Cone snail species off the Brazilian coast and their venoms: a review and update

Abstract

The genus Conus includes over 900 species of marine invertebrates known as cone snails, whose venoms are among the most powerful described so far. This potency is mainly due to the concerted action of hundreds of small bioactive peptides named conopeptides, which target different ion channels and membrane receptors and thus interfere with crucial physiological processes. By swiftly harpooning and injecting their prey and predators with such deadly cocktails, the slow-moving cone snails guarantee their survival in the harsh, competitive marine environment. Each cone snail species produces a unique venom, as the mature sequences of conopeptides from the venoms of different species share very little identity. This biochemical diversity, added to the numerous species and conopeptides contained in their venoms, results in an immense biotechnological and therapeutic potential, still largely unexplored. That is especially true regarding the bioprospection of the venoms of cone snail species found off the Brazilian coast - a region widely known for its biodiversity. Of the 31 species described in this region so far, only four - Conus cancellatus, Conus regius, Conus villepinii, and Conus ermineus - have had their venoms partially characterized, and, although many bioactive molecules have been identified, only a few have been actually isolated and studied. In addition to providing an overview on all the cone snail species found off the Brazilian coast to date, this review compiles the information on the structural and pharmacological features of conopeptides and other molecules identified in the venoms of the four aforementioned species, paving the way for future studies.

Keywords:
Conus; Cone snail; Brazilian coast; Venom; Conopeptides; Conotoxins

Background

The genus Conus Linnaeus, 1758, a member of the Conidae J. Fleming, 1822 family, comprises a group of marine venomous snails, with more than 900 species recognized to date [1]. The so-called cone snails are distributed throughout the globe, predominantly in tropical waters. These animals usually dwell near reefs, burying in the sand during the day and hunting at night.

Cone snails are usually divided into three groups according to feeding habit: the vermivorous group, which feeds on worms; the molluscivorous group, which hunts other gastropods; and the piscivorous group, the fish-hunting cone snails. Among the immense diversity of cone snail species, those belonging to the piscivorous group pose a greater threat to humans, being responsible for serious and sometimes fatal accidents [2]. Nevertheless, molluscivorous and vermivorous cone snails also produce toxins that can be harmful to vertebrate systems, at the very least under experimental conditions [3, 4].

Through millions of years of evolution, cone snails developed a highly sophisticated and well-conserved venom apparatus for prey capture and defense against predators [5, 6]. It consists of a duct, where the venom is synthesized and stored, a bulb, which transfers venom from the duct [7], and a hollow, harpoonlike radula tooth, which enables the fast and efficient delivery of venom into the prey [8].

A shell of the piscivorous species Conus ermineus and a live specimen of the vermivorous Conus regius and its radula tooth are shown in Figures 1A, 1B, and 1C, respectively. Both species, although not endemic, are found off the Brazilian coast and the exploration of their venoms will be discussed in this review.

Figure 1.
Examples of cone snail species found off the Brazilian coast. (A) Conus ermineus shell. MNRJ 8741. 70.2 x 40.4 mm. (B) Conus regius live specimen. Rio de Janeiro National Museum collection (MNRJ) 9704. 47 mm. Photos by Paulo Márcio Costa. (C) Conus regius radula tooth. MNRJ 9608. Optical microscopy photo (200x) by Renata Gomes.

The composition of the venom produced by cone snails depends on the purpose it must serve, that is, defense or predation, and the protein expression pattern along the venom duct is closely related to the type of venom produced [9]. Regardless, cone snail venoms are highly efficient weapons because their major components - small toxic peptides with about 10 to 45 amino acid residues named conopeptides - have ion channels and membrane receptors as canonical pharmacological targets [10, 11]. There have been also some reports about the presence of other bioactive compounds in cone snail venoms with hormone-like [12-14], proteolytic [15-17], hyaluronidase [18, 19] and phospholipase [20] activities, in addition to small non-peptidic molecules such as neurotransmitters [21, 22]. However, these cone snail venom components are less studied than conopeptides.

Conopeptides act with a high degree of specificity, selectively disturbing crucial physiological processes that involve electrical signaling and signal transduction as a whole. For instance, the venom of fish-hunting species that use the hook-and-line strategy contains conopeptides that act synergistically to quickly immobilize and paralyze the prey - the “lightening-strike” and motor cabals, respectively [23]. This cocktail simultaneously inhibits the inactivation of neuronal Na⁺ channels and blocks skeletal muscle Na⁺ channels, K⁺ channels, presynaptic Ca²⁺channels, and nicotinic acetylcholine receptors (nAChRs) [24]. Those who employ the “net-engulfment” strategy first release a “nirvana” cabal into the water, which contains an insulin-like peptide and other components that numb the fish, to which follows the injection of the paralytic motor cabal [13]. Thus, by producing a biochemically engineered venom that acts in different systems, cone snails guarantee their survival in the diverse and very competitive marine environment.

Traditionally, conopeptides are divided into two broad groups: disulfide-poor peptides, a minor group whose members have a single disulfide bond or even none at all; and disulfide-rich peptides that contain two or more disulfide bonds, also known as conotoxins [10]. The first group includes conopeptides with canonical targets, hormone-like conopeptides, and conopeptides whose targets remain unknown [25]. A list of disulfide-poor conopeptide families, along with their known/potential targets, is displayed in Table 1.

Table 1.
Disulfide-poor conopeptides and their known targets.

Family	General target
Conantokins0	NMDA receptor
Conorfamides0	RFamide receptor, ASIC channels, nAChR
Conolysins0	Cellular membranes
Conophans0	ND
Conomaps0	ND
Conomarphins0	ND
Cono-NPYs0	ND
Contulakins0	Neurotensin receptor
Hormone-like conopeptides0 (Elevenin, PH4)	ND
Contryphans1	K+ and Ca2+ channels
ConoGAYs1	ND
ConoCAPs1	ND
Conopressins1	Vasopressin/oxytocin receptor

• ⁰
  No disulfide bond; ¹one disulfide bond; ND: not determined.

The conotoxins group is much more complex, as each cone snail species produces a unique venom containing hundreds of different such peptides. This uniqueness arises mainly from the fact that, except for the number and position of cysteine residues, which are conserved among conotoxins that share the same cysteine framework and disulfide connectivity, the mature sequences of conotoxins from different species share very little identity [10]. Nevertheless, the gene superfamilies - defined by conserved signal sequences - that encode such a diversity of conotoxins are relatively few, and a same cysteine framework can be shared by different superfamilies [26]. Conotoxins differ also in the length of the loops formed by the residues that flank the conserved cysteine residues, in a classification system known as loop class [27]. They can be also classified according to three-dimensional structure, with different folds (A-L and Kunitz fold) and sub-folds being determined mainly by disulfide connectivity [27]. However, conotoxins that belong to different gene superfamilies and display different disulfide patterns, loop classes, and 3D structures can have the same target, which would classify them into the same pharmacological family [26, 27]. As an overlap between these classification schemes does not always take place, the task to group the ever-growing number of conotoxins described into simple categories is an impossible one.

The gene superfamilies, cysteine frameworks, pharmacological families, and targets of conotoxins described so far are listed in Table 2. For simplicity’s sake, we chose to overlook the other classification systems in this list. We must highlight that Table 2 does not include conodipines, which display phospholipase (PLA₂) activity, and con-ikot-ikot, which target a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors, both dimeric cysteine-rich conotoxins that do not fall into known gene superfamilies [20, 28].

Table 2.
Gene superfamily, cysteine framework, general targets, and pharmacological families of conotoxins according to ConoServer [26].

Superfamily	Cysteine framework	Pharmacological family	General Target
A	I (CC-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
		ρ	Alpha1-adrenoceptors (GPCR)
	II (CCC-C-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
	IV (CC-C-C-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
		κ	K+ channels
		μ	Na+ channels
	VI/VII (C-C-CC-C-C)	δ, μ	Na+ channels
		γ	Pacemaker channels
		κ	K+ channels
		ω	Ca2+ channels
	XIV (C-C-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
		κ	K+ channels
	XXII (C-C-C-C-C-C-C-C)	ND	ND
B2	VIII (C-C-C-C-C-C-C-C-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
		σ	Serotonin receptor
B3	XXIV (C-CC-C)	ND	ND
D	IV (CC-C-C-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
		κ	K+ channels
		μ	Na+ channels
	XIV (C-C-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
		κ	K+ channels
	XV (C-C-CC-C-C-C-C)	ND	ND
	XX (C-CC-C-CC-C-C-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
	XXIV (C-CC-C)	ND	ND
	XXVIII (C-C-C-CC-C-C-C-C-C)	ND	ND
E	XXII (C-C-C-C-C-C-C-C)	ND	ND
G	XIII (C-C-C-CC-C-C-C)	ND	ND
I1	VI/VII (C-C-CC-C-C)	δ, μ	Na+ channels
		γ	Pacemaker channels
		κ	K+ channels
		ω	Ca2+ channels
	XI (C-C-CC-CC-C-C)	ι	Na+ channels
		κ	K+ channels
	XXI (CC-C-C-C-CC-C-C-C)	ND	ND
I2	VI/VII (C-C-CC-C-C)	δ, μ	Na+ channels
		γ	Pacemaker channels
		κ	K+ channels
		ω	Ca2+ channels
	XI (C-C-CC-CC-C-C)	ι	Na+ channels
		κ	K+ channels
	XII (C-C-C-C-CC-C-C)	ND	ND
	XIII (C-C-C-CC-C-C-C)	ND	ND
	XIV (C-C-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
		κ	K+ channels
I3	VI/VII (C-C-CC-C-C)	δ, μ	Na+ channels
		γ	Pacemaker channels
		κ	K+ channels
		ω	Ca2+ channels
	XI (C-C-CC-CC-C-C)	ι	Na+ channels
		κ	K+ channels
J	XIV (C-C-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
		κ	K+ channels
K	XXIII (C-C-C-CC-C)	ND	ND
L	XIV (C-C-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
		κ	K+ channels
	XXIV (C-CC-C)	ND	ND
M	I (CC-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
		ρ	Alpha1-adrenoceptors (GPCR)
	II (CCC-C-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
	III (CC-C-C-CC)	α	Nicotinic acetylcholine receptors (nAChR)
		ι, μ	Na+ channels
		κ	K+ channels
	IV (CC-C-C-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
		κ	K+ channels
		μ	Na+ channels
	VI/VII (C-C-CC-C-C)	δ, μ	Na+ channels
		γ	Pacemaker channels
		κ	K+ channels
		ω	Ca2+ channels
	IX (C-C-C-C-C-C)	ND	ND
	XIV (C-C-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
		κ	K+ channels
	XVI (C-C-CC)	ND	ND
	XXXII (C-CC-C-C-C)	ND	ND
N	XV (C-C-CC-C-C-C-C)	ND	ND -
O1	I (CC-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
		ρ	Alpha1-adrenoceptors (GPCR)
	VI/VII (C-C-CC-C-C)	δ, μ	Na+ channels
		γ	Pacemaker channels
		κ	K+ channels
		ω	Ca2+ channels
	IX (C-C-C-C-C-C)	ND	ND
	XII (C-C-C-C-CC-C-C)	ND	ND
	XIV (C-C-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
		κ	K+ channels
	XVI (C-C-CC)	ND	ND
	XXIX (CCC-C-CC-C-C)	ND	ND
O2	I (CC-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
		ρ	Alpha1-adrenoceptors (GPCR)
	VI/VII (C-C-CC-C-C)	δ, μ	Na+ channels
		γ	Pacemaker channels
		κ	K+ channels
		ω	Ca2+ channels
	XII (C-C-C-C-CC-C-C)	ND	ND
	XIV (C-C-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
		κ	K+ channels
	XV (C-C-CC-C-C-C-C)	ND	ND
	XVI (C-C-CC)	ND	ND
O3	VI/VII (C-C-CC-C-C)	δ, μ	Na+ channels
		γ	Pacemaker channels
		κ	K+ channels
		ω	Ca2+ channels
	XVI (C-C-CC)	ND	ND
P	IX (C-C-C-C-C-C)	ND	ND
	XVI (C-C-CC)	ND	ND
Q	VI/VII (C-C-CC-C-C)	δ, μ	Na+ channels
		γ	Pacemaker channels
		κ	K+ channels
		ω	Ca2+ channels
	XVI (C-C-CC)	ND	ND
R	XIV (C-C-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
		κ	K+ channels
S	VIII (C-C-C-C-C-C-C-C-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
		σ	Serotonin receptor
	XXXIII (C-C-C-C-C-C-C-C-C-C-C-C)	ND	ND
T	I (CC-C-C)	α	Nicotinic acetylcholine receptors (nAChR)
		ρ	Alpha1-adrenoceptors (GPCR)
	V (CC-CC)	ε	Ca2+ channels or G protein receptors
		μ	Na+ channels
	X (CC-C[PO]C)	ε	Ca2+ channels or G protein receptors
	XVI (C-C-CC)	ND	ND
U	VI/VII (C-C-CC-C-C)	δ, μ	Na+ channels
		γ	Pacemaker channels
		κ	K+ channels
		ω	Ca2+ channels
V	XV (C-C-CC-C-C-C-C)	ND	ND
Y	VI/VII (C-C-CC-C-C)	δ, μ	Na+ channels
		γ	Pacemaker channels
		κ	K+ channels
		ω	Ca2+ channels
	XVII (C-C-CC-C-CC-C)	ND	ND
Undetermined	XVIII (C-C-CC-CC)	ND	ND
	XIX (C-C-C-CCC-C-C-C-C)	ND	ND
	XXV (C-C-C-C-CC)	ND	ND
	XXVI (C-C-C-C-CC-CC)	ND	ND
	XXVII (C-C-C-CCC-C-C)	ND	ND
	XXX (C-C-CCC-C-C-C-CC)	ND	ND

