(realized by Danilo Casasola & Stefano Data)
Spider are the largest group of venomous animals and the most abundant terrestrial predators, with an estimated 100,000 extant species. Their remarkable success is due in large part to their ingenious exploitation of silk and the evolution of pharmacologically complex venoms that ensure rapid subjugation of prey. Most spider venoms are dominated by disulfide-rich peptides that typically have high affinity and specificity for particular subtypes of ion channels and receptors. The vast majority of spiders employ a lethal cocktail to rapidly subdue their prey, which are often many times their own size. However, despite their fearsome reputation, less than a handful of these insect assassins are harmful to humans
Spider venoms are predicted to contain by far the largest number of pharmacologically active peptide toxins, making them a valuable resource for new drug discovery.
Here we review the structure and pharmacology of spider-venom peptides that are being used as leads for the development of therapeutics against a wide range of pathophysiological conditions including cardiovascular disorders, chronic pain, inflammation, and erectile dysfunction.
Toxins (Basel). 2010 Dec;2(12):2851-71
Although spider venoms are a complex cocktail containing salt, proteins and organic molecules, peptides are the primary components of spider venoms. Based on the number of described spider species and a relatively conservative estimate of the complexity of their venom it has been estimated that the potential number of unique spider venom peptides could be upwards of 12 million.
In addition to the well known neurotoxic effects of spider venoms, they contain peptides with antiarrhythmic, antimicrobial, analgesic, antiparasitic, cytolytic, haemolytic, and enzyme inhibitory activity and antitumor activity, (such as the crude venom of Macrothele raveni, even if the responsible component has not been yet identified).
The majority of characterized spider-venom peptides target voltage-gated potassium(KV), calcium (CaV) or sodium (NaV) channels, as well as some interact with ligand-gated channels (e.g., purinergic receptors).
Inhibitor Cystine Knot Structure
As we said, the major component of spider venoms are peptides, wich have generally been considered poor candidates for human therapeutics because of their susceptibility to proteolytic degradation in vivo and their limited penetration of intestinal mucosa. Therefore, how can they have an effect in other organisms without being simply degradated?
The reason of their resistance is a particular structure known as inhibitor cystine knot (ICK). The inhibitor cystine knot (ICK) is defined as an antiparallel β sheet stabilized by a cystine knot.
An inhibitor cysteine knot (ICK) is a protein structural motif containing three disulfide bridges. Along with the sections of polypeptide between them, two disulfides form a loop through which the third disulfide bond (linking the 3rd and 6th cysteine in the sequence) passes, forming a knot and giving rise to the alternative name of knotting for this motif. The motif is common in invertebrate toxins such as those from arachnids and mollusks. The ICK motif is a very stable protein structure which is resistant to heat denaturation and proteolysis. ICK peptide components of venoms target voltage-gated ion channels but members of the family also act as antibacterial and haemolytic agents. Because of their stability, ICK motifs are being developed as possible therapeutics.
The inhibitor cystine knot (ICK) motif comprises an antiparallel β sheet stabilized by a cystine knot. β strands are shown in orange and the six cysteine residues that form the cystine knot are labeled 1–6. The two “outer” disulfide bonds are shown in green and the “inner” disulfide bridge is red.
The cystine knot of the 37-residue spider-venom peptide ω-hexatoxin-Hv1a. The cystine knot comprises a ring formed by two disulfides (green) and the intervening sections of polypeptide backbone (gray), with a third disulfide (red) piercing the ring to create a pseudo-knot. The hydrophobic core of the toxin consists primarily of the two central disulfide bridges connected to the β strands. Key functional residues in ICK toxins are often located in the β hairpin that projects from the central disulfide-rich core of the peptide.
Cysteine knot converts ICK toxins into hyperstable mini-proteins with tremendous chemical, thermal, and biological stability, resistant to extremes of pH, organic solvents, and high temperatures. ICK peptides are typically stable in human serum for several days and have half-lives in simulated gastric fluid of >12 hours.
With the exception of those with antibacterial/antifungal activity, all of the spider-venom peptides to be discussed in this review contain an ICK motif.
