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International audience; The melanocortin 4 receptor (MC4R) plays a role in energy homeostasis and represents a target for treating energy balance disorders. For decades, synthetic ligands have been derived from MC4R endogenous agonists and antagonists, such as setmelanotide used to treat rare forms of genetic obesity. Recently, animal venoms have demonstrated their capacity to provide melanocortin ligands with toxins from a scorpion and a spider. Here, we described a cone snail toxin, N-CTX-Ltg1a, with a nanomolar affinity for hMC4R but unrelated to any known toxins or melanocortin ligands. We then derived from the conotoxin the linear peptide HT1-0, a competitive antagonist of G s , G 15 , and β-arrestin2 pathways with a low nanomolar affinity for hMC4R. Similar to endogenous ligands, HT1-0 needs hydrophobic and basic residues to bind hMC4R. Altogether, it represents the first venom-derived peptide of high affinity on MC4R and paves the way for the development of new MC4R antagonists.

Background and purpose. Venomous animals express numerous Kunitz-type peptides. The mambaquaretin-1 (MQ1) recently identified from the Dendroaspis angusticeps venom is the most selective antagonist of the arginine-vasopressin V2 receptor (V2R) and the unique Kunitz-type peptide active on a GPCR. We aimed to exploit other mamba venoms to enlarge the V2R-Kunitz peptide family and get insight into the MQ1 molecular mode of action. Experimental approach. We used a bio-guided screening assay to identify novel MQs and placed them phylogenetically. Several newly identified MQs were produced by solid phase peptide synthesis. They were characterized in vitro by binding and functional tests andin vivo by diuresis measurement in rats. Key results. Eight additional MQs were identified with nanomolar affinities for the V2R, all antagonists. MQs form a new subgroup in the Kunitz family, close to the V2R non-active dendrotoxins and to 2 V2R active cobra toxins. Sequence comparison between active and non-active V2R Kunitz peptides highlighted 5 specific V2R positions. Four of them are involved in V2R activity and belong to the 2 large MQ1 loops. We finally determined that 8 positions, part of these 2 loops, interact with the V2R. The variant MQ1-K39A showed specificity for the human versus the rat V2R . Conclusions and implications. A third function and mode of action is now associated with the Kunitz-peptides. The number of MQ1 residues involved in V2R binding is large and may explain its absolute selectivity. MQ1-K39A represents the first step in the improvement of the MQ1 design for medicinal perspective.

Over the two last decades, venom toxins have been explored as alternatives to opioids to treat chronic debilitating pain. At present, approximately 20 potential analgesic toxins, mainly from spider venoms, are known to inhibit with high affinity the NaV1.7 subtype of voltage-gated sodium (NaV) channels, the most promising genetically validated antinociceptive target identified so far. The present study aimed to consolidate the development of phlotoxin 1 (PhlTx1), a 34-amino acid and 3-disulfide bridge peptide of a Phlogiellus genus spider, as an antinociceptive agent by improving its affinity and selectivity for the human (h) NaV1.7 subtype. The synthetic homologue of PhlTx1 was generated and equilibrated between two conformers on reverse-phase liquid chromatography and exhibited potent analgesic effects in a mouse model of NaV1.7-mediated pain. The effects of PhlTx1 and 8 successfully synthetized alanine-substituted variants were studied (by automated whole-cell patch-clamp electrophysiology) on cell lines stably overexpressing hNaV subtypes, as well as two cardiac targets, the hCaV1.2 and hKV11.1 subtypes of voltage-gated calcium (CaV) and potassium (KV) channels, respectively. PhlTx1 and D7A-PhlTx1 were shown to inhibit hNaV1.1–1.3 and 1.5–1.7 subtypes at hundred nanomolar concentrations, while their affinities for hNaV1.4 and 1.8, hCaV1.2 and hKV11.1 subtypes were over micromolar concentrations. Despite similar analgesic effects in the mouse model of NaV1.7-mediated pain and selectivity profiles, the affinity of D7A-PhlTx1 for the NaV1.7 subtype was at least five times higher than that of the wild-type peptide. Computational modelling was performed to deduce the 3D-structure of PhlTx1 and to suggest the amino acids involved in the efficiency of the molecule. In conclusion, the present structure–activity relationship study of PhlTx1 results in a low improved affinity of the molecule for the NaV1.7 subtype, but without any marked change in the molecule selectivity against the other studied ion channel subtypes. Further experiments are therefore necessary before considering the development of PhlTx1 or synthetic variants as antinociceptive drug candidates.

Introduction It is estimated that there are about 170,000 venomous species throughout the world. The venom produced by their glands contains hundreds of bioactive compounds. These natural libraries open complete new therapeutic avenues. Heart failure is a cardiac disease associated with morbidities. Recent progress in medicine allowed to develop treatments, yet they are often not highly efficient and possess harmful side effects. Given that venom compounds are pharmacologically active, they can be considered as a source of life-saving therapeutics. Objective The aim of our study is to find therapeutic effects of venom compounds to treat hypertension which can lead to heart failure. Method Black Mamba snake venom fractionations were performed by using cation exchange and RP-HPLC techniques. The obtained fractions and purified compounds were investigated for their ability to displace 3H-NMS from muscarinic type 2 receptors (M2R). One particular snake peptide, found to be active, was de novo sequenced and synthesized. This peptide was studied in vivo by invasive hemodynamics to evaluate the main cardiovascular parameters (n = 5, 100 μg/kg) and ex vivo on isolated mesenteric and aorta arteries (n = 6; 0.8 nM, 8 nM, 80 nM and 8 μM). Wistar rats that were 10–12 weeks old were used for this study. Results After consecutive fractionations of the venom, we succeeded to identify a unique peptide with major hypotension effects. Once injected intravenously in vivo, invasive hemodynamic studies highlighted a significant arterial blood pressure decrease (−65%, P Conclusion In this study, we demonstrated the hypotensive effect of an isolated peptide from Black Mamba snake. These results confirm that venom compounds represent a promising opportunity to develop new drugs.