• ND: not determined.

The diversity of mature conopeptides is further increased by post-translational modifications (PTMs) other than the formation of disulfide bonds, with the most common being C-terminal amidation, proline hydroxylation, and glutamate gamma-carboxylation [26]. It has been proposed that the unusually high frequency of PTMs in conopeptides is related to the pressure cone snails suffer to rapidly adapt their venoms to face environmental challenges [29]. It is, therefore, reasonable to infer that such modifications might have functional consequences, affecting the structure, potency, and even the selectivity of conopeptides, both naturally-occurring ones and synthesized analogs. In addition to PTMs, other mechanisms such as variable peptide processing and the maintenance of the propeptide region in the mature sequences of some conopeptides further increase their diversity [30].

By virtue of their specificity, diversity, and abundance, conopeptides are superb pharmacological tools and have great therapeutic potential. For example, the conotoxin ω-MVIIA, an N-type calcium channel blocker isolated from the venom of Conus magus, had its synthetic version - ziconotide (Prialt^®) - approved by the Food and Drug Administration (FDA) for treatment of severe chronic pain [31]. Other conopeptides have been assessed as drug leads for a number of conditions that include pain, epilepsy, and diabetes, among others [32]. A few have reached the pre-clinical stage of development, for instance: RgIA4 (KCP-400), an analog of the α-RgIA from the venom of Conus regius, is an α9 α10 nAChR blocker that counteracts neuropathic pain [33]; and mini-Ins, a minimal insulin analog based on the insulin-like peptide found in the venom of Conus geographus that has been tested for type-I diabetes [34].

Considering the astounding diversity and potential of conopeptides, these molecules remain still very much unexplored [11, 35]. The advent of “omic” techniques furthered the identification of new conopeptides considerably, for their isolation and characterization through traditional chromatographic methods are laborious and slow. By combining transcriptomic screenings and modern proteomic techniques, the full content of both dissected and milked cone snail venoms can be determined up to the PTM profile of mature peptides [30]. Nevertheless, relatively few such molecules have been identified so far, and even fewer isolated and characterized, the vast majority from the venoms of Indo-Pacific species [26]. The larger distribution of cone snail species in this biogeographic region might account at least in part for this predominance, but other regions such as the Western Atlantic coast are no less relevant and are home to a considerable number of species [36].

The Brazilian coast, for instance, is known for its privileged marine biodiversity, with diverse continental biogeographic regions and oceanic islands (Figure 2) [37] that favor the establishment of large populations of mollusks such as those of the genus Conus. To date, 31 species of cone snails have been described off the Brazilian coast, with 18 of them being endemic [38-40] (Table 3). Although, at first, these species were all classified as belonging to the genus Conus, it is now accepted that many of them actually belong to the genus Conasprella Thiele, 1929 [36]. Notwithstanding, these are all predatory, venomous snails.

Figure 2.
Biogeographical division of the Brazilian coast according to the distribution of marine prosobranch species from shallow waters proposed by Barroso et al. [37].

Table 3
Conus and Conasprella species found off the Brazilian coast.

Species - Accepted names	Biogeographical distribution (Brazilian coast)
Conus Linnaeus, 1758	🟩 🟥 🟦
Conus (Attenuiconus) eversoni, Petuch 1987*	🟥 □ △
Conus (Brasiliconus) scopulorum, Van Mol, Tursch & Kempf 1971*	🟥 ◯
Conus (Chelyconus) ermineus, Born 1778	🟩 🟥 🟦
Conus (Dauciconus) cancellatus, Hwass in Bruguière 1792	🟩 🟥 🟦 🟨
Conus (Dauciconus) villepinii, P. Fisher & Bernardi 1857	🟥 🟦 ⬡
Conus (Dauciconus) riosi, Petuch 1986*	🟥
Conus (Dauciconus) hazinorum, Petuch & Myers 2014*	◇
Conus (Dauciconus) abrolhoensis, Petuch 1987*	◇
Conus (Dauciconus) cargilei, Coltro 2004*	◇
Conus (Dauciconus) pseudocardinalis, Coltro 2004*	🟥
Conus (Dauciconus) ziczac archetypus, Crosse 1865#	🟥
Conus (Dauciconus) fonsecai, Petuch & Berschauer 2016*#	🟥
Conus (Dauciconus) smoesi, Petuch & Berschauer 2016*#	🟥
Conus (Dauciconus) mariaodeteae, Petuch & Berschauer 2014*#	🟥
Conus (Dauciconus) tonisii, Petuch & Berschauer 2014*#	🟦
Conus (Lamniconus) petestimpsoni, Petuch & Berschauer 2016*	🟥 🟦 🟨
Conus (Lamniconus) clerii, Reeve 1844	🟦
Conus (Lamniconus) lemniscatus, Reeve 1849	🟦 🟨
Conus (Lamniconus) carcellesi, Martins 1945	🟥 🟦
Conus (Sandericonus) carioca, Petuch 1986*	🟥 🟦 ◯ □ △
Conus (Stephanoconus) regius, Gmelin 1791
Conasprella Thiele, 1929
Conasprella (Coltroconus) iansa, Petuch 1979*	🟥
Conasprella (Coltroconus) bodarti, Coltro 2004*	🟥
Conasprella (Coltroconus) delucai, Coltro 2004*	🟥
Conasprella (Coltroconus) schirrmeisteri, Coltro 2004*	🟥
Conasprella (Dalliconus) mazei, Deshayes 1874	🟩 🟥 🟦 🟨
Conasprella (Kohniconus) centurio, Born 1778	🟥 🟦
Conasprella (Ximeniconus) jaspidea, Gmelin 1791	🟩 🟥 🟦
Conasprella (Ximeniconus) pusio, Hwass in Bruguière 1792	🟥 🟦
Conasprella (Ximeniconus) mindana, Hwass in Bruguière 1792	🟥 🟦
Conasprella (Ximeniconus) henckesi, Coltro 2004*	🟥

• Biogeographical distribution colored according to the division depicted in Figure 2. Accepted names according to the World Register of Marine Species (WoRMS).
• *Endemic species; ^#also accepted as members of the genus Poremskiconus, Petuch 2013, now considered a synonym of the subgenus Dauciconus, Cotton 1945.

Probably because the vast majority of these animals are often restricted to small geographic areas, the venoms of cone snail species found off the Brazilian coast remain mostly unexplored [39]. As a result, only four of the species found in Brazil - C. cancellatus, C. regius, C. villepinii, and C. ermineus - have had their venoms partially characterized, and, although many conopeptides have been identified in these venoms, only a few have been isolated and characterized. This review focuses on the compilation of all knowledge regarding the biochemical and pharmacological properties described for the conopeptides and other toxic components identified in the venoms of the aforementioned species thus far. Searches were performed on PubMed, ConoServer, and WoRMS databases, using either the names of the species - for PubMed and WoRMS - or the names of the toxins - for ConoServer. The last searches were conducted on August 13, 2022.

Conus cancellatus

A subspecies from the vermivorous C. cancellatus - known as C. cancellatus cancellatus or C. austini - found in the Caribbean Sea, Colombia, Gulf of Mexico, Venezuela, and off the Brazilian coast, had six peptides identified in its venom so far (Table 4). Of these, four are conotoxins that were isolated by reverse-phase high performance liquid chromatography (RP-HPLC) and had their primary sequences and molecular mass determined by Edman degradation and matrix-assisted laser desorption/ionization - time of flight (MALDI-ToF), respectively.

Table 4.
Conopeptides identified in the venom of Conus cancellatus.

Name (protein card)	MM	Sequence	G. Sf	Cys F	References
γ-AsVIIA (P0147)*	3282.61	TCKQKGEGCSLDV(Gla)CCSSSCKPGGPLFDFDC	O2	VI/VII	[41]
κ-like-AsXIVA (P02828)*	2886.35	GGVGRCIYNCMNSGGGLNFIQCKTMCY	ND	XIV	[42]
κ-like-AsXIVB (P02830)*	3154.51	WDVDQCIYYCLNGVVGYSYTECQTMCT	ND	XIV	[41]
As25a/b (P05524)*	2678.05	CKC(O)SCNFNDVTENCKCCIFRQ(O)(nh2)	ND	XXV	[43]
As1a/b (P08700)#	1632.08	RIKKPIFIAFPRF(nh2)	Conorfamide	NA	[44]
As2a/b (P08701)#	1660.10	RIRKPIFIAFPRF(nh2)	Conorfamide	NA	[44]

• *Conotoxins and ^#disulfide-poor conopeptides. MM: molecular mass; G. Sf: gene superfamily, Cys F: cysteine framework according to ConoServer [26]. (Gla): gamma carboxylic glutamic acid; (O): 4-hydroxyproline; (nh2): C-terminal amidation; ND: not determined; NA: not applicable. All sequences were determined at protein level.

