Regulatory Toxicology and Pharmacology 70 (2014) 197–202

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Regulatory Toxicology and Pharmacology

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Commentary

Conotoxins and their regulatory considerations

http://dx.doi.org/10.1016/j.yrtph.2014.06.0270273-2300/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author. Fax: +1 (808) 965 3542.E-mail address: jbingham@hawaii.edu (J.-P. Bingham).

Parashar Thapa, Michael J. Espiritu, Chino C. Cabalteja, Jon-Paul Bingham ⇑Department of Molecular Biosciences and Bioengineering, College of Tropical Agriculture and Human Resources, University of Hawai’i, Honolulu, HI 96822, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 20 June 2014Available online 8 July 2014

Keywords:ConotoxinsPeptidesCyclic peptidesRegulationSelect agents

Venom derived peptides from marine cone snails, conotoxins, have demonstrated unique pharmacolog-ical targeting properties that have been pivotal in advancing medical research. The awareness of theirtrue toxic origins and potent pharmacological nature is emphasized by their ‘select agent’ classificationby the US Centers for Disease Control and Prevention. We briefly introduce the biochemical and pharma-cological aspects of conotoxins, highlighting current advancements into their biological engineering, andprovide details to the present regulations that govern their use in research.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction to conotoxins

The venom of Conus sea snails has evolved as an efficient meansof prey incapacitation and as an effective defense mechanism(Armishaw and Alewood, 2005). The deadly effects of this venom-ous cocktail are a direct result of conotoxins, small disulfide-richpeptides that are potent antagonists of a range neuronal receptorsand ion channels (Table 1). These bioactive peptides expressselectivity towards their target receptors and are even able todiscriminate between receptor subtypes. These properties arebeing utilized to design receptor-modulating ligands with poten-tial therapeutic applications.

Conotoxins are typically about 10–40 amino acids in length, butexhibit many motifs that are normally found in larger proteins.These structural features are stabilized by disulfide bonds generatedfrom the abundance of cysteine moieties in its primary structure(Armishaw and Alewood, 2005; Bingham et al., 2005). Interestingly,these cysteine residues are found in predictable locations within aconotoxin’s sequence, giving rise to loops of known amino acidlengths, which influence their bioactivity (Bingham et al., 2005;Espiritu et al., 2014).

Although disulfide bonds play a major role in determining cono-toxin structure activity relationships, assessing how they influencebioactivity of conotoxins should be handled on a case-by-case basisas they stabilize peptide structure in varying degrees.

The number of disulfide isomers possible within a conotoxin isdirectly correlated with an increasing number of cysteines within apeptide sequence (Kaas et al., 2010). Notably, when the disulfide

bond connectivity of a-conotoxin AuIB was rearranged, the result-ing ribbon isomer exhibited greater bioactivity than the globularnative toxin (Dutton et al., 2002). This finding is valuable to cono-toxin bioengineering as it expands the repertoire of modifiableconotoxins to include non-native isomers.

Despite their great sequence diversity, the cysteine frameworksof these bioactive peptides are quite predictable. For majority ofconotoxins containing four cysteines, their cysteine framework isgiven by the formula (X)n1CiCii(X)n2Ciii(X)n3Civ(X)n4 (Binghamet al., 2005). Interestingly, the number of amino acids in n2 andn3 of these conotoxin’s cysteine frameworks can have profoundconsequences in their selectivity toward a specific receptor class.Generally, a-conotoxins with a 3/5 cysteine framework (n2 = 3and n3 = 5) will target muscle nAChRs (Gray et al., 1981) whileconotoxins with a 4/7 framework (n2 = 4 and n3 = 7) will bespecific for neuronal nAChRs (Armishaw and Alewood, 2005;Bingham et al., 2010, 2012; Gray et al., 1981). This has importantimplications regarding conotoxin classification.

Conotoxins can be systematically ordered into superfamiliesand further organized into families (sometimes referred to asclasses). Superfamilies are based on disulfide bond framework pat-terns, while families are founded on their pharmacological target(Armishaw and Alewood, 2005; Terlau and Olivera, 2004). Cur-rently, there are twenty main conotoxin superfamilies and withinthese inclusive categories lie their respective families, oftendenoted as a single Greek letter prefix. These categories are rapidlyexpanding with the discovery of new conotoxins (refer to Table 1).

Along with conotoxins, cyclotides are another category ofnatural products that are garnering immense interest due to theirtherapeutic potential. Cyclotides are disulfide rich plant peptidecharacterized by head to tail cyclization and six conserved cysteine

Table 1The classification of conotoxins based on their pharmacological families (adapted from Kaas et al., 2012).