Spider Toxins with Analgesic Potential
Normal nociceptive pain is a key adaptive response that limits our exposure to potentially damaging or life-threatening events. In contrast, aberrant long-lasting pain transforms this adaptive response into a debilitating and often poorly managed disease. More than one-quarter of adults suffer from chronic pain and the percentage rises as the population gets older.
Recently, a number of ion channels have been shown to be critical players in the pathophysiology of pain, and in many cases the most potent and selective blockers of these channels are spider-venom peptides.
Modulators of Acid Sensing Ion Channels
Acid sensing ion channels (ASICs) are proton-gated sodium channels that open in response to low pH. They belong to the epithelial sodium channel/degenerin (ENaC/DEG) superfamily and to date, seven ASIC subunits have been identified.
ASIC1a is the most abundant ASIC subunit in the central nervous system (CNS) and it has the highest affinity for protons. It has been implicated as a novel therapeutic target for a broad range of pathophysiological conditions including pain, ischemic stroke, depression, and autoimmune and neurodegenerative diseases such as multiple sclerosis, Huntington’s Disease, and Parkinson’s Disease. Inhibitors of ASIC1a might therefore be therapeutically valuable for some of these conditions.
The only potent and specific inhibitor of ASIC1a that has been identified to date is π-theraphotoxin-Pc1a (π-TRTX-Pc1a), a 40-residue ICK peptide isolated from the venom of the Trinidad chevron tarantula Psalmopoeus cambridgei.
π-TRTX-Pc1a was shown to be an effective analgesic, comparable to morphine, but only when administered intrathecally or by intracerebroventricular injection. Thus, even if it is now far to be a useful therapeutic analgesic, it can be a model for developing mimetic molecules for managing severe chronic pain in patients who are intolerant or refractory to other treatments.
Modulators of Voltage-Gated Sodium Channels
Voltage-gated sodium (NaV) channels provide a current pathway for the rapid depolarization of excitable cells that is required to initiate an action potential.
Of the nine NaV subtypes, NaV1.3, NaV1.7, and NaV1.8 are involved in pain signaling, but NaV1.7 has emerged as perhaps the best validated pain target in human.
Gain-of-function mutations in the gene encoding the α subunit of NaV1.7 (SCN9A) underlie two painful neuropathies known as paroxysmal extreme pain disorder (PEPD) and inherited erythromelalgia (IE), whereas loss-of-function mutations in SCN9A result in a congenital indifference to all forms of pain. Remarkably, apart from their complete inability to sense pain, partial loss of smell (hyposmia) is the only other sensory impairment in individuals with this channelopathy.
The preferential expression of NaV1.7 in peripheral sensory and sympathetic neurons makes it an ideal target for novel analgesics. Recent studies have revealed that spider venoms may provide an excellent source of such subtype-specific blockers.
However, according to ArachnoServer, only three peptide toxins have been isolated thus far with activity against vertebrate NaV1.7 channel. All of these toxins were isolated from members of the Theraphosidae family (commonly known as tarantulas).
Spider-venom peptides with submicromolar potency against NaV1.3, NaV1.7, or NaV1.81.
The most potent known blocker of human NaV1.7, is β-TRTX-Tp2a (Protoxin II).
Modulators of P2X Receptors
P2X purinergic receptors are ATP-gated non-selective ion channels permeable to Na+, K+ and Ca2+. Currently, seven subunits (P2X1-7) are known and functional P2X channels are formed by association of these subunits to form homomeric or heteromeric dimers.
P2X3 is the best-studied subtype with regards to pain. Consistent with its localization on ascending nociceptive sensory neurons, it has been found to be involved in acute pain, inflammatory pain, chronic neuropathic pain, visceral pain, migraine pain, and cancer pain. A potent and selective modulator of P2X3 was isolated from the venom of the central Asian spider Geolycosa sp. and named purotoxin-1 (PT1). http://www.ncbi.nlm.nih.gov/pubmed/20437566
Ann Neurol. 2010 May.
