortin receptors, mean (µM) ± SEM (n=3-10).

  in the binding of HBD3 on melanocortin receptors[START_REF] Nix | Molecular and Functional Analysis of Human β-Defensin 3 Action at Melanocortin Receptors[END_REF] . In the same study, the authors described nanomolar affinities on melanocortin receptors (42 nM on hMC1R and 110 nM on hMC4R), but more recently, Ericson et al. published micromolar affinities, 7 µM on hMC1R and 100 µM on hMC4R 59 . According to Ericson et al., this difference may be explained by a discrepancy between protocols. We used herein the same methodology as Ericson et al. but with a longer incubation time (3 hours versus 1 hour)

3 for N-BUTX-Ptr1a. This last remark was of particular interest as in a previous study, Nix et al. demonstrated the role of large patches of positively charged residues

ACS Paragon Plus EnvironmentJournal of Medicinal Chemistry

was supported by the University of Paris-Sud. JT was funded by GOC2319N and GOA4919N (F.W.O.

Vlaanderen) and CELSA/17/047 (BOF, KU Leuven). SP is a postdoctoral fellow supported by KU Leuven funding (PDM/19/164).

FUNDING

This work was supported by the European FP7 project 278346 2011-2015 and by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01. A PhD grant (S.R.)

respectively by uppercase and lowercase letters, a number to distinguish different toxins with similar activity and a lowercase letter for isoforms. In this study, uppercase nu greek letter (N) was chosen as new descriptor of toxins active on melanocortin receptors.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: LC-MS analysis of five representative toxins with their cystein patterns, produced by solid phase chemical synthesis; analysis of 4 representative toxins with their cystein patterns, produced by recombinant expression; quality assessment at each stage of N-TRTX-Preg1a and N-BUTX-Ptr1a production in Fmoc solid-phase chemical synthesis; activity of 100 μM N-TRTX-Preg1a on ion channels; activity of 100 μM N-BUTX-Ptr1a on ion channels; cysteine pattern diversity for toxins containing an even number of cysteines identified in the Venomics bank; cysteine pattern diversity for toxins containing an odd number of cysteine residues identified in the Venomics bank; experimental NMR data used for the calculations, and final structural statistics for the ten models representative of the solution structure of N-TRTX-Preg1a; experimental NMR data used for the calculations, and final structural statistics for the ten models representative of the solution structure of N-BUTX-Ptr1a

AUTHOR INFORMATION

Corresponding Author *E-mail: Nicolas.gilles@cea.fr

ACKNOWLEDGMENT

We specifically thank Dr. Philipe Robin, lecturer at the Université Paris-Sud, and Mrs. Marion Chaigneau for technical support.