Lachlan D. Rash

Pharmacology and biochemistry of spider venoms.

Spider venoms represent an incredible source of biologically active substances which selectively target a variety of vital physiological functions in both insects and mammals. Many toxins isolated from spider venoms have been invaluable in helping to determine the role and diversity of neuronal ion channels and the process of exocytosis. In addition, there is enormous potential for the use of insect specific toxins from animal sources in agriculture. For these reasons, the past 15-20 years has seen a dramatic increase in studies on the venoms of many animals, particularly scorpions and spiders. This review covers the pharmacological and biochemical activities of spider venoms and the nature of the active components. In particular, it focuses on the wide variety of ion channel toxins, novel non-neurotoxic peptide toxins, enzymes and low molecular weight compounds that have been isolated. It also discusses the intraspecific sex differences in given species of spiders.

A new sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensory neurons

From a systematic screening of animal venoms, we isolated a new toxin (APETx2) from the sea anemone Anthopleura elegantissima, which inhibits ASIC3 homomeric channels and ASIC3‐containing heteromeric channels both in heterologous expression systems and in primary cultures of rat sensory neurons. APETx2 is a 42 amino‐acid peptide crosslinked by three disulfide bridges, with a structural organization similar to that of other sea anemone toxins that inhibit voltage‐sensitive Na+ and K+ channels. APETx2 reversibly inhibits rat ASIC3 (IC50=63 nM), without any effect on ASIC1a, ASIC1b, and ASIC2a. APETx2 directly inhibits the ASIC3 channel by acting at its external side, and it does not modify the channel unitary conductance. APETx2 also inhibits heteromeric ASIC2b+3 current (IC50=117 nM), while it has less affinity for ASIC1b+3 (IC50=0.9 μM), ASIC1a+3 (IC50=2 μM), and no effect on the ASIC2a+3 current. The ASIC3‐like current in primary cultured sensory neurons is partly and reversibly inhibited by APETx2 with an IC50 of 216 nM, probably due to the mixed inhibitions of various co‐expressed ASIC3‐containing channels.

Spider-Venom Peptides as Therapeutics

Spiders are the most successful venomous animals and the most abundant terrestrial predators. 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. Spider venoms are conservatively predicted to contain more than 10 million bioactive peptides, making them a valuable resource for 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.

Venomics: a new paradigm for natural products-based drug discovery

The remarkable potency and pharmacological diversity of animal venoms has made them an increasingly valuable source of lead molecules for drug and insecticide discovery. Nevertheless, most of the chemical diversity encoded within these venoms remains uncharacterized, despite decades of research, in part because of the small quantities of venom available. However, recent advances in the miniaturization of bioassays and improvements in the sensitivity of mass spectrometry and NMR spectroscopy have allowed unprecedented access to the molecular diversity of animal venoms. Here, we discuss these technological developments in the context of establishing a high-throughput pipeline for venoms-based drug discovery.

The receptor site of the spider toxin PcTx1 on the proton-gated cation channel ASIC1a.

Acid‐sensing ion channels (ASICs) are excitatory neuronal cation channels, involved in physiopathological processes related to extracellular pH fluctuation such as nociception, ischaemia, perception of sour taste and synaptic transmission. The spider peptide toxin psalmotoxin 1 (PcTx1) has previously been shown to inhibit specifically the proton‐gated cation channel ASIC1a. To identify the binding site of PcTx1, we produced an iodinated form of the toxin (125I‐PcTx1YN) and developed a set of binding and electrophysiological experiments on several chimeras of ASIC1a and the PcTx1‐insensitive channels ASIC1b and ASIC2a. We show that 125I‐PcTx1YN binds specifically to ASIC1a at a single site, with an IC50 of 128 pm, distinct from the amiloride blocking site. Results obtained from chimeras indicate that PcTx1 does not bind to ASIC1a transmembrane domains (M1 and M2), involved in formation of the ion pore, but binds principally on both cysteine‐rich domains I and II (CRDI and CRDII) of the extracellular loop. The post‐M1 and pre‐M2 regions, although not involved in the binding site, are crucial for the ability of PcTx1 to inhibit ASIC1a current. The linker domain between CRDI and CRDII is important for their correct spatial positioning to form the PcTx1 binding site. These results will be useful for the future identification or design of new molecules acting on ASICs.

Production of Recombinant Disulfide-Rich Venom Peptides for Structural and Functional Analysis via Expression in the Periplasm of E. coli

Disulfide-rich peptides are the dominant component of most animal venoms. These peptides have received much attention as leads for the development of novel therapeutic agents and bioinsecticides because they target a wide range of neuronal receptors and ion channels with a high degree of potency and selectivity. In addition, their rigid disulfide framework makes them particularly well suited for addressing the crucial issue of in vivo stability. Structural and functional characterization of these peptides necessitates the development of a robust, reliable expression system that maintains their native disulfide framework. The bacterium Escherichia coli has long been used for economical production of recombinant proteins. However, the expression of functional disulfide-rich proteins in the reducing environment of the E. coli cytoplasm presents a significant challenge. Thus, we present here an optimised protocol for the expression of disulfide-rich venom peptides in the periplasm of E. coli, which is where the endogenous machinery for production of disulfide-bonds is located. The parameters that have been investigated include choice of media, induction conditions, lysis methods, methods of fusion protein and peptide purification, and sample preparation for NMR studies. After each section a recommendation is made for conditions to use. We demonstrate the use of this method for the production of venom peptides ranging in size from 2 to 8 kDa and containing 2–6 disulfide bonds.

A Dynamic Pharmacophore Drives the Interaction between Psalmotoxin-1 and the Putative Drug Target Acid-Sensing Ion Channel 1a

Acid-sensing ion channel 1a (ASIC1a) is a primary acid sensor in the peripheral and central nervous system. It has been implicated as a novel therapeutic target for a broad range of pathophysiological conditions including pain, ischemic stroke, depression, and autoimmune diseases such as multiple sclerosis. The only known selective blocker of ASIC1a is π-TRTX-Pc1a (PcTx1), a disulfide-rich 40-residue peptide isolated from spider venom. π-TRTX-Pc1a is an effective analgesic in rodent models of acute pain and it provides neuroprotection in a mouse model of ischemic stroke. Thus, understanding the molecular basis of the π-TRTX-Pc1a–ASIC1a interaction should facilitate development of therapeutically useful ASIC1a blockers. We therefore developed an efficient bacterial expression system to produce a panel of π-TRTX-Pc1a mutants for probing structure-activity relationships as well as isotopically labeled toxin for determination of its solution structure and dynamics. We demonstrate that the toxin pharmacophore resides in a β-hairpin loop that was revealed to be mobile over a wide range of time scales using molecular dynamics simulations in combination with NMR spin relaxation and relaxation dispersion measurements. The toxin-receptor interaction was modeled by in silico docking of the toxin structure onto a homology model of rat ASIC1a in a restraints-driven approach that was designed to take account of the dynamics of the toxin pharmacophore and the consequent remodeling of side-chain conformations upon receptor binding. The resulting model reveals new insights into the mechanism of action of π-TRTX-Pc1a and provides an experimentally validated template for the rational design of therapeutically useful π-TRTX-Pc1a mimetics.

Inhibition of voltage‐gated Na+ currents in sensory neurones by the sea anemone toxin APETx2

BACKGROUND AND PURPOSE APETx2, a toxin from the sea anemone Anthropleura elegantissima, inhibits acid‐sensing ion channel 3 (ASIC3)‐containing homo‐ and heterotrimeric channels with IC50 values < 100 nM and 0.1–2 µM respectively. ASIC3 channels mediate acute acid‐induced and inflammatory pain response and APETx2 has been used as a selective pharmacological tool in animal studies. Toxins from sea anemones also modulate voltage‐gated Na+ channel (Nav) function. Here we tested the effects of APETx2 on Nav function in sensory neurones.

Chemical Synthesis, 3D Structure, and ASIC Binding Site of the Toxin Mambalgin-2†

Mambalgins are a novel class of snake venom components that exert potent analgesic effects mediated through the inhibition of acid-sensing ion channels (ASICs). The 57-residue polypeptide mambalgin-2 (Ma-2) was synthesized by using a combination of solid-phase peptide synthesis and native chemical ligation. The structure of the synthetic toxin, determined using homonuclear NMR, revealed an unusual three-finger toxin fold reminiscent of functionally unrelated snake toxins. Electrophysiological analysis of Ma-2 on wild-type and mutant ASIC1a receptors allowed us to identify α-helix 5, which borders on the functionally critical acidic pocket of the channel, as a major part of the Ma-2 binding site. This region is also crucial for the interaction of ASIC1a with the spider toxin PcTx1, thus suggesting that the binding sites for these toxins substantially overlap. This work lays the foundation for structure-activity relationship (SAR) studies and further development of this promising analgesic peptide.

Neurotoxic activity of venom from the Australian Eastern mouse spider (Missulena bradleyi) involves modulation of sodium channel gating

Mouse spiders represent a potential cause of serious envenomation in humans. This study examined the activity of Missulena bradleyi venom in several in vitro preparations. Whilst female M. bradleyi venom at doses up to 0.05 μl ml−1 failed to alter twitch or resting tension in all preparations used, male venom (0.02 and 0.05 μl ml−1) produced potent effects on transmitter release in both smooth and skeletal neuromuscular preparations. In the mouse phrenic nerve diaphragm preparation, male M. bradleyi venom (0.02 μl ml−1) caused rapid fasciculations and an increase in indirectly evoked twitches. Male venom (0.02 and 0.05 μl ml−1) also caused a large contracture and rapid decrease in indirectly evoked twitches in the chick biventer cervicis muscle, however had no effect on responses to exogenous ACh (1 mM) or potassium chloride (40 mM). In the chick preparation, contractile responses to male M. bradleyi venom (0.05 μl ml−1) were attenuated by (+)‐tubocurarine (100 μM) and by tetrodotoxin (TTX, 1 μM). Both actions of male M. bradleyi venom were blocked by Atrax robustus antivenom (2 units ml−1). In the unstimulated rat vas deferens, male venom (0.05 μl ml−1) caused contractions which were inhibited by a combination of prazosin (0.3 μM) and P2X‐receptor desensitization (with α,β‐methylene ATP 10 μM). In the rat stimulated vas deferens, male venom (0.05 μl ml−1) augmented indirectly evoked twitches. Male venom (0.1 μl ml−1) causes a slowing of inactivation of TTX‐sensitive sodium currents in acutely dissociated rat dorsal root ganglion neurons. These results suggest that venom from male M. bradleyi contains a potent neurotoxin which facilitates neurotransmitter release by modifying TTX‐sensitive sodium channel gating. This action is similar to that of the δ‐atracotoxins from Australian funnel‐web spiders.