Conotoxin family Definition Genesuperfamily

Cysteineframework

References

a (ALPHA) Nicotinic acetylcholine receptor(nAChR)

A, B3, D, J, L,M, S

I, II, III, IV,VIII, XIV, XX,XXV

Corpuz et al. (2005), Gray et al. (1981), Imperial et al. (2006), Liu et al. (2008),Loughnan et al. (2009), Luo et al. (2013), Peng et al. (2006), Santos et al.(2004)

c (GAMMA) Neuronal pacemaker cationcurrents (inward cation current)

O1, O2 VI/VII Fainzilber et al. (1998), McIntosh et al. (1995), Zhangsun et al. (2006)

d (DELTA) Voltage gated Na Channel (agonist,delay inactivation)

O1 VI/VII Fainzilber et al. (1991), McIntosh et al. (1995)

e (EPISILON) Presynaptic calcium channels or Gprotein coupled presynapticreceptor

T V Rigby et al. (1999), Walker et al. (1999)

I (IOTA) Voltage gated Na Channel (agonist,no delayed inactivation)

I1, M III, XI Buczek et al. (2007), Corpuz et al. (2005), Jimenez et al. (2003)

j (KAPPA) Voltage gated K Channel (blocker) A, I2, J, M,O1

III, IV, VI/VII,XI, XIV

Buczek et al. (2005b), Imperial et al. (2006), McIntosh et al. (1995), Santoset al. (2004), Terlau et al. (1996a)

l (MU) Voltage gated Na Channel(antagonist, blocker)

M, O1, T III, IV, V, VI,VII

Corpuz et al. (2005), Cruz et al. (1985), McIntosh et al. (1995), Walker et al.(1999)

q (RHO) Alpha 1 adrenoreceptor (GPCR) A I Santos et al. (2004), Sharpe et al. (2001)r (SIGMA) Serotonin gated ion channels

(GPCR)S VIII England et al. (1998), Liu et al. (2008)

T (TAU) Somatostatine receptor T V Petrel et al. (2013), Walker et al. (1999)v (CHI) Neuronal noradrenaline

transporterT X Sharpe et al. (2001), Walker et al. (1999)

x (OMEGA) Voltage gated calcium channel O1, O2 VI/VII, XVI,XXVI

Kerr and Yoshikami (1984), McIntosh et al. (1995), Zhangsun et al. (2006)

198 P. Thapa et al. / Regulatory Toxicology and Pharmacology 70 (2014) 197–202

residues arranged in a knotted topology (Craik et al., 2012; Dalyet al., 2009). Cyclotides have insecticidal properties and functionin plants as host-defense agents (Craik, 2012). In addition to theirnatural function cyclotides have been reported to posses anti-HIV,anticancer and hemolytic activities (Daly et al., 2009; Henriquesand Craik, 2010). Like conotoxins, cyclotides contain a high fre-quency of cysteine residues in their sequence. The presence ofdisulfide bonds confers considerable stability to these naturalproducts thereby making them worthwhile research tools forstructural studies and as scaffolds for stable delivery of drugs(Poth et al., 2013; Schroeder et al., 2013). The idea of mergingthe notable pharmacological properties of cyclotides with the spec-ificity of conotoxins has drawn much attention to the bioengineer-ing of cyclic forms of conotoxins (Bingham et al., 2012).

2. Biological spectrum of conotoxins

2.1. Phyla selectivity

One of the most important and well known aspects of conotox-ins is their ability to selectively target ion channel subtypes (Terlauand Olivera, 2004). This dynamic receptor–ligand interaction canbe utilized to understand subtle differences in the normal physiol-ogy of various ion channel subtypes. As such, conotoxins areviewed as valuable resources for phyla selective receptor probes.Phyla selectivity is commonly seen amongst individual conotoxins,representing their native predatory preferences (Terlau andOlivera, 2004). Many peptides isolated from a piscivorous (fish eat-ing) cone snail such as Conus magus, will only display activity inreceptor subtypes related to higher order organisms, whereas mol-luscivorous (mollusk eating) cone snails tend to produce conotox-ins which target mollusk receptor subtypes (Bergeron et al., 2013).This type of selectivity can be greatly utilized in terms of creatingsafe and effective pesticides. Potential pesticides produced fromthe venoms of molluscivorous cone snails may provide compoundsthat could be naturally degraded by microorganisms without pro-ducing harmful byproducts or causing environmental damage(Bruce et al., 2011).

2.2. General and clinical pharmacological activity of conotoxins

A third feature of conotoxins is their nature to exhibit a highamount of biological activity (see Table 2). To date, x-ConotoxinMVIIA (SNX-111) is the only United States Food and Drug Admin-istration (FDA) approved conotoxin for use as a therapeutic and isknown for being extremely potent, displaying bioactivity in thelow nanomolar range (7.6 nM IC50) (Lee et al., 2010; Oliveraet al., 1987). However, this type of activity is not unique to thissingle peptide, as many conotoxins appear to be exceedinglypotent. Examples of nanomolar potency among conotoxins includebut are not limited to: the lO-conotoxin MrVIA (IC50 = 345 nM)(Safo et al., 2000; Terlau et al., 1996b) and the a-conotoxin MIC(Ki = 248.7 nM) (Kapono et al., 2013).

x-Conotoxin MVIIA, otherwise known as Ziconotide, has under-gone extensive clinical testing (FDA, 2006). Pharmacokinetics onIntravenous (IV) and Intrathecal (IT) injections of this peptide hasbeen established. Classical distribution, metabolism, and excretionstudies were undertaken before Ziconotide was entered into clini-cal trials. Ziconotide is the first non-opioid IT treatment for themanagement of chronic refractory pain and is known by its clinicalname Prialt� (PRImary ALTernative to morphine). The therapeuticis designed to be delivered intrathecally and should not beadministered via any other Route of Administration (RoA). Severalpublications describing the safety and efficacy of Prialt� have beenpublished and can be studied for more comprehensive information.