The first conotoxin isolated from the venom of C. cancellatus is a 31-residue peptide named AsVIIA [41]. It was classified as a γ-conotoxin from the O2 superfamily with a VI/VII cysteine framework. This somewhat rare pharmacological family of conotoxins, which have a conserved -(Gla)CCS- motif in their primary structures, modulates neuronal pacemaker cation currents in mollusks [45]. Toxins belonging to this family have been identified in three molluscivorous [46-48] and one vermivorous species [49] apart from C. cancellatus. Similar to other γ-conotoxins such as TxVIIA from Conus textile [46] and γ-PnVIIA, from C. pennaceus [49], AsVIIA induced foot shrinking in the freshwater snail Pomacea paludosa after intramuscular (i.m.) injection [41]. This biological activity towards mollusks and the structural similarities between AsVIIA and other γ-conotoxins suggest that they share the same unidentified pacemaker channels as possible targets [41].

Another two conotoxins were isolated from the C. cancellatus venom by the same research group - AsXIVA and AsXIVB. These 27-residue peptides share about 40% identity and belong to cysteine framework XIV, although their gene superfamily and pharmacological family remain unknown [42]. AsXIVA exhibited sequence similarity with VilXIVA, a conotoxin from C. villepinii whose 3D structure resembles that of K⁺ channel blockers [50]. In addition, AsXIVA may have two Lys/Tyr dyads, a motif identified in K⁺ channel blockers [51, 52], suggesting that this conotoxin might also target these channels. On the other hand, AsXIVB exhibited sequence similarity with FlfXIVB, a conotoxin from the vermivorous Conus floridanus floridensis (Conus anabathrum) [50], which suggests that both have the same - yet undetermined - pharmacological target. Both AsXIVA and AsXIVB increased scratching and grooming activities in mice after intracranial (i.c.) injection, although the latter provoked a more noticeable and durable effect, inducing also body and rear limbs extension and tail curling [42].

A 23-amino acid conotoxin named As25a and its post-translationally modified variant As25b, in which Pro4 and Pro23 are hydroxylated, have been also isolated from the venom of C. cancellatus [43]. At first, this conotoxin and its modified counterpart had been named As24a and As24b, but they were renamed because of a nomenclature switch of their cysteine framework from XXIV to XXV. Although As25a has not yet been classified into any conotoxin superfamily, it has some sequence similarity with conotoxins from the M-superfamily, which can target Na⁺ channels, K⁺ channels, pacemaker channels, and nAChRs [53]. As25a (i.c.) induced hind limb paralysis and death in mice [43].

In addition to the aforementioned conotoxins, two conopeptides from the conorfamide family were isolated from the C. cancellatus venom: conorfamides As1a and As2a and their non-amidated counterparts, As1b and As2b. They all had their molecular masses determined by MALDI-ToF and were sequenced by mass spectrometry (MS)-based de novo sequencing, followed by confirmation through Edman degradation. These nearly identical 13-residue peptides were isolated through an activity-guided fractionation assay assessing α7 nAChR activity and their sequences suggested an association with the conorfamide family, whose targets include also acid-sensing ion channels (ASIC) [44, 45]. All four peptides inhibited α7- and muscle-type nAChRs, but the amidated isoforms were more potent than the non-amidated ones. In addition, As1a and As2a were more potent against α7 when compared with muscle-type nAChRs, and their effects on the former were slowly reversible while those on the latter were quickly reversible [44]. As1a and As2a inhibited the desensitization of rat ASIC1a and, to a lesser degree, ASIC3 channels, which resulted in a sustained current at low pH. The non-amidated variants As1b and As2b were mostly ineffective, except for a discrete inhibition of ASIC1a by As1b [44]. These results add to the evidence pointing to a crucial role of PTMs in the activity of conopeptides.

Conus regius

C. regius is a vermivorous species found in the Western Atlantic (north Florida), Gulf of Mexico, and off the Brazilian coast. Thirty-three conotoxins have been identified in the venom of this species to date (Table 5).

Table 5.
Conotoxins identified in the venom of Conus regius.

Name (protein card)	MM	Sequence	G. Sf	Cys F	References
Rg9.1 (P01509)Δ	4021.66	FCGQACSSVKCPKKCFCHPEEKVCYREMRTKERD	P	IX	[54]
RgXIA (P01508)ρ	4695.30	CQAYGESCCSAVVRCCDPNAVCCQYPEDAVCVTRGYCRPPATVLT	I1	XI	[55]
α-RgIA (P02585)Δ	1570.79	GCCSDPRCRYRCR	A	I	[56, 57]
α-Reg1b/c (P00028)ρ	1347.51	GCCSD(O)RCKHQC(nh2)	A	I	[57]
α-Reg1d (P00029)ρ	1332.50	GCCSDPRCKHEC(nh2)	A	I	[57]
α-Reg1f (P00031)ρ	1569.84	DYCCRR(O)(O)CTLIC(nh2)	A	I	[57]
α-RegIIA (P00032)ρ	1663.88	GCCSHPACNVNNPHIC(nh2)	A	I	[57]
α-RgIB (P05941)ρ	2700.98	TWEECCKNPGCRNNHVDRCRGQV	ND	I	[58]
Reg3a (P08570)ρ	1814.97	GCC(O)(O)QWCG(O)DCTS(O)CC	M	III	[57, 59]
Reg3b (P07504)ρ	1669.99	CCTALCSRYHCLPCC	M	III	[59]
Reg3c (P08572)ρ	1787.09	CCAF(O)QWCGAGCIV(O)CC	M	III	[59]
Reg3d (P08573)ρ	1767.95	LCC(O)(O)Q(O)CG(O)DCAS(O)CC	M	III	[59]
Reg3e (P08533)ρ	1738.17	KCCMRPICTC(O)CCIGP	M	III	[57, 59]
Reg3f (P08536)ρ	1821.20	GCCPFPACTTHIICRCC(nh2)	M	III	[57, 59]
Reg3g (P08560)ρ	1699.09	CCMALCSRYHCLPCC(nh2)	M	III	[57, 59]
Reg3h (P08538)ρ	1209.42	GCCS(O)WNCIQLRAC(O)CC(O)N(nh2)	M	III	[57, 59]
Reg3i (P0DPJ6)ρ	1831.19	CCAIRLCNVYLCGSCC(O)	M	III	[59]
Reg3j (P08561)ρ	1827.12	GCCS(O)WNCIQLRACGCC	M	III	[59]
Reg3k (P08534)ρ	1767.27	KCCMRPICMC(O)CCIGP(nh2)	M	III	[59]
Reg3l (P08541)ρ	1982.42	RCCPMPGCFAGPFCPCCPV	M	III	[57, 59]
Reg3m (P08543)ρ	ND	IVRCCSATCK(X)SCVCCF	M	III	[57, 59]
Reg3.5 (P08544)Δ	1399.71	CCMRPVCTCPCCS	M	III	[59]
Reg3.6 (P08546)Δ	1899.31	GCCPYPKCIHVTFCKCC	M	III	[59]
Reg3.7 (P08548)Δ	1776.06	CCPYPSCIDIPFCDCC	M	III	[59]
Reg3.8 (P08550)Δ	1825.20	CCPFPMCYQVPHCPCC	M	III	[59]
Reg3.9 (P08552)Δ	1737.08	CCTIWNCVQLPGCPCC	M	III	[59]
Reg3.10 (P08554)Δ	2132.55	CCHPNLCIGRSALGRKCTCC	M	III	[59]
Reg3.11(P08556)Δ	1592.77	CCRIEFCDEDCGCC	M	III	[59]
Reg3.12 (P08558)Δ	1490.63	CCEVGWCDSGCECC	M	III	[59]
Reg3.14 (P08562)Δ	1828.15	CCNWPRCNVYLCGPCC	M	III	[59]
Reg3.15 (P08564)Δ	1507.84	CCPIQGCILGCTPCC	M	III	[59]
Reg3.16 (P08566)Δ	1703.95	CCYEEECPPSCKLCC	M	III	[59]
Reg3.17 (P08568)Δ	1450.70	CCTGQCHICWPCC	M	III	[59]

• ρ: sequences determined at protein level; Δ: sequences deduced from precursor. MM: molecular mass; G. Sf: gene superfamily; Cys F: cysteine framework according to ConoServer [26]; O: 4-hydroxyproline; nh2: C-terminal amidation; X: unidentified amino acid; ND: not determined.

The first conotoxin identified in the venom of C. regius is a 34-residue peptide named Rg9.1, whose sequence was deduced from its precursor cDNA [54]. Although its pharmacological target remains unknown, Rg9.1 was classified as a member of the P superfamily, cysteine framework IX, which harbors TxIXA, a conotoxin from the molluscivorous C. textile that causes spasms in mice [60].

Soon after, the same research group identified a 44-residue peptide that belongs the I1 superfamily, cysteine framework XI, named RgXIA [55]. This conotoxin was isolated through RP-HPLC and had its molecular mass and sequence determined through MALDI-ToF and de novo sequencing, respectively. Although its biological activity is still under investigation, RgXIA is homologous to BtX [61] and ViTx [62], κ-conotoxins from Conus betulinus and Conus virgo, respectively, that modulate vertebrate K⁺ channels.

The best-studied conotoxins identified in the venom of C. regius are the α-conotoxins RgIA, RegIIA, and RgIB, which target nAChRs. As potent modulators of different neuronal and muscular isoforms of these receptors [63], α-conotoxins are useful tools in the investigation of chronic pain and inflammation, having been extensively employed in these fronts.

RgIA is an α-conotoxin from the A superfamily, cysteine framework I, and loop class 4/3. The sequence of the mature 13-residue peptide was first deduced from the nucleotide sequence [56], and later confirmed by Edman degradation when RgIA was isolated by RP-HPLC along four other similar α4/3 conotoxins - Reg1b/c, Reg1d, Reg1e, and Reg1f - in a study focused on the post-translational hydroxylation of conopeptides [57]. It is now accepted that RgIA and Reg1e are most likely the same peptide, as the only difference in their mature sequences is the absence of an arginine residue at the C-terminus in the latter. Analysis by nuclear magnetic resonance (NMR) of the 3D structure of RgIA (Figure 3A) and a few synthesized analogues revealed that this conotoxin assumes a globular (fold A), two-loop backbone architecture, with the residues Asp5, Pro6, and Arg7 in the loop 1 being important for the interaction between the toxin and α9α10 nAChRs, although selectivity to this receptor is actually determined by the Arg9 in the loop 2 [64, 65].

Figure 3.
Three-dimensional structures of conotoxins. (A) Three-dimensional structure of RgIA, an α-conotoxin from the Conus regius venom, classified into A superfamily, cysteine framework I, and fold A. The residues involved in the interaction with α9α10 nAChRs - D5, P6, and R7, and the residue that determines selectivity - R9, are highlighted. (B) Three-dimensional structure of the cis-isomer of EVIA, a δ-conotoxin from Conus ermineus venom, classified into O1 superfamily, cysteine framework VI/VII, and fold C. The L12-P13 residues, which are connected by a peptide bond that shows a 1:1 cis/trans isomerism, are highlighted. Both structures are represented as stick models with surface models on the background. The images were produced using PyMOL [66], from the models 2JUS for RgIA [64] and 1GIZ for EVIA [67] deposited on the Protein Data Bank (PDB - https://www.rcsb.org/).

