​ Lab LEADER Denis Servent 01 69 08 52 02 denis.servent@cea.fr

​DISCOVERY OF IMAGING AGENTS AND DRUG CANDIDATES PEPTIDE ENGINEERING  PHARMACOLOGICAL STUDIES  IN VIVO PROOF OF CONCEPT AND BIODISTRIBUTION STUDIES DETECTION OF NEUROTOXINS

LAB MEMBERS 

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Biodiversity as a source of theranostic agents  

Nature is a vast reservoir of bioactive substances that have evolved over millions of years to confer properties beneficial to human health. Our expertise lies in mastering the entire value chain required to transform a natural molecule into both a therapeutic candidate and an imaging agent. The process begins with the identification of a therapeutic target associated with an unmet medical need. We focus primarily on the family of G protein-coupled receptors (GPCRs). Hit compounds are identified through screening approaches and subsequently synthesized using solid-phase peptide synthesis (SPPS), followed by in vitro pharmacological characterization and in vivo evaluation in relevant disease models.

This comprehensive workflow has been specifically adapted to animal toxins, which are disulfide-rich peptides. Our preferred sources of natural compounds include venoms, as well as the Venomics peptide library, which comprises 3,600 synthetic toxins (Reynaud et al., 2020; Droctove et al., 2020; Goncalves et al., 2019; Ciolek et al., 2017; Van Baelen et al., 2023). Numerous animal toxins targeting GPCRs of therapeutic interest have been identified, characterized, and patented. For the most promising candidates, biological properties have been further optimized through molecular engineering to generate advanced therapeutic leads.

The Venomics screening strategy: This unique bank of 3600 toxins coming from the omics study of 200 venomous animals allows high throughput screening (HTS) resulting in the discovery of several original peptides interacting with G-protein coupled receptors (GPCRs).

Contact: Nicolas Gilles; nicolas.gilles@cea.fr​

Peptide engineering

The laboratory has developed strong expertise in peptide chemistry and engineering, with the capacity to routinely synthesize and optimize complex peptide sequences, including long peptides of natural or endogenous origin. We have extensive experience in solid-phase peptide synthesis (SPPS) of both linear and highly cross-linked peptides, enabling precise control over sequence design and molecular architecture. We are particularly specialized in the synthesis of cysteine-rich peptides containing multiple disulfide bridges. For these structurally constrained systems, we have optimized folding protocols that allow fine control of oxidative folding and disulfide bond pairing, ensuring correct tertiary structure formation and preservation of biological activity. These optimized folding strategies are essential for the functional characterization of toxins and other structurally complex bioactive peptides.

To define and optimize the pharmacological profiles of peptides studied in the laboratory, we combine SPPS with advanced peptide engineering approaches. These include systematic sequence modifications for structure–activity relationship (SAR) studies, deimmunization strategies, and pharmacodynamic/pharmacokinetic optimizations. Our platform also enables multiple, site-specific functionalizations and diverse chemical derivatizations.

Our dedicated chemistry platforms enable the routine design and synthesis of peptide-based molecular tools, supported by state-of-the-art infrastructure, including two automated SPPS synthesizers and five analytical and preparative HPLC systems. Modern coupling strategies combined with integrated biochemical approaches allow us to produce highly pure and structurally defined peptide constructs suitable for mechanistic studies, imaging applications, and therapeutic development.​

Contact : Nicolo Tonali ; nicolo.tonali@cea.fr

Pharmacological studies

The pharmacological characterization of the interactions between the different peptides (animal toxins, endogenous peptides...) or small molecules (phycotoxins) studied in the laboratory and their respective molecular targets is performed using in-vitro, ex-vivo and in-vivo experiments. These studies are crucial in the understanding of the mode of action of these molecules and to assess their potential development as imaging agents or innovative drugs, according to their pharmacological profile. In addition, the laboratory operates within a fully integrated cellular, biochemical, and biophysical research environment dedicated to molecular and cellular neuroscience to study for example the Aβ1-42 oligomerization process.