2.3. Oral bioavailability

A second feature of conotoxins is their poor oral bioavailability.Many peptides and biologic based compounds are degraded easilyby the digestive tract due to their susceptibility to peptidases,making them virtually ineffective in terms of oral delivery. A sec-ond reason for the poor oral bioavailability of conotoxins (alongwith most peptides) is their large size. According to Lipinski’s ruleof five compounds over 500 Daltons are typically seen as ineffec-tive orally due to poor membrane and paracellular permeability.These properties have provided a justifiable means for the current

P. Thapa et al. / Regulatory Toxicology and Pharmacology 70 (2014) 197–202 199

‘‘relaxed’’ regulatory handling of material as ingestion is not seenas an immediate threat to health (Hamman et al., 2005).

The problem with poor bioavailability of conotoxins may soonbe solved by current research on N- to C-terminal cyclizationstrategies. N- to C-terminal cyclization of peptides has shown toproduce characteristics of increasing stability and even increasedabsorption in disulfide rich compounds containing the cysteineknot motif such as Kalata B1 (Clark and Craik, 2012). Thistechnique, when applied to synthetic conotoxins, could potentiallyprovide these peptides with the properties necessary for oral bio-availability. These new N- to C-terminal cyclized conotoxins maythen require a re-evaluation of handling and regulation standards.

3. Synthesis of conotoxins

3.1. Biosynthesis of conotoxin

Each Conus species has a venom profile that is unique and hasminimal overlap with other species. Given that there are 100–200unique peptide in each species, the �700 living Conus species havea possible repertoire of 50,000 to >140,000 different conotoxins/conopeptides (Olivera, 1997, 2006; Olivera and Teichert, 2007).Conotoxin biosynthesis involves a single open reading frame, tran-scribed to mRNA and translated into a prepropeptide approximately80–100 amino acids in length (Buczek et al., 2005a). The prepropep-tide is cleaved by proteases to give a final mature peptide devoid ofthe prepro-region. The signal sequence from a given gene superfam-ily is highly conserved. In x-conotoxins MVIIA and GVIA the 22amino acid containing pre region has no variation and the 23 aminoacid containing pro region has 13% divergence (3 out the 23 aminoacid are different). With the exception of conserved cysteine resi-dues, the mature peptide is highly variable. In x-conotoxins MVIIAand GVIA 16 of the 22 non cysteine residues are different resultingin a 73% divergence in the mature toxin region (Olivera, 1997).Conotoxins are also heavily post-translationally modified (PTM).PTM’s seen in conotoxin are proteolytic processing of propeptideto mature peptide, disulfide bridge formation, hydroxylation,C-terminal amidation, carboxylation, bromination, epimerization,cyclization, sulfation and O-glycosylation (Buczek et al., 2005a).The hypervariable peptide region in conjugation with PTMs providegreat diversity to cone snail venom.

3.2. Chemical synthesis of conotoxin

The synthesis of conotoxins is largely undertaken through solidphase peptide synthesis (SPPS), however conotoxins have also beenproduced recombinantly (Bruce et al., 2011). SPPS remains thedominant technique due to its simplicity, automation and versatil-ity. It can be used in concert with other synthetic techniques such aschemically directed oxidation strategies of disulfide bonds andnative chemical ligation. Such benefits allow for the investigation

Table 2Examples of various conotoxins indicating their lethal dose (LD50) and specific activity.

Name Amino acid sequence Lethal dose 50

a-GI ECCNPACGRHYSC⁄ 12 lg/kg ip.a-MI GRCCHPACGKNYSC⁄

Ac1.1B NGRCCHPACGKHFSC⁄ 38 ± 9 lg/kg ia-SI ICCNPACGPKYSC⁄

a-EI RDOCCYHPTCNMSNPQIC⁄

l-GIIIA RDCCTOOKKCKDRQCKOQRCCA⁄ 340 lg/kg ip.l-GIIIB RDCCTOORKCKDRRCKOMKCCA⁄ 110 lg/kg ip.Conantokin G GEccLQcNQcLIRcKSN⁄ 11 ng/g ic.

ip. = intraperitoneal injection; ic. = intracerebral injection; ⁄ = C-terminal amidation; O =a The unit of activity is the quantity of material needed to cause death of a 20 g moub In a-conotoxin SI up to 30 nmol the effects are minimal, 0.2 nmol caused death in a

of a greater diversity of synthetic isomers, chemical modificationsto enhance desirable pharmacokinetic properties, and avoid com-plications with recombinant expression and folding. Further desir-able qualities of SPPS include the ability to incorporate non-nativeand PTM amino acids with ease, automated combination synthesesto obtain epitope information such as alanine walks, and epitopereplacement using stabilized backbone scaffolds (Daly and Craik,2009).