RgIA showed a dose-dependent antinociceptive action in rats, as it increased the paw withdrawal threshold that had been reduced by chronic constriction injury of the sciatic nerve [68]. Moreover, it affected the peripheral immune response to nerve injury by reducing the number of choline acetyltransferase-immunoreactive cells, ED1-immunoreactive macrophages, and CD2-immunoreactive T cells at the injury site [68]. This same neuropathic injury model was used in another study that investigated the analgesic effect of RgIA [69]. It was observed that the repeated administration (i.m.) of this conotoxin into the ipsilateral paw significantly prevented the development of pain hypersensitivity in injured rats, increasing their pain threshold, relieving the postural imbalance caused by the injury in up to 80%, and protecting the ipsilateral sciatic nerve against morphological derangements [69]. In addition, the treatment reduced the edema and inflammatory infiltrate - particularly CD86+ cells - observed in the constricted nerve, prevented the reduction in the somatic area of L4-L5 dorsal root ganglia (DRG) neurons induced by the constriction injury to the sciatic nerve, and inhibited the activation of astrocytes and microglia in the dorsal horn of the spinal cord [69]. RgIA was also effective against chemotherapy-induced neuropathy, as the concomitant intraperitoneal (i.p.) administration of this toxin induced both analgesic and neuroprotective effects in rats treated with oxaliplatin (i.m.) - a drug often employed in the treatment of colorectal cancer - for three weeks [70]. RgIA reduced mechanical hypersensitivity and the sensitivity to cold noxious stimuli in these animals, in addition to partially preventing the morphological changes induced by this drug in L4-L5 DRG neurons [70]. It has been recently shown that RgIA reduces the damage caused by dextran sodium sulfate-induced colitis in mice: the subcutaneous (s.c.) injection of this conotoxin reversed the severity of the disease in various aspects, including a reduction in the colonic levels of TNF-α, which suggests that α9α10 nAChRs may be involved in pro-inflammatory mechanisms that take place in colitis [71].

Besides modulating nicotinic receptors, RgIA has other targets: it inhibited high-voltage-activated (HVA) Ca²⁺ currents in rat DRG neurons via activation of γ-aminobutyric acid (GABA)B G-protein coupled receptors and Ca_v2.2 (N-type) channels expressed in baclofen-sensitive Xenopus oocytes [72]. The participation of GABAB receptors in the modulation of HVA Ca²⁺ channels by RgIA was further confirmed when the activity of this conotoxin was significantly reduced in GABAB-knockdown DRG neurons and also when RgIA failed to inhibit Ca_v2.2 currents in HEK cells in the absence of the two subunits of GABAB [73].

The α-conotoxin RegIIA was isolated along RgIA in the same RP-HPLC process [57, 74]. Although it also belongs to the A superfamily and cysteine framework I, this 16-residue peptide is classified into loop class 4/7 instead. RegIIA is nearly identical to OmIA, an α-conotoxin from the molluscivorous Conus omaria, the only difference being that the latter has an extra glycine residue at the C-terminal position [74]. It is also highly homologous to GIC [75] and GID [76], two α4/7 conotoxins isolated from the venom of the fish-hunting C. geographus [57]. Unlike its counterparts, RegIIA was found to be primarily a potent antagonist of α3β4 nAChRs, and, to a lesser degree, of α3β2 and α7 nAChRs, which are the usual targets of α4/7 conotoxins [74]. The 3D structure of RegIIA obtained by NMR revealed that, like RgIA, it belongs to the classical fold A category, with a distribution of charged, polar, and hydrophobic residues in its surface that could account for its unique selectivity [74]. The effect of RegIIA on both α3β4 and α7 receptors was completely abolished when the asparagine residue at position 9 in the loop II was replaced by alanine [77]. More importantly, a potent α3β4 nAChRs-selective antagonist ([N11A,N12A]RegIIA) was synthesized by replacing the asparagine residues at positions 11 and 12 by alanine, shedding some light into the differences between the interaction of RegIIA with its various targets [77]. There is also a difference regarding the interaction between RegIIA and nAChR from different species, but this species-specificity does not encompass all the subtypes targeted by this toxin [78]. For instance, RegIIA and the analogue [N11A,N12A]RegIIA blocked human and rat α3β4 nAChRs equally. On the other hand, the native toxin completely blocked the current evoked by the rat α3β2 subtype while only reducing that from the human α3β2 subtype [78]. Surprisingly, this difference in selectivity was associated to the amino acid residue in the position 198 of the α3 subunit: a glutamine in the rat subtype and a proline and the human subtype [78]. As the α3 subunit is shared between the α3β4 and α3β2 subtypes, it stands to reason that the differences regarding the species-specificity shown by RegIIA towards these subtypes is strongly influenced by the type of β subunit interacting with α3.

RgIB, yet another nAChR-blocker, completes the list of α-conotoxins already described in the venom of C. regius thus far [58]. It was identified by MALDI-ToF following the RP-HPLC fractionation of the crude venom and sequenced through electrospray-ionization quadrupole time-of-flight (ESI-Q-ToF) mass spectrometry. This 23-residue toxin is the largest conotoxin identified in this venom, having been classified as a member of the cysteine framework I group, although its loop class and gene superfamily remain unknown. At high doses, the injection (i.c.) of RgIB in mice induced hyperactivity, while lower doses led to respiratory difficulties. In addition, α-RgIB partially blocked - in an irreversible manner - the slow-desensitizing ionic currents from differentiated PC12 neurons, which express α3β4 and/or α3β4α5 nAChRs [58].

Last but not least, a large group of mini-M conotoxins - a subclass of M-conotoxins that have either one (M1), two (M2) or three (M3) residues in the third loop - was identified in the venom of C. regius [57, 59]. Thirteen of them - Reg3a-m - were isolated through RP-HPLC and sequenced by Edman degradation [57, 59]. Although they all belong to cysteine framework III, there is very little homology between their primary sequences, which fall into eight different loop classes: 3/3/1, 3/4/2, 4/1/1, 4/2/3, 4/3/3, 4/4/2, 4/5/1, and 5/3/3. In addition, these toxins present various degrees of PTMs, which, combined to the differences in their sequences, suggests they modulate different, yet undetermined targets. The structure of Reg3b, solved by NMR, revealed that the toxin assumes a compact, globular fold that comprises a series of turns [59]. The same research group identified 12 other mini-M conotoxins in the transcriptome of the C. regius venom, named Reg3.5-12, and 3.14-17 [59]. Their sequences were proved as diverse as those of their isolated counterparts, adding the loop classes 3/2/2, 4/3/1, 4/3/2, and 4/9/1 to those already described for the hypervariable C. regius mini-M conotoxins.

Conus villepinii

This vermivorous species is found along the Brazilian coast, Florida, West Indies, and Uruguay. It had 12 toxins identified in its venom to date (Table 6).

Table 6.
Conopeptides identified in the venom of Conus villepinii.

Name (Protein card)	MM	Sequence	G. Sf	Cys F	References
VilXIVA (P01619)*ρ	2873.35	GGLGRCIYNCMNSGGGLSFIQCKTMCY	R	XIV	[50]
VilXIVB (P08492)*Δ	3160.53	WDVDQCMYYCLTGVVGYSYTECETMCT	R	XIV	[79]
VilXIVC (P08495) *Δ	3159.54	WDVDQCMYYCLTGVVGYSYTECQTMCT	R	XIV	[79]
Vil14.8*Δ§	ND	GGLGRCIYNCMNSGGGLSFIQCKTMCY	R	XIV	[79]
Vil14.9*Δ§	ND	GGVEQCIYNCLTGYIGRSYIQCKTMCT	R	XIV	[79]
Vil14.10*Δ§	ND	GGVEQCIYNCLTGYIGGSYIQCKTMCT	R	XIV	[79]
Vil14.11*Δ§	ND	WDVDQCMYYCLTGVLEYSYTECETMCT	R	XIV	[79]
Vil14.12*Δ§	ND	WDVDQCIYYCLTGVVGYSYTECETMCT	R	XIV	[79]
γ-conopressin-vil (P01307)#ρ	1018.12	CLIQDCP(Gla)G(nh2)	Conopressins	NA	[80]
conoCAP-Vila (P07533)#ρ	1148.27	PFCNSFGCYN(nh2)	conoCAPs	NA	[81]
conoCAP-Vilb (P07532)#Δ	1001.14	VFCNGFTGCG(nh2)	conoCAPs	NA	[81]
conoCAP-Vilc (P07534)# Δ	1143.30	LFCNGYGGCRG(nh2)	conoCAPs	NA	[81]

• *Conotoxins and ^#disulfide-poor conopeptides. ρ: sequences determined at protein level; Δ: sequences deduced from precursor. MM: molecular mass; G. Sf: gene superfamily; Cys F: cysteine framework according to ConoServer [26]. §: no ConoServer protein card, name and sequence obtained from the publication itself [77]. (Gla): gamma carboxylic glutamic acid; (nh2): C-terminal amidation; ND: not determined; NA: not applicable.

The first conotoxin identified in the venom of C. villepinii is a 27-residue peptide named VilXIVA, isolated by size exclusion (SE) followed by RP-HPLC and sequenced by Edman degradation [50]. Its four cysteine residues are arranged in a previously unreported framework, then classified as framework XIV. This same framework was identified in FlfXIVA and FlfXIVB, conotoxins from the venom of C. anabathrum whose cDNA precursor revealed a signal sequence that defined a new gene superfamily, then named R superfamily [79]. Through this conserved signal sequence, seven additional R/XIV conotoxins were cloned from the cDNA of C. villepinii venom: VilXIVB, VilXIVC, and Vil14.8-12 [79]. In addition to belonging to the same superfamily and cysteine framework, the aforementioned C. villepinii conotoxins share the same loop class, 3/11/3. Three of them - vil14.8-10 - share high sequence identity with VilXIVA, while the other ones display high similarity with the R-conotoxins from C. anabathrum venom. A molecular model of VilXIVA based on NMR data revealed that this conotoxin has a well-defined three-dimensional structure in solution, which resembles that of K⁺ channel blockers isolated from scorpion venoms [50]. Nevertheless, framework XIV toxins belonging to other gene superfamilies can have different targets; for instance, LtXIVA - an αL-conotoxin from the vermivorous Conus literatus - inhibited neuronal nAChRs [82]. The actual target of the C. villepinii R-toxins remains to be determined.

In addition to the aforementioned conotoxins, single-disulfide conopeptides were identified in the C. villepinii venom. For instance, a 9-residue vasopressin/oxytocin-like peptide, named γ-conopressin-vil, was isolated from this venom by SE- and RP-HPLC and sequenced through Edman degradation [80]. The eighth residue in this conopressin is a gamma-carboxyglutamate, a unique feature that distinguishes γ-conopressin-vil from other conopressins described so far [12, 83, 84]. NMR spectroscopy data revealed that γ-conopressin-vil goes through Ca²⁺-mediated conformational changes as a result of the gamma-carboxyglutamate in its structure. The net charge of this residue (-2) could affect the electrostatic surface of γ-conopressin-vil, and, consequently, the way it binds to its yet undetermined target [80].

Another three disulfide-poor conopeptides were identified in the venom of C. villepinii, all crustacean cardioactive peptide (CCAP)-like toxins named conoCAP-Vila, conoCAP-Vilb, and conoCAP-Vilc [81]. The 10-residue conoCAP-Vila was isolated through SE- and RP-HPLC and sequenced by Edman degradation, while its counterparts were identified through cloning of the multi-peptide conoCAP precursor. ConoCAP-Vila is a hydrophobic peptide with three aromatic residues that showed high sequence identity with other known CAPs. In addition to decreasing the heart rate and causing arrhythmia in Drosophila melanogaster larvae, ConoCAP-Vila decreased the mean arterial blood pressure and heart rate of rats [79]. It also promoted a time-dependent and irreversible decrease in the amplitude of systolic Ca²⁺ transients and in the contractile activity of rat cardiac myocytes, although it had no effect on L-type Ca²⁺ currents [81].