Experimental approaches

1.       Binding of ligands on various receptors (GPCRs, nicotinic acetylcholine receptors (nAChRs), ion channels)

Our ability to label (¹²⁵I, ³H, ¹⁴C) and use radioactive molecules allows us to perform binding assays to characterize the fundamental parameters of toxin/target interactions by kinetic (Kon, Koff), saturation (K_D, Bmax) or competition experiments (IC₅₀, Ki). In addition, binding assays allow to study the mode of action (competitive/non-competitive antagonism, allostery) and the determination of the pharmacological profile (subtype and species selectivity) of studied compounds (Blanchet et al., 2017; Petrel et al., 2013).

2.       Functionnal characterization of Ligand-GPCRs interaction

The GPCR ligands we investigate in the laboratory can exert different effects on their target receptors, either activating them (agonists), blocking them (antagonists) or modulating them (allosteric modulators). To characterize their pharmacological properties, we employ a range of functional assays developed in-house, most of which are based on fluorescence- or luminescence-based techniques compatible with 96- and 384-well microplate formats.

We quantify second messengers such as cAMP and inositol monophosphate (IP1), as well as downstream kinase activation, including ERK1/2 MAP kinases, using TR-FRET-based assays with commercially available HTRF kits. Upstream signaling events are further investigated using BRET-based approaches to assess miniGs recruitment (biosensors developed by N. Lambert, Augusta University), G protein activation (M. Garcia Marcos, Boston University), and β-arrestin recruitment (biosensors developed in our laboratory).

Functional characterization of the interactions between ligands and GPCR

3.       Ion channels : in-vitro/ex-vivo/in-vivo electrophysiology

The effects of bioactive molecules are evaluated on the various ion channels (mainly sodium channel subtypes) and ligand-gated ion channels (various nAChR subtypes) involved in the functioning of sensory and neuromuscular systems, both under physiological and pathological conditions, by using multiscale approaches (Molgo et al., 2020) (Goncalves et al., 2019) (Benoit et al., 2019) (Goncalves et al., 2018) (Araoz et al., 2015).

• In-vitro and ex-vivo electrophysiology

• Two-electrode voltage-clamp recording from Xenopus laevis oocytes expressing exogenous receptors and ion channels
• Patch-clamp recordings from cell lines stably expressing exogenous ion channels
• Intracellular microelectrode recording from rodent neuromuscular junctions
• Patch-clamp recordings from rodent dorsal root ganglion (DRG) neurons
  

• In-vitro/ex-vivo mechanical recordings of rodent muscle contraction
• In-vivo electrophysiological recordings from rodent sensory and neuromuscular systems
  
   

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Multiscale electrophysiological characterization of ion channel-interacting molecules.

These complementary approaches make it possible to address the question of the mode of action of neurotoxins, active for example at the neuro-muscular junction, by identifying (i) their action at the pre- or post-synaptic level, (ii) their molecular targets (NaV, CaV, Kv channels, nAChR) or (iii) their activating or inhibiting properties. Moreover, this multiscale characterization can be exploited to evaluate the specificity of the molecules developed in the laboratory and the absence of potential neuromuscular side-effects. 

Research Projects

• ​​Mambaquaretin-Vasopressin V2R interaction

The most illustrative example of our research in toxin-derived therapeutic agent is mambaquaretin, a toxin identified in the venom of the green mamba Dendroaspis angusticeps. Given the huge affinity and selectivity of this toxin for the vasopressine V2 receptor and its established potential in several kidney diseases (refractory ascites, hyponatremia, polycystic kidney dieases), mambaquaretin was optimized to generate MQ232 (Stanajic-Petrovic et al. 2025), a therapeutic candidate developed by the spin-off company V4Cure (https://v4cure.com/).

