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Chapter 30. Polyamine Spider Toxins: Unique Pharmacological Tools
Chapter 30. Polyamine Spider Toxins: Unique Pharmacological Tools Nicholas A. Saccomanol, Robert A. Volkmannl, Hunter Jackson2 and Thomas N. Parks2 1Pfizer Central Research, Groton, Connecticut 06340 2Natural Product Sciences, Inc. 420 Chipeta Way Suite 240, Salt Lake City, Utah 84108 Introduction - The use of naturally derived venom constituents as research tools has allowed scientists t o describe and understand the pharmacology and physiology of many important biological systems (1). Long-standing interest in spider venoms has primarily centered around the relatively large proteinaceous toxins (>3Kd) isolated from exotic spiders belonging t o Latrodectus, Loxosceles and Atrax. genera (2). However, recent research from the laboratories of N. Kawai, P.N.R. Usherwood, and B.A. Tashmukhamedov has focused o n the isolation and pharmacology of an exciting new class of low molecular weight (
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6-Philanthotoxin (9, an insect neurotoxin structurally and pharmacologically related to the spider polyamines was found in the venom of the digger wasp Philanthus triangulurn and subsequently assigned the structure 6 (17,18). The philanthotoxin molecule has a spermine nucleus appended to an N-n-butyryltyrosineresidue by an arnide bond.
Chap. 30
Polyamine Spider ?bxlns
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H!?
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H
Q
OH
Svnthesis of Polvamine Spider Toxins -The chemistry involved in the preparation of many of the naturally occurring polyamines has been summarized (19). Because polyamine spider venoms have highly polar hydroxyaryl and amino acid residues, synthetic strategy i s dictated by the appropriate selection of chemically compatible basic amine, phenol and amino acid protecting groups. The polyamine backbones are routinely assembled in a stepwise fashion using amine alkylations, reductive aminations and acrylonitrile additions. Standard peptide coupling reactions are employed for the formation o f amide bonds. This general strategy has been utilized for the preparation o f NSTX-3 (lJ (20), JSTX-3 (1)(21), argiotoxin-636 (1)(13,22-24), -659 (A) (13,23), -673 (I)(23), and 8-philanthotoxin (PTX-433) (6) (17,18). Synthesis i s necessary t o unambiguously verify structural assignments and t o provide generous quantities of these and related toxins for continued biological investigations. Polvamines Spider Venoms as Excitatow Amino Acid (EAA) Antagonists - Interest in the polyamine spider toxins is a result of the observation that these molecules affect those synapses at which an excitatory amino acid (glutamate or aspartate) i s the neurotransmitter (vide infra). The invertebrate neuromuscular junction is a well recognized example of an EAA synapse at which functional antagonism will elicit paralysis. In addition, most excitatory synapses in mammalian brain use glutamate or aspartate as neurotransmitters, suggesting that EAA's are intimately involved in brain function (4,5). Moreover, increasingly strong and detailed evidence relating EAA function t o important neurological disorders (e.g. stroke damage and Alzheimer's disease) now exists (25). Compounds that affect EAA function, particularly those that antagonize the action o f such transmitters, are therefore o f considerable agricultural (insect control) and therapeutic interest. Excitatory amino acids produce synaptically-mediated depolarization of neurons by acting on specific receptors. The depolarizing effect produced by activation of excitatory synapses is referred t o as an excitatory postsynaptic potential (EPSP) or excitatory postsynaptic current (EPSC). On the basis o f extensive biochemical, pharmacological and electrophysiological data, three main classes of EAA receptor (named for their selective activation by known agonists) are recognized: N-methyl-D-aspartate receptors (NMDA) and the quisqualate and kainate receptors (both non-NMDA). The NMDA and non-NMDA receptors appear t o have distinct functions (4,s).
