Cone Snail Venomics to Drugs: a Review B

ISSN No.: 2454- 2024 (online)

International Journal of Technical Research & Science CONE SNAIL VENOMICS TO DRUGS: A REVIEW B. Saghya Infant Shofia, G. S. Selvam, K. Jayakumar E-Mail Id: [email protected], [email protected], [email protected] School of Biological Sciences, Madurai Kamaraj University, Madurai, India

Abstract-Cone snails are slow-moving marine gastropods under the phylum molluscs. They are carnivorous and use venom to kill their prey. The venom of cone snails consists of most complex mixtures of toxins in the animal kingdom. These toxins are mostly peptidic in nature and so they are called as conopeptides or conotoxins. Such a small droplet of this venom causes a great damage to its prey and the same impressive destructive power of venoms has been turned into a life-saving drugs. One such molecule, ω-conotoxin is now an FDA-approved drug to treat chronic pain, for cancer and post-surgical pain. In addition, several other cone snail compounds are being investigated for the treatment of neuropathic pain, epilepsy, cardiac infarction, and neurological diseases. Conotoxins, rich in di - sulfide bonds are small peptides of 10 to 35 residues has high and specific effect on various receptors in pain pathway, voltage or ligand gated ion channels and nerve transmission. Due to these properties conotoxins have been used as molecular tool in the study of ion channels and receptors and as potential pharmaceuticals.Thus, conus venoms are now regarded as pharmacological treasures leading to drug discovery. Key words: cone snails, conus venom, conotoxins, conopeptides, ion channels.

Abbreviations

FDA – Food and Drug Administration; NMD – Neuromuscular Disorders; NMDA – N-methyl D-aspartate; GLA – Gamma carboxy glutamate; BBB – Blood Brain Barrier

1. INTRODUCTION

Discovering drugs from natural products emerges as a rich and abundant source of novel compounds. Drugs from traditional plants with medicinal uses and marine organisms find a conspicuous place in treating various diseases [88-90]. In recent years researchers are interested to study about the toxins from venomous animals which give rise to the therapeutic lead in drug discovery. Venoms has been turned into a life-saving drugs as these toxins cannot cross the epithelial layers including the blood brain barrier, and because of its hydrophilic nature it cannot spread through tissues. It has to be locally administered by intravenous, intramuscular, subcutaneous or epidural injection which reduces the risk of side effects [2]. The biological actions of toxin from venomous animals include pain, paralysis or even cause death. Venom helps the animal for prey capture and defense. The special features of the toxins or venom is that it remains stable against chemical degradation or enzymatic process as it undergoes post translational modifications. The specificity of the toxins is because of the presence of cysteine patterns which is the highly conserved class among the toxins. Venoms have various pharmacological activities because of the presence of protease inhibitors and stabilizing agents [1]. The components of the venom help the animal to survive in a specific environment. Toxins are being used for diagnostic, therapeutic purposes and as a chemical tool in scientific research field to study the molecular mechanisms. The active components of venoms are mostly protein in nature and exert a very high potency because of its tight bonding contact with their target receptors [12, 13] Studying about the venom gives three levels of information (i) Conserved cysteine patterns - helps in identifying the entire protein and genome domain with same structural signature as toxins use this to recognize their physiological targets. (ii) Evolutionary relationship - The folds present in the toxin structure are shared by non-toxic proteins or peptides which give insights on an evolutionary relationship (iii) Genes involved in cell process and venom production

2. CONE SNAILS (CONUS)

Cone snails are slow moving marine invertebrates belonging to the genus Conus, phylum Mollusca and class Gastropoda in Linnean classification based on their radular morphology and dietary habits [93] (Fig. 2.1, 2.2). Cone snails are 0.5 to 8.5 inches long and are nocturnal whereas throughout the day they are found buried under the rocks sand [80, 92]. Cone snails propels with its muscular foot on the floor of the ocean.

International Journal of Technical Research & Science

Fig. 2.1 Cone Snails: Conusbetulinus Collected from Gulf of Mannar region

Fig. 2.2Conus Species in Atlantic and Pacific Oceans. (PC: http://otlibrary.com/conus-snail/)

3. HABITAT

The habitat of cone snails are coral reefs and shallow sandy waters found in Western Atlantic, Indian and Pacific oceans but they cannot survive in fresh water. Cone snails prefer a unique environment depending on its diet and other survival factors [80]. Cone snails maintain a symbiotic relationship with corals.

