toxins Review From Mollusks to Medicine: A Venomics Approach for the Discovery and Characterization of Therapeutics from Terebridae Peptide Toxins Aida Verdes 1,2,3, Prachi Anand 1, Juliette Gorson 1,2,3, Stephen Jannetti 1,2, Patrick Kelly 1,2, Abba Leffler 1,4, Danny Simpson 1,5, Girish Ramrattan 1 and Mandë Holford 1,2,3,* 1 Hunter College, The City University of New York, Belfer Research Building, 413 E. 69th Street, New York, NY 10021, USA; [email protected] (A.V.); [email protected] (P.A.); [email protected] (J.G.); [email protected] (S.J.); [email protected] (P.K.); [email protected] (A.L.); [email protected] (D.S.); [email protected] (G.R.) 2 The Graduate Center, City University of New York, 365 5th Ave, New York, NY 10016, USA 3 Sackler Institute for Comparative Genomics, Invertebrate Zoology, American Museum of Natural History, Central Park West & 79th St, New York, NY 10024, USA 4 Sackler Institute of Graduate Biomedical Sciences, New York University School of Medicine 550 1st Avenue, New York, NY 10016, USA 5 Tandon School of Engineering, New York University 6 MetroTech Center, Brooklyn, NY 11201, USA * Correspondence: [email protected]; Tel.: +1-212-896-0449 Academic Editor: Stephen Mackessy Received: 3 March 2016; Accepted: 7 April 2016; Published: 19 April 2016 Abstract: Animal venoms comprise a diversity of peptide toxins that manipulate molecular targets such as ion channels and receptors, making venom peptides attractive candidates for the development of therapeutics to benefit human health. However, identifying bioactive venom peptides remains a significant challenge. In this review we describe our particular venomics strategy for the discovery, characterization, and optimization of Terebridae venom peptides, teretoxins. Our strategy reflects the scientific path from mollusks to medicine in an integrative sequential approach with the following steps: (1) delimitation of venomous Terebridae lineages through taxonomic and phylogenetic analyses; (2) identification and classification of putative teretoxins through omics methodologies, including genomics, transcriptomics, and proteomics; (3) chemical and recombinant synthesis of promising peptide toxins; (4) structural characterization through experimental and computational methods; (5) determination of teretoxin bioactivity and molecular function through biological assays and computational modeling; (6) optimization of peptide toxin affinity and selectivity to molecular target; and (7) development of strategies for effective delivery of venom peptide therapeutics. While our research focuses on terebrids, the venomics approach outlined here can be applied to the discovery and characterization of peptide toxins from any venomous taxa. Keywords: venomics; Terebridae; teretoxins; peptide toxins; animal venom; venom peptides; drug development; drug discovery; peptide therapeutics; drug delivery 1. Introduction Medicinal treatments have a storied history tied to natural products discovery and development. Natural products derived from plants and animals have been the source of traditional medicine for millennia, and more recently have become major sources of chemical diversity as drug leads, driving research efforts in pharmaceutical drug discovery and development [1,2]. The ascendancy of natural products was acknowledged with the awarding of the 2015 Nobel Prize in Physiology or Toxins 2016, 8, 117; doi:10.3390/toxins8040117 www.mdpi.com/journal/toxins Toxins 2016, 8, 117 2 of 30 Medicine for the discovery of two revolutionary therapies based on natural compounds, Avermectin and Artemisinin. Avermectin has helped to nearly eradicate parasitic worm diseases such as river blindness and lymphatic filariasis, while Artemisinin represents the most effective treatment for malariaToxins known 2016, to8, 117 date [3]. The impact of these natural products on improving global human2 of 29 health is incalculable. Artemisinin. Avermectin has helped to nearly eradicate parasitic worm diseases such as river Theblindness journey and from lymphatic natural filariasis, product while discovery Artemisinin to therapy represents has the largely most effective focused treatment on small for chemical compoundsmalaria such known as Avermectinto date [3]. The and impact Artemisinin; of these natural however, products natural on improving peptides global are human increasingly health being investigatedis incalculable. as drug leads in pharmaceutical research [4]. In particular, peptides found in venomous organisms areThe a journey very promising from natural source product for discovery drug discovery. to therapy Successfulhas largely focused examples on small of drugs chemical developed from venomcompounds peptides such include as Avermectin Captopril