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Pharmacologv of Polvamine Spider Toxins: Invertebrate Nervous Systems Blockade o f neuromuscular transmission in invertebrates by polyamine spider toxins reveals their glutamate antagonist activity (26-28). For example, JSTX-3 (1)blocks the excitatory postsynaptic potential (EPSP) elicited by iontophoretically applied glutamate at synapses i n the lobster neuromuscular junction where an excitatory amino acid i s thought t o be the neurotransmitter. In this preparation, JSTX is reported t o produce an irreversible, voltage-independent blockade at very low concentrations (28). 125I-JSTX blocks neuromuscular transmission in the lobster (29) and binds exclusively t o regions of the sacrolemma opposed t o axon terminals (30). In studies published prior to the synthesis of 2 the JSTX used presumably consisted of a group of related toxins partially purified from extracts of whole venom glands by gel filtration and Sephadex chromatography (26). No data on the biological activities of any other components of Nephila clavata venom, save the single toxin ultimately identified as JSTX-3 (3, have been published. However, given the large number of components in this venom, it is unlikely that the effects attributed t o the non-synthetic JSTX arose from a single toxin (31). The effects of JSTX have been less intensively studied in other invertebrate systems. In the giant synapse o f the squid stellate
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ganglion, venom gland extracts block EPSP’s and glutamate-induced depolarization without affecting electrically evoked antidromic (presynaptic) responses (32). It is not clear whether the toxin binds t o the normal attachment site of glutamate on t he receptor molecule or to an external site on the associated ion channel, thereby preventing entry o f ions (28). Therefore, it is uncertain from the available literature whether JSTX produces i t s antagonism o f crustacean neuromuscular transmission via competitive or non-competitive mechanisms. A venom-gland extract from Argiope lobata blocks neuromuscular transmission and the effects of applied glutamate in locust muscle (33). Similar activity was observed i n venoms from other orb-weaving spiders and active fractions were isolated from A. lobata venom by gel chromatography (34). This toxin was further used for purification of glutamate receptors from crab muscle. The affinity-purified fraction was inserted into artificial membranes and shown t o produce pharmacologically specific glutamate-induced ion conductances (35). One of the several active components of Argiope lobata venom was subsequently identified as argiopine 3 (1 5,34). This toxin was determined by electrophysiological methods t o a c t functionally as a postsynaptic open-channel blocker (KD = 10-7M) in blowfly larval muscle (36). Argiope aurantia venom contains t w o distinct classes of toxins that paralyze grasshoppers, cockroaches, flies and moths (37). Five toxins with molecular weights below 1.000 daltons 659 (4J and 673 (5J and toxins above 5000 daltons have been including argiotoxins-636 (3, isolated (13). In housefly paralysis assays, synthetic argiotoxin-659 has an EDSOof 0.9 pmollmg, whereas synthetic argiotoxin-636 exhibits an EDSOof 3.3 pmollmg. In in vitro assays, however, the potencies of the t w o toxins families are similar. The effects o f low molecular weight toxins from the venoms of Argiope trifasciata, Argiope florida and Araneus gemma on EAA-mediated synaptic transmission in the locust retractor unguis muscle have also been studied (38-40). These toxins block neurally evoked muscle twitch, the junctional potential elicited by glutamate iontophoresis, and the voltage-clamped EPSP in a slowly reversing fashion. Single channel studies of locust muscle suggest that the low molecular weight orb-weaver toxins (principally argiotoxins -636, -659 and -673) are very potent noncompetitive antagonists of EAA receptor channel complexes. Their principal effect is a usedependent blockade of open ion channels, although some action on closed channels is probable (41,3). The combination, in orb-weaver venom, of a high concentration of free glutamate, which activates the receptor-associated channels, wi th a potent open-channel non-competitive antagonist t 4 or 3 provides the spider wi th a sophisticated and powerful mechanism for immobi Iizi ng insect prey (42). The effects of 6-philanthotoxin (6J on a variety of insects have been extensively studied (17). This toxin causes reversible paralysis, apparently by blocking quisqualate-sensitive junctional and extra-junctional glutamate receptors on skeletal muscle. The specificity of 6 for a particular subtype of glutamate receptor has not yet been demonstrated. Pharmacoloqv of Polvamine Spider Toxins: Vertebrate Nervous Svstems - Polyamine spider venoms also act on glutaminergic synapses in vertebrate systems. Partially purified JSTX blocks the responses o f CA1 pyramidal neurons i n rat hippocampus both t o stimulation o f t h e appropriate afferent neurons (Schaeffer collaterals) and t o direct application of glutamate (43). 2,4-Dihydroxyphenylacetylasparagine, a common structural f eat ure o f JSTX (2)and inhibits binding of 3H-L-glutamate t o rat membranes with an IC50 of 10-sM argiotoxin-636 (3, (44). JSTX also inhibits uptake of 3H-glutamate i nt o rat brain synaptosomes (45). The physiological actions of JSTX were assessed by recording EAA responses in voltage-clamped single rat hippocampal pyramidal neurons in culture (46). JSTX from partially purified gland extract completely blocked neuronal responses t o quisqualic acid (QA) and kainic acid (KA) in a dose dependent (but use independent manner) a t concentrations o f 10-12 t o 10-8M. Voltage dependent Na+ and K + currents and glycine dependent CI- currents were not affected by the toxin. Because JSTX was ineffective in blocking neuronal responses t o L-aspartate, it appears that this toxin has little antagonist action a t NMDA receptors. In catfish lateral line organ, partially purified JSTX abolishes afferent impulses elicited by focal application of glutamate (47). In studies w i t h t he dogfish retina, purified JSTX
