Structure-Function Relations of Natural Toxins and Nerve Agents: An

Chapter 1

Structure-Function Relations of Natural Toxins and Nerve Agents: An Overview 1

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Anthony T. T u and William Gaffield 1

Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870 Western Regional Research Center, U.S. Department of Agriculture, Albany, CA 94710

Downloaded by 60.240.224.88 on November 29, 2015 | http://pubs.acs.org Publication Date: December 20, 1999 | doi: 10.1021/bk-2000-0745.ch001

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Nature has endowed mankind with sources of an extraordinarilyricharray of complex molecular structures and assemblies possessing functions that range from gene regulation and signal transduction to photosynthesis and protein biosynthesis (1). The creation of diverse collections of chemical structures followed by critical selection of specific molecules exhibiting a desired biological property allows the evolution of new molecular functions. As the structures and molecular mechanisms involved in the biological processes are elaborated and understood, the insights gleaned, as well as the processes themselves, can be combined with the tools of the physical and biological sciences to create new and novel molecules that incorporate functions found neither in Nature or the laboratory. Inspection of the Bioactive Natural Products Database shows that the origin of approximately 30,000 biologically active natural products is derivedfromfour primary kingdoms: bacteria (33%), plants (27%), fungi (26%), and animals (13%) (2). Recent years have witnessed the isolation of increased numbers of bioactive compounds from fungal and marine organisms; however, the potential for the discovery of new natural products is farfromexhausted and compounds of natural origin continue to represent a key source for the discovery of important leads. Furthermore, although combinatorial chemistry and high-throughput screening are the methods of choice for lead-structure optimization, a combinatorial library will not provide the structural diversity inherent in a library generatedfromnatural products. As an example, natural products possessing physiological properties such as those of constituents of snake, spider, and scorpion venoms, can assist in creating targeted libraries of potential drug candidates (3). Venom peptides and proteins that act

©2000 American Chemical Society

In Natural and Selected Synthetic Toxins; Tu, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1999.

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upon mammalian physiological processes are extremely site-specific in their actions and this specificity is highly valuable in performing as a lead therapeutic or biological probe. Thus, although a protein isolated and purified from snake venom might be therapeutically useful, more likely it will serve as a lead for drug development. Alternatively, a venom protein can function as a biological probe that delineates the molecular surface of its receptor binding site, thus providing key insights that enable the design and development of simpler molecules that mimic the desired receptor site and have the potential to serve as drugs. The biological implications of natural toxins that are derived from marine, fungal, microbial, plant, and animal sources, along with selected synthetic nerve gases and doping agents, are described in this ACS Symposium Series volume under four subject headings (for earlier natural toxin symposia, see ref. 4,5). Although the nerve gases and doping agents are not naturally occurring, their development was stimulated by the goal of preparing large quantities of inexpensive substances that have toxic or enhanced performance properties of potency and selectivity greater than those of naturally occurring materials. Marine, Fungal, and Microbial Toxins Certain marine species contain deadly toxins; the puffer fish, which is a Japanese culinary delicacy, accumulates the potent neurotoxin tetrodotoxinfromdietary dinoflagellates who, in turn, obtain the toxinfrommarine bacteria whereas corals of the Palythoa genus contain palytoxin, a toxin that depolarizes excitable tissue and ruptures blood cells. Extracts from these corals were employed by natives of the Hawaiian Islands for hunting and warfare; however, the relative inaccessibility of marine organisms limited their applicability, resulting in greater use of amphibian and other terrestrial poisons in hunting by early cultures. A coral reef is a treasure chest of novel marine organisms, both toxic and benign. T. Higa (Japan) describes the isolation and characterization of numerous cytotoxic and bioactive compounds from coral reef sponges that are prime candidates for anti-tumor drugs and immunostimulatory compounds. Pardaxin is a unique substance releasedfroma Red Sea flatfish that repels a predatory shark. P.H. Lazarovici (Israel) reports the cellular and molecular mechanism of pardaxin effects on cellular signaling. Serious contamination of foodstuffs often results from exposure to fungi and their constituent mycotoxins. A notorious example is the fungus Claviceps purpurea that produces ergot alkaloids which can contaminate rye and other cereal grains resulting in induction of ergotism in consumers. Other fimgal toxins are more insidious, inducing liver or gastrointestinal tumors over a period of years. Prominent among the latter toxins are the aflatoxins which were originally identified in 1960 following the deaths of thousands of turkeys. No direct evidence exists that liver disease in humans can be caused by ingestion of aflatoxins although a high incidence of liver cancer has occurred in