4. Conotoxins used as structural tools

Conotoxins are currently known to be of great benefit for use asmolecular probes due to their ability to selectively interact withreceptor subtypes. A prominent example of this is x-conotoxinMVIIA, which has been cited as a probe in over 2000 scientificpublications (Terlau and Olivera, 2004). The potential for activityand structural investigation is being fostered by the bioconjugationof visually traceable media such as biotin and fluorophores(Bingham et al., 2009). Many examples of the usefulness of cono-toxins as biological probes exist in literature and include receptorsubtypes in calcium channels, potassium channels, sodium chan-nels, 5-HT3 receptors, NMDA receptors and more (Bingham et al.,2010; Kaas et al., 2012).

5. Conopeptide based therapeutics in the pipeline

In January 2013 Kineta� a Seattle based Biotech Companyannounced that it had acquired the developmental rights toa-conotoxin RgIA (U2902) from the University of Utah researchfoundation. a-Conotoxin RgIA was isolated by the team ofDr. Olivera and found to selectively block a9a10 nAChRs (Ellisonet al., 2008, 2006). Kineta� intends to develop a-conotoxin RgIAas a non-narcotic treatment for severe pain. Currently a-conotoxinRgIA is in preclinical stage of development and Kineta� statesthat early results have been promising. Several conotoxins likex-conotoxin CVID (Leconotide; Relevare Pharmaceuticals), Conan-tokin-G (CGX 1007; Cognetix), Contulakin-G (CGX 1160; Cognetix),and Xen2174 (structural analog of v-conotoxin MrIA; XenomePharmaceuticals) have reached various stages of clinical trial test-ing (Essack et al., 2012). So far Prialt� remains the only FDAapproved drug derived from conotoxin.

6. Present regulations regarding conotoxin usage

6.1. USA Federal regulations

The Federal Select Agent Program is a collaborative venturebetween the Division of Select Agents and Toxins of the Centersfor Disease Control and Prevention (CDC) and the AgriculturalSelect Agents Program under the Animal and Plant Health Inspec-tion Services. As stated on the select agent website ‘‘The Federal

Specific activity (units/nmol)a References

1.5 Cruz et al. (1978)3.8 McIntosh et al. (1982)

p. Liu et al. (2007)NAb Zafaralla et al. (1988)0.36 Martinez et al. (1995)

Sato et al. (1983)Sato et al. (1983)Rivier et al. (1987)

4 trans-hydroxyproline; c = gamma-caboxy glutamic acid.se in 20 min.-conotoxins GI and MI.

200 P. Thapa et al. / Regulatory Toxicology and Pharmacology 70 (2014) 197–202

Select Agent Program oversees the possession, use and transfer ofbiological select agents and toxins, which have the potential topose a severe threat to public, animal or plant health or to animalor plant products’’. The federal select agent programs contribute tonational security by performing tasks such as maintaining a data-base of select agents, and inspecting entities that use, possess ortransfer select agents. In addition the program also develops poli-cies and regulations regarding select agents and ensures that theseregulations are being implemented.

The program in accordance to 42 USC 262a and 7 USC 8401defines select agents and toxins as a ‘‘subset of biological agentsand toxins that the Departments of Health and Human Services(HHS) and Agriculture (USDA) have determined to have the poten-tial to pose a severe threat to public health and safety, to animal orplant health, or to animal or plant products’’. A subset of the selectagents and toxins has been classified as Tier 1 select agents andtoxins. Tier 1 consists of biologic agents and toxins that possessthe maximum risk of being misused to inflict mass causality andposes a high level of threat to public health and safety, furtherthese agents could also be used to negatively impact the economyand cause damage to essential public infrastructure. Some of theTier 1 select agents and toxins are Botulism neurotoxin, Ebolavirus, Marburg virus, Bacillus anthracis (anthrax) and Rinderpestvirus. A complete list of select agents and Tier 1 select agentsand toxins can be found at National select agent registry or onthe select agent list website at http://www.selectagents.gov/ (lastupdated 4/2/2014).

An important question to answer is how the select agent pro-gram determines which agents or toxins to include in the HHSselect agent list. The ‘‘Public Health Security and BioterrorismPreparedness and Response Act’’ considers the impact on humanhealth after exposures, the severity of contagiousness, the possi-ble delivery routes of exposure, the availability and efficacy ofdrug therapies and immunizations, and special criteria asdeemed necessary by the secretary to protect more susceptibleor vulnerable demographics (e.g., children, senior citizens, etc.)before including an agent or toxin in the HHS list. The list isreviewed after every two years and republished with the neces-sary amendments.

The CDC states that based on the available experimental datamajority of known conotoxins do not possess sufficient acute tox-icity to generate a considerable threat to human health. Further-more the CDC acknowledges that conotoxins are invaluableresearch tools and have the potential to be developed into humantherapeutics. Nevertheless, the CDC mentions that based on theavailable data one subclass of conotoxin referred to as ‘‘shortparalytic alpha conotoxins, illustrated by a-conotoxin GI anda-conotoxin MI, do possess sufficient acute toxicity by multipleroutes of exposure, biophysical stability, ease of synthesis, andavailability. The conotoxins that remain on the HHS list will be lim-ited to the short, paralytic alpha conotoxins containing the follow-ing amino acid sequence X1CCX2PACGX3X4X5X6CX7’’ (see Table 1).’’Where X1 is any amino acid or amino acid deletion, the cysteinesare present in a disulfide bond between C1 and C3 and C2 andC4 giving the conotoxin its stable globular form, X2 is Asparagineor Histidine, P is Proline, A is Alanine, G is Glycine, X3 is Arginineor Lysine, X4 is Asparagine, Histidine, Lysine, Arginine, Tyrosine,Phenylalanine or Tryptophan, X5 is Tyrosine, Phenylalanine, orTryptophan, X6 is Serine, Threonine, Glutamate, Aspartate, Gluta-mine, or Asparagine, X7 is any amino acid(s) or amino acid deletion.

