Venomics as a Drug Discovery Platform: Identifying Conopeptides with Pharmacological Activity

Cone snail venom is a mixture of disulfide-constrained peptides (conotoxins), hormone-like peptides, and proteins that have been ‘weaponized’ for predation and defense. Venom peptides, or conopeptides, have efficiently evolved to bind receptors and ion channels that modulate the neuromuscular, cardiovascular, and central nervous systems in prey species. With over 850 species of cone snails, each with unique venom concoctions, cone snail venom is a valuable source of novel pharmacological probes and potential drug leads. However, the complexity of the venom poses a challenge for drug discovery. Contributing to the complexity is 1) a wide range in molecular weight 2) peptide hyper-variability by post-translational modifications and 3) many potential molecular targets to pursue. In this research, a ‘venomics’ approach was employed for the global identification of venom components. This ‘venomics’ methodology combines RNAseq data from the venom duct and proteomic data from raw injected venom to identify novel conopeptides. This project was a data-driven effort to define the venom components of the cone snail, Conus purpurascens, and to stimulate further hypothesis-driven studies. First, 21 new base conopeptides were identified from the injected venom of Conus purpurascens, a fish-hunting cone snail native to the Pacific coast of Central America. The molecular targets were projected based on homology to previously characterized conopeptides. The newly identified conopeptides included α-conotoxin, α-PID. Alpha-conotoxins are inhibitory ligands of nicotinic acetylcholine receptors (nAChRs), and the most ubiquitous venom components across the Conus genus. Ligands of nAChRs are clinically important for addiction, cognitive disorders, neurodegenerative diseases, and pain. Functional characterization of α-PID and three other α-conotoxins was performed to test their activity on different nAChR subtypes using heterologous receptor expression and molecular modeling techniques. A unique insulin-like peptide (Con-Ins P1) was also identified and was the first instance of an insulin-like peptide identified directly from injected venom. This research demonstrates how discovery-based ‘venomics’ workflows can be used to yield novel peptides with pharmacological applications and stimulate further hypothesis-driven experiments

Doctor of Philosophy

dissertationCone snails (genus Conus) have attracted scientific interest for the great neuropharmacological potential of their venoms to treat chronic pain, which consist of a complex mixture of peptides known as conotoxins. For discovery purposes, we have carried out a survey of the venom-ducts of 22 Conus species using next generation high throughput RNAseq (NGS). In silico analyses of these data are complicated because paralogous conotoxin precursors display both highly conserved, as well as hyper varied regions. As a result, NGS-based discovery involves an inherent trade off between fidelity of transcript assembly and sensitivity towards novel discovery. On the one hand, overly lenient assembly parameters create a few, long, but misassembled chimeric transcripts, which lessen the true discovery potential of NGS. On the other hand, overly stringent assembly parameters can mistake sequencing artifacts as novel discoveries. Moreover, many new conotoxins likely remain undiscovered. This fact can complicate homology-based discovery efforts using tools such as BLAST because reference databases may lack homologous peptides, leading to false negative results. With these problems in mind, I developed a comprehensive pipeline for discovery of conotoxins and their modification enzymes from high throughput RNAseq data. My pipeline includes (1) simulation software for benchmarking purposes, (2) a ‘partial extension pipeline' that employs a novel kmerization tool called Taxonomer to rapidly cluster and taxonomically classify reads prior to assembly, and (3) a discovery engine that can identify novel conotoxins even when they lack significant homologs. Collectively, my pipeline maximizes the discovery potential of Conus RNAseq data, identifying on average ~ 30% more full length toxins per sample than any other than approach in use today

Novel conopeptides of largely unexplored Indo Pacific <i>Conus</i> sp.

Cone snails are predatory creatures using venom as a weapon for prey capture and defense. Since this venom is neurotoxic, the venom gland is considered as an enormous collection of pharmacologically interesting compounds having a broad spectrum of targets. As such, cone snail peptides represent an interesting treasure for drug development. Here, we report five novel peptides isolated from the venom of Conus longurionis, Conus asiaticus and Conus australis. Lo6/7a and Lo6/7b were retrieved from C. longurionis and have a cysteine framework VI/VII. Lo6/7b has an exceptional amino acid sequence because no similar conopeptide has been described to date (similarity percentage C. asiaticus, has a typical framework III Cys arrangement, classifying the peptide in the M-superfamily. Asi14a, another peptide of C. asiaticus, belongs to framework XIV peptides and has a unique amino acid sequence. Finally, AusB is a novel conopeptide from C. australis. The peptide has only one disulfide bond, but is structurally very different as compared to other disulfide-poor peptides. The peptides were screened on nAChRs, NaV and KV channels depending on their cysteine framework and proposed classification. No targets could be attributed to the peptides, pointing to novel functionalities. Moreover, in the quest of identifying novel pharmacological targets, the peptides were tested for antagonistic activity against a broad panel of Gram-negative and Gram-positive bacteria, as well as two yeast strains

