From a Cone Snail Toxin to a Competitive MC4R Antagonist.

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 Gs, G15, and ß-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.

MT9, a natural peptide from black mamba venom antagonizes the muscarinic type 2 receptor and reverses the M2R-agonist-induced relaxation in rat and human arteries.

All five muscarinic receptors have important physiological roles. The endothelial M2 and M3 subtypes regulate arterial tone through direct coupling to Gq or Gi/o proteins. Yet, we lack selective pharmacological drugs to assess the respective contribution of muscarinic receptors to a given function. We used mamba snake venoms to identify a selective M2R ligand to investigate its contribution to arterial contractions. Using a bio-guided screening binding assay, we isolated MT9 from the black mamba venom, a three-finger toxin active on the M2R subtype. After sequencing and chemical synthesis of MT9, we characterized its structure by X-ray diffraction and determined its pharmacological characteristics by binding assays, functional tests, and ex vivo experiments on rat and human arteries. Although MT9 belongs to the three-finger fold toxins family, it is phylogenetically apart from the previously discovered muscarinic toxins, suggesting that two groups of peptides evolved independently and in a convergent way to target muscarinic receptors. The affinity of MT9 for the M2R is 100 times stronger than that for the four other muscarinic receptors. It also antagonizes the M2R/Gi pathways in cell-based assays. MT9 acts as a non-competitive antagonist against acetylcholine or arecaine, with low nM potency, for the activation of isolated rat mesenteric arteries. These results were confirmed on human internal mammary arteries. In conclusion, MT9 is the first fully characterized M2R-specific natural toxin. It should provide a tool for further understanding of the effect of M2R in various arteries and may position itself as a new drug candidate in cardio-vascular diseases.

A new Kunitz-type snake toxin family associated with an original mode of interaction with the vasopressin 2 receptor.

BACKGROUND AND PURPOSE: Venomous animals express numerous Kunitz-type peptides. The mambaquaretin-1 (MQ1) peptide identified from the Dendroaspis angusticeps venom is the most selective antagonist of the arginine-vasopressin V2 receptor (V2R) and the only unique Kunitz-type peptide active on a GPCR. We aimed to exploit other mamba venoms to enlarge the V2R-Kunitz peptide family and gain 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. MQs were produced by solid-phase peptide synthesis and characterized in vitro by binding and functional tests and in 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 two V2R-active cobra toxins. Sequence comparison between active and non-active V2R Kunitz peptides highlighted five positions, among which four are involved in V2R interaction and belong to the two large MQ1 loops. We finally determined that eight positions, part of these two loops, interact with the V2R. The variant MQ1-K39A showed a higher affinity for the hV2R, but not for the rat V2R. CONCLUSIONS AND IMPLICATIONS: A new function and mode of action is 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 from a medicinal perspective.

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

Animal venoms are rich in hundreds of toxins with extraordinary biological activities. Their exploitation is difficult due to their complexity and the small quantities of venom available from most venomous species. We developed a Venomics approach combining transcriptomic and proteomic characterization of 191 species and identified 20,206 venom toxin sequences. Two complementary production strategies based on solid-phase synthesis and recombinant expression in Escherichia coli generated a physical bank of 3597 toxins. Screened on hMC4R, this bank gave an incredible hit rate of 8%. Here, we focus on two novel toxins: N-TRTX-Preg1a, exhibiting an inhibitory cystine knot (ICK) motif, and N-BUTX-Ptr1a, a short scorpion-CSαß structure. Neither N-TRTX-Preg1a nor N-BUTX-Ptr1a affects ion channels, the known targets of their toxin scaffolds, but binds to four melanocortin receptors with low micromolar affinities and activates the hMC1R/Gs pathway. Phylogenetically, these two toxins form new groups within their respective families and represent novel hMC1R agonists, structurally unrelated to the natural agonists.

Evaluation of the Spider (Phlogiellus genus) Phlotoxin 1 and Synthetic Variants as Antinociceptive Drug Candidates.

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.

Green mamba peptide targets type-2 vasopressin receptor against polycystic kidney disease.

Polycystic kidney diseases (PKDs) are genetic disorders that can cause renal failure and death in children and adults. Lowering cAMP in cystic tissues through the inhibition of the type-2 vasopressin receptor (V2R) constitutes a validated strategy to reduce disease progression. We identified a peptide from green mamba venom that exhibits nanomolar affinity for the V2R without any activity on 155 other G-protein-coupled receptors or on 15 ionic channels. Mambaquaretin-1 is a full antagonist of the V2R activation pathways studied: cAMP production, beta-arrestin interaction, and MAP kinase activity. This peptide adopts the Kunitz fold known to mostly act on potassium channels and serine proteases. Mambaquaretin-1 interacts selectively with the V2R through its first loop, in the same manner that aprotinin inhibits trypsin. Injected in mice, mambaquaretin-1 increases in a dose-dependent manner urine outflow with concomitant reduction of urine osmolality, indicating a purely aquaretic effect associated with the in vivo blockade of V2R. CD1-pcy/pcy mice, a juvenile model of PKD, daily treated with 13 [Formula: see text]g of mambaquaretin-1 for 99 d, developed less abundant (by 33%) and smaller (by 47%) cysts than control mice. Neither tachyphylaxis nor apparent toxicity has been noted. Mambaquaretin-1 represents a promising therapeutic agent against PKDs.

Pharmacological characterization of zinc and copper interaction with the human alpha(1A)-adrenoceptor.

Metal ions have a major role in human health, and interact with many classes of receptors including the G-protein coupled receptors. In the peripheral system, zinc mainly accumulates in the soft prostate organ and, with copper, influences prostate disease progression, from normal to hypertrophic or cancerous states. The development of these pathologies may be influenced by the α(1A)-adrenoceptor, the principal regulator of prostate tonicity. There is currently no information on possible interactions between metals and the α(1A)-adrenoceptor. We therefore studied the effects of several mono- and divalent ions on this receptor subtype using binding and functional experiments performed on expressed cloned human α(1A)-adrenoceptor. Regardless of the counter anion used, Zn(2+) and Cu(2+) interact with α(1A)-adrenoceptor with apparent affinities in the low micromolar range. In addition, using specific binding experiments, we established that these ions acted as negative allosteric ligands on prazosin/α(1A)-adrenoceptor interaction, but in a different manner from the allosteric modulator 5-(N-ethyl-N-isopropyl)-amiloride, suggesting distinct mode of interaction. In addition, the presence of Cu(2+) weakly decreased epinephrine affinity, whereas the addition of Zn(2+) shifted to the left the epinephrine binding curve, revealing a positive allosteric effect but only on half of the binding site. Finally, cell-based functional experiments demonstrated that Zn(2+) and Cu(2+) antagonized epinephrine activation in an insurmountable manner, by reducing agonist efficacy without any shift in the epinephrine activation curves. This study shows the interactions between metal ions and the α(1A)-adrenoceptor with affinities compatible with physiological concentrations and suggests that zinc and copper may have a biological role in prostate function.