4. ANATOMY OF CONUS

The internal and external anatomy of conus is clearly explained in Fig. 4.1. The parts of the internal anatomy are essential for movement and capturing of prey. 1. Proboscis – used for hunting by injecting the venom into prey. 2. Siphon – used for detecting prey and it is also a tool for respiration 3. Eye stalks. 4. Mouth – This can be extended to engulf its prey. 5. Foot – a long muscular foot that allows the snail to move. pg. 573 www.ijtrs.com www.ijtrs.org Paper Id: IJTRS-V2-I8-003 Volume 2 Issue IX, September 2017 @2017, IJTRS All Right Reserved ISSN No.: 2454- 2024 (online)

International Journal of Technical Research & Science

Fig. 4.1 External and Internal Anatomy of Conus. (PC: http://zoologybe.blogspot.in/2011/01/cone-snail-conus- by-zachary-kaye.html) 1. Probocis 2. Siphon 3. Eye stalks 4. Mouth 5. Foot.

5. FEEDIND AND HUNTING

Cone snails are carnivores in nature and they are divided into three groups (i) Cone snails that eat fish are called piscivores (ii) those that eat worms are called vermivores and (iii) those species which eat molluscs are called molluscivores. Conus bury itself in sand and detect its prey using a chemosensor organ called osphradium. The prey will be stung by the siphon which will make the prey paralyzed within seconds‟after which the cone snails will open its mouth widely and swallows the prey completely and digest it with the help of the venom components (Fig. 5.1). The predators of cone snails are hermite crabs and horseshoe crabs, they do not have many predators because they defend themselves by using their proboscis to kill the predator or protect themselves by staying inside their hard shell [80, 92].

Fig. 5.1 Feeding and Hunting Process of Cone Snails. (PC: http://otlibrary.com/conus-snail/)

6. VENOM SYSTEM

Cone snails paralyze the prey by using the venom which is present in the venom bulb, a modified salivary gland. Cone snails consist of harpoons which can be reloaded with venom for every attack (Fig. 6.1). Thus harpoons are pg. 574 www.ijtrs.com www.ijtrs.org Paper Id: IJTRS-V2-I8-003 Volume 2 Issue IX, September 2017 @2017, IJTRS All Right Reserved ISSN No.: 2454- 2024 (online)

International Journal of Technical Research & Science considered as modified teeth stored in the radular sac, like a disposable, hypodermic needle filled with venom to attack the prey [80].

Fig. 6.1 Structure of the Venom System (PC: http://otlibrary.com/conus-snail/)

7. CONOTOXINS

The toxins present in the venom of cone snails are a source of neuro - pharmacologically active peptides named conotoxins. According to Olivera, the peptides present in the venom play a major role in protecting themselves from predators, prey capture and some other biological interactions [16]. More than 500 species of cone snails have been reported till date and interestingly conotoxins consist of most complex mixture of toxins that is the venom of each species consists of 50 to 200 different components that is more than 50,000 different conopeptides are present of which only less than 0.1% is pharmacologically characterized [15, 56-58, 4]. Peptides found in conotoxins are mostly short strings of amino acids (8-35 amino acids in length) and are rich in disulphide bonds which are solely responsible for the rigid shape of the peptide and thus it increases the binding ability of conotoxins with its specific targets [17, 18, 59-60]. Reports have been given that by purification and characterization of the venom from Conusgeographus and Conus magus, the pisivorous species have three pharmacologically distinct classes of toxins. They are α conotoxins that acts on acetylcholine receptors; µ conotoxins that acts on skeletal muscle sodium channels and ω conotoxins that acts on neuronal calcium channels [7-11]. Conotoxins also contains conantokins, a group of peptides that are not disulphide rich, large polypeptides (>10KDa) or small molecules such as biologically active amines. The target of many peptide toxins are ion channels and receptors and thus according to Cruz et al the list of macromolecular targets of these peptides includes six types of ion channels and receptors: Acetylcholine receptors (α, αA and ψ conotoxins), NMDA receptors (conotakins), Sodium (µ, µO and δ conotoxins), calcium (ω conotoxins) and potassium (κ conotoxins) channels, vasopressin (conopressins) and neurotensin (contulateins) receptors [19]. Peptidic toxins have a complex structure with many foldings that keep them stable and resistent to enzymatic actions and provides strong bonding to their targets [14, 41]. Conotoxins are classified into six different classes based on their stuctures. They are α conotoxins, ω conotoxins, δ conotoxins, µ conotoxins, κ conotoxins and contulakins [61]. Table-7.1 Various type of Conotoxins Conopeptides Conus species Target Stage Use Reference Blocks neuronal type α conotoxin Conusvictoriae Preclinical Effective against pain. [66, 93] nicotonic Ach receptors. Inhibits α-1 adrenergic Rho conotoxin Conustulipa Preclinical Non - competitive inhibitor [78] receptors Blocks N-type calcium Reported to have a better ω-conotoxin Conus cactus Channel specific sub- Stage II therapeutic index than [94] type. PrialtTM Significant pain relief to ω-conotoxin Conus magus N-type calcium channels Stage III [95-98] patients in clinical trials. Conusmarmoreo inhibits neuronal χ-conopeptides Preclinical Treats neuropathic pain [78] us noradrenaline transporter