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portions of Africa where large amounts of aflatoxins are consumed by a population often afflicted with hepatitis. In addition to presenting a small, albeit real, long-term hazard to mankind, mycotoxins offer the potential for inducing fetal abnormalities as demonstrated in laboratory rodents. R. J. Molyneux (US) describes isolation and characterization of anti-aflatoxigenic constituents of tree nuts, such as pistachios, almonds, and walnuts. The perception of extreme hazards due to aflatoxin contamination has been exacerbated recently by reports that Iraq has placed die mycotoxin in ballistic warheads and bombs. These missiles are speculated to serve as potential sources of economic and political intimidation through the possible dispersal and contamination of food crops that become either non-consumable or unexportable. Current research on the newest class of mycotoxins, the fumonisins, is reported by W. T. Shier (US). His research suggests that the most abundant fumonisin, FBi, binds covalently to protein and starch during processing of foods in a manner that may allow the release of a biologically active form of the toxin upon digestion.

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3

C0 H 2

FUMONISIN B i The occurrence of pathogenic bacteria in diverse environmental, food processing, and industrial sources has necessitated a sensitive and selective assay for their detection and identification. T. Krishnamurthy (US) has distinguished a number of pathogenic bacteria from their non-pathogenic counterparts by the application of either electrospray ionization or matrix assisted laser desorption ionization mass spectroscopy. Plant Toxins Primitive cultures differentiated edible from poisonous plants by a process of trial and error. After appropriate experimentation, they began to exploit the naturally


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occurring toxins for materials useful in hunting, euthanasia, and murder. From the perspective of the plant, toxins are produced presumably for the purpose of deterring predatory herbivores. Pyrrolizidine alkaloids (e.g., diester derivatives of retronecine) are potent hepatotoxins and carcinogens isolated from a variety of plants that at low levels can cause genetic damage, cirrhosis, and possibly cancer. The mode of action of these toxic alkaloids occurs via a cytochrome P-450 mediated oxidation which produces a highly active intermediate. Subsequently, the intermediate dehydropyrrolizidine mediates the interstrand cross-linking of DNA by forming a bis-alkylated adduct. R. J. Huxtable (US) reports that human exposure to pyrrolizidine alkaloids in the United States occurs primarily through intentional consumption of herbs thought to provide health benefits. Thus, although comfrey has been shown to contain dangerous levels of toxic pyrrolizidine alkaloids, the herb is still available in some US health-food stores. J. A. Edgar (Australia) states that even though pyrrolizidine alkaloids do not normally occur in plants consumed by humans, they may enter the human food chain from foodproducing animals that eat plants containing the alkaloids. For example, humans may be exposed potentially to pyrrolizidine alkaloids upon consuming eggs, milk, or honey.

RETRONECINE

A number of polyhydroxylated indolizidine alkaloids that resemble natural carbohydrates but which incorporate a nitrogen atom in place of an oxygen ring atom, were observed to function as glycosidase inhibitors,i.e., they interfere with the functioning of certain glycosidases that assist in the removal or cleavage of carbohydrate residuesfromglycoproteins. A related group of «ortropane alkaloids, the calystegines, has been identified by R. J. Nash (UK) in various fruits and vegetables. In particular, both the occurrence of calystegines (e.g., calystegine B 2 ) in potatoes and the likelihood that their effects would be expressed as gastrointestinal distress and neurological disorders suggests that the calystegines might account for certain toxicological aspects normally attributed to steroidal alkaloid glycosides. Several known and new alkaloids of varied structures have been characterized from Australian pasture grasses by S. M. Colegate (Australia) that might be responsible for outbreaks of neurological and sudden death intoxication in range animals.