and® Artemisinin;, based on ahowever, venom natural peptide peptides from the are Brazilian increasingly viper being and used investigated as drug leads in pharmaceutical research [4]. In particular, peptides found in venomous to treat hypertension [5,6]; exenatide (marketed as Byetta®), based on the Gila monster venom and used organisms are a very promising source for drug discovery. Successful examples of drugs developed ® as an anti-diabeticfrom venom peptides agent [7 include]; and ziconotideCaptopril®, based (Prialt on ),a basedvenom onpeptide a venom from the peptide Brazilian from viper the and predatory cone snailusedConus to treat magus hypertensionand used [5,6]; to exenatide treat chronic (marketed pain as [ 8Byetta,9]. Most®), based venom on the peptides Gila monster are disulfide-richvenom and varyand in used length as an from anti‐ 12–30diabetic residues agent [7]; inand cone ziconotide snails (Prialt to 40–80®), based residues on a venom in terebrids, peptide from scorpions, the and snakes [predatory10–12]. The cone relatively snail Conus small magus size and and used the stabilityto treat chronic provided pain by [8,9]. disulfide Most venom bridges peptides that characterize are disulfide‐rich and vary in length from 12–30 residues in cone snails to 40–80 residues in terebrids, natural peptides make them ideal candidates for drug leads. Venom peptides are predominantly being scorpions, and snakes [10–12]. The relatively small size and the stability provided by disulfide investigatedbridges for that the characterize development natural of drugpeptides therapies make them targeted ideal candidates to ion channels for drug and leads. receptors Venom [12–16]. Due to technologicalpeptides are predominantly constraints, being such investigated as size and for ease the development of collection, of drug venomous therapies organisms targeted to like ion snakes and scorpionschannels haveand receptors been traditionally [12–16]. Due to singled technological out forconstraints, drug discovery such as size research. and ease of However, collection, recent advancesvenomous in next-generation organisms like sequencing snakes and (NGS) scorpions techniques have been and traditionally improvements singled in proteomicout for drug methods have alloweddiscovery venom research. research However, to recent expand advances and include in next‐generation neglected sequencing venomous (NGS) invertebrates techniques and with great improvements in proteomic methods have allowed venom research to expand and include neglected potential, such as the conoideans (Figure1)[17–19]. venomous invertebrates with great potential, such as the conoideans (Figure 1) [17–19]. Figure 1. From mollusks to medicine. Overview of venomics approach for discovery, characterization, Figure 1. From mollusks to medicine. Overview of venomics approach for discovery, characterization, and development of therapeutics from Terebridae venom peptides. This strategy begins with a and developmentphylogenetic ofdelimitation therapeutics of venomous from Terebridae terebrid lineages venom to identify peptides. the species This that strategy are producing begins with a phylogeneticvenom delimitationto subdue their of prey venomous (shown in terebrid red); in lineagesyellow, identification to identify of the teretoxins species through that are omics producing venom(genomics, to subdue transcriptomics, their prey (shown proteomics); in red); in ingreen, yellow, synthesis identification and structural of teretoxins characterization through of omics (genomics,teretoxins; transcriptomics, in blue, bioactivity proteomics); assays and inidentification green, synthesis of molecular and targets; structural and in characterizationpink, peptide of teretoxins;optimization in blue, and bioactivity development assays of delivery and identification methods for potential of molecular terebrid therapeutics. targets; and in pink, peptide optimization and development of delivery methods for potential terebrid therapeutics. Toxins 2016, 8, 117 3 of 30 The Conoidea superfamily (cone snails, terebrids, and turrids s.l.) is an extremely diverse group of predatory marine neogastropods divided into 16 families, with several lineages characterized by having a venom apparatus used for predation [10,19,20]. The genus Conus, the most extensively studied among the conoideans, and from which the drug ziconotide (Prialt®) was discovered, includes species that produce very complex venoms with thousands of unique venom peptides, known as conotoxins or conopeptides [21–27]. As such, it is not surprising that conotoxins have been considerably studied for several decades. However, the ~700 described species of cone snails represent far less than half of the over