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hyperpolarizes horizontal cells, blocks their response t o light and antagonizes glutamate-induced depolarization of these cells in a slowly reversible manner (48). Interestingly, JSTX acts as an agonist at "on-bipolar" cells in a slowly reversible manner, opening the same ion channels as does glutamate. Thus, Nephila venom appears capable o f exerting distinct and opposite effects at t w o classes of E A A receptor-channel complexes in vertebrates (48). A variety of venoms have been screened for their ability t o block synaptic transmission a t glutaminergic synapses in the chick cochlear nucleus (49). Argiotoxin-659 (A),isolated from Argiope aurantia, produced a readily reversible use-independent blockade o f transmission. Similarly, whole venom from A. trifasciata suppresses both spontaneous and sound-evoked electrical activity in guinea pig cochlear nerve (50). Synthetic argiotoxin-636 (3, -659 (4J and JSTX-3 (2J are functional antagonists o f both NMDA and non-NMDA classes o f glutamate receptor in rat brain. In addition, these materials greatly increase the binding of glycine, apparently by increasing the number (Bmax) o f strychnine-insensitive binding sites (51). Occupation of the strychnine-insensitive glycine binding site (found predominantly i n rat forebrain) appears t o be a prerequisite for activation of the NMDA receptor (52). It i s not yet certain whether modulation of this site by polyamine spider toxins underlies their glutamate antagonist activity. New Directions in Venom Research: Funnel Web Spider Toxins - A number of toxins from the funnel-web spider Agelenopsis aperta paralyze insects (53). These toxins contain low molecular weight acylpolyamine constituents (a - agatoxins), at least six 36-37 amino acid residue peptides (p agatoxins; reportedly sodium channel activators), and several larger polypeptides (oagatoxins; potent irreversible blockers o f synaptic transmission, presumably by action a t presynaptic calcium channels) (54). Other high molecular weight toxins found in the venom of the funnel web spider, Hololena curta, and the primitive hunting spider, Plectreurys tristes, are similar t o the o-agatoxins in inhibiting insect neuromuscular transmission (55,56). A (5Kd) toxin isolated from the venom of Hololena curta produces potent irreversible blockade of synaptic transmission by action on the postsynaptic neuron (50). Antidromic stimulation and responses to E A A agonists are also blocked by this toxin.
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Two classes o f toxins from Agelenopsis aperta venom that block transmission in the chick cochlear nucleus assay have also been studied (49,57). A multi-component fraction identified as "AG-l", containing polypeptides o f 4-7 Kd irreversibly blocks synaptic transmission at very low doses without affecting antidromic stimulation or postsynaptic responses t o applied EAA agonists. This fraction i s likely t o contain many i f not all of the "p'' and "a"-agatoxins (53). AG-1's action was shown t o be use independent and i s antagonized by extracellular calcium (58). In a dose-dependent manner, AG-1 potently blocks the binding of 1*5I-o-conotoxin (a calcium antagonist peptide from the venom o f the marine snail, Conus geographus) t o rat brain. However, AG-1 markedly increases binding o f the dihydropyridine calcium antagonist 3Hnitrendipine t o brain membranes, apparently by increasing the number of binding sites (59). The second fraction from Agelenopsis aperta contains polyamine toxins of e l Kd, whose structures have not been reported. This low molecular weight fraction, AG-2, suppresses synaptic transmission in the chick cochlear nucleus i n a fully-reversible, dose-dependent and useindependent manner (57). In sharp contrast t o AG-1, AG-2 substantially increases binding of 1251o-conotoxin but decreases the binding of 3H-nitrendipine t o rat brain (59). AG-2, administered intravenously or intracerebroventricullarly, protects rats against otherwise-lethal doses o f the convulsants, kainic acid, picrotoxin or bicuculline. A t doses effective in preventing lethal seizures, AG-2 produces only mild sedation in test animals (60). Conclusion -To date, venoms from only a handful o f the estimated one hundred thousand spider species have been studied in any detail. The results o f these studies already suggest that considerable structural and functional diversity exists within the polyamine toxin class and that these compounds differ significantly from previously-known classes o f EAA and calcium antagonists. For these reasons, it seems likely that spider venoms will continue, for some time, t o provide novel molecules useful for pharmaceutical and pesticide research.