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Therapeutic development of V2R-specific Mambaquaretin for the treatment of kidney diseases

Contact: Nicolas Gilles; nicolas.gilles@cea.fr

• ​​​Innovative Antibodies targeting GPCRs

We are developing a platform for the discovery and optimization of antibodies targeting GPCRs for therapeutic applications. Antibodies offer several advantages over small synthetic molecules, which are often associated with undesirable side effects. In particular, antibodies display high target specificity, prolonged plasma half-life, and the ability to stabilize distinct receptor conformational states. We combine yeast surface display and deep mutational scanning technologies developed at SiMoS to identify and optimize nanobodies (VHHs) directed against membrane receptors. Special attention is given to preserving the correct structural presentation of receptors using innovative systems such as extracellular vesicles and nanodiscs.

Among the receptors of interest, we focus on the apelin receptor to develop antagonist antibodies for the treatment of colorectal cancer and glioblastoma, as well as agonist antibodies for cardiovascular and renal diseases, including heart failure and chronic kidney disease.​

GPCRs-targeting antibodies development by Yeast-Surface Display

Contact : Xavier.iturrioz@cea.fr

• ​​​Animal toxins targeting ion channels as cancer imaging agents

Ion channels, particularly voltage-gated sodium channels (Navs) and acid-sensing ion channels (ASICs), are increasingly recognized as key contributors to cancer biology. They are implicated in tumor progression through mechanisms involving cell migration, invasion, angiogenesis, and proliferation. The overexpression of specific channel subtypes across various cancer types highlights their potential as both biomarkers and therapeutic targets.

We have leveraged the high affinity, remarkable selectivity, and favorable pharmacokinetic properties of animal toxins targeting these ion channels to develop fluorescent and radiolabeled probes for their visualization in vitro and in vivo in tumoral processes (Baudat et al. 2025 & 2026).​

Animal toxins as theranostic agents in cancers

Contact : Denis Servent; denis.servent@cea.fr​

• ​​​Pharmacology of nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) and acetylcholinesterase (AChE) are key regulators of synaptic transmission in the central and peripheral nervous systems, mediating neuromuscular signaling and modulating neurotransmitter release. Beyond the nervous system, nAChRs are also expressed in non-neuronal cells and tissues, including epithelial and immune cells, where they fulfill diverse physiological functions.

Given their major pathophysiological roles, the development of novel nicotinic ligands and acetylcholinesterase inhibitors is of strong interest for human health, as well as for veterinary and agricultural applications. Marine phytoplankton constitutes a valuable source of structurally original bioactive compounds arising from the diversity of secondary metabolism.

Our research focuses on muscle and neuronal nAChRs to (i) characterize the mechanisms of action of marine natural products and synthetic compounds as potential leads for neurological disorders and cancer, (ii) develop countermeasures against organophosphorus nerve agents, (iii) identify novel bioactive molecules from marine and freshwater phytoplankton, and (iv) design innovative detection methodologies for dual-use threat agents.

Example 1. Bio-guided discovery of ingrilimine, a novel pro-apoptotic cyclic imine with high affinity for α7 nAChR

Ingrilimine, discovered and purified in trace amounts from Vulcanodinium rugosum (strain IFR-VRU-01), was structurally characterized by NMR as a novel cyclic imine toxin. Ingrilimine displays cytotoxic and pro-apoptotic activities against human cancer cell lines and shows a dual activity on the human α7 nicotinic acetylcholine receptor, blocking or activating it in a concentration-dependent manner. These features identify ingrilimine as a promising lead for developing new therapeutics targeting cancers and neurodegenerative diseases associated with α7 nicotinic acetylcholine receptor dysfunction.