A perspective on toxicology of Conus venom peptides

Abstract The evolutionarily unique and ecologically diverse family Conidae presents fundamental opportunities for marine pharmacology research and drug discovery. The focus of this investigation is to summarize the worldwide distribution of Conus and their species diversity with special reference to the Indian coast. In addition, this study will contribute to understanding the structural properties of conotoxin and therapeutic application of Conus venom peptides. Cone snails can inject a mix of various conotoxins and these venoms are their major weapon for prey capture, and may also have other biological purposes, and some of these conotoxins fatal to humans. Conus venoms contain a remarkable diversity of pharmacologically active small peptides; their targets are an iron channel and receptors in the neuromuscular system. Interspecific divergence is pronounced in venom peptide genes, which is generally attributed to their species specific biotic interactions. There is a notable interspecific divergence observed in venom peptide genes, which can be justified as of biotic interactions that stipulate species peculiar habitat and ecology of cone snails. There are several conopeptides used in clinical trials and one peptide (Ziconotide) has received FDA approval for treatment of pain. This perspective provides a comprehensive overview of the distribution of cone shells and focus on the molecular approach in documenting their taxonomy and diversity with special reference to geographic distribution of Indian cone snails, structure and properties of conopeptide and their pharmacological targets and future directions

Identification and Characterization of a Novel Family of Cysteine-Rich Peptides (MgCRP-I) from Mytilus galloprovincialis

We report the identification of a novel gene family (named MgCRP-I) encoding short secreted cysteine-rich peptides in the Mediterranean mussel Mytilus galloprovincialis. These peptides display a highly conserved pre-pro region and a hypervariable mature peptide comprising six invariant cysteine residues arranged in three intramolecular disulfide bridges. Although their cysteine pattern is similar to cysteines-rich neurotoxic peptides of distantly related protostomes such as cone snails and arachnids, the different organization of the disulfide bridges observed in synthetic peptides and phylogenetic analyses revealed MgCRP-I as a novel protein family. Genome- and transcriptome-wide searches for orthologous sequences in other bivalve species indicated the unique presence of this gene family in Mytilus spp. Like many antimicrobial peptides and neurotoxins, MgCRP-I peptides are produced as pre-propeptides, usually have a net positive charge and likely derive from similar evolutionary mechanisms, that is, gene duplication and positive selection within the mature peptide region; however, synthetic MgCRP-I peptides did not display significant toxicity in cultured mammalian cells, insecticidal, antimicrobial, or antifungal activities. The functional role of MgCRP-I peptides in mussel physiology still remains puzzling

Characterization of peptides derived from marine organisms

Casey Schmidt studied peptides from marine organisms in a range of different aspects focusing on their potential use as drug leads. She investigated a previously described peptide from the venom of a cone snail and the relationship between its three-dimensional structure and its function. She also discovered and characterized five new peptides from the stony coral Heliofungia actiniformis