This provides the potential to generate thousands of peptidecombinations that may possess biological activity, an approachthat has already been tested using synthetic peptide combinatoriallibraries (Armishaw et al., 2010). Short paralytic a-conotoxins arelisted in the HHS select agents and toxin list but they are not con-sidered Tier 1 select agents.

6.2. Permissible amounts as defined by the CDC

An ‘‘entity’’, which is defined as ‘‘any government agency(Federal, State, or local) academic institution, corporation, com-pany, partnership, society, association, firm, sole proprietorship,or other legal entity’’ by the CDC select agent registry (CDC,2014a), is allowed to possess up to 100 mg of a short, paralytica-conotoxin (CDC, 2014b). This amount is only applicable tobiologically active conotoxins that have been folded in theirthree-dimensional conformation. Non-functional conotoxins areexcluded from this restriction. Conotoxins possess a high amountof cysteines in their sequence and unless these cysteines areoxidized to form disulfide bonds the conotoxins will not possessbiological activity. Thus, the 100 mg restriction does not apply todisulfide reduced conotoxin sequences lacking oxidized cysteines,or resin-bound synthetic peptides.

6.3. FDA approval process and requirements

The FDA approval process, especially in consideration of trans-lational research (from a university setting to a clinical one) con-sists of three important stages that typically have extensivetimelines (on average it takes approximately 12 years for the entireprocess). These stages include: preclinical research done by thesubmitting entity (commercial or university) and the successivefiling of an Investigational New Drug Application or IND, Phase I,II, and III clinical trials, and the New Drug Application (NDA) or Bio-logic License Application (BLA) (FDA, 2014a).

Preclinical data necessary for the filing of an IND includes threebroad areas of information. The first area of information is the ani-mal pharmacology and toxicology studies. These studies are oftendone on mice or other mammals and must demonstrate that thecompound is safe for human use. The second area is the manufac-turing information. This information must include compound com-position, information about the manufacturer itself, compoundstability, and demonstrate that the company is able to maintain ahigh degree of reproducibility. The final area of information isthe clinical protocols and investigator information. This informa-tion must include detailed protocols for the clinical study andstates what risks may be involved. This final area also determineswhether the physician(s) in charge of the study is/are qualified tomeet the study’s requirements. Lastly, this area dictates that thecompany must demonstrate ‘‘commitments to obtain informedconsent from the research subjects, to obtain review of the studyby an institutional review board (IRB), and to adhere to the inves-tigational new drug regulations’’ (FDA, 2014b).

Once the IND has been accepted, 30 days must pass before thephase I clinical trial can begin, in order to allow the FDA to com-plete a review on the prospective study (FDA, 2014a). Clinical trialstypically consist of three phases: phase I, phase II, and phase III(NIH, 2008). Phase I trials consists of a small group of patientsand is undertaken to evaluate safety, effective dosage range, andidentify any side effects, a process that takes approximately 1 year.In phase II trials the drug candidate is given to a larger amount ofpatients and further reviewed for safety for a longer time period,approximately 2 years. Finally in phase III trials the drug candidateis further reviewed for safety in a large group of patients and com-pared to the current standard of therapy if one exists, a processthat takes approximately 3 years. Occasionally phase IV trialsmay be conducted post-marketing to gather additional informationon various populations.

Once the trials have concluded and the data has been ade-quately reviewed the drug sponsor may file for an NDA or BLAdepending on whether the compound is of natural or synthetic ori-gin. The NDA includes ‘‘full information on manufacturing specifi-cations, stability and bioavailability data, method of analysis of

P. Thapa et al. / Regulatory Toxicology and Pharmacology 70 (2014) 197–202 201

each of the dosage forms the sponsor intends to market, packagingand labeling for both physician and consumer, and the results ofany additional toxicological studies not already submitted in theInvestigational New Drug application’’ (FDA, 2014a). Once this pro-cess has completed the drug may be marketed to the public. Prialt�

(x-conotoxin MVIIA) endured this same venture, yet as a peptidedrug it faced additional challenges in its approval status and man-ufacturing process (Bingham et al., 2010).

7. Future direction for conotoxin regulation – bioengineeredconotoxins

Conotoxins and conopeptides have been the subject of exten-sive bioengineering and chemical manipulation (Bingham et al.,2012). Their ease of synthesis has positioned them as primarychoice among peptide chemists who manipulate the nativepeptide to enhance stability and activity. The resulting bioengi-neered peptide with augmented properties has proved to be aninvaluable tool in studies pertaining to structural activityrelationships, drug leads and ion channel characterization. Thedevelopment of bioengineered conotoxins presents special casesand equivocal situations that may require amendments to thecurrent regulations.

