Engineering NaV1.7 Inhibitory JzTx-V Peptides with a Potency and Basicity Profile Suitable for Antibody Conjugation To Enhance Pharmacokinetics.

Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel NaV1.7, is being pursued to address the unmet medical need with respect to chronic pain and the rising opioid epidemic. As part of early research efforts on this front, we have previously developed NaV1.7 inhibitory peptide-antibody conjugates with tarantula venom-derived GpTx-1 toxin peptides with an extended half-life (80 h) in rodents but only moderate in vitro activity (hNaV1.7 IC50 = 250 nM) and without in vivo activity. We identified the more potent peptide JzTx-V from our natural peptide collection and improved its selectivity against other sodium channel isoforms through positional analogueing. Here we report utilization of the JzTx-V scaffold in a peptide-antibody conjugate and architectural variations in the linker, peptide loading, and antibody attachment site. We found conjugates with 100-fold improved in vitro potency relative to those of complementary GpTx-1 analogues, but pharmacokinetic and bioimaging analyses of these JzTx-V conjugates revealed a shorter than expected plasma half-life in vivo with accumulation in the liver. In an attempt to increase circulatory serum levels, we sought the reduction of the net +6 charge of the JzTx-V scaffold while retaining a desirable NaV in vitro activity profile. The conjugate of a JzTx-V peptide analogue with a +2 formal charge maintained NaV1.7 potency with 18-fold improved plasma exposure in rodents. Balancing the loss of peptide and conjugate potency associated with the reduction of net charge necessary for improved target exposure resulted in a compound with moderate activity in a NaV1.7-dependent pharmacodynamic model but requires further optimization to identify a conjugate that can fully engage NaV1.7 in vivo.

GsMTx4-D provides protection to the D2.mdx mouse.

Duchenne muscular dystrophy is a life-limiting muscle disease that has no current effective therapy. Despite mounting evidence that dysregulation of mechanosensitive ion channels is a significant contributor to dystrophy pathogenesis, effective pharmacologic strategies targeting these channels are lacking. GsMTx4, and its enantiomer GsMTx4-D, are peptide inhibitors of mechanosensitive channels with identical activity. In previous studies, acute in vitro application of GsMTx4 to dystrophic murine muscle effectively reduced the excess MSC dependent calcium influx linked to contraction-induced muscle damage. Here we sought to determine if in vivo treatment with GsMTx4-D proffered benefit in the D2.mdx mouse. GsMTx4-D showed a 1-week half-life when administered by subcutaneous injection over four weeks. Informed by these results, D2.mdx mice were then treated by a subcutaneous injection regimen of GsMTx4-D for six weeks followed by determination of muscle mass, muscle susceptibility to eccentric contraction injury and multiple histological indicators of disease progression. The mice showed a reduction in the loss of muscle mass and a decrease in susceptibility to contraction induced injury. These protective effects were realized without reduction in fibrosis, supporting a model where GsMTx4-D acts directly on muscle cells. We propose GsMTx4-D represents a promising new therapy to slow disease progression and may complement other therapies such as anti-inflammatory agents and gene-replacement strategies.

Acid Sensing Ion Channel 1a (ASIC1a) Mediates Activity-induced Pain by Modulation of Heteromeric ASIC Channel Kinetics.

Chronic muscle pain is acutely worsened by exercise. Acid sensing ion channels (ASIC) are heteromeric channels expressed in muscle sensory neurons that detect decreases in pH. We have previously shown ASIC3 is important in activity-induced hyperalgesia. However, ASICs form heteromers with ASIC1a being a key component in sensory neurons. Therefore, we studied the role of ASIC1a in mice using behavioral pharmacology and genetic deletion in a model of activity-induced hyperalgesia. We found ASIC1a-/- mice developed mechanical hyperalgesia similar to wild-type mice, but antagonism of ASIC1a, with psalmotoxin, prevented development of mechanical hyperalgesia in wild-type mice, but not in ASIC1a-/- mice. To explain this discrepancy, we then performed electrophysiology studies of ASICs and examined the effects of psalmotoxin on ASIC heteromers. We expressed ASIC1a, 2 and 3 heteromers or ASIC1 and 3 heteromers in CHO cells, and examined the effects of psalmotoxin on pH sensitivity. Psalmotoxin significantly altered the properties of ASIC hetomeric channels. Specifically, in ASIC1a/2/3 heteromers, psalmotoxin slowed the kinetics of desensitization, slowed the recovery from desensitization, and inhibited pH-dependent steady-state desensitization, but had no effect on pH-evoked current amplitudes. We found a different pattern in ASIC1a/3 heteromers. There was a significant leftward shift in the pH dose response of steady-state desensitization and decrease in pH-evoked current amplitudes. These results suggest that blockade of ASIC1a modulates the kinetics of heteromeric ASICs to prevent development of activity-induced hyperalgesia. These data suggest ASIC1a is a key subunit in heteromeric ASICs and may be a pharmacological target for treatment of musculoskeletal pain.