Influence of the Conformation of Conotoxins on their Bioactivity

In the last decade peptides filled the gap between small molecule drugs and biologics as therapeutics. A rich source of such peptides represents the venom of different animals evolved over millions of years. Conotoxins are small, disulfide-rich neuropeptides isolated from cone snails of the genus Conus, which act on different biological targets like ion channels or receptors. These toxins contain a complex cocktail of bioactive substances and are utilized for self-defence or hunting prey. With over 700 species known, these marine invertebrates reveal a large potential of novel pharmacologically active molecules, e.g. for the treatment of severe and chronic pain. Although peptide synthesis is established nowadays, preparation and characterization of disulfide-rich peptides is still a challenge. The formation of different disulfide isomers is one main issue facilitating the elucidation of such disulfide-rich peptides and proteins. The aim of this work was to investigate the folding of small multiple disulfide-bridged peptides under different conditions and perform structure-activity relationship studies of the resulting products. Within the scope of this project different conotoxins were synthesized by solidphase peptide synthesis. Disulfide formation was achieved by different methods i.e. oxidative self-folding, oxidation in Ionic Liquids and successive oxidation in combination with an orthogonal protecting group strategy. The oxidized conotoxins were systematically investigated and characterized with different analytical methods. Elucidation of the final three-dimensional structure was performed by NMR spectroscopy. Furthermore, determination of the disulfide connectivity was investigated by different methods of mass spectrometric analysis (LC-ESI and MALDI) after partial reduction and derivatization. The results of all applied analytical methods revealed their advantages and limitations for the discrimination of different disulfide isomers within conotoxins depending on primary amino acid sequence. Biological testing of the synthesized peptides on ion channels was performed in parallel with computational studies to enlighten the structure-activity relationships. The work presented in this thesis significantly extends the knowledge about conotoxins and disulfide formation, their impact with respect to biological activity, and the challenging characterization and structure elucidation procedures.Auswirkung der Faltung von Conotoxinen auf ihre Biologische Aktivität In den letzten Jahrzehnten haben Peptidtherapeutika begonnen, die Lücke zwischen niedermolekularen Arzneimitteln und Biopharmazeutika zu schließen. Eine reiche Quelle für solche Peptide stellen die Giftstoffe verschiedener Tiere dar, die sich über Millionen von Jahren entwickeln konnten. Conotoxine sind kleine, disulfidverbrückte, neuroaktive Peptide, die verschiedene biologische Ziele wie Ionenkanäle und Rezeptoren beeinflussen und aus dem Gift der Kegelschnecke der Gattung Conus isoliert werden. Dieses Gift besteht aus einem komplexen Cocktail aus bioaktiven Substanzen und wird von der Schnecke zur Selbstverteidigung oder zur Jagd eingesetzt. Mit über 700 bekannten Spezies bergen diese wirbellosen Meerestiere ein großes Potential für neuartige, pharmakologisch wirksame Moleküle, z.B. zur Behandlung von akuten und chronischen Schmerzen. Obwohl die Peptidsynthese heutzutage etabliert ist, stellt die Herstellung und Charakterisierung von disulfidverbrückten Peptiden immer noch eine Herausforderung dar. Hierbei ist die Bildung von unterschiedlichen disulfidverbrückten Peptidisomeren ein Hauptproblem, welches die Aufklärung disulfidverbrückter Peptide und Proteine erschwert. Das Ziel dieser Arbeit war es, die Faltung von kleinen, mehrfach disulfidverbrückten Peptide unter verschiedenen Bedingungen zu analysieren und Struktur-Aktivitätsuntersuchungen der erhaltenen Produkte durchzuführen. Im Rahmen dieses Projektes wurden unterschiedliche Conotoxine mittels Festphasenpeptidsynthese hergestellt. Zur Ausbildung der Disulfidbrücken wurden verschiedene Methoden eingesetzt: oxidative Selbstfaltung, Oxidation in ionischen Flüssigkeiten und stufenweise Oxidation in Verbindung mit einer orthogonalen Schutzgruppenstrategie. Die oxidierten Conotoxine wurden anschließend funktionell untersucht und durch verschiedene analytische Methoden charakterisiert. Mittels NMR-Spektroskopie wurden die dreidimensionalen Strukturen der erhaltenen Peptide aufgeklärt. Weiterhin konnte nach partieller Reduktion und Derivatisierung die Disulfidverbrückung durch verschiedene massenspektrometrische Verfahren (LC-ESI und MALDI) bestimmt werden. Die Ergebnisse zeigen die jeweiligen Vorteile und Limitierungen der verwendeten analytischen Methoden hinsichtlich der Unterscheidung verschiedener disulfidverbrückter Conotoxinisomere in Abhängigkeit von ihrer primären Aminosäuresequenz. Parallel zu den biologischen Testungen der hergestellten Peptide wurden computerbasierte Untersuchungen durchgeführt um ihre Struktur-Aktivitätsbeziehungen zu analysieren. Die vorgestellte Dissertation erweitert das Wissen über Conotoxine und die Bildung von Disulfidbrücken mit ihren Auswirkungen auf die biologische Aktivität und demonstriert darüber hinaus die schwierige Strukturaufklärung und Charakterisierung

Characterizing Venom Gene Expression, Function and Species Diversity in Predatory Marine Snails of the Terebridae

The Terebridae is a group of predatory marine snails that use their venom to feed on marine annelids. Similar to other venomous organisms, the Terebridae have evolved over millions of years, diverging from their closest relative in the Paleocene era. This thesis investigates what is driving terebrid evolution and species diversification by applying a multidimensional approach

Small Packages, Big Returns: Uncovering the Venom Diversity of Small Inverebrate Conoidean Snails

Venomous organisms used in research were historically chosen based on size and availability. This opportunity-driven strategy created a species bias in which snakes, scorpions, and spiders became the primary subjects of venom research. Increasing technological advancements have enabled interdisciplinary studies using genomics, transcriptomics, and proteomics to expand venom investigation to animals that produce small amounts of venom or lack traditional venom producing organs. One group of non-traditional venomous organisms that have benefitted from the rise of -omic technologies is the Conoideans. The Conoidean superfamily of venomous marine snails includes, the Terebridae, Turridae (s.l), and Conidae. Conoidea venom is used for both predation and defense, and therefore under strong selection pressures. The need for conoidean venom peptides to be potent and specific to their molecular targets has made them important tools for investigating cellular physiology and bioactive compounds that are beneficial to improving human health. A convincing case for the potential of Conoidean venom is made with the first commercially available conoidean venom peptide drug Ziconotide (Prialt®), an analgesic derived from Conus magus venom that is used to treat chronic pain in HIV and cancer patients. Investigation of conoidean venom using -omics technology provides significant insights into predator-driven diversification in biodiversity and identifies novel compounds for manipulating cellular communication, especially as it pertains to disease and disorders