Antiarrhythmic Drugs from Spider Venoms
Mechanosensitive channels (MSCs), sometimes referred to as stretch-activated channels, are found in all cells, but mechanosensitivity is best viewed as a phenotype rather than a genotype. Only two selective inhibitors of MSCs have been isolated, namely M-theraphotoxin-Gr1a (M-TRTX-Gr1a) and κ-TRTX-Gr2a from the venom of the tarantula Grammostola rosea;
M-TRTX-Gr1a is a significantly more potent inhibitor of MSCs, with a Kd of 630 nM in rat astrocytes, and it has proved to be a valuable tool for study of MSCs.
Atrial fibrillation, the most common cardiac arrhythmia to occur in humans, is associated with passive stretching of the atrial chamber caused by mechanical dysfunction of the heart. The stretching causing arrhythmias seems to be dominated by inward cationic currents; hence, the blockade of SA-CAT channels should reduce the extent of the abnormalities in the heart beat.
Block of these channels presumably explains the remarkable observation that M-TRTX-Gr1a suppresses atrial fibrillation in dilatated rabbit heart. This suggests that MSCs might be a novel target for antiarrhythmic agents. M-TRTX-Gr1a itself is unlikely to be a useful therapeutic agent because of its unusual mode of action. It does not interact directly with MSCs, since an enantiomer comprised entirely of D-amino acids is equipotent with the native peptide. Rather, the peptide perturbs the channel-bilayer boundary by partitioning into the membrane, and this membrane-disrupting activity presumably also underlies its antimicrobial activity. Nevertheless, M-TRTX-Gr1a is likely to be a useful tool for determining the potential of MSCs as a therapeutic target for the treatment of pathologies as diverse as cardiac arrhythmias, spinal cord damage, muscular dystrophy, and gliomas.
J Cell Sci. 2004 May.
Spider Toxins for Treating Erectile Dysfunction
Penile erection is a complex process initiated by activation of parasympathetic pelvic nerves, resulting in arterial dilatation followed by relaxation of corpora cavernosa. Nitric oxide (NO) plays a major role in the generation and maintenance of intracavernous pressure and penile erection. NO, which is released from nitrergic nerves within the trabecular and arterial tissues as well as by the endothelial tissue of penile arteries, exerts its relaxing action by activating soluble guanylyl cyclase. This causes an increase in intracellular cGMP which relaxes the smooth muscles of the cavernous body and results in penile erection.
There are several drugs on the market today for treatment of erectile dysfuncion (ED), including sildenafil (Viagra®), tadalafil (Cialis®), and vardenafil (Levitra®). These drugs all affect phosphodiesterase type 5 (PDE5), which is present in large amounts in the penis. Inhibition of PDE5 leads to increased levels of cGMP and hence increased blood flow to the penis. The aforementioned PDE5-blocking drugs have similar side effects including headache, flushing, dyspepsia, nasal congestion, impaired vision, photophobia and blurred vision. Hence, there is a need for better drugs with fewer side effects for the treatment of ED.
In South America, humans bitten by the “armed-spider” Phoneutria nigriventer experience a variety of symptoms including priapism. The toxin responsible for this effect, δ-ctenitoxin-Pn2a (δ-CNTX-Pn2a; Tx2-6) was isolated in 1992 and subsequently found to modulate the activity of NaV channels. δ-CNTX-Pn2a is a 48-residue toxin with five disulfide bonds; it is unclear whether it contains an ICK motif. The toxin has a complex pharmacology that results in inhibition of NaV channel inactivation and a hyperpolarizing shift in the channel activation potential. Intracerebroventricular injection of δ-CNTX-Pn2a into mice results in scratching, hypersalivation, lachrymation, sweating, and agitation followed by spastic paralysis of the anterior and posterior extremities and death. However, subcutaneous injection of δ-CNTX-Pn2a into rats induced erection, and rats with severely depressed erectile function could be normalized by subcutaneous administration of δ-CNTX-Pn2a. A minimum dose of only 0.006 μg/kg was required to cause an erection in mice when injected directly into the corpus cavernosum; at this dose, no local and systemic collateral toxic effects were observed and the erection was lost after 120–140 min.