Conus ermineus

Conus ermineus is the only piscivorous species described off the Brazilian coast to date, being also found off the Caribbean, the northern coast of South America, and off the African coast. The venom from this species has been fairly well-explored, and a large number of molecules were identified, mostly through transcriptomic analysis. Amongst all the conopeptides reported to be present in the venom of this species, 24 have had their sequences published and are listed in Table 7.

Table 7.
Conopeptides identified in the venom of Conus ermineus.

Name (protein card)	MM	Sequence	G. Sf	Cys F.	References
α-EI (P00050)*ρ	2093.36	RD(O)CCYHPTCNMSNPQIC(nh2)	A	I	[85]
α-EIIA (P04069)*ρ	1774.03	(Z)T(O)GCCWNPACVKNRC(nh2)	A	I	[86]
α-EIIB (P07538)*ρ	1754.99	(Z)T(O)GCCWHPACGKNRC(nh2)	A	I	[87]
E1.1(P03001)*Δ	1787.96	DPCCSNPACNVNNPQIC	A	I	[88]
E1.2(P08455)*Δ	1757.00	QTPGCCWHPACGKNRC	A	I	[88]
E1.3(P08471)*Δ	1999.14	DDCCPDPSCRQNHPELCA	A	I	[88]
αA-EIVA (P01635)*ρ	3096.42	GCCGPY(O)NAACH(O)CGCKVGR(O)(O)YCDR(O)SGG(nh2)	A	IV	[89]
αA-EIVB (P01740)*ρ	3100.40	GCCGKY(O)NAACH(O)CGCTVGR(O)(O)YCDR(O)SGG(nh2)	A	IV	[89]
E4.1(P08469)*Δ	4055.75	DCCGVKLDMCHPCLCNNSCKQGQGKKRVWEMMKATD	A	IV	[88]
δ-EVIA (P01561)*ρ	3287.85	DDCIK(O)YGFCSLPILKNGLCCSGACVGVCADL(nh2)	O1	VI/VII	[90]
δ-EVIB (P01575)*Δ	2972.48	EACY(O)(O)GTFCGIK(O)GLCCSELCLPAVCVG(nh2)	O1	VI/VII	[91]
E6.1(P03273)*Δ	3618.22	ATSNRPCKPKGRKCFPHQKDCCNKTCTRSKCP	O1	VI/VII	[88]
E6.2 (P03274)*Δ	2777.14	QCTPHGGSCGLVSTCCGRCSVPRNKCE	O1	VI/VII	[88]
E5.1(P08457)*Δ	1423.70	DCCPEKMWCCPL	T	V	[88]
E5.2(P08461)*Δ	2756.09	TEHFPLMIWVDCCPAYDCCVPDSD	T	V	[88]
E5.3(P08463)*Δ	1416.61	GPCCFSNPYCCNL	T	V	[88]
E20.1 (P08483)*Δ	5070.71	AVIATCHPNYPGSPWGRCCTTKMCGSVCCNYAHCSCVYHSDMGDGCSC	D	XX	[88]
E22.1 (P08485)*Δ	8255.59	WPRLTDSDCELGRNMHITCKQLDQCGVIEKKDGQLTCKLRCKCKPGKRCLRKENIDWSDITTRIYHCPWP	E	XXII	[88]
Conantokin-E1 (P03534)#Δ	3139.19	GE(Gla)(Gla)HSKYQ(Gla)CLR(Gla)IRVNNVQQ(Gla)C	Conantokin	NA	[92]
Conantokin-E2 (P08574)#Δ	2034.16	SSEEDIELIEALEESGKR	Conantokin	NA	[86]
Conkunitzin-E3 (P08477)#Δ	8944.91	DTVPGLSALTVDDDTVPDVCRQPLEVGPCKAAYPRYYYNHASDTCQLFYYGGCNGNENRFEDFSGCLFTCIYPWMAALGY	Conkunitzin	NA	[88]
Conkunitzin-E4 (P08479)#Δ	4318.87	CHLPPETGMCRAYIPMHFYNATLGRCQGFIYGGCNGNDN	Conkunitzin	NA	[88]
Conkunitzin-E5 (P08481)#Δ	9869.11	CCHPPCEYGERCVSANRRERRHRHNNVCVPVRCLFQARRGRCLNFDRRYHFNTLTMSCTRVHTGACYGRNNRFSSSENCELTCAP	Conkunitzin	NA	[88]
Con-ikot-ikot-E1 (P08474)#Δ	8043.11	DDCCIGNTYGCLKRRPGQEHEQVMPCKHEATIRCPGSDIDGCCPGYATCMSIFAKDNLIPAHYHCEKRPCYT	Con-ikot-ikot	NA	[88]

• *Conotoxins and ^#disulfide-poor conopeptides. MM: molecular mass; G. Sf: gene superfamily; Cys F: cysteine framework according to ConoServer [26]. (Gla): gamma carboxylic glutamic acid; (Z): pyroglutamic acid; (O): 4-hydroxyproline; (nh2): C-terminal amidation; ND: not determined; NA: not applicable; ρ: sequences determined at protein level; Δ: sequences deduced from precursor.

A few conotoxins from the A superfamily were isolated from the venom of C. ermineus. The first one to be described was EI, an 18-aminoacid α4/7-conotoxin with four cysteine residues arranged into framework I, isolated from the milked venom of this species through RP-HPLC [83]. As expected, the injection (i.m.) of nanomolar concentrations of EI into fish led to paralysis; in mice, it caused muscle weakness, which eventually evolved to paralysis and death [83]. Binding assays revealed that this α-conotoxin selectively binds the α/δ site of Torpedo nAChRs, and, although this is also its preferred site in mammalian receptors, EI can also bind the α/γ site in the latter [85]. It has been recently shown that: (i) the point mutations of residues His7, Pro8, Met12, and Pro15 into alanine significantly reduced the effect of EI on α1β1δε nAChRs; (ii) the replacement of a critical serine residue at position 13 by alanine increased the potency of the toxin against this muscle-type nAChR while reducing its effect on the neuronal α3β2 and α3β4 subtypes; and (iii) the potency against α1β1δε nAChRs was related to the Arg1-Asn2-Hyp3 residues at the N-terminus of the toxin, as the deletion of these residues in the analogue ^△1-3EI caused total loss of effect on this subtype [93].

Another α-conotoxin from the A superfamily, named EIIA, was identified in the venom of C. ermineus by an MS-based ‘fishing’ technique, using common features of α-conotoxins as hooks [84]. This 16-residue peptide, which belongs to cysteine framework I and loop class 4/4, is highly homologous to PIB, an α-conotoxin from the fish-hunting, Eastern Pacific species Conus purpurascens that selectively blocks muscle-type α1β1δε nAChRs [94].

Similarly, the synthetic EIIA was found to be highly selective for the muscle-type nAChR present in Torpedo membranes, distinguishing between its two acetylcholine binding sites [86]. A nearly identical isoform of EIIA, named EIIB, was identified in this venom through affinity-selection mass spectrometry, and they differ from each other only in the residues H8N and G12V [87]. As expected, radioligand binding assays revealed that a synthetic EIIB homologue bound with high affinity to Torpedo nAChRs [87].

Two αA-conotoxins named EIVA and EIVB, which also belong to the A superfamily, were isolated from the milked venom of C. ermineus by RP-HPLC [89]. These nearly identical 30-residue peptides were classified into cysteine framework IV, loop class 7/2/1/7, and fold I [11], being homologous to the αA-conotoxin PIVA from C. purpurascens, an antagonist of both α/δ and α/g sites in muscle-type nAChRs from mice [95]. The functional characterization of the synthetic EIVA through electrophysiology and binding assays in Torpedo and mouse nAChRs revealed that it also interacts with both α/δ and α/γ sites, though with higher affinity than PIVA [89, 96]. The higher potency of EIVA can be the result of structural differences between this toxin and its C. purpurascens counterpart: not only EIVA has four additional residues in its C-terminus but also a more hydrophobic, protruding region in which the residue at position 18 is a valine instead of the aspartic acid present in PIVA, as revealed by the 3D structures of both toxins solved by NMR [96].

Members from the O1 superfamily have also been found in the venom of C. ermineus. A δ-conotoxin named EVIA was isolated from this venom through RP-HPLC in multiple steps and sequenced by Edman degradation [90]. This 32-residue peptide, classified into cysteine framework VI/VII, loop class 6/9/3/3, differs considerably from other δ-conotoxins described in cone snail venoms [67], unlike δ-EVIB, a 29-peptide previously identified in the venom of C. ermineus through cDNA cloning [91]. The 3D structure of δ-EVIA solved by NMR (Figure 3B) showed that it assembles into the stable inhibitor cysteine-knot (ICK) motif [95], found not only in other fold C conotoxins from the O1 superfamily but also in toxic peptides from other kingdoms [97]. However, because of its unusually long and disordered 2^nd loop, δ-EVIA exhibits a 1:1 cis/trans isomerism in the Leu12-Pro13 peptide bond, which most likely affects the way the toxin interacts with its binding site [67]. δ-EVIA is unique because, unlike most δ-conotoxins already described, it is a selective modulator of neuronal Na⁺ channels in vertebrates, a feature shared only by δ-CnIVD from Conus consors [98]. It increased the excitability of frog neuromuscular preparations by increasing the duration of nerve action potentials without affecting those directly elicited in the muscle tissue [90]. Furthermore, it delayed the decay of Na⁺ currents recorded from frog myelinated axons and spinal neurons [90]. The selectivity of δ-EVIA for neuronal Na⁺ channels was confirmed by the observation that only the mammalian neuronal isoforms rNav1.2a, rNav1.3, rNav1.6, and mNav1.7 had their fast inactivation inhibited by the toxin, while that of the skeletal muscle rNav1.4 and the cardiac hNav1.5 isoforms remained unaltered [90, 99]. The interaction between δ-EVIA and the Na⁺ channel was further elucidated when the toxin was found to be active in a chimera formed by the replacement of domains I and/or IV of the muscle Na1.4 by those of the neuronal Nav1.7 [99]. In addition, molecular dynamics and docking data showed that the voltage sensor of domain IV and the 5^th transmembrane segment (S5) of domain I form the binding site to δ-EVIA in the Na⁺ channel [99].

A set of eleven conotoxins were identified through a combination of nanoNMR spectroscopy, liquid chromatography, and mass spectrometry in a study focused on the intraspecies variability of the milked venom from eight specimens of C. ermineus [100]. Although the actual sequences of these peptides, named EIB, EIB[O8], EIC, EIIB, EIIB[O2], EIIB[O2,8], EIIC, EIIIA, EIVA[P5,7,13], EVIIA, and EVIIA[O22] have not been deposited, some of them appear to differ only in the absence or presence of certain PTMs [100]. It is worthy of note that, at least for the specimens of C. ermineus evaluated in the aforementioned study, the composition of the venom varied considerably among different specimens, while remaining fairly constant in individual specimens throughout time [100].