International Journal of Technical Research & Science Conusgeographu Binds to neurotensin Short term management of Contulakin-G Stage II [77] s receptor post-operative pain Inhibits NMDA receptor Conusgeographu Control of seizures in Conantokin-G (NR2B Stage II [77] s intractable epilepsy subtype Inhibits NMDA receptor Effective in injury induced Conantokin-T Conustulipa (NR2A Stage II [77] pain and NR2B) subtypes

7.1 Brief History of the Discovery of Conotoxins

About 300 years ago a Dutch naturalist first noted the lethality of cone snails [6], from then it was known as predators for many years [30] after which in the year 1970 analysis and isolation of active compounds from the venom ofConuscalifornicus[31] and Conusgeographus [32]were reported. Characterization of cone snail toxins was started almost half a century ago [83–85] and has blossomed into a successful research field [86, 87]. Later in 1981, Gray et al [33] did biochemical studies and described the structure and function of several conotoxins extracted from Conusgeographus. In recent years, Olivera group had identified numerous toxins from many conus species, such as Conus magus, Conusstriatus, andConus textile [34, 35].

7.2 Structure and Function of Conotoxins

Conotoxins are mainly characterized by a three domain structure which consists of: a highly conserved signal sequence which is considered to be a diagnostic character, a more variable pro-region and a hyper-variable mature sequence. The mature toxin is a disulphide rich peptide with cysteine pattern and is found to be highly conserved in each superfamily [36].

8. NICOTINIC ACETYLCHOLINE RECEPTORS

Nicotinic acetylcholine receptors are pentamericcation channels that open in response to acetylcholine binding. The muscle nicotinic acetylcholine receptors in adults are the major neurotransmitter receptor at the neuromuscular junction and it is the target for many paralyzing toxins [49]. Toxins from many conus species have given rise to α conotoxins which act as antagonists mainly targets the nerve and muscle nicotinic acetylcholine receptors for prey capture [20, 21]. The first toxin isolated was α conotoxins from the venom of conus species and it was designated as alpha because they had the same action as the alpha neurotoxin that was isolated from the venom of snake (eg. Alpha Bungarotoxin) that inhibits the muscle type nicotinic receptor. The α conotoxins GI, GIA, GII from Conusgeographus and MI from Conus magus are homologous peptides with 13 and 15 amino acids respectively and contains two disulphide bridges. The main function of muscle type α conotoxins is to inhibit the post synaptic region at neuromuscular junction which results in paralysis and death. The mechanism involved in causing paralysis is that α conotoxins bind to the alpha subunit of the nicotinic acetylcholine ligand gated ion channel and thus blocks the binding of acetylcholine and nicotine. The neuronal type α conotoxins does not cause paralysis since its target is not nicotinic receptor at neuromuscular junction instead they target neuronal type nicotinic receptors in the brain and on peripheral sensory neurons [62-66]. Each α conotoxins has its unique electrostatic interactions and hydrogen bonds that are responsible for its strong affinity and selectivity [49]. These peptides could be of interest in the treatment of anxiety, Parkinson‟s disease, pain, hypertension, cancer and also muscle relaxants [22].

9. POTASSIUM CHANNEL TOXINS

Potassium channels are four domain membrane proteins that selectively transport potassium (K+) ions across the cell membrane. These ions play a key role in regulating the physiology of excitability and non - excitability of the cell [45]. Several toxins isolated from the venom of conus species affect this potassium channel. The first discovered was κ conotoxin PVIIA a 27 amino acid toxin from the venom of Conuspurpurascens targets and inhibits the potassium channel [46, 71,72] through the functional dyad comprising Lys7 and Phe9 plus Lys25 [47]. This toxin has been chemically synthesized in a biologically active form. The folding of κ conotoxin is similar to that of calcium channel blocking ω conotoxin [25].