High yield production and refolding of the double-knot toxin, an activator of TRPV1 channels.

A unique peptide toxin, named double-knot toxin (DkTx), was recently purified from the venom of the tarantula Ornithoctonus huwena and was found to stably activate TRPV1 channels by targeting the outer pore domain. DkTx has been shown to consist of two inhibitory cysteine-knot (ICK) motifs, referred to as K1 and K2, each containing six cysteine residues. Beyond this initial characterization, however, the structural and functional details about DkTx remains elusive in large part due to the lack of a high yielding methodology for the synthesis and folding of this cysteine-rich peptide. Here, we overcome this obstacle by generating pure DkTx in quantities sufficient for structural and functional analyses. Our methodology entails expression of DkTx in E. coli followed by oxidative folding of the isolated linear peptide. Upon screening of various oxidative conditions for optimizing the folding yield of the toxin, we observed that detergents were required for efficient folding of the linear peptide. Our synthetic DkTx co-eluted with the native toxin on HPLC, and irreversibly activated TRPV1 in a manner identical to native DkTx. Interestingly, we find that DkTx has two interconvertible conformations present in a 1∶6 ratio at equilibrium. Kinetic analysis of DkTx folding suggests that the K1 and K2 domains influence each other during the folding process. Moreover, the CD spectra of the toxins shows that the secondary structures of K1 and K2 remains intact even after separating the two knots. These findings provide a starting point for detailed studies on the structural and functional characterization of DkTx and utilization of this toxin as a tool to explore the elusive mechanisms underlying the polymodal gating of TRPV1.

Systemic envenomation caused by the wandering spider Phoneutria nigriventer, with quantification of circulating venom.

INTRODUCTION: Bites by Phoneutria spp. spiders are common in Brazil, although only 0.5-1% result in severe envenomation, with most of these occurring in children. Cases of systemic envenomation in adults are very unusual, and no serum venom levels have been previously quantified in these cases. CASE REPORT: A 52-year-old man was bitten on the neck by an adult female Phoneutria nigriventer. Immediately after the bite, there was intense local pain followed by blurred vision, profuse sweating, tremors, and an episode of vomiting; 1-2 h post bite the patient showed agitation and a blood pressure of 200/130 mmHg, and was given captopril and meperidine. Upon admission to our service 4 h post bite (time zero - T0), his blood pressure was 130/80 mmHg with a heart rate of 150 beats/min, mild tachypnea, agitation, cold extremities, profuse sweating, generalized tremors, and priapism. The patient was treated with antivenom, local anesthetic, and fluid replacement. Most of the systemic manifestations disappeared within 1 h after antivenom. Laboratory blood analyses at T0, T1, T6, T24, and T48 detected circulating venom by ELISA only at T0, before antivenom infusion (47.5 ng/mL; cut-off, 17.1 ng/mL); his serum blood sugar was 163 mg/dL at T0. The patient was discharged on the second day with a normal arterial blood pressure and a follow-up evaluation revealed no sequelae. CONCLUSION: This is the first report of confirmed moderate/severe envenoming in an adult caused by P. nigriventer with the quantification of circulating venom.

Distinct primary structures of the major peptide toxins from the venom of the spider Macrothele gigas that bind to sites 3 and 4 in the sodium channel.

Six peptide toxins (Magi 1-6) were isolated from the Hexathelidae spider Macrothele gigas. The amino acid sequences of Magi 1, 2, 5 and 6 have low similarities to the amino acid sequences of known spider toxins. The primary structure of Magi 3 is similar to the structure of the palmitoylated peptide named PlTx-II from the North American spider Plectreurys tristis (Plectreuridae). Moreover, the amino acid sequence of Magi 4, which was revealed by cloning of its cDNA, displays similarities to the Na+ channel modifier delta-atracotoxin from the Australian spider Atrax robustus (Hexathelidae). Competitive binding assays using several 125I-labelled peptide toxins clearly demonstrated the specific binding affinity of Magi 1-5 to site 3 of the insect sodium channel and also that of Magi 5 to site 4 of the rat sodium channel. Only Magi 6 did not compete with the scorpion toxin LqhalphaIT in binding to site 3 despite high toxicity on lepidoptera larvae of 3.1 nmol/g. The K(i)s of other toxins were between 50 pM for Magi 4 and 1747 nM for Magi 1. In addition, only Magi 5 binds to both site 3 in insects (K(i)=267 nM) and site 4 in rat brain synaptosomes (K(i)=1.2 nM), whereas it showed no affinities for either mammal binding site 3 or insect binding site 4. Magi 5 is the first spider toxin with binding affinity to site 4 of a mammalian sodium channel.