Further insights into this astounding intraspecies variability were obtained through a comparison of the transcriptomes of three specimens of C. ermineus from different islands in Cabo Verde, which revealed that only about 20% of the inferred mature conotoxins were present in the venoms of all three individuals [88]. Moreover, venom composition and expression levels varied significantly along the venom duct: in terms of diversity, the distal region was found to be richer, while the proximal region exhibited higher expression levels, particularly of conopeptides from the A superfamily [88]. Although many known and unassigned superfamilies were represented in the transcriptomes of the venom ducts of these C. ermineus specimens, members from the superfamilies O1, O2, M, and T were present in larger numbers [88]. The sequences of 11 novel conotoxins - four from the A superfamily (E1.1, E1.2, E1.3, and E4.1), two from the O1 superfamily (E6.1 and E6.2), three from the T superfamily (E5.1, E5.2, and E5.3), one from the D superfamily (E20.1), and one from the E superfamily (E22.1) - were deduced based on their precursors [86] (Table 7).

In addition to the aforementioned conotoxins, five disulfide-poor conopeptides were identified in the venom of C. ermineus by the same transcriptomic analysis [88] and had their precursor-derived sequences published in the ConoServer [26]: conantokin-E2, conkunitzin-E3-E5, and con-ikot-ikot-E1 (Table 7). In a previous study, a conantokin named conantokin-E1 was cloned from the genomic DNA of C. ermineus along con-P, an identical conantokin from the venom of the closely related species C. purpurascens [92]. These 24-residue conopeptides present five gamma carboxylic glutamic acids and a long inter-cysteine loop in their structures. As con-P was found to be an antagonist of N-methyl-D-aspartate (NMDA) receptors [92], it is safe to assume that conantokin-E1 has the same target.

Finally, larger proteins were also identified in the venom of C. ermineus. The milked venom of this species, whose major protein component was found to be a hyaluronidase named Hyal-E, exhibited fibrinogenolytic and gelatinolytic activity [17]. In addition, an MS analysis identified an angiotensin-converting enzyme-1 (ACE-1) and an endothelin-converting enzyme-1 (ECE-1) in the milked venom of this species [101]. Although their roles in the envenomation need further clarification, the fact that these enzymes are present in the injected venom point to them being relevant in some way.

Conclusion

In spite of the many different species of venomous animals, found in almost every ecosystem of our planet - from snails and fish to insects and arthropods, not to mention reptiles - they all have in common the fact that the toxins contained in their venoms have immeasurable biotechnological and therapeutic value, by virtue of their pharmacological targets. Cone snail venoms are not an exception to that rule, for they contain a powerful cocktail of bioactive molecules that target mainly ion channels and membrane receptors, which are crucial players of a number of vital physiological processes.

But perhaps the most striking feature of cone snail venoms is their unmatched uniqueness. Hundreds of cone snail species have been described to date, and every single one of them produces a different venom containing hundreds of different conotoxins, among other no less important molecules. This diversity is the result of the remarkable ability these animals have to adapt their venom composition to different circumstances imposed by the environment, as well as of the variation in the mature protein sequences of conotoxins from different species.

In this review, we sought to assemble the information available on cone snail species found in the various biogeographic regions that form the Brazilian coast, focusing on the structural and pharmacological features of the toxins already identified in their venoms. Although only four species, out of the 31 described off the Brazilian coast to date, have had their venoms at least partially explored, a large number of conotoxins and other conopeptides were identified and some of them extensively characterized, attesting to the potential contained in these venoms.

In fact, Brazilian biodiversity has already provided Captopril - a potent angiotensin-converting enzyme inhibitor based on a peptide isolated from the venom of the snake Bothrops jararaca - as a successful drug to the market. More to the point, an ω-conotoxin from the venom of the Pacific cone snail species C. magus is the biological source of the analgesic Prialt^®, as previously discussed here. Thus, the thorough study of cone snail species found in Brazil holds considerable promise, for their largely untapped venoms could be the source of another biodiversity-derived drug.

Acknowledgments

We would like to thank Dr. Renata dos Santos Gomes for generously allowing us to use the photos of the cone snail specimens and radula tooth. We would also like to thank Dr. Juliana Barbosa Coitinho for building the images of the EIVA and RgIA structures.