The potency and specificity of δ-CNTX-Pn2a makes it an attractive lead molecule for the development of new therapeutics for ED treatment that might have fewer side effects than current drugs. The pharmacology inherent in δ-CNTX-Pn2a may be widespread in spider venoms, at least amongst members of the Ctenidae family (which currently comprises 475 species).
Antibacterial and Antifungal Toxins
The introduction of antibiotics in the 1930s and 1940s was in large part responsible for the dramatic decline in the mortality rate from communicable diseases in developed countries. However, bacteria are remarkably proficient at adapting to environmental stresses, and they have evolved at least one mechanism of resistance for all 17 classes of antibiotics that have been developed to date. The recent widespread emergence of antibiotic resistance in clinically important bacterial pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis, combined with a dramatic decrease in the rate of development of new antibiotics, has led some to suggest that we may be approaching the post-antibiotic era. While this may overstate the problem, there is nevertheless an urgent need to develop new antimicrobials with novel mechanisms of action.
The recent successful introduction of the lipopeptide antibiotic daptomycin has rekindled interest in antimicrobial peptides. Daptomycin is a newly-approved antibacterial agent, the first lipopeptide agent to be released onto the market. Its spectrum of activity is limited to Gram-positive organisms, including a number of highly resistant species. It is a nautral product derived from Streptomyces roseosporus. The drug was originally developed by Lilly in the early 1980s but was abandoned after early clinical trials. Cubist Pharmaceuticals licensed daptomycin in 1997 and began new clinical trials with the agent in 1999.
To date, 40 membrane-acting antimicrobial peptides (MAMPs) have been isolated from the venom of four different families of araneomorphs, suggesting that antimicrobial activity is widespread in this infraorder of spiders. These MAMPs often have a wide range of antimicrobial activities, with some toxins active against Gram-positive and Gram-negative bacteria as well as fungal pathogens such as Candida albicans.
MAMPs differ from most other spider-venom peptides in their structure and mode of action. Rather than utilizing the ICK fold common to most spider toxins, MAMPs are α-helical amphipathic peptides that interact with and perturb cell membranes to yield their antimicrobial effects. This mode of action can be potentially problematic from a therapeutic perspective since MAMPs that interact nonspecifically with cell membranes are cytolytic. Indeed, this property is likely to be the basis of their biological function in spider venoms. Although it has been proposed that MAMPs might protect the spider’s venom apparatus against infection their primary role is more likely to be as membrane disrupting agents that augment the activity of the disulfide-rich neurotoxic peptides by facilitating their spread.
It has been shown that the cytolytic activity of at least some spider-venom MAMPs can be minimized by truncation without significantly disrupting their antimicrobial activity. Nevertheless, the therapeutic use of these peptides is likely to be limited by their inherent susceptibility to proteolysis, which is likely to result in short gut and plasma half-lives. Whether this problem can be solved by strategies such as cyclization or grafting key sequence elements onto more stable ICK scaffolds remains to be seen.
LyeTx I, an antimicrobial peptide isolated from the venom of Lycosa erythrognatha, known as wolf spider, has been synthesised and its structural profile studied by using the CD and NMR techniques. LyeTx I has shown to be active against bacteria (Escherichia coli and Staphylococcus aureus) and fungi (Candida krusei and Cryptococcus neoformans) and able to alter the permeabilisation of L: -alpha-phosphatidylcholine-liposomes (POPC) in a dose-dependent manner. In POPC containing cholesterol or ergosterol, permeabilisation has either decreased about five times or remained unchanged, respectively. These results, along with the observed low haemolytic activity, indicated that antimicrobial membranes, rather than vertebrate membranes seem to be the preferential targets. However, the complexity of biological membranes compared to liposomes must be taken in account. Besides, other membrane components, such as proteins and even specific lipids, cannot be discarded to be important to the preferential action of the LyeTx I to the tested microorganisms. The secondary structure of LyeTx I shows a small random-coil region at the N-terminus followed by an alpha-helix that reached the amidated C-terminus, which might favour the peptide-membrane interaction. The high activity against bacteria together with the moderate activity against fungi and the low haemolytic activity have indicated LyeTx I as a good prototype for developing new antibiotic peptides.