Example 2. Shortening the gap on the discovery of nicotinic ligands: from scratch to functional molecular networks

A target-fishing methodology was developed to discover new ligands directed against nAChRs to shorten the time-gap between environmental bioactives screening and the physicochemical and functional characterization of novel chemical scaffolds active on nAChRs generating functional molecular networks to enrich the universe of nicotinic-ligands chemical structural diversity. The latter is an outcome from our toxin detection methodologies developed in our laboratory.​

Example 3. Functional evaluation of MTDL countermeasure compounds

Organophosphorus nerve agents (OPNAs) irreversibly inhibit acetylcholinesterase (AChE), causing acetylcholine accumulation and overstimulation of muscarinic and nicotinic receptors at cholinergic synapses. This cholinergic crisis leads to seizures, muscle paralysis, respiratory failure, and death. Current treatments combine an AChE reactivator, atropine (muscarinic antagonist), and diazepam (anticonvulsant), but they do not directly address nicotinic receptor dysfunction, which plays a major role in neuromuscular and respiratory impairment.

Within the ANR MULTIDOTE project, we developed multi-target-directed antidotes (MTDAs) designed to both reactivate AChE and modulate nicotinic receptors. These hybrid molecules couple an AChE reactivator to a nicotinic ligand via a carbon linker. Among ~50 synthesized compounds, several showed in vitro efficacy by reactivating OPNA-inhibited AChE and exerting antagonistic activity at the α7 nicotinic receptor.

Contact : Romulo Araoz ; romulo.araoz@cea.fr

• ​​Deciphering the role and toxicity of Aβ1-42 various oligomers in Alzheimer disease

In addition, the laboratory operates within a fully integrated cellular, biochemical, and biophysical environment dedicated to molecular and cellular neuroscience. It includes controlled cell culture facilities supporting the maintenance and differentiation of SH-SY5Y human neuroblastoma cells into stable neuron-like phenotypes. These models are rigorously validated by immunofluorescence imaging, RT-qPCR profiling of neuronal, inflammatory, and synaptic markers, and functional metabolic assays, ensuring robustness for studying Aβ1-42–induced perturbations across defined aggregation states. The unit also has strong expertise in amyloid aggregation, with established protocols to generate and characterize monomeric, oligomeric, and fibrillar Aβ species using fluorescence spectroscopy, aggregation kinetics assays, circular dichroism, solution-state NMR, transmission electron microscopy, dynamic light scattering, and conformation-specific immunoassays. Biochemistry platforms include UV–Vis and fluorescence spectrophotometers, microplate readers for kinetic and binding studies, and SDS-PAGE/Western blot systems for quantifying soluble and oligomeric Aβ species. Advanced imaging facilities comprise high-resolution widefield and confocal microscopy equipped with near-infrared modules, enabling sensitive in cellulo detection of intracellular Aβ1-42 oligomers using NIR peptide probes. These capabilities are complemented by dedicated computational resources for quantitative image analysis, aggregation-kinetic modeling, and integrative correlation of oligomer detection with neurodegenerative and neuroinflammatory readouts (Herrera et al. 2025).​

Contact : Nicolo Tonali ; nicolo.tonali@cea.fr

In-vivo proof of concept and biodistribution studies

In order to perform in-vivo proof of concept of the potency of the molecules studied in our laboratory, we have developed an expertise in animal models to evaluate their physiological impact, in particular on cardiovascular and renal physiology or for their anti-nociceptive property. In addition, their biodistribution within the body, or their safety/toxicity profile can be evaluated. For this purpose, a wide range of materials and expertise is accessible within our rodent facilities (Servent et al., 2021):

• Syringe/cannula/osmotic pump… to explore and compare various route of administration (intravenous, intraperitoneal, subcutaneous, intranasal, intra-tracheal, intracerebroventricular),
• Metabolic cages in order to evaluate elimination of molecule by urine and feces over time,
• BioDAQ food intake monitoring system to collect and record consumption of individual animal,
• Isoflurane anaesthetic stations enabling refinement of experimental procedures,
• Surgical expertise allowing the development of pathological study models,
• Authorization for the administration of radiolabeled molecules in rodents to observe (by digital autoradiography) and quantify (by scintillation) tissue distribution of molecules over time and characterize their pharmacokinetic properties (Oosterlaken et al. 2025).​
  

 

Biodistribution analysis using digital autoradiography. Lung distribution of radiolabeled nanoparticle 3 months after intra-tracheal administration in mouse (left) and biodistribution of ³H-pinnatoxin-G in rat embryos after intravenous injection of the toxin in pregnant rats (right).