References

• 1. Editorial Board W. World Register of Marine Species (WoRMS). Available online: http://www.marinespecies.org
  » http://www.marinespecies.org
• 2. Rice RD, Halstead BW. Report of fatal cone shell sting by Conus geographus linnaeus. Toxicon. 1968 Feb;5(3):223-4.
• 3. Shon KJ, Grilley MM, Marsh M, Yoshikami D, Hall AR, Kurz B, Gray WR, Imperial JS, Hillyard DR, Olivera BM. Purification, Characterization, Synthesis, and Cloning of the Lockjaw Peptide from Conus purpurascens Venom. Biochemistry. 1995 Apr 18;34(15):4913-8.
• 4. Neves JLB, Imperial JS, Morgenstern D, Ueberheide B, Gajewiak J, Antunes A, Robinson SD, Espino S, Watkins M, Vasconcelos V, Olivera BM. Characterization of the First Conotoxin from Conus ateralbus, a Vermivorous Cone Snail from the Cabo Verde Archipelago. Mar Drugs. 2019 Jul 24;17(8):432.
• 5. Taylor J, Kantor Y, Sysoev A. Foregut anatomy, feeding mechanisms, relationships and classification of the Conoidea (=Toxoglossa)(Gastropoda). Bull Nat Hist Museum London. 1993 Nov;59(2):125-70.
• 6. Puillandre N, Samadi S, Boisselier M-C, Sysoev AV, Kantor YI, Cruaud C, Couloux A, Bouchet P. Starting to unravel the toxoglossan knot: Molecular phylogeny of the “turrids” (Neogastropoda: Conoidea). Mol Phylogenet Evol. 2008 Jan;47:1122-34.
• 7. Haddad Junior V, Coltro M, Simone LRL. Report of a human accident caused by Conus regius (Gastropoda, Conidae). Rev Soc Bras Med Trop. 2009 Aug;42(4):446-8.
• 8. Salisbury SM, Martin GG, Kier WM, Schulz JR. Venom kinematics during prey capture in Conus: the biomechanics of a rapid injection system. J Exp Biol. 2010 Mar 1;213(5):673-82.
• 9. Dutertre S, Jin A-H, Vetter I, Hamilton B, Sunagar K, Lavergne V, Dutertre V, Fry BG, Antunes A, Venter DJ, Alewood PF, Lewis RJ. Evolution of separate predation- and defence-evoked venoms in carnivorous cone snails. Nat Commun. 2014 Mar 24(3521);5:3521.
• 10. Terlau H, Olivera BM. Conus Venoms: A Rich Source of Novel Ion Channel-Targeted Peptides. Physiol Rev. 2004 Jan 1;84:41-68.
• 11. Akondi KB, Muttenthaler M, Dutertre S, Kaas Q, Craik DJ, Lewis RJ, Alewood PF. Discovery, Synthesis, and Structure-Activity Relationships of Conotoxins. Chem Rev. 2014;114:5815-47.
• 12. Cruz LJ, de Santos V, Zafaralla GC, Ramilo CA, Zeikus R, Gray WR, Olivera BM. Invertebrate vasopressin/oxytocin homologs. Characterization of peptides from Conus geographus and Conus straitus venoms. J Biol Chem. 1987 Nov 25; 262(33):15821-4.
• 13. Safavi-Hemami H, Gajewiak J, Karanth S, Robinson SD, Ueberheide B, Douglass AD, Schelegel A, Imperial JS, Watkins M, Bandyopadhyay PK, Yandell M, Li M, Purcell AW, Norton RS, Ellgaard L, Olivera BM. Specialized insulin is used for chemical warfare by fish-hunting cone snails. Proc Natl Acad Sci. 2015 Feb 10;112(6):1743-8.
• 14. Robinson SD, Li Q, Bandyopadhyay PK, Gajewiak J, Yandell M, Papenfuss AT, Purcell AW, Norton RS, Safavi-Hemami H. Hormone-like peptides in the venoms of marine cone snails. Gen Comp Endocrinol. 2017 Apr 1;244:11-8.
• 15. Pali E, Tangco O, Cruz L. The venom duct of Conus geographus: some biochemical and histologic studies. Bull Philip Biochem Soc. 1979;2:30-51.
• 16. Milne TJ, Abbenante G, Tyndall JDA, Halliday J, Lewis RJ. Isolation and Characterization of a Cone Snail Protease with Homology to CRISP Proteins of the Pathogenesis-related Protein Superfamily. J Biol Chem. 2003 Aug 15;278(33):31105-10.
• 17. Möller C, Vanderweit N, Bubis J, Marí F. Comparative analysis of proteases in the injected and dissected venom of cone snail species. Toxicon. 2013 Apr;65:59-67.
• 18. Violette A, Leonardi A, Piquemal D, Terrat Y, Biass D, Dutertre S, Noguier F, Ducancel F, Stocklin R, Krizaj I, Favreau P. Recruitment of Glycosyl Hydrolase Proteins in a Cone Snail Venomous Arsenal: Further Insights into Biomolecular Features of Conus Venoms. Mar Drugs. 2012;10(2):258-80.
• 19. Mӧller C, Clark E, Safavi-Hemami H, DeCaprio A, Marí F. Isolation and characterization of Conohyal-P1, a hyaluronidase from the injected venom of Conus purpurascens J Proteomics. 2017 Jul 5;164:73-84.
• 20. McIntosh JM, Ghomashchi F, Gelb MH, Dooley DJ, Stoehr SJ, Giordani AB, Naisbitt SR, Olivera BM. Conodipine-M, a Novel Phospholipase A2 Isolated from the Venom of the Marine Snail Conus magus J Biol Chem. 1995 Feb 24;270(8):3518-26.
• 21. Neves JLB, Lin Z, Imperial JS, Antunes A, Vasconcelos V, Olivera BM, Schmidt EW. Small Molecules in the Cone Snail Arsenal. Org Lett. 2015 Oct 16;17(20):4933-5.
• 22. Torres JP, Lin Z, Watkins M, Salcedo PF, Baskin RP, Elhabian S, Savafi-Hemami H, Taylor D, Tun J, Concepcion GP, Saguil N, Yanagihara AA, Fang Y, McArthur JR, Tae HS, Finol-Urdaneta RK, Ozpolat BD, Olivera BM, Schmidt EW. Small-molecule mimicry hunting strategy in the imperial cone snail, Conus imperialis Sci Adv. 2021 Mar 12;7(11):eabf2704.
• 23. Olivera BM. Conus Venom Peptides: Reflections from the Biology of Clades and Species. Ann Rev Ecol Syst. 2002;33:25-47.
• 24. Terlau H, Shon KJ, Grilley M, Stocker M, Stühmer W, Olivera BM. Strategy for rapid immobilization of prey by a fish-hunting marine snail. Nature. 1996 May 9;381(6578):148-51.
• 25. Lebbe EKM, Tytgat J. In the picture: Disulfide-poor conopeptides, a class of pharmacologically interesting compounds. J Venom Anim Toxins Incl Trop Dis. 2016 Nov 7;22:30.
• 26. Kaas Q, Yu R, Jin A-H, Dutertre S, Craik DJ. ConoServer: updated content, knowledge, and discovery tools in the conopeptide database. Nucleic Acids Res. 2012 Jan;40:D325-30.
• 27. Mansbach R, Travers T, McMahon B, Fair J, Gnanakaran S. Snails In Silico: A Review of Computational Studies on the Conopeptides. Mar Drugs. 2019 Mar;17(3):145.
• 28. Walker CS, Jensen S, Ellison M, Matta JA, Lee WY, Imperial JS, et al. A Novel Conus Snail Polypeptide Causes Excitotoxicity by Blocking Desensitization of AMPA Receptors. Curr Biol. 2009 Jun 9;19(11):900-8.
• 29. Buczek O, Bulaj G, Olivera BM. Conotoxins and the posttranslational modification of secreted gene products. Cell Mol Life Sci. 2005 Dec;62(24):3067-79.
• 30. Dutertre S, Jin A, Kaas Q, Jones A, Alewood PF, Lewis RJ. Deep Venomics Reveals the Mechanism for Expanded Peptide Diversity in Cone Snail Venom. Mol Cell Proteomics. 2013 Feb;12(2):312-29.
• 31. Miljanich GP. Ziconotide: Neuronal Calcium Channel Blocker for Treating Severe Chronic Pain. Curr Med Chem. 2004 Dec;11(23):3029-40.
• 32. Bjørn-Yoshimoto WE, Ramiro IBL, Yandell M, McIntosh JM, Olivera BM, Ellgaard L, Safavi-Hemami H. Curses or Cures: A Review of the Numerous Benefits Versus the Biosecurity Concerns of Conotoxin Research. Biomedicines. 2020 Jul 22;8(8):235.
• 33. Romero HK, Christensen SB, Di Cesare Mannelli L, Gajewiak J, Ramachandra R, Elmslie KS, Vetter DE, Ghelardini C, Iadonato SP, Mercado JL, Olivera BM, Mcintoch JM. Inhibition of α9α10 nicotinic acetylcholine receptors prevents chemotherapy-induced neuropathic pain. Proc Natl Acad Sci U S A. 2017 Mar 7;114(10):E1825-32.
• 34. Xiong X, Menting JG, Disotuar MM, Smith NA, Delaine CA, Ghabash G, Agrawal R, Wang X, He X, Fisher SJ, MacRaild CA, Norton RS, Gajewiak J, Forbes BE, Smith BJ, Safavi-Hemami H, Olivera B, Lawrence MC, Chou DHC. A structurally minimized yet fully active insulin based on cone-snail venom insulin principles. Nat Struct Mol Biol. 2020;27:615-24.
• 35. Biass D, Violette A, Hulo N, Lisacek F, Favreau P, Stöcklin R. Uncovering Intense Protein Diversification in a Cone Snail Venom Gland Using an Integrative Venomics Approach. J Proteome Res. 2015 Feb 6;14(2):628-38.
• 36. Puillandre N, Duda TF, Meyer C, Olivera BM, Bouchet P. One, four or 100 genera? A new classification of the cone snails. J Molluscan Stud. 2015 Feb;81(1):1-23.
• 37. Barroso CX, Lotufo TM da C, Bezerra LEA, Matthews-Cascon H. A biogeographic approach to the insular marine ‘prosobranch’ gastropods from the southwestern Atlantic Ocean. J Molluscan Stud. 2016;82:558-63.
• 38. Gomes RS. Taxonomia e morfologia de representantes da família Conidae (mollusca, gastropoda, neogastropoda) na costa brasileira [Thesis]. Rio de Janeiro, Brasil: Universidade Federal do Rio de Janeiro; 2004.
• 39. Petuch EJ, Myers RF. Additions to the Cone Shell Faunas (Conidae and Conilithidae) of the Cearaian and Bahian Subprovinces, Brazilian Molluscan Province. Xenophora Taxon. 2014 Jul;4:30-43.
• 40. Petuch E, Berschauer D. Six New Species of Gastropods (Fasciolariidae, Conidae, and Conilithidae) from Brazil. Festivus. 2016 Apr;48:257-66.
• 41. Zugasti-Cruz A, Maillo M, López-Vera E, Falcón A, Cotera EPH de la, Olivera BM, Aguilar MB. Amino acid sequence and biological activity of a γ-conotoxin-like peptide from the worm-hunting snail Conus austini Peptides. 2006 Mar;27(3):506-11.
• 42. Zugasti-Cruz A, Aguilar MB, Falcón A, Olivera BM, Heimer de la Cotera EP. Two new 4-Cys conotoxins (framework 14) of the vermivorous snail Conus austini from the Gulf of Mexico with activity in the central nervous system of mice. Peptides. 2008 Feb;29(2):179-85.
• 43. Aguilar MB, Zugasti-Cruz A, Falcón A, Batista CVF, Olivera BM, Heimer de la Cotera EP. A novel arrangement of Cys residues in a paralytic peptide of Conus cancellatus (jr. syn.: Conus austini), a worm-hunting snail from the Gulf of Mexico. Peptides. 2013 Mar;41:38-44.
• 44. Jin A, Cristofori-Armstrong B, Rash LD, Román-González SA, Espinosa RA, Lewis RJ, Alewood PF, Vetter I. Novel conorfamides from Conus austini venom modulate both nicotinic acetylcholine receptors and acid-sensing ion channels. Biochem Pharmacol. 2019 Jun;164:342-8.
• 45. Jin AH, Muttenthaler M, Dutertre S, Himaya SWA, Kaas Q, Craik DJ, Lewis RJ, Alewood PF. Conotoxins: Chemistry and Biology. Chem Rev. 2019 Nov 13;119(21):11510-49.
• 46. Fainzilber M, Gordon D, Hasson A, Spira ME, Zlotkin E. Mollusc-specific toxins from the venom of Conus textile neovicarius. Eur J Biochem. 1991 Dec 5;202(2):589-95.
• 47. Nakamura T, Yu Z, Fainzilber M, Burlingame AL. Mass spectrometric-based revision of the structure of a cysteine-rich peptide toxin with γ-carboxyglutamic acid, TxVIIA, from the sea snail, Conus textile. Protein Sci. 1996 Mar;5(3):524-30.
• 48. Bernáldez J, Jiménez S, González L, Ferro J, Soto E, Salceda E, Chávez D, Aguilar MB, Licea-Navarro A. A New Member of Gamma-Conotoxin Family Isolated from Conus princeps Displays a Novel Molecular Target. Toxins (Basel). 2016 Feb 5;8(2):39.
• 49. Fainzilber M, Nakamura T, Lodder JC, Zlotkin E, Kits KS, Burlingame AL. γ-Conotoxin-PnVIIA, A γ-Carboxyglutamate-Containing Peptide Agonist of Neuronal Pacemaker Cation Currents. Biochemistry. 1998 Feb 10;37(6):1470-7.
• 50. Möller C, Rahmankhah S, Lauer-Fields J, Bubis J, Fields GB, Marí F. A Novel Conotoxin Framework with a Helix−Loop−Helix (Cs α/α) Fold. Biochemistry. 2005 Dec 13;44(49):15986-96.
• 51. Dauplais M, Lecoq A, Song J, Cotton J, Jamin N, Gilquin B, Roumestand C, Vita C, de Medeiros CL, Rowan EG, Harvey AL, Ménez A. On the Convergent Evolution of Animal Toxins. J Biol Chem. 1997 Feb 14;272(7):4302-9.
• 52. Savarin P, Guenneugues M, Gilquin B, Lamthanh H, Gasparini S, Zinn-Justin S, Ménez A. Three-Dimensional Structure of κ-Conotoxin PVIIA, a Novel Potassium Channel-Blocking Toxin from Cone Snails. Biochemistry. 1998 Apr 21;37(16):5407-16.
• 53. Corpuz GP, Jacobsen RB, Jimenez EC, Watkins M, Walker C, Colledge C, Garret JE, McDougal O, Li W, Gray WR, Hillyard DR, Rivier J, Mcintoch JM, Cruz LJ, Olivera BM. Definition of the M-Conotoxin Superfamily: Characterization of Novel Peptides from Molluscivorous Conus Venoms. Biochemistry. 2005 Jun 7;44(22):8176-86.