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References 1.
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"Handbook of Natural Toxins," Vol. 1-4, A.T. Tu, R.F. Keeler and M.C. Hardegree, Eds.. Marcel Dekker, Inc., New York. 1984. "Handbook of Natural Toxins," Vol. 2, A.T. Tu, Ed., Marcel Dekker Inc., New York, 1984, Chapters 12-14. H. Jackson and P.N.R. Usherwood, Trends in Neurosciences, 11,278 (1988). o, 263-302 (1987). Trends in Neuroscience, l G. Johnson, Ann. Rep. Med. Chem., 24,(1989). R.K. Atkinson and P. Walker, Aust J. Exp Biol., 63,555 (1985). 5. Bettini and M. Maroli, Handbook of Experimental Pharmacology, Vol. 48, "Arthropod Venoms," S Bettini, Ed.,SpringerVerlag, New York, New York, 1978, p. 149-212. C.R. Geren and G.V. Odell. "Handbook of Natural Toxins," Vol. 2, A.T. Tu, Ed., Marcel Dekker, Inc., New York. 1984, p. 441. L.D. Foil, J.L. Fra2ierandB.R. Norment,Toxicon.c, 347(1979). Y. Aramaki. T. Yasuhara, T. Higashijima, M. Yoshioka. A. Miwa, N. Kawai and T. Nakajima. Proc. Japan Acad., 62 Ser. B, 359(1986). Y. Aramaki, T. Yasuhara, T. Higashijima, A. Miwa, N. Kawai and T. Nakajima, Biomed. Res., 8, 167 (1987). Y. Aramaki, T. Yasuhara, T. Higashijima, A. Miwa, N. Kawai and T. Nakajima, Biomed. Res., 8,241 (1987). M.E.Adams, R.L.Carney, F.E. Enderlin, E.T. Fu, M.A. Jarema, J.P. Li, C.A. Miller, D.A. Schooley, M.J. Shapiro, and V.J. Venema, Biochem. Biophys. Re<. Commun., 148,678 (1987). T. Budd, P. Clinton, A. Dell, I.R. Duce, 5.1. Johnson, D.L.J. Quicke, G.W. Taylor, P.N.R. Usherwood and G.Usoh, Brain Res.,B, 30 (1988). E.V. Grishin, T.M. Volkova, AS. Arsenlev, 0 . 5 . Reshetova, V.V. Onoprienko, L.G. Magazanic, S.M. Antonov and I.M. Fedorova, Bioorgan Khim., l2, 1121 (1986). P.N.R. Usherwood, I.R. Duce, A. Dell and G.W. Taylor, European Patent At0 208 523 (1986). T. Piek, R. H. Fokkens, H. Karst, C. Kruk, A. Lind, 1. van Marle, T. Nakajima, N.M.M. Nibbering, H. Shinozaki. W. Spanjer and Y.C. Tong, in "Neurotox '88: Molecular Basis of Drug & Pesticide Action." G.G. Lunt, Ed., Elsevier SciencePublishersBV,NewYork, 1988, p. 61. A.T. Eldefrawi, M.E. Eldefrawi, K. Konno, N.A. Mansour. K. Nakanishi, E. Oltz and P.N.R. Usherwood, Proc. Natl. Acad.Sci. USA.85,4910(1988). B. Ganem,Acc. Chem. Res.,lS, 290(1982). T. Teshima, T. Wakamiya. Y. Aramaki, T. Nakajima, N. Kawai and T. Shiba, Tetrahedron Lett., 28, 3509 (1987). Y. Hashimoto, Y. Endo, K. Shudo, Y. Aramaki, N. Kawai and T. Nakajima, Tetrahedron Lett., 28,351 1 (1987). T.L. Shih, 1. Ruiz-Sanchez and H. Mrozik, Tetrahedron Lett., 28,6015 (1987). V.J. Jasys, P.R. Kelbaugh, D.M. Nason. D Phillips, N.A. Saccomano and R.A. Volkmann, Tetrahedron Lett., 29, 6223 (1988). E.A. Elin,B.F.deMasedo, E.V. Onoprienko, N.E. Osokinaand 0.8. Tikhomirova, Bioorg. Khim.,l4,704(1988). D. W. Choi, Neuron, 1,623 (1988). N. Kawai.A. Miwaand T. Abe, Brain Res., 247,169(1982). T. Abe, N. Kawai, and A. Miwa, J. Physiol. (London), 243 (1983). A. Miwa, N. Kawai, M. Saito, H. Pan-Hou and M. Yoshioka, J. Neurophysiol., 319 (1987). K. Hagiwara, Y. Aramaki, K. Shimaraki, N. Kawai