Contact : Mathilde Keck ; mathilde.keck@cea.fr​

Araoz R, Barnes P, Sechet V, Delepierre M, Zinn-Justin S, Molgo J, Zakarian A, Hess P, Servent D. 2020. Cyclic imine toxins survey in coastal european shellfish samples: Bioaccumulation and mode of action of 28-O-palmitoyl ester of pinnatoxin-G. first report of portimine-A bioaccumulation. Harmful Algae  98, 101887. 10.1016/j.hal.2020.101887.

Araoz R, Ouanounou G, Iorga BI, Goudet A, Alili D, Amar M, Benoit E, Molgo J, Servent D. 2015. The neurotoxic effect of 13, 19-didesmethyl and 13-desmethyl spirolide C phycotoxins is mainly mediated by nicotinic rather than muscarinic acetylcholine receptors. Toxicol. Sci.  147, 156-167. 10.1093/toxsci/kfv119.

Baudat, R. Montnach, J. Benoit, E. Zoukimian, C. Carvalhosa, C. Beroud, R. Waard, M. Servent, D. 2025. Cyanine 5-huwentoxin-IV as a novel imaging probe to detect hNa(v)1.7 channel overexpressed in non-small cell lung cancer. Biochem. Pharmacol. 242, 117203

Baudat R, Diochot S, Lange L, Lingueglia E, Benoit E, Servent D. 2026. Toxin peptides targeting acid-sensing ion channels: Opportunities for cancer diagnosis and therapy. Biomed Pharmacother. 18; 196:119129. doi: 10.1016/j.biopha.2026.119129

Benoit E, Couesnon A, Lindovsky J, Iorga BI, Araoz R, Servent D, Zakarian A, Molgo J. 2019. Synthetic Pinnatoxins A and G Reversibly Block Mouse Skeletal Neuromuscular Transmission In Vivo and In Vitro. Mar Drugs  17. 10.3390/md17050306.

Blanchet G, Alili D, Protte A, Upert G, Gilles N, Tepshi L, Stura EA, Mourier G, Servent D. 2017. Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins. Sci. Rep.  7, 2701. 10.1038/s41598-017-02953-0.

Ciolek J, Reinfrank H, Quinton L, Viengchareun S, Stura EA, Vera L, Sigismeau S, Mouillac B, Orcel H, Peigneur S, Tytgat J, Droctove L, Beau F, Nevoux J, Lombes M, Mourier G, De Pauw E, Servent D, Mendre C, Witzgall R, Gilles N. 2017. Green mamba peptide targets type-2 vasopressin receptor against polycystic kidney disease. Proc. Natl. Acad. Sci. U S A  114, 7154-7159. 10.1073/pnas.1620454114.

Goncalves TC, Benoit E, Kurz M, Lucarain L, Fouconnier S, Combemale S, Jaquillard L, Schombert B, Chambard JM, Boukaiba R, Hessler G, Bohme A, Bialy L, Hourcade S, Beroud R, De Waard M, Servent D, Partiseti M. 2019. From identification to functional characterization of cyriotoxin-1a, an antinociceptive toxin from the spider Cyriopagopus schioedtei. Br. J. Pharmacol.  176, 1298-1314. 10.1111/bph.14628.

Goncalves TC, Boukaiba R, Molgo J, Amar M, Partiseti M, Servent D, Benoit E. 2018. Direct evidence for high affinity blockade of NaV1.6 channel subtype by huwentoxin-IV spider peptide, using multiscale functional approaches. Neuropharmacology  133, 404-414. 10.1016/j.neuropharm.2018.02.016.

