Pain therapeutics from cone snail venoms: From Ziconotide to novel non-opioid pathways.

There have been numerous attempts to develop non-opioid drugs for severe pain, but the vast majority of these efforts have failed. A notable exception is Ziconotide (Prialt®), approved by the FDA in 2004. In this review, we summarize the present status of Ziconotide as a therapeutic drug and introduce a wider framework: the potential of venom peptides from cone snails as a resource providing a continuous pipeline for the discovery of non-opioid pain therapeutics. An auxiliary theme that we hope to develop is that these venoms, already a validated starting point for non-opioid drug leads, should also provide an opportunity for identifying novel molecular targets for future pain drugs. This review comprises several sections: the first focuses on Ziconotide as a therapeutic (including a historical retrospective and a clinical perspective); followed by sections on other promising Conus venom peptides that are either in clinical or pre-clinical development. We conclude with a discussion on why the outlook for discovery appears exceptionally promising. The combination of new technologies in diverse fields, including the development of novel high-content assays and revolutionary advancements in transcriptomics and proteomics, puts us at the cusp of providing a continuous pipeline of non-opioid drug innovations for pain. SIGNIFICANCE: The current opioid epidemic is the deadliest drug crisis in American history. Thus, this review on the discovery of non-opioid pain therapeutics and pathways from cone snail venoms is significant and timely.

Deep venomics reveals the mechanism for expanded peptide diversity in cone snail venom.

Cone snails produce highly complex venom comprising mostly small biologically active peptides known as conotoxins or conopeptides. Early estimates that suggested 50-200 venom peptides are produced per species have been recently increased at least 10-fold using advanced mass spectrometry. To uncover the mechanism(s) responsible for generating this impressive diversity, we used an integrated approach combining second-generation transcriptome sequencing with high sensitivity proteomics. From the venom gland transcriptome of Conus marmoreus, a total of 105 conopeptide precursor sequences from 13 gene superfamilies were identified. Over 60% of these precursors belonged to the three gene superfamilies O1, T, and M, consistent with their high levels of expression, which suggests these conotoxins play an important role in prey capture and/or defense. Seven gene superfamilies not previously identified in C. marmoreus, including five novel superfamilies, were also discovered. To confirm the expression of toxins identified at the transcript level, the injected venom of C. marmoreus was comprehensively analyzed by mass spectrometry, revealing 2710 and 3172 peptides using MALDI and ESI-MS, respectively, and 6254 peptides using an ESI-MS TripleTOF 5600 instrument. All conopeptides derived from transcriptomic sequences could be matched to masses obtained on the TripleTOF within 100 ppm accuracy, with 66 (63%) providing MS/MS coverage that unambiguously confirmed these matches. Comprehensive integration of transcriptomic and proteomic data revealed for the first time that the vast majority of the conopeptide diversity arises from a more limited set of genes through a process of variable peptide processing, which generates conopeptides with alternative cleavage sites, heterogeneous post-translational modifications, and highly variable N- and C-terminal truncations. Variable peptide processing is expected to contribute to the evolution of venoms, and explains how a limited set of ∼ 100 gene transcripts can generate thousands of conopeptides in a single species of cone snail.

Large-scale discovery of conopeptides and conoproteins in the injectable venom of a fish-hunting cone snail using a combined proteomic and transcriptomic approach.

Predatory marine snails of the genus Conus use venom containing a complex mixture of bioactive peptides to subdue their prey. Here we report on a comprehensive analysis of the protein content of injectable venom from Conus consors, an indo-pacific fish-hunting cone snail. By matching MS/MS data against an extensive set of venom gland transcriptomic mRNA sequences, we identified 105 components out of ~400 molecular masses detected in the venom. Among them, we described new conotoxins belonging to the A, M- and O1-superfamilies as well as a novel superfamily of disulphide free conopeptides. A high proportion of the deduced sequences (36%) corresponded to propeptide regions of the A- and M-superfamilies, raising the question of their putative role in injectable venom. Enzymatic digestion of higher molecular mass components allowed the identification of new conkunitzins (~7 kDa) and two proteins in the 25 and 50 kDa molecular mass ranges respectively characterised as actinoporin-like and hyaluronidase-like protein. These results provide the most exhaustive and accurate proteomic overview of an injectable cone snail venom to date, and delineate the major protein families present in the delivered venom. This study demonstrates the feasibility of this analytical approach and paves the way for transcriptomics-assisted strategies in drug discovery.

Alpha-conopeptides specifically expressed in the salivary gland of Conus pulicarius.

To date, studies conducted on cone snail venoms have attributed the origins of this complex mixture of neuroactive peptides entirely to gene expression by the secretory cells lining the lumen of the venom duct. However, specialized tissues such as the salivary glands also secrete their contents into the anterior gut and could potentially contribute some venom components injected into target animals; evidence supporting this possibility is reported here. Sequence analysis of a cDNA library created from a salivary gland of Conus pulicarius revealed the expression of two transcripts whose predicted gene products, after post-translational processing, strikingly resemble mature conopeptides belonging to the alpha-conotoxin family. These two transcripts, like alpha-conotoxin transcripts, putatively encode mature peptides containing the conserved A-superfamily cysteine pattern (CC-C-C) but the highly conserved A-superfamily signal sequences were not present. Analysis of A-superfamily members expressed in the venom duct of the same C. pulicarius specimens revealed three putative alpha-conotoxin sequences; the salivary gland transcripts were not found in the venom duct cDNA library, suggesting that these alpha-conotoxins are salivary gland specific. Therefore, expression of conotoxin-like gene products by the salivary gland could potentially add to the complexity of Conus venoms.