and T. Nakajima, Chem. Pharm. Bull., 36,1233 (1988). K. Shimazaki, K. Hagiwara, Y. Hirata, T. Nakajimaand N. Kawai, Neuroscience Lett.,84,173 (1988). T. Toki, T. Yasuhara, Y. Aramaki, K. 0sawa.A. Miwa, N. Kawai and T. Nakajima, Biomed. Res., 9,421 (1988). N. Kawai, 5. Yamagishi, M. Saito and K. Furuya. Brain Res., 278,346 (1983). P.8. Usmanov, D. Kalikulov, N. Shadyeva and B.A. Tashmukhamedov, Dokl. Akad. Nauk. SSSR, 273,1017 (1983). P.B. Usmanov, D. Kalikulov, N.G. Shadyeva, A.B. Nenilin and B.A. Tashmukhamedov, Toxicon, 11.528 (1985). B.A. Tashmukhamedov, E.M. Makhumudova, P.B. Usmanov and I. Kazakov, Gen. Physiol. Biophys., 4, 625 ( 1985). L.G. Magaranik, S.M. Antonov, I.M. Federova, T.M. Volkova and E.V. Grishin, "Receptors and Ion Channels," Y.A. Ovchinnikovand F. Hucho, Eds., DeGruyter, New York, New York. 1987, p. 305-312. M.E.Adams, F.E. Enderlin and D.A. Schooley, SOC. Neurosci. Abstr., 11.946 (1986). 7 241 (1 984). P.N.R. Usherwood, I.R. Duce and P. Boden. J. Physiol. (Paris), -9 A. Bateman, P. 8oden.A. Dell, I.R. Duce, D.L.J. Quickeand P.N.R. Usherwood, Brain Rer..m, 237 (1985). P.N.R. Usherwoodand I.R. Duce. Neurotoxicol, 5,239 (1985). C.J. Kerry, R.L. Ramsey.M.5.P. Sansom and P.N.R. Usherwood, Brain Res., 312 (1988). S.L. Early and E.K. Michaelis, Toxicon, 25,433 (1987). M. Saito, N. Kawai, A. Miwa, H. Pan-Hou and M.Yoshioka, Brain Res., 346,397 (1985). H. Pan-Hou, Y. Suda, M. Sumi, M. Yoshioka and N. Kawai, Neurosci. Lett., gl, 199 (1987). H. Pan-Hou, Y. Suda, M. Sumi, M. Yoshioka and N. Kawai, Brain Res., a,354(1989). N. Akaike, N. Kawai, N.I. Kiskin, E.M. Kljuchko, O.A. Krishtal and A. Ya. Tsyndrenko, Neurosci Lett., 79, 326 ( 1987). T. Nagai.5. Obaraand N. Kawai, Brain Res., 300,183 (1984). R.A. Shiellsand G. Falk, Neuroscience Lett.,77,221 (1987). H. Jackson, M.R. Urnes, W.R. Gray and T.N. Parks, 5oc. Neurosci. Abstr., 11,107 (1985). H. Cousillas, K.S. Coleand B.M. Johnstone, Hear. Res..36,213 (1988). M.J. Pagnorri, N.A. Saccomano, M.F. Gullak. R.A.Volkmannand E.E. Mena,Soc. Neurosci. Abstr., 14,482 (1988). N.W. Kleckner and R. Dingledine, Science, 241,835 (1988). M.E. Adams, V.P. Bindokas, L. Hasegawa and V.J. Venema. SOC. Neurosci Abstr., 14.30 (1988). V.P. Bindokas and M.E. Adams. Soc. Neurosci Abstr., 14.30 (1988). C.W. Bowers, H.S. Phillips, P. Lee, Y.N. Jan, L.Y. Jan, Proc. Nat. Acad. Sci. USA, 84,3506 (1987). W.D. Branton, L. Kolton, Y.N. Jan and L.Y. Jan, J. Neurosci., 4195 (1987).
m,
s,
z,
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57. H. Jackson, M.R. Urnes, W.R. Gray and T.N. Parks, "Excitatory Amino Acid Transmission," D. Lodge, Ed., A.R. Liss Inc., New York, 1987,p. 51-54. 58. H. Jackson, M.R. Urnes, W.R. Gray and T.N. Parks, SOC. Neurosci Abstr., l2,730(1986). 59. L.M. Kerr, F. Filloux, J.K. Wamsley, T.N. Parksand H. Jackson, SOC. Neurosci Abstr.,s, 102 (1987). 60. H. Jackson and T.N. Parks, SOC. Neurosci Abstr., 1078 (1987).
s,
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Copyright 0 1989 by Academic Press. Inc. All rights of reproduction in any form resewed.