Amino Acids. 2010.
In Vivo Applications
In the preceding sections we surveyed a number of spider-venom peptides with potential therapeutic applications. With the exception of the MAMPs that have antimicrobial/antifungal activity, all of these peptides are stabilized by an inhibitor cystine knot motif. Thus, they are unlikely to suffer from one of the major problems typically associated with peptide drugs, namely rapid proteolytic degradation. As any surviving bite victim will attest, the devastating in vivo efficacy of the 42-residue lethal NaV channel toxin from the Australian funnel-web spider plainly demonstrates how minute quantities of ICK peptides can be effective in humans. Should rapid proteolysis prove to be an issue for a spider-venom peptide of therapeutic interest, strategies such as D-amino acid substitution of susceptible residues, cyclization to reduce conformational flexibility, and protection of the termini via C-terminal amidation or use of N-terminal pyroglutamate could be employed to improve proteolytic resistance. Indeed, spiders routinely employ C-terminal amidation as a protection strategy, with ~12% of all known spider toxins containing this posttranslational modification.
Because of their inherent proteolytic resistance, the plasma half-life of ICK peptides is likely to be determined by the rate at which they are cleared by glomerular filtration, which efficiently removes small proteins and peptides that are not bound to carrier proteins such as serum albumin. However, there are a variety of strategies that can be employed to reduce peptide clearance rates, such as increasing the peptide mass by PEGylation, conjugation to carrier proteins, or by making peptides more hydrophobic in order to enhance their association with serum albumin; the latter approach can also cause hydrophobic depoting, which provides sustained release from a subcutaneous injection.
For acute life-threatening conditions as well as chronic conditions such as persistent pain, subcutaneous injection is likely to be an acceptable route of peptide-drug administration. In certain pathologies where quality of life is dramatically reduced, intrathecal administration may even be a viable option if the difficulties associated with peptide delivery are outweighed by the benefits of treatment. Generally, however, oral delivery is likely to be desirable. The intrinsic stability of ICK peptides is likely to facilitate the development of oral delivery strategies since they will presumably have much longer gut and plasma residence times than typical peptides. Moreover, spider-venom ICK peptides are small enough to consider alternative routes of administration such as intranasal, transdermal, and pulmonary.
An alternative but complementary approach is to develop small-molecule mimetics of spider-venom peptides. The epitope (pharmacophore) that mediates the interaction of these peptides with their cognate receptors or ion channels can be remarkably small. For example, the interaction between the spider-venom peptide ω-hexatoxin-Hv1a and invertebrate CaV channels is mediated by a pharmacophore comprising only three spatially contiguous residues with a solvent-accessible surface area of ~200 Å, which approximates the typical solvent-accessible surface area of a small drug. As long as a high-quality structure of the peptide is available, this enables ab initio design of nonpeptide mimetics, identification of small molecule mimetics via in silico screening of chemical libraries, or a combination of these approaches.
ChemMedChem. 2013 Mar.
Over a period of more than 300 million years, spiders have evolved an extensive library of bioactive peptides. Moreover, in contrast with man-made combinatorial peptide libraries, spider-venom peptides have been pre-optimized for high affinity and selectivity against a diverse range of molecular targets. It is therefore not surprising that numerous spider-venom peptides have been characterized that potently and selectively modulate the activity of a diverse range of therapeutic targets. These include peptides that target NaV channels, ASICs, MSCs, and purinergic receptors as well as peptides with antimalarial and antimicrobial activity. Most of these peptides contain an ICK motif, and their extraordinary stability provides a variety of delivery options for therapeutic administration. Only a small fraction of spider-venom peptides have been characterized, and continued technical advances in the venoms-based drug discovery process are likely to uncover many new therapeutic leads from spider venoms.