Identification, structural and pharmacological characterization of τ-CnVA, a conopeptide that selectively interacts with somatostatin sst₃ receptor

Abstract

Conopeptides are a diverse array of small linear and reticulated peptides that interact with high potency and selectivity with a large diversity of receptors and ion channels. They are used by cone snails for prey capture or defense. Recent advances in venom gland transcriptomic and venom peptidomic/proteomic technologies combined with bioactivity screening approaches lead to the identification of new toxins with original pharmacological profiles. Here, from transcriptomic/proteomic analyses of the Conus consors cone snail, we identified a new conopeptide called τ-CnVA, which displays the typical cysteine framework V of the T1-conotoxin superfamily. This peptide was chemically synthesized and its three-dimensional structure was solved by NMR analysis and compared to that of TxVA belonging to the same family, revealing very few common structural features apart a common orientation of the intercysteine loop. Because of the lack of a clear biological function associated with the T-conotoxin family, τ-CnVA was screened against more than fifty different ion channels and receptors, highlighting its capacity to interact selectively with the somatostatine sst₃ receptor. Pharmacological and functional studies show that τ-CnVA displays a micromolar (Ki of 1.5 μM) antagonist property for the sst3 receptor, being currently the only known toxin to interact with this GPCR subfamily.

Introduction

Marine snails of the genus Conus are predatory gastropods that use the sophisticated arsenal of biologically active peptide present in their venom to subdue their prey or for self defense [1]. These peptide toxins have been selected through evolutionary processes to interact with a large variety of physiologically-relevant molecular targets, such as voltage-gated and ligand-gated ion channels [2], [3], [4], [5]. Even if some conopeptides possess none or only one disulfide bond, most conotoxins present in Conus venoms are disulfide-rich peptides with 12–35 residues that display a high frequency of post-translational modifications. Until now, more than 1500 non-redundant conopeptide sequences have been reported, covering less than 1% of their estimated natural diversity. These conopeptides belong to 19 gene superfamilies, each of them can include one or several cysteine frameworks and pharmacological profiles. For example, more than one hundred sequences of T-conotoxins belonging to the framework V were currently listed in the ConoServer database [6]. In addition, 140 non-redundant sequences were recently identified in the frame of bioinformatics and molecular evolution studies [7], [8] revealing a high degree of sequence variability in the mature peptides (10–36 residues in length with very low sequence identity aside from the four Cys) suggesting a broad biological function, while the signal peptide sequences were more conserved [9]. High affinity and specificity of conotoxins make them not only useful tools for neurophysiological studies but also a largely unexploited resource of novel therapeutic agents [5], [10], [11], [12]. The most well-known example of conotoxin-based drug is the ω-conotoxin MVIIA from Conus magus (known as the “magician cone”) that is used clinically under the name ziconotide for the treatment of severe chronic neuropathic pain [13]. Until now, a small number of conotoxins have been described to interact with G-protein coupled receptors (GPCRs) [14], [15], [16], despite the major physiological role of this receptor superfamily which is the target of approximately 30% of the drugs actually on the market [17]. This includes five conopressins interacting with the vasopressin receptors [18], [19], [20], [21], as for example the conopressin-T which is a selective V1a receptor antagonist [22]. In addition, three contulakins belonging to the class C gene superfamily and interacting with neurotensin receptors were identified in the venoms of C. geographus [23] and C. litteratus [24] and the antinociceptive effect of contulakin-G has led to its evaluation in clinical phase II [25], [26]. Furthermore, ρ-TIA conotoxin was purified from the C. tulipa venom [27] and found to be an allosteric antagonist of the α_1A-adrenoceptor [28]. Finally, a patent reports preliminary results on the activity of some conotoxins on GPCRs such as, melanocortin or somatostatin receptors [29].

The hormone somatostatin, also named SRIF (somatotropin release-inhibiting factor), is an endogenous tetradecapeptide cyclized by one disulfide bond that is produced by the hypothalamus and has been reported to support numerous physiological functions by inhibiting the release of several secondary hormones such as glucagon, insulin, gastrin, secretin, and growth hormone [30]. The effects of somatostatin are related to its interaction with five human somatostatin receptors subtypes (hsst1-5) belonging to the class A4 of the G-proteins coupled receptors superfamily [31], [32]. The somatostatin hormone has a nanomolar affinity toward the five sst-receptor subtypes but its low selectivity and very short half-life in the plasma limits considerably its therapeutic potential [33], explaining the intensive research in the last decades of SRIF analogs as therapeutic agents [34].

Due, on the one hand to the lack of selective and potent ligands for many GPCR subtypes including sst-receptors and on the other hand, to the extraordinary diversity of toxins present in animal venoms, we have developed a screening strategy based on binding experiments in order to identify new toxins active on this receptor superfamily. In the present study, we described the identification from transcriptomic and peptidomic analysis of the fish-hunting C. consors venom [35] of a toxin, called τ-CnVA, which may belong to the T1-conotoxin superfamily. This toxin was chemically synthesized by solid phase peptide synthesis, refolded and its three-dimensional structure was solved by NMR analysis. Furthermore, using electrophysiological and binding experiments on more than fifty different receptors and ion channels in the frame of a pharmacological profiling study, the property of τ-CnVA to interact selectively with the somatostatin sst3 receptor subtype was demonstrated. Pharmacological and functional studies show that τ-CnVA displays antagonist property for the sst3 receptor, being the first toxin known to interact selectively with this GPCR subfamily. Finally, among six other T1-conotoxins that have been synthesized and pharmacologically characterized, only LiC32 from the vermivorus Conus lividus with poor sequence homology compared to the others, revealed a micromolar affinity on sst3-receptor (Ki of 3.5 μM), highlighting that no direct correlation exists between T1-conotoxins and a somatostatin sst3 function.