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6-Philanthotoxin (9, an insect neurotoxin structurally and pharmacologically related to the spider polyamines was found in the venom of the digger wasp Philanthus triangulurn and subsequently assigned the structure 6 (17,18). The philanthotoxin molecule has a spermine nucleus appended to an N-n-butyryltyrosineresidue by an arnide bond.
Chap. 30
Polyamine Spider ?bxlns
Saccomano,Volkmann, Jackson, Parks
H
H!?
289
H
Q
OH
Svnthesis of Polvamine Spider Toxins -The chemistry involved in the preparation of many of the naturally occurring polyamines has been summarized (19). Because polyamine spider venoms have highly polar hydroxyaryl and amino acid residues, synthetic strategy i s dictated by the appropriate selection of chemically compatible basic amine, phenol and amino acid protecting groups. The polyamine backbones are routinely assembled in a stepwise fashion using amine alkylations, reductive aminations and acrylonitrile additions. Standard peptide coupling reactions are employed for the formation o f amide bonds. This general strategy has been utilized for the preparation o f NSTX-3 (lJ (20), JSTX-3 (1)(21), argiotoxin-636 (1)(13,22-24), -659 (A) (13,23), -673 (I)(23), and 8-philanthotoxin (PTX-433) (6) (17,18). Synthesis i s necessary t o unambiguously verify structural assignments and t o provide generous quantities of these and related toxins for continued biological investigations. Polvamines Spider Venoms as Excitatow Amino Acid (EAA) Antagonists - Interest in the polyamine spider toxins is a result of the observation that these molecules affect those synapses at which an excitatory amino acid (glutamate or aspartate) i s the neurotransmitter (vide infra). The invertebrate neuromuscular junction is a well recognized example of an EAA synapse at which functional antagonism will elicit paralysis. In addition, most excitatory synapses in mammalian brain use glutamate or aspartate as neurotransmitters, suggesting that EAA's are intimately involved in brain function (4,5). Moreover, increasingly strong and detailed evidence relating EAA function t o important neurological disorders (e.g. stroke damage and Alzheimer's disease) now exists (25). Compounds that affect EAA function, particularly those that antagonize the action o f such transmitters, are therefore o f considerable agricultural (insect control) and therapeutic interest. Excitatory amino acids produce synaptically-mediated depolarization of neurons by acting on specific receptors. The depolarizing effect produced by activation of excitatory synapses is referred t o as an excitatory postsynaptic potential (EPSP) or excitatory postsynaptic current (EPSC). On the basis o f extensive biochemical, pharmacological and electrophysiological data, three main classes of EAA receptor (named for their selective activation by known agonists) are recognized: N-methyl-D-aspartate receptors (NMDA) and the quisqualate and kainate receptors (both non-NMDA). The NMDA and non-NMDA receptors appear t o have distinct functions (4,s).