Conotoxins contain numerous post-translational modifications(Espiritu et al., 2014). These PTMs are responsible for phyla andreceptor selectivity. Removing some or all of the PTMs can changereceptor selectivity (Bergeron et al., 2013). Through bioengineer-ing, a native conotoxin not active in mammalian receptors can bemodified to have activity in mammalian receptors. Peptidomemet-ics has further advanced the field of conopeptide bioengineering.Allowing researchers to incorporate synthetic residues or combineactive regions of various peptides, giving them the ability to createa new peptide with enhanced properties (Donevan and McCabe,2000). With the advent of native chemical ligation (NCL) the link-ing of a peptide’s N-terminus to its C-terminus has been success-fully performed (Clark et al., 2010). Conotoxins, with theirnumerous cysteine residues, present themselves as a prime targetfor peptide backbone cyclization. Cyclization is known to enhancethe stability of conotoxins. This synthetic strategy has been appliedto Vc1.1, a-conotoxins AuIB, RgIA and MII using NCL; the cyclizedanalogs have been shown increased oral bioavailability and sys-temic stability (Armishaw et al., 2011; Clark et al., 2005, 2010;Ellison et al., 2008). In addition to the use of synthetic chemistry,cyclization has also been accomplished through advanced molecu-lar biology techniques such as interim mediated ligation, which is apotential route for future recombinantly-produced cyclic conotox-ins (Sancheti and Camarero, 2009). Another approach to increasethe in vivo stability is using cystathionine and dicarba linkages inreplacement to essential disulfide bonds. This approach has beensuccessfully applied in a-conotoxin ImI (MacRaild et al., 2009).Enhancing oral stability of conotoxins provides a new route ofadministration and hence requires a reassessment of current cono-toxin directives.

Bioengineering of conotoxins has undoubtedly opened newchallenges for regulation. A potential new approach to regulatingconotoxins is to distinguish native and non-native peptides. Com-bining all forms of conotoxins in one all-encompassing umbrella isnot the way forward. Distinguishing between various types ofconotoxins will allow better classification and undoubtedly leadto enhanced regulation.

Conflict of interest

Authors state that there is no conflict of interest.

Acknowledgments

We are indebted to sponsors for supporting our work inconotoxins, these include the University of Hawaii Sea GrantCollege Program and USDA TSTAR (# 2009-34135-20067) & HATCH(HAW00595-R) Programs.

References

Armishaw, C.J., Alewood, P.F., 2005. Conotoxins as research tools and drug leads.Curr. Protein Pept. Sci. 6, 221–240.

Armishaw, C.J. et al., 2011. Improving the stability of alpha-conotoxin AuIB throughN-to-C cyclization: the effect of linker length on stability and activity atnicotinic acetylcholine receptors. Antioxid. Redox Signal. 14, 65–76.

Armishaw, C.J. et al., 2010. A synthetic combinatorial strategy for developing alpha-conotoxin analogs as potent alpha7 nicotinic acetylcholine receptorantagonists. J. Biol. Chem. 285, 1809–1821.

Bergeron, Z.L. et al., 2013. A ‘conovenomic’ analysis of the milked venom from themollusk-hunting cone snail Conus textile – the pharmacological importance ofpost-translational modifications. Peptides 49, 145–158.

Bingham, J.P. et al., 2012. Drugs from slugs. Part II – conopeptide bioengineering.Chem. Biol. Interact. 200, 92–113.

Bingham, J.P. et al., 2005. Optimizing the connectivity in disulfide-rich peptides:alpha-conotoxin SII as a case study. Anal. Biochem. 338, 48–61.

Bingham, J.P. et al., 2009. Synthesis of an iberiotoxin derivative by chemicalligation: a method for improved yields of cysteine-rich scorpion toxin peptides.Peptides 30, 1049–1057.

Bingham, J.P. et al., 2010. Drugs from slugs – past, present and future perspectives ofomega-conotoxin research. Chem. Biol. Interact. 183, 1–18.

Bruce, C. et al., 2011. Recombinant conotoxin, TxVIA, produced in yeast hasinsecticidal activity. Toxicon 58, 93–100.

Buczek, O. et al., 2005a. Conotoxins and the posttranslational modification ofsecreted gene products. Cell. Mol. Life Sci. 62, 3067–3079.

Buczek, O. et al., 2007. Structure and sodium channel activity of an excitatory I1-superfamily conotoxin. Biochemistry 46, 9929–9940.

Buczek, O. et al., 2005b. Characterization of D-amino-acid-containing excitatoryconotoxins and redefinition of the I-conotoxin superfamily. FEBS J. 272, 4178–4188.

CDC, 2014. General FAQ’s about Select Agents and Toxins, vol. 2014.CDC, 2014. National Select Agent Registry, vol. 2014.Clark, R.J., Craik, D.J., 2012. Engineering cyclic peptide toxins. Methods Enzymol.

503, 57–74.Clark, R.J. et al., 2005. Engineering stable peptide toxins by means of backbone

cyclization: stabilization of the alpha-conotoxin MII. Proc. Natl. Acad. Sci. U.S.A.102, 13767–13772.