Herrera MG, Ciccone L, Moleiro LH, Tonali N, Dodero VI. 2025. Endogenous Aβ and Exogenous Wheat Gluten Nanostructures: Understanding Peptide Self-Assembly in Disease. ACS Nano. 19(34):30688-30719. doi: 10.1021/acsnano.5c01662

Molgo J, Schlumberger S, Sasaki M, Fuwa H, Louzao MC, Botana LM, Servent D, Benoit E. 2020. Gambierol Potently Increases Evoked Quantal Transmitter Release and Reverses Pre- and Post-Synaptic Blockade at Vertebrate Neuromuscular Junctions. Neuroscience  439, 106-116. 10.1016/j.neuroscience.2019.06.024.

Oosterlaken, M. Rogliardo, A. Lipina, T. Lafon, P. A. Tsitokana, M. E. Keck, M. Cahuzac, H. Prieu-Serandon, P. Diem, S. Derieux, C. Camberlin, C. Lafont, C. Meyer, D. Chames, P. Vandermoere, F. Marin, P. Prezeau, L. Servent, D. Salahpour, A. Ramsey, A. J. Becamel, C. Pin, J. P. Kniazeff, J. Rondard, P. (2025) Nanobody therapy rescues behavioural deficits of NMDA receptor hypofunction. Nature 645 (8079), 262-270

Petrel C, Hocking HG, Reynaud M, Upert G, Favreau P, Biass D, Paolini-Bertrand M, Peigneur S, Tytgat J, Gilles N, Hartley O, Boelens R, Stocklin R, Servent D. 2013. Identification, structural and pharmacological characterization of tau-CnVA, a conopeptide that selectively interacts with somatostatin sst3 receptor. Biochem. Pharmacol.  85, 1663-71. S0006-2952(13)00219-0 [pii]

Reynaud S, Ciolek J, Degueldre M, Saez NJ, Sequeira AF, Duhoo Y, Bras JLA, Meudal H, Cabo Diez M, Fernandez Pedrosa V, Verdenaud M, Boeri J, Pereira Ramos O, Ducancel F, Vanden Driessche M, Fourmy R, Violette A, Upert G, Mourier G, Beck-Sickinger AG, Morl K, Landon C, Fontes C, Minambres Herraiz R, Rodriguez de la Vega RC, Peigneur S, Tytgat J, Quinton L, De Pauw E, Vincentelli R, Servent D, Gilles N. 2020. A Venomics Approach Coupled to High-Throughput Toxin Production Strategies Identifies the First Venom-Derived Melanocortin Receptor Agonists. J Med Chem  63, 8250-8264. 10.1021/acs.jmedchem.0c00485.

Servent D, Malgorn C, Bernes M, Gil S, Simasotchi C, Herard AS, Delzescaux T, Thai R, Barbe P, Keck M, Beau F, Zakarian A, Dive V, Molgo J. 2021. First evidence that emerging pinnatoxin-G, a contaminant of shellfish, reaches the brain and crosses the placental barrier. Sci Total Environ  790, 148125. 10.1016/j.scitotenv.2021.148125.

 Stanajic-Petrovic, G., Keck, M., Barbe, P., Urman, A., Correia, E., Isnard, P., Duong Van Huyen, J. P., Chmeis, K., Diarra, S. S., Palea, S., Theodoro, F., Nguyen, A. L., Castelli, F., Pruvost, A., Zhao, W., Mendre, C., Mouillac, B., Bienaime, F., Robin, P., Kessler, P., Llorens-Cortes, C., Servent, D., Nozach, H., Maillere, B., Guo, D., Truillet, C., and Gilles, N. 2025. MQ232, A Snake Toxin Derivative for Treatment of Hyponatremia and Polycystic Kidney Diseases. J Am Soc Nephrol 36, 181-192

Van Baelen, A. C., Iturrioz, X., Chaigneau, M., Kessler, P., Llorens-Cortes, C., Servent, D., Gilles, N., and Robin, P. 2023. Characterization of the First Animal Toxin Acting as an Antagonist on AT1 Receptor. Int J Mol Sci 24

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