-
Pharmacologv of Polvamine Spider Toxins: Invertebrate Nervous Systems Blockade o f neuromuscular transmission in invertebrates by polyamine spider toxins reveals their glutamate antagonist activity (26-28). For example, JSTX-3 (1)blocks the excitatory postsynaptic potential (EPSP) elicited by iontophoretically applied glutamate at synapses i n the lobster neuromuscular junction where an excitatory amino acid i s thought t o be the neurotransmitter. In this preparation, JSTX is reported t o produce an irreversible, voltage-independent blockade at very low concentrations (28). 125I-JSTX blocks neuromuscular transmission in the lobster (29) and binds exclusively t o regions of the sacrolemma opposed t o axon terminals (30). In studies published prior to the synthesis of 2 the JSTX used presumably consisted of a group of related toxins partially purified from extracts of whole venom glands by gel filtration and Sephadex chromatography (26). No data on the biological activities of any other components of Nephila clavata venom, save the single toxin ultimately identified as JSTX-3 (3, have been published. However, given the large number of components in this venom, it is unlikely that the effects attributed t o the non-synthetic JSTX arose from a single toxin (31). The effects of JSTX have been less intensively studied in other invertebrate systems. In the giant synapse o f the squid stellate
290
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ganglion, venom gland extracts block EPSP’s and glutamate-induced depolarization without affecting electrically evoked antidromic (presynaptic) responses (32). It is not clear whether the toxin binds t o the normal attachment site of glutamate on t he receptor molecule or to an external site on the associated ion channel, thereby preventing entry o f ions (28). Therefore, it is uncertain from the available literature whether JSTX produces i t s antagonism o f crustacean neuromuscular transmission via competitive or non-competitive mechanisms. A venom-gland extract from Argiope lobata blocks neuromuscular transmission and the effects of applied glutamate in locust muscle (33). Similar activity was observed i n venoms from other orb-weaving spiders and active fractions were isolated from A. lobata venom by gel chromatography (34). This toxin was further used for purification of glutamate receptors from crab muscle. The affinity-purified fraction was inserted into artificial membranes and shown t o produce pharmacologically specific glutamate-induced ion conductances (35). One of the several active components of Argiope lobata venom was subsequently identified as argiopine 3 (1 5,34). This toxin was determined by electrophysiological methods t o a c t functionally as a postsynaptic open-channel blocker (KD = 10-7M) in blowfly larval muscle (36). Argiope aurantia venom contains t w o distinct classes of toxins that paralyze grasshoppers, cockroaches, flies and moths (37). Five toxins with molecular weights below 1.000 daltons 659 (4J and 673 (5J and toxins above 5000 daltons have been including argiotoxins-636 (3, isolated (13). In housefly paralysis assays, synthetic argiotoxin-659 has an EDSOof 0.9 pmollmg, whereas synthetic argiotoxin-636 exhibits an EDSOof 3.3 pmollmg. In in vitro assays, however, the potencies of the t w o toxins families are similar. The effects o f low molecular weight toxins from the venoms of Argiope trifasciata, Argiope florida and Araneus gemma on EAA-mediated synaptic transmission in the locust retractor unguis muscle have also been studied (38-40). These toxins block neurally evoked muscle twitch, the junctional potential elicited by glutamate iontophoresis, and the voltage-clamped EPSP in a slowly reversing fashion. Single channel studies of locust muscle suggest that the low molecular weight orb-weaver toxins (principally argiotoxins -636, -659 and -673) are very potent noncompetitive antagonists of EAA receptor channel complexes. Their principal effect is a usedependent blockade of open ion channels, although some action on closed channels is probable (41,3). The combination, in orb-weaver venom, of a high concentration of free glutamate, which activates the receptor-associated channels, wi th a potent open-channel non-competitive antagonist t 4 or 3 provides the spider wi th a sophisticated and powerful mechanism for immobi Iizi ng insect prey (42). The effects of 6-philanthotoxin (6J on a variety of insects have been extensively studied (17). This toxin causes reversible paralysis, apparently by blocking quisqualate-sensitive junctional and extra-junctional glutamate receptors on skeletal muscle. The specificity of 6 for a particular subtype of glutamate receptor has not yet been demonstrated. Pharmacoloqv of Polvamine Spider Toxins: Vertebrate Nervous Svstems - Polyamine spider venoms also act on glutaminergic synapses in vertebrate systems. Partially purified JSTX blocks the responses o f CA1 pyramidal neurons i n rat hippocampus both t o stimulation o f t h e appropriate afferent neurons (Schaeffer collaterals) and t o direct application of glutamate (43). 2,4-Dihydroxyphenylacetylasparagine, a common structural f eat ure o f JSTX (2)and inhibits binding of 3H-L-glutamate t o rat membranes with an IC50 of 10-sM argiotoxin-636 (3, (44). JSTX also inhibits uptake of 3H-glutamate i nt o rat brain synaptosomes (45). The physiological actions of JSTX were assessed by recording EAA responses in voltage-clamped single rat hippocampal pyramidal neurons in culture (46). JSTX from partially purified gland extract completely blocked neuronal responses t o quisqualic acid (QA) and kainic acid (KA) in a dose dependent (but use independent manner) a t concentrations o f 10-12 t o 10-8M. Voltage dependent Na+ and K + currents and glycine dependent CI- currents were not affected by the toxin. Because JSTX was ineffective in blocking neuronal responses t o L-aspartate, it appears that this toxin has little antagonist action a t NMDA receptors. In catfish lateral line organ, partially purified JSTX abolishes afferent impulses elicited by focal application of glutamate (47). In studies w i t h t he dogfish retina, purified JSTX