10. SODIUM CHANNEL TOXINS

Voltage sensitive sodium channels which are essential for electrical signaling in cells are four domain membrane proteins. Scientists have reported that mutations in this sodium channel are the causes for many genetic diseases which include epilepsy and migraines [48]. Different types of conotoxins act on the voltage sensitive sodium pg. 576 www.ijtrs.com www.ijtrs.org Paper Id: IJTRS-V2-I8-003 Volume 2 Issue IX, September 2017 @2017, IJTRS All Right Reserved ISSN No.: 2454- 2024 (online)

International Journal of Technical Research & Science channels. The best studied and first identified peptides that are found to be the inhibitors of sodium channels is µ conotoxins [7]. These µ conotoxins are globular in structure with 16-25 amino acid peptides and possess three disulphide bonds. They act upon sodium channels in muscle and also to a very limited extent in neurons. µ Conotoxins binds with sodium channel and inhibits the influx of sodium ions into the cell which makes the organism paralyzed [69]. µ Conotoxins which act on neuromuscular sodium channels might be useful to treat NMD [20]. GIIIA, GIIIB and GIIIC are µ conotoxins isolated from the venom of the species Conusgeographus are hydroxyproline rich basic peptides with 22 amino acids. These peptides contain 3 hydroxyprolines and 6 cysteine residues and possess either an exposed Arg or Lys in loop 2 that is important for high affinity interactions with the sodium channels [48, 69]. Cone snail venoms contain two other classes of sodium channel toxins. (i) µO conotoxins and (ii) δ conotoxins. The µO conotoxinsMrVIA and MrVIB which are isolated from conusmarmoreus [28] is a group of peptides which block sodium currents of voltage sensitive sodium channels. They are 31 amino acid peptides that mainly block the sodium conductance in aplysia neurons. Though these peptides exhibit a pharmacological action similar to that of the µ conotoxins, they are structurally unrelated [23, 48]. The δ conotoxins isolated from mollusc and fish hunting cone snails also targets sodium channels [48] with a core of disulphide bridges but are found to be unusual chemically as they lack hydrophobic amino acids in the interior but are found to be present in the surrounding solvent. Researchers have suspected that the peptides may dissolve in the lipid membrane by binding to it laterally and thus interfere in the opening and closing of sodium channels [70].

11. CALCIUM CHANNEL TOXINS

Voltage gated calcium channels are structurally related to voltage gated sodium channels. These channels are mainly involved in the influx of calcium ions that are required for contraction of muscle and release of neurotransmitters. According to their electrophysiological and pharmacological characteristics calcium channels have been classified into L-, N-, P-, Q-, T- and R- types [42]. The ω conotoxins are the most potent ichthyotoxins isolated from fish hunting cone snails with 24-30 amino acids and three disulphide bonds. The first isolated ω conotoxins was GVIA from Conusgeographus [26, 27] and MVIIA, MVIIC and MVIID from Conus magus venom. They are known as „shaker peptides‟ as they induce continuous tremors in mice when injected intracerebrally. Conotoxin from conusgeographus block the neuromuscular junction of skeletal muscle and act by blocking calcium channels without interfering with cellular action potential [67, 68]. Among the conotoxins the most selective inhibitor is the ω conotoxins because of its therapeutic potential in the management of severe pain. The synthetic peptide SNX-III isolated from the venom of snake corresponds to the sequence of a ω conopeptides MVIIA obtained from the venom of Conus magus and it acts as a highly potent and selective antagonist of N-type calcium channels [24].

12. NMDA RECEPTORS

N-methyl D-aspartate (NMDA) receptors are tetrameric ligand gated ion channels with high calcium permeability that mediates fast excitatory neurotransmission in the central nervous system. They play an important role in the pathophysiology of central nervous system. NMDA receptors are a subtype of glutamate receptors. Till date it has been reported that only cone snails have yielded venom peptides targeting NMDA receptors [50-52]. Conantokins are linear peptides with 7, 22 amino acids which inhibits NMDA receptors [76] as it targets the major excitatory receptors, the glutamate receptors in the central nervous system. The first conantokin was isolated from Conusgeographus. All conantokins are linear peptides except the conantokin isolated from Conusradiatus which has a three amino acid disulphide bridged loop near the C-terminus [20]. Unlike the conotoxins, the conantokins have no disulphide bonds but their structural ability has been derived from post translationally modified glutamic residues present as γ carboxy glutamate (GLA) [73-75].