Clark, R.J. et al., 2010. The engineering of an orally active conotoxin for thetreatment of neuropathic pain. Angew. Chem. Int. Ed. Engl. 49, 6545–6548.

Corpuz, G.P. et al., 2005. Definition of the M-conotoxin superfamily:characterization of novel peptides from molluscivorous Conus venoms.Biochemistry 44, 8176–8186.

Craik, D.J., 2012. Host-defense activities of cyclotides. Toxins 4, 139–156.Craik, D.J. et al., 2012. Cyclotides as a basis for drug design. Expert Opin. Drug

Discov. 7, 179–194.Cruz, L.J. et al., 1978. Purification and properties of a myotoxin from Conus

geographus venom. Arch. Biochem. Biophys. 190, 539–548.Cruz, L.J. et al., 1985. Conus geographus toxins that discriminate between neuronal

and muscle sodium channels. J. Biol. Chem. 260, 9280–9288.Daly, N.L., Craik, D.J., 2009. Design and therapeutic applications of cyclotides. Future

Med. Chem. 1, 1613–1622.Daly, N.L. et al., 2009. Discovery, structure and biological activities of cyclotides.

Adv. Drug Deliv. Rev. 61, 918–930.Donevan, S.D., McCabe, R.T., 2000. Conantokin G is an NR2B-selective competitive

antagonist of N-methyl-D-aspartate receptors. Mol. Pharmacol. 58, 614–623.Dutton, J.L. et al., 2002. A new level of conotoxin diversity, a non-native disulfide

bond connectivity in alpha-conotoxin AuIB reduces structural definition butincreases biological activity. J. Biol. Chem. 277, 48849–48857.

Ellison, M. et al., 2008. Alpha-RgIA, a novel conotoxin that blocks the alpha9alpha10nAChR: structure and identification of key receptor-binding residues. J. Mol.Biol. 377, 1216–1227.

Ellison, M. et al., 2006. Alpha-RgIA: a novel conotoxin that specifically and potentlyblocks the alpha9alpha10 nAChR. Biochemistry 45, 1511–1517.

England, L.J. et al., 1998. Inactivation of a serotonin-gated ion channel by apolypeptide toxin from marine snails. Science 281, 575–578.

Espiritu, M.J. et al., 2014. Incorporation of post-translational modified amino acidsas an approach to increase both chemical and biological diversity of conotoxinsand conopeptides. Amino Acids 46, 125–151.

Essack, M. et al., 2012. Conotoxins that confer therapeutic possibilities. Mar. Drugs10, 1244–1265.

Fainzilber, M. et al., 1991. Mollusc-specific toxins from the venom of Conus textileneovicarius. Eur. J. Biochem. 202, 589–595.

Fainzilber, M. et al., 1998. Gamma-conotoxin-PnVIIA, a gamma-carboxyglutamate-containing peptide agonist of neuronal pacemaker cation currents.Biochemistry 37, 1470–1477.

202 P. Thapa et al. / Regulatory Toxicology and Pharmacology 70 (2014) 197–202

FDA, PRIALT (ziconotide intrathecal infusion), 2006.FDA, 2014. How Drugs are Developed and Approved, vol. 2014.FDA, 2014. Investigational New Drug (IND) Application, vol. 2014.Gray, W.R. et al., 1981. Peptide toxins from Conus geographus venom. J. Biol. Chem.

256, 4734–4740.Hamman, J.H. et al., 2005. Oral delivery of peptide drugs: barriers and

developments. BioDrugs 19, 165–177.Henriques, S.T., Craik, D.J., 2010. Cyclotides as templates in drug design. Drug

Discov. Today 15, 57–64.Imperial, J.S. et al., 2006. A novel conotoxin inhibitor of Kv1.6 channel and nAChR

subtypes defines a new superfamily of conotoxins. Biochemistry 45, 8331–8340.

Jimenez, E.C. et al., 2003. Novel excitatory Conus peptides define a new conotoxinsuperfamily. J. Neurochem. 85, 610–621.

Kaas, Q. et al., 2010. Conopeptide characterization and classifications: an analysisusing ConoServer. Toxicon 55, 1491–1509.

Kaas, Q. et al., 2012. ConoServer: updated content, knowledge, and discovery toolsin the conopeptide database. Nucleic Acids Res. 40, D325–D330.

Kapono, C.A. et al., 2013. Conotoxin truncation as a post-translational modificationto increase the pharmacological diversity within the milked venom of Conusmagus. Toxicon 70, 170–178.

Kerr, L.M., Yoshikami, D., 1984. A venom peptide with a novel presynaptic blockingaction. Nature 308, 282–284.

Lee, S. et al., 2010. Analgesic effect of highly reversible omega-conotoxin FVIA on Ntype Ca2+ channels. Mol. Pain 6, 1744–8069.

Liu, L. et al., 2007. Two potent alpha3/5 conotoxins from piscivorous Conusachatinus. Acta Biochim. Biophys. Sin. (Shanghai) 39, 438–444.

Liu, L. et al., 2008. Identification of a novel S-superfamily conotoxin fromvermivorous Conus caracteristicus. Toxicon 51, 1331–1337.