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hyperpolarizes horizontal cells, blocks their response t o light and antagonizes glutamate-induced depolarization of these cells in a slowly reversible manner (48). Interestingly, JSTX acts as an agonist at "on-bipolar" cells in a slowly reversible manner, opening the same ion channels as does glutamate. Thus, Nephila venom appears capable o f exerting distinct and opposite effects at t w o classes of E A A receptor-channel complexes in vertebrates (48). A variety of venoms have been screened for their ability t o block synaptic transmission a t glutaminergic synapses in the chick cochlear nucleus (49). Argiotoxin-659 (A),isolated from Argiope aurantia, produced a readily reversible use-independent blockade o f transmission. Similarly, whole venom from A. trifasciata suppresses both spontaneous and sound-evoked electrical activity in guinea pig cochlear nerve (50). Synthetic argiotoxin-636 (3, -659 (4J and JSTX-3 (2J are functional antagonists o f both NMDA and non-NMDA classes o f glutamate receptor in rat brain. In addition, these materials greatly increase the binding of glycine, apparently by increasing the number (Bmax) o f strychnine-insensitive binding sites (51). Occupation of the strychnine-insensitive glycine binding site (found predominantly i n rat forebrain) appears t o be a prerequisite for activation of the NMDA receptor (52). It i s not yet certain whether modulation of this site by polyamine spider toxins underlies their glutamate antagonist activity. New Directions in Venom Research: Funnel Web Spider Toxins - A number of toxins from the funnel-web spider Agelenopsis aperta paralyze insects (53). These toxins contain low molecular weight acylpolyamine constituents (a - agatoxins), at least six 36-37 amino acid residue peptides (p agatoxins; reportedly sodium channel activators), and several larger polypeptides (oagatoxins; potent irreversible blockers o f synaptic transmission, presumably by action a t presynaptic calcium channels) (54). Other high molecular weight toxins found in the venom of the funnel web spider, Hololena curta, and the primitive hunting spider, Plectreurys tristes, are similar t o the o-agatoxins in inhibiting insect neuromuscular transmission (55,56). A (5Kd) toxin isolated from the venom of Hololena curta produces potent irreversible blockade of synaptic transmission by action on the postsynaptic neuron (50). Antidromic stimulation and responses to E A A agonists are also blocked by this toxin.
-
Two classes o f toxins from Agelenopsis aperta venom that block transmission in the chick cochlear nucleus assay have also been studied (49,57). A multi-component fraction identified as "AG-l", containing polypeptides o f 4-7 Kd irreversibly blocks synaptic transmission at very low doses without affecting antidromic stimulation or postsynaptic responses t o applied EAA agonists. This fraction i s likely t o contain many i f not all of the "p'' and "a"-agatoxins (53). AG-1's action was shown t o be use independent and i s antagonized by extracellular calcium (58). In a dose-dependent manner, AG-1 potently blocks the binding of 1*5I-o-conotoxin (a calcium antagonist peptide from the venom o f the marine snail, Conus geographus) t o rat brain. However, AG-1 markedly increases binding o f the dihydropyridine calcium antagonist 3Hnitrendipine t o brain membranes, apparently by increasing the number of binding sites (59). The second fraction from Agelenopsis aperta contains polyamine toxins of e l Kd, whose structures have not been reported. This low molecular weight fraction, AG-2, suppresses synaptic transmission in the chick cochlear nucleus i n a fully-reversible, dose-dependent and useindependent manner (57). In sharp contrast t o AG-1, AG-2 substantially increases binding of 1251o-conotoxin but decreases the binding of 3H-nitrendipine t o rat brain (59). AG-2, administered intravenously or intracerebroventricullarly, protects rats against otherwise-lethal doses o f the convulsants, kainic acid, picrotoxin or bicuculline. A t doses effective in preventing lethal seizures, AG-2 produces only mild sedation in test animals (60). Conclusion -To date, venoms from only a handful o f the estimated one hundred thousand spider species have been studied in any detail. The results o f these studies already suggest that considerable structural and functional diversity exists within the polyamine toxin class and that these compounds differ significantly from previously-known classes o f EAA and calcium antagonists. For these reasons, it seems likely that spider venoms will continue, for some time, t o provide novel molecules useful for pharmaceutical and pesticide research.
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