5

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CALYSTEGINEB2 Mankind has long been interested in plant extracts that control fertility and either induce abortion or labor depending upon the period since conception. K. E. Panter (US) describes natural toxins from poisonous range plants that affect reproductive function in livestock. His review focuses on three groups of plants that contain specific classes of toxins affecting different aspects of reproduction; locoweeds from Astragalus and Oxytropis, labdane resin acids, e.g., the abortifacient isocupressic acid,fromPonderosa pine, and quinolizidine and piperidine alkaloidsfromLupinus spp. The Greek philosopher Dioscorides recommended a decoction known as 'abortion wine' that contained a Veratrum sp. as one of several ingredients. Several Western US plains and mountain Native American tribes employed a decoction preparedfromthe roots of Veratrum californicum as a contraceptive rather than as an abortifacient. In the 1960s, the steroidal alkaloids jervine and its 11-deoxo analog, cydopamine, were shown to be responsible for induction of cyclopia and related congenital malformations in sheep that had ingested V, californicwn on a specific day of their gestation. W. Gaffield (US) reports recent molecular biological research that establishes cyclopamine's selective and potent inhibition of Sonic hedgehog signaling. This pathway is responsible for critical aspects of neural tube development that upon disruption will result in cyclopia and other craniofacial defects.

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Fifteenth century explorers were astonished to observe Cuban natives inserting smouldering leavesfromtobacco plants into their nostrils and mouths. Five centuries later, nearly 500,000 Americans die annually from smoking related afflictions and approximately 60% of all hospitalizations are attributed to illnesses related to smoking. K. Stone (US) describes the DNA damaging properties of stable free radicals derived from cigarette tar extracts. Ricin possesses a sinister history of usage exemplified by its presumed administration to an unsuspecting victim via a needle-tipped umbrella in a highly publicized international espionage incident of the 1970s. R. G. Wiley (US) describes the application of ricin and related toxic plant lectins as tools in molecular neurosurgery for producing highly selective neural lesions. Sheep and goats in various regions of the world are susceptible to seasonal hepatogenous photosensitization. The photosensitivity is caused by accumulation of a photodynamic compound, phylloerythrin, a porphyrin derived from chlorophyll degradation, whose excretion is prevented by certain types of liver damage. A. Flâoyen (Norway) discusses several plant-derived natural toxins that cause liver damage or dysfunction in livestock resulting in retention of phylloerythrin.

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PHYLLOERYTHRIN



Animal Toxins

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A comparison of highly poisonous substances reveals that amphibian and reptile toxins are among Nature's most potent. Snake envenomation is often fatal to a victim; thus, snake venom has been feared since the beginning of human history. However, humans were puzzled as to how such small amounts of venom could exert such powerful effects on biological systems. Toxins derived from poisonous frogs have a fascinating ethnopharmacological history and were used by primitive cultures as arrow poisons. Studies of the mode of action of these poisons have contributed immensely to our understanding of neuropharmacology. Snake venom toxins are effective tools in medical applications as demonstrated by the development of angiotensin-converting enzyme (ACE) inhibitors starting from a component of venom. Other research has employed snake venoms in the development of platelet aggregation and blood clotting inhibitors. Recently, scientists have been engaged in more sophisticated investigations targeted at delineating the molecular basis of toxic action. The secondary structure of snake venom cardiotoxins as revealed by nuclear magnetic spectroscopy is discussed by T. K. S. Kumar (Taiwan) and the crystal structure of snake venom phospholipase A, that possesses presynaptic toxicity, is described by Z. Lin (China). Illustrating the potential beneficial effects of advanced technology in elaborating the mechanism of snake venom constituents, F. S. Markland (US) reports the application of disintegrin in blocking the adhesion of human breast cancer cells to several extracellular matrix proteins. Thus, disintegrin, isolated from the venom of the Southern Copperhead snake, inhibits in vivo progression by a combination of anti-tumor and anti-angiogenic activities. Among all lizards, only the species of Heloderma (Gila monster) is venomous. The Gila monster produces a venom approximately as toxic as that of the Western Diamondback rattlesnake. A. T. Tu (US) reports the chemical and functional aspects of Gila monster toxins, with particular emphasis on toxic enzymes.

Nerve Agents and Doping Compounds The chemical warfare agents, commonly known as nerve gases, are not gases but polar organic liquids at ambient conditions. Most of these nerve gases are esters similar in structure to organophosphorus insecticides that irreversibly react