13. CONTULAKINS, CONTRYPHANS AND OTHER CONOPEPTIDES A number of other conopeptides are being developed as drugs for the treatment of various neurological conditions [53]. Contulakin G is a glycosylated 15 amino acid conopeptides isolated from the venom of Conusgeographus [77] is being developed for short term management of post - operative pain and another type of conotoxin has been identified to be a non - competitive inhibitor of α-1 adrenergic receptors and is named as rho-conotoxin [78]. Another type of conopeptides has been isolated from the venom of the Conusmarmoreus and named as chi conopeptides. This chi conopeptides act as reversible non - competitive inhibitors of the neuronal noradrenalin transporter and it has been developed for the treatment of neuropathic pain [78]. Contryphans are the smallest bioactive conopeptides with 8 or 9 amino acids and just one disulphide bond. It has been isolated from the venom of Conusventricosus and Conusregius. They are distinguished by their numerous post pg. 577 www.ijtrs.com www.ijtrs.org Paper Id: IJTRS-V2-I8-003 Volume 2 Issue IX, September 2017 @2017, IJTRS All Right Reserved ISSN No.: 2454- 2024 (online)

International Journal of Technical Research & Science translational modifications. The disease target for contryphans is not yet known but this peptide promotes body tremor and mucous secretion when injected in fish, suggesting that they may have biological activity related to endocrine or neuronal functions [79].

14. ION CHANNELS AS DRUG TARGETS AND EMERGENCE OF CONOTOXINS FOR DRUG DEVELOPMENT

Ion channels are class of membrane proteins that play crucial roles in cellular physiology, neuronal signaling and muscle contractility [43]. These voltage dependent ion channels are intrinsic membrane proteins that play a very important role in communicating to the excitable cells rapidly. Venoms of cone snails are found to be a reservoir of millions of bioactive peptides with highly diverse sequences and structures. These are having numerous clinical importances because the ion channels are their main target for its function to emerge [44, 61]. Toxins that interact with specific regions in ion channels can be used as structural templates to study the ion channels. Ion channels have structural and functional similarities but even within a class of ion channels they have significant differences that can be targeted in drug applications [39]. The most successful venomic project is the development of PRIALT, the first conotoxin derived drug. This analgesic is a synthetic version of a ω conotoxin isolated from the venom of Conus magus. The emergence of Ziconotide, artificial toxin or peptide has led to the increased investigation of cone snail peptides in drug development [3, 29]. The main advantage of Ziconotide is that it has only limited ability to cross the BBB with reduced potential for serious side-effects; it must be administered intrathecally to patients. This spinal route of administration permits Ziconotide to reach its maximum local concentration in a short time, which encourages a rapid onset of analgesia [82]. The target of Ziconotide is to inhibit the N type calcium channel, which is involved in the pain pathway. Ziconotide is administered in case of acute and chronic pain caused by HIV, cancer, post - operative pain. It has been found to be efficient in patients who have no response to opioid drugs. The only listed side effect is hypotension, and this may be due to the block caused by the drug prialt on neurons of calcium channels that regulate the blood pressure [3, 82]. Apart from this there are other conotoxin derived peptides that have been reached clinical trials at various stages. It includes (i). Contulakin G – neurotensin receptor, (ii). Χ MrIA–norepinephrine receptor, (iii). Α Vc1.1 – nicotinic receptor, (iv). Conantokin G – NMDA receptor, (v). κ PVIIA – potassium channel, (vi). µO MrVIB – Sodium channel. These conotoxins have a potential therapeutic for pain but they are being evaluated for epilepsy or myocardial infarctions [29]. Twedeet al [37] also have cited several other conotoxins with cardio - protective and neuroprotective properties namely conantokins, µ, ω and κ conotoxins.

CONCLUSION

“Poison kills the poison”, the famous proverb has been the underlying basis for the researchers in finding the novel bioactive metabolites from living organisms. Venom peptides have evolved to target ion channels using a diversity of structures and modes of interaction. It has been reported that >99% of venom peptides are not characterized pharmacologically still and most of the toxins that have been identified targets the ion channels. The conotoxins are the large family of peptides that has received increasing attention because of their unique actions on excitable membranes and chemical synapses. The drugs obtained from the conotoxins are found to be neurologically active as it reveals the complexity of voltage gated and ligand gated ion channels. Thus venom peptides remain as an untapped source of new probes for research and leads to novel therapeutics.

ACKNOWLEDGEMENT

This work was supported by UGC-MaulanaAzad National Fellowship [grant no: F117.1/201516/MANF201517PON48888/(SAIII/Website)]

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