• 54. Braga MCV, Freitas J, Yamane T, Radis-Baptista G. A P-Superfamily toxin encoded from Conus regius cDNA. Submitted (AUG-2002) to the EMBL/GenBank/DDBJ databases. 2002.
• 55. Braga MCV, Konno K, Portaro FCV, Carlos de Freitas J, Yamane T, Olivera BM. Mass spectrometric and high performance liquid chromatography profiling of the venom of the Brazilian vermivorous mollusk Conus regius: feeding behavior and identification of one novel conotoxin. Toxicon. 2005;45:113-22.
• 56. Ellison M, Haberlandt C, Gomez-Casati ME, Watkins M, Elgoyhen AB, McIntosh JM, Olivera BM. α-RgIA: A Novel Conotoxin That Specifically and Potently Blocks the α9α10 nAChR†^,‡. Biochemistry. 2006;45(5):1511-7.
• 57. Franco A, Pisarewicz K, Moller C, Mora D, Fields GB, Marí F. Hyperhydroxylation: A New Strategy for Neuronal Targeting by Venomous Marine Molluscs. 2006;43:83-103.
• 58. Braga MCV, Nery AA, Ulrich H, Konno K, Sciani JM, Pimenta DC. α-RgIB: A Novel Antagonist Peptide of Neuronal Acetylcholine Receptor Isolated from Conus regius Venom. Int J Pept. 2013;2013:543028.
• 59. Franco A, Dovell S, Möller C, Grandal M, Clark E, Marí F. Structural plasticity of mini‐M conotoxins - expression of all mini‐M subtypes by Conus regius FEBS J. 2018 Mar285(5):887-902.
• 60. Lirazan MB, Hooper D, Corpuz GP, Ramilo CA, Bandyopadhyay P, Cruz LJ, Olivera BM. The Spasmodic Peptide Defines a New Conotoxin Superfamily. Biochemistry. 2000 Feb 2239(7):1583-8.
• 61. Fan CX, Chen XK, Zhang C, Wang LX, Duan KL, He LL, Cao Y, Liu SY, Zhong MN, Ulens C, Tytgat J, Chen JS, Chi CW, Zhou Z. A Novel Conotoxin from Conus betulinus, κ-BtX, Unique in Cysteine Pattern and in Function as a Specific BK Channel Modulator. J Biol Chem. 2003 Apr 11;278(15):12624-33.
• 62. Kauferstein S, Huys I, Lamthanh H, Stöcklin R, Sotto F, Menez A, Tytgat J, Mebs D. A novel conotoxin inhibiting vertebrate voltage-sensitive potassium channels. Toxicon. 2003 Jul;42(1):43-52.
• 63. McIntosh JM, Santos AD, Olivera BM. Conus Peptides Targeted to Specific Nicotinic Acetylcholine Receptor Subtypes. Annu Rev Biochem. 1999;68:59-88.
• 64. Ellison M, Feng ZP, Park AJ, Zhang X, Olivera BM, McIntosh JM, Norton RS. α-RgIA, a Novel Conotoxin That Blocks the α9α10 nAChR: Structure and Identification of Key Receptor-Binding Residues. J Mol Biol. 2008 Apr 4;377(4):1216-27.
• 65. Clark RJ, Daly NL, Halai R, Nevin ST, Adams DJ, Craik DJ. The three-dimensional structure of the analgesic α-conotoxin, RgIA. FEBS Lett. 2008 Mar 5;582(5):597-602.
• 66. Schrödinger L, DeLano W. PyMOL. Available from: http://www.pymol.org/pymol.2020
  » http://www.pymol.org/pymol.2020
• 67. Volpon L, Lamthanh H, Barbier J, Gilles N, Molgó J, Ménez A, Lancelin JM. NMR Solution Structures of δ-Conotoxin EVIA from Conus ermineus That Selectively Acts on Vertebrate Neuronal Na+ Channels. J Biol Chem. 2004 May 14;279(20):21356-66.
• 68. Vincler M, Wittenauer S, Parker R, Ellison M, Olivera BM, McIntosh JM. Molecular mechanism for analgesia involving specific antagonism of α9α10 nicotinic acetylcholine receptors. Proc Natl Acad Sci U S A. 2006 Nov 21;103(47):17880-4.
• 69. Di Cesare Mannelli L, Cinci L, Micheli L, Zanardelli M, Pacini A, McIntosh MJ, Ghelardini C. α-Conotoxin RgIA protects against the development of nerve injury-induced chronic pain and prevents both neuronal and glial derangement. Pain. 2014 Oct;155(10):1986-95.
• 70. Pacini A, Micheli L, Maresca M, Branca JJV, McIntosh JM, Ghelardini C, Mannelli LDC. The α9α10 nicotinic receptor antagonist α-conotoxin RgIA prevents neuropathic pain induced by oxaliplatin treatment. Exp Neurol. 2016 Aug;282:37-48.
• 71. AlSharari SD, Toma W, Mahmood HM, Michael McIntosh J, Imad Damaj M. The α9α10 nicotinic acetylcholine receptors antagonist α-conotoxin RgIA reverses colitis signs in murine dextran sodium sulfate model. Eur J Pharmacol. 2020 Sep 15;883:173320.
• 72. Callaghan B, Haythornthwaite A, Berecki G, Clark RJ, Craik DJ, Adams DJ. Analgesic α-Conotoxins Vc1.1 and Rg1A Inhibit N-Type Calcium Channels in Rat Sensory Neurons via GABAB Receptor Activation. J Neurosci. 2008 Oct 22;28(43):10943-51.
• 73. Cuny H, de Faoite A, Huynh TG, Yasuda T, Berecki G, Adams DJ. γ-Aminobutyric Acid Type B (GABAB) Receptor Expression Is Needed for Inhibition of N-type (Cav2.2) Calcium Channels by Analgesic α-Conotoxins. J Biol Chem. 2012 Jul 6;287(28):23948-57.
• 74. Franco A, Kompella SN, Akondi KB, Melaun C, Daly NL, Luetje CW, Alewood PF, Craik DJ, Adams DJ, Marí F. RegIIA: An α4/7-conotoxin from the venom of Conus regius that potently blocks α3β4 nAChRs. Biochem Pharmacol. 2012 Feb 1;83(3):419-26.
• 75. McIntosh JM, Dowell C, Watkins M, Garrett JE, Yoshikami D, Olivera BM. α-Conotoxin GIC from Conus geographus, a Novel Peptide Antagonist of Nicotinic Acetylcholine Receptors. J Biol Chem. 2002 Sep 13;277(37):33610-5.
• 76. Nicke A, Loughnan ML, Millard EL, Alewood PF, Adams DJ, Daly NL, Craik DJ, Lewis RJ. Isolation, Structure, and Activity of GID, a Novel α4/7-Conotoxin with an Extended N-terminal Sequence. J Biol Chem. 2003 Jan 31;278(5):3137-44.
• 77. Kompella SN, Hung A, Clark RJ, Marí F, Adams DJ. Alanine Scan of α-Conotoxin RegIIA Reveals a Selective α3β4 Nicotinic Acetylcholine Receptor Antagonist. J Biol Chem. 2015 Jan 9;290(2):1039-48.
• 78. Kompella SN, Cuny H, Hung A, Adams DJ. Molecular Basis for Differential Sensitivity of α -Conotoxin RegIIA at Rat and Human Neuronal Nicotinic Acetylcholine Receptors. Mol Pharmacol. 2015 Dec;88(6):993-1001.
• 79. Möller C, Dovell S, Melaun C, Marí F. Definition of the R-superfamily of conotoxins: Structural convergence of helix-loop-helix peptidic scaffolds. Peptides. 2018;107:75-82.
• 80. Möller C, Marí F. A vasopressin/oxytocin-related conopeptide with γ-carboxyglutamate at position 8. Biochem J. 2007 Jun 15;404(Pt 3):413-9.
• 81. Möller C, Melaun C, Castillo C, Díaz ME, Renzelman CM, Estrada O, Kuch U, Lokey S. Functional Hypervariability and Gene Diversity of Cardioactive Neuropeptides. J Biol Chem. 2010 Dec 24;285(52):40673-80.
• 82. Peng C, Tang S, Pi C, Liu J, Wang F, Wang L, Zhou W, Xu A. Discovery of a novel class of conotoxin from Conus litteratus, lt14a, with a unique cysteine pattern. Peptides. 2006 Sep;27(9):2174-81.
• 83. Dutertre S, Croker D, Daly NL, Andersson Å, Muttenthaler M, Lumsden NG, Craik DJ, Alewood PF, Guillon G, Lewis RJ. Conopressin-T from Conus tulipa Reveals an Antagonist Switch in Vasopressin-like Peptides. J Biol Chem. 2008 Mar 14;283(11):7100-8.
• 84. Giribaldi J, Ragnarsson L, Pujante T, Enjalbal C, Wilson D, Daly NL, Lewis RJ, Dutertre S. Synthesis, Pharmacological and Structural Characterization of Novel Conopressins from Conus miliaris Mar Drugs. 2020 Mar 6;18(3):150.
• 85. Martinez JS, Olivera BM, Gray WR, Craig AG, Groebe DR, Abramson SN, McIntosh JM. alpha-Conotoxin EI, A New Nicotinic Acetylcholine Receptor Antagonist with Novel Selectivity. Biochemistry. 1995 Nov 7;34(44):14519-26.
• 86. Quinton L, Servent D, Girard E, Molgó J, Le Caer J-P, Malosse C, Haidar EA, Lecoq A, Gilles N, Chamot-Rooke J. Identification and functional characterization of a novel α-conotoxin (EIIA) from Conus ermineus Anal Bioanal Chem. 2013 Jun;405(15):5341-51.
• 87. Echterbille J, Gilles N, Araóz R, Mourier G, Amar M, Servent D, De Pauw E, Quinton L. Discovery and characterization of EII B, a new α-conotoxin from Conus ermineus venom by nAChRs affinity capture monitored by MALDI-TOF/TOF mass spectrometry. Toxicon. 2017 May;130:1-10.
• 88. Abalde S, Tenorio MJ, Afonso CML, Zardoya R. Conotoxin Diversity in Chelyconus ermineus (Born, 1778) and the Convergent Origin of Piscivory in the Atlantic and Indo-Pacific Cones. Genome Biol Evol. 2018 Oct 1;10(10):2643-62.
• 89. Jacobsen R, Yoshikami D, Ellison M, Martinez J, Gray WR, Cartier GE, Shon KJ, Groebe DR, Abramson SN, Olivera BM, McIntosh JM. Differential Targeting of Nicotinic Acetylcholine Receptors by Novel αA-Conotoxins. J Biol Chem. 1997 Sep 5;272(36):22531-7.
• 90. Barbier J, Lamthanh H, Le Gall F, Favreau P, Benoit E, Chen H, Gilles N, Ilan N, Heinemann SH, Gordon D, Ménez A, Molgó J. A δ-Conotoxin from Conus ermineus Venom Inhibits Inactivation in Vertebrate Neuronal Na+ Channels but Not in Skeletal and Cardiac Muscles. J Biol Chem. 2004 Feb 6;279(6):4680-5.
• 91. Bulaj G, DeLaCruz R, Azimi-Zonooz A, West P, Watkins M, Yoshikami D, Olivera BM. δ-Conotoxin Structure/Function through a Cladistic Analysis. Biochemistry. 2001 Nov 6;40(44):13201-8.
• 92. Gowd KH, Twede V, Watkins M, Krishnan KS, Teichert RW, Bulaj G, Olivera BM. Conantokin-P, an unusual conantokin with a long disulfide loop. Toxicon. 2008 Aug 1;52(2):203-13.
• 93. Ning J, Ren J, Xiong Y, Wu Y, Zhangsun M, Zhangsun D, Zhu X, Luo S. Identification of Crucial Residues in α-Conotoxin EI Inhibiting Muscle Nicotinic Acetylcholine Receptor. Toxins (Basel). 2019 Oct;11(10):603.
• 94. López-Vera E, Jacobsen RB, Ellison M, Olivera BM, Teichert RW. A novel alpha conotoxin (α-PIB) isolated from C. purpurascens is selective for skeletal muscle nicotinic acetylcholine receptors. Toxicon. 2007 Jun;49(8):1193-9.
• 95. Teichert RW, López-Vera E, Gulyas J, Watkins M, Rivier J, Olivera BM. Definition and Characterization of the Short αA-Conotoxins: A Single Residue Determines Dissociation Kinetics from the Fetal Muscle Nicotinic Acetylcholine Receptor. Biochemistry. 2006 Jan 31;45(4):1304-12.
• 96. Chi SW, Park KH, Suk JE, Olivera BM, McIntosh JM, Han KH. Solution Conformation of αA-conotoxin EIVA, a Potent Neuromuscular Nicotinic Acetylcholine Receptor Antagonist from Conus ermineus J Biol Chem. 2003 Oct 24;278(43):42208-13.
• 97. Gracy J, Le-Nguyen D, Gelly J-C, Kaas Q, Heitz A, Chiche L. KNOTTIN: the knottin or inhibitor cystine knot scaffold in 2007. Nucleic Acids Res.2007 Jan;36(Database isse):D314-9.
• 98. Peigneur S, Paolini-Bertrand M, Gaertner H, Biass D, Violette A, Stöcklin R, Favreau P, Tytgat J, Hartley O. δ-Conotoxins Synthesized Using an Acid-cleavable Solubility Tag Approach Reveal Key Structural Determinants for NaV Subtype Selectivity. J Biol Chem. 2014 Dec 19;289(5):35341-50.
• 99. Tietze D, Leipold E, Heimer P, Böhm M, Winschel W, Imhof D, Heinemann SH, Tietze AA. Molecular interaction of δ-conopeptide EVIA with voltage-gated Na+ channels. Biochim Biophys Acta. 2016 Sep;1860(9):2053-63.
• 100. Rivera-Ortiz JA, Cano H, Marí F. Intraspecies variability and conopeptide profiling of the injected venom of Conus ermineus Peptides. 2011 Feb;32(2):306-16.
• 101. Safavi-Hemami H, Möller C, Marí F, Purcell AW. High molecular weight components of the injected venom of fish-hunting cone snails target the vascular system. J Proteomics. 2013 Oct 8;91:97-105.

• Availability of data and materials

  Not applicable.

• Funding

  HBF is the recipient of a Ph.D. fellowship from the Coordination for the Improvement of Higher Education Personnel (CAPES) and FVC is the recipient of a postdoctoral fellowship (PNPD) from CAPES. The present study was also supported by a FAPES-Universal 21/2018 grant - no. 105/2019 (granted to SGF).

• Ethics approval

  Not applicable.

• Consent for publication

  Not applicable.

Data availability

Data citations

1. Editorial Board W. World Register of Marine Species (WoRMS). Available online: http://www.marinespecies.org

Publication Dates

• Publication in this collection
  27 Jan 2023
• Date of issue
  2023

History

• Received
  15 Aug 2022
• Accepted
  14 Dec 2022