Loughnan, M.L. et al., 2009. Novel alpha D-conopeptides and their precursorsidentified by cDNA cloning define the D-conotoxin superfamily. Biochemistry48, 3717–3729.

Luo, S. et al., 2013. A novel inhibitor of alpha9alpha10 nicotinic acetylcholinereceptors from Conus vexillum delineates a new conotoxin superfamily. PLoSOne 8, 30.

MacRaild, C.A. et al., 2009. Structure and activity of (2,8)-dicarba-(3,12)-cystinoalpha-ImI, an alpha-conotoxin containing a nonreducible cystine analogue. J.Med. Chem. 52, 755–762.

Martinez, J.S. et al., 1995. Alpha-conotoxin EI, a new nicotinic acetylcholine receptorantagonist with novel selectivity. Biochemistry 34, 14519–14526.

McIntosh, J.M. et al., 1995. A new family of conotoxins that blocks voltage-gatedsodium channels. J. Biol. Chem. 270, 16796–16802.

McIntosh, M. et al., 1982. Isolation and structure of a peptide toxin from the marinesnail Conus magus. Arch. Biochem. Biophys. 218, 329–334.

NIH, 2008. FAQ ClinicalTrials.gov – Clinical Trial Phases, vol. 2014.Olivera, B.M., 1997. E.E. Just Lecture, 1996. Conus venom peptides, receptor and ion

channel targets, and drug design: 50 million years of neuropharmacology. Mol.Biol. Cell 8, 2101–2109.

Olivera, B.M., 2006. Conus peptides: biodiversity-based discovery and exogenomics.J. Biol. Chem. 281, 31173–31177.

Olivera, B.M. et al., 1987. Neuronal calcium channel antagonists. Discriminationbetween calcium channel subtypes using omega-conotoxin from Conus magusvenom. Biochemistry 26, 2086–2090.

Olivera, B.M., Teichert, R.W., 2007. Diversity of the neurotoxic Conus peptides: amodel for concerted pharmacological discovery. Mol. Interv. 7, 251–260.

Peng, C. et al., 2006. Discovery of a novel class of conotoxin from Conus litteratus,lt14a, with a unique cysteine pattern. Peptides 27, 2174–2181.

Petrel, C. et al., 2013. Identification, structural and pharmacological characterizationof tau-CnVA, a conopeptide that selectively interacts with somatostatin sst3receptor. Biochem. Pharmacol. 85, 1663–1671.

Poth, A.G. et al., 2013. Cyclotides as grafting frameworks for protein engineeringand drug design applications. Biopolymers 100, 480–491.

Rigby, A.C. et al., 1999. A conotoxin from Conus textile with unusualposttranslational modifications reduces presynaptic Ca2+ influx. Proc. Natl.Acad. Sci. U.S.A. 96, 5758–5763.

Rivier, J. et al., 1987. Total synthesis and further characterization of the gamma-carboxyglutamate-containing ‘‘sleeper’’ peptide from Conus geographus venom.Biochemistry 26, 8508–8512.

Safo, P. et al., 2000. Distinction among neuronal subtypes of voltage-activatedsodium channels by mu-conotoxin PIIIA. J. Neurosci. 20, 76–80.

Sancheti, H., Camarero, J.A., 2009. ‘‘Splicing up’’ drug discovery. Cell-basedexpression and screening of genetically-encoded libraries of backbone-cyclized polypeptides. Adv. Drug Deliv. Rev. 61, 908–917.

Santos, A.D. et al., 2004. The A-superfamily of conotoxins: structural and functionaldivergence. J. Biol. Chem. 279, 17596–17606.

Sato, S. et al., 1983. The amino acid sequences of homologous hydroxyproline-containing myotoxins from the marine snail Conus geographus venom. FEBSLett. 155, 277–280.

Schroeder, C.I. et al., 2013. Recent progress towards pharmaceutical applications ofdisulfide-rich cyclic peptides. Curr. Protein Pept. Sci. 14, 532–542.

Sharpe, I.A. et al., 2001. Two new classes of conopeptides inhibit the alpha1-adrenoceptor and noradrenaline transporter. Nat. Neurosci. 4, 902–907.

Terlau, H., Olivera, B.M., 2004. Conus venoms: a rich source of novel ion channel-targeted peptides. Physiol. Rev. 84, 41–68.

Terlau, H. et al., 1996a. Strategy for rapid immobilization of prey by a fish-huntingmarine snail. Nature 381, 148–151.

Terlau, H. et al., 1996b. MicroO-conotoxin MrVIA inhibits mammalian sodiumchannels, but not through site I. J. Neurophysiol. 76, 1423–1429.

Walker, C.S. et al., 1999. The T-superfamily of conotoxins. J. Biol. Chem. 274, 30664–30671.

Zafaralla, G.C. et al., 1988. Phylogenetic specificity of cholinergic ligands: alpha-conotoxin SI. Biochemistry 27, 7102–7105.

Zhangsun, D. et al., 2006. Novel O-superfamily conotoxins identified by cDNAcloning from three vermivorous Conus species. Chem. Biol. Drug Des. 68, 256–265.