Breakdown of the blood-brain barrier and neuropathological changes induced by Phoneutria nigriventer spider venom.

The blood-brain barrier (BBB) is responsible for selective flux of substances between blood and brain. The selective permeability of the BBB is crucial for the maintenance of the brain microenvironment homeostasis, and alterations in the barrier may be involved in many pathophysiological processes. Phoneutria nigriventer armed spider venom produces excitatory signals and symptoms in humans, and its recognized neurotoxic action suggests a potential ability to alter BBB permeability. The aim of the present study was to investigate the capacity of P. nigriventer venom (PNV) in promoting BBB breakdown in adult rats. After intravenous injection of 850 micro g/kg of the whole venom, BBB lesions were evaluated after 18 h to 9 days by ultrastructural methods using the extracellular tracer lanthanum nitrate. Clinical signs and symptoms of rats showed acute neurotoxicity, with some of the animals presenting convulsions, but which were clinically resolved by 12 h post-envenoming. The results showed that PNV is able to increase BBB permeability, particularly in the hippocampus. Changes were first detected in arterioles and post-capillary venules 18 h to 5 days after venom inoculation. The increased permeation of the extracellular tracer peaked on day 1, representing about 42% of the examined vessels (P<0.01). This appeared to occur by both transendothelial and intercellular routes, i.e., by pinocytic transport and through interendothelial junctions. Concomitantly, the surrounding tissue showed vasogenic edema and swollen astrocytic processes, without inflammatory infiltrates. The peak of the edema occurrence was observed on day 3, in about 60% of the vessels (P<0.001). Enhanced capillary permeability was observed on day 9, and affected 36% of all capillaries (P<0.05). The affected capillaries were characterized by increased number of pinocytotic vesicles, which, in addition, were filled with the extracellular tracer, but without visible transport through the interendothelial pathway. This study demonstrates that systemic PNV inoculation induces BBB breakdown through trans- and paracellular routes. It is concluded that BBB breakdown is an event not associated with the acute neurotoxicity exhibited by the rats.

Mechanism of induction of complement susceptibility of erythrocytes by spider and bacterial sphingomyelinases

We have recently shown that the sphingomyelinase toxins P1 and P2 from the venom of the spider Loxosceles intermedia induce complement (C)-dependent lysis of autologous erythrocytes by induction of the cleavage of cell surface glycophorins through activation of an endogenous metalloproteinase facilitating the activation of the alternative pathway of C. Phospholipase D (PLD) from Corynebacterium pseudotuberculosis shows some degree of homology with the spider sphingomyelinases and can induce similar clinical symptoms to those observed after spider envenomation. The aim of this study was to investigate if the bacterial PLD-induced haemolysis of human erythrocytes was C dependent and if cleavage of glycophorins occurred. We show here that haemolysis of both PLD- and P1-treated human erythrocytes was C dependent, but while PLD-mediated haemolysis was dependent on activation of the classical pathway of C, P1 induced lysis via both the classical and alternative pathways. P1, but not PLD, induced cleavage of glycophorins and no change in expression of complement regulators was induced by either of the toxins. In both cases, annexin V binding sites were exposed, suggesting that the membrane asymmetry had been disturbed causing exposure of phosphatidylserine to the cell surface. Our results suggest that C susceptibility induced by L. intermedia and C. pseudotuberculosis PLD is a result of exposure of phosphatidylserine, and the higher potency of P1 toxin can be explained by its additional effect of cleavage of glycophorins.

The spider toxin omega-Aga IIIA defines a high affinity site on neuronal high voltage-activated calcium channels.

The spider toxin omega-agatoxin IIIA (omega-Aga-IIIA) is a potent inhibitor of high voltage-activated calcium currents in the mammalian brain. To establish the biochemical parameters governing its action, we radiolabeled the toxin and examined its binding to native and recombinant calcium channels. In experiments with purified rat synaptosomal membranes, both kinetic and equilibrium data demonstrate one-to-one binding of omega-Aga-IIIA to a single population of high affinity sites, with K(d) = approximately 9 pm and B(max) = approximately 1.4 pmol/mg protein. Partial inhibition of omega-Aga-IIIA binding by omega-conotoxins GVIA, MVIIA, and MVIIC identifies N and P/Q channels as components of this population. omega-Aga-IIIA binds to recombinant alpha(1B) and alpha(1E) calcium channels with a similar high affinity (K(d) = approximately 5-9 pm) in apparent one-to-one fashion. Results from recombinant alpha(1B) binding experiments demonstrate virtually identical B(max) values for omega-Aga-IIIA and omega-conotoxin MVIIA, providing further evidence for a one-to-one stoichiometry of agatoxin binding to calcium channels. The combined evidence suggests that omega-Aga-IIIA defines a unique, high affinity binding site on N-, P/Q-, and R-type calcium channels.

Binding sites and actions of Tx1, a neurotoxin from the venom of the spider Phoneutria nigriventer, in guinea pig ileum.

Tx1, a neurotoxin isolated from the venom of the South American spider Phoneutria nigriventer, produces tail elevation, behavioral excitation and spastic paralysis of the hind limbs after intracerebroventricular injection in mice. Since Tx1 contracts isolated guinea pig ileum, we have investigated the effect of this toxin on acetylcholine release, as well as its binding to myenteric plexus-longitudinal muscle membranes from the guinea pig ileum. [125I]-Tx1 binds specifically and with high affinity (Kd = 0.36 +/- 0.02 nM) to a single, non-interacting (nH = 1.1), low capacity (Bmax 1.1 pmol/mg protein) binding site. In competition experiments using several compounds (including ion channel ligands), only PhTx2 and PhTx3 competed with [125I]-Tx1 for specific binding sites (K0.5 apparent = 7.50 x 10(-4) g/l and 1.85 x 10(-5) g/l, respectively). PhTx2 and PhTx3, fractions from P. nigriventer venom, contain toxins acting on sodium and calcium channels, respectively. However, the neurotoxin PhTx2-6, one of the isoforms found in the PhTx2 pool, did not affect [125I]-Tx1 binding. Tx1 reduced the [3H]-ACh release evoked by the PhTx2 pool by 33%, but did not affect basal or KCl-induced [3H]-ACh release. Based on these results, as well as on the homology of Tx1 with toxins acting on calcium channels (omega-Aga IA and IB) and its competition with [125I]-omega-Cono GVIA in the central nervous system, we suggest that the target site for Tx1 may be calcium channels.

Mechanisms of alpha-latrotoxin action.

The major component of black widow spider venom, alpha-latrotoxin, triggers massive exocytosis in a variety of neurosecretory cells. An important trigger for exocytosis is the calcium influx via alpha-latrotoxin-induced channels in biological membranes. However, this mechanism fails to explain exocytosis which occurred in the complete absence of extracellular calcium. Recently, sophisticated biochemical and molecular techniques have led to the discovery of novel alpha latrotoxin-binding membrane receptors: neurexins and latrophilin/CIRL (calcium-independent receptor for alpha-latrotoxin). Neurexins are single transmembrane proteins which bind to alpha-latrotoxin in a calcium-dependent manner and also interact with the synaptic vesicle protein, synaptotagmin. On the other hand, latrophilin is a seven-transmembrane protein and belongs to the family of G-protein-coupled receptors. The multitude of effects of alpha-latrotoxin on exocytosis in different cell systems and the nature of its membrane targets are discussed in this article. The molecular details of how alpha-latrotoxin binding is transduced eventually to exocytosis remain to be elucidated.

Selective presynaptic insectotoxin (alpha-latroinsectotoxin) isolated from black widow spider venom.

A homogenous protein of 120,000 mol. wt isolated from black widow spider (Lactrodectus mactans tredecimguttatus) venom and referred to as alpha-latroinsectotoxin was highly potent (4 nM) in the induction of an increase of the frequency of miniature excitatory postsynaptic potentials in blowfly (Calliphora vicina) larvae neuromuscular preparations. In the frog nerve ending, however, even 50 nM alpha-latroinsectotoxin failed to affect transmitter release. Pretreatment of insect preparations with alpha-latrotoxin or frog preparations with alpha-latroinsectotoxin did not prevent the specific effect of consequent applications of alpha-latroinsectotoxin (insect) and alpha-latrotoxin (frog), respectively. The binding of labelled [125I]alpha-latroinsectotoxin to insect and [125I]alpha-latrotoxin to bovine membrane preparations was saturable and highly specific. The presynaptic effect, but not the binding of alpha-latroinsectotoxin, was dependent on the presence of divalent cations in the external medium. Mg2+ could readily substitute for Ca2+ and increase of transmitter release induced by alpha-latroinsectotoxin also occurred in Ca(2+)-free solutions. Pretreatment of preparations with 300 micrograms/ml concanavalin A completely abolished both the presynaptic effect of alpha-latroinsectotoxin and its binding to insect membrane preparations. Thus, the phenomenology of alpha-latroinsectotoxin action on insects resembles in general that described for the action of alpha-latrotoxin on vertebrates. The selectivity of alpha-latrotoxin and alpha-latroinsectotoxin seems to be due to differences in the structure of neurotoxin receptors in nerve endings of vertebrates and insects, although the mode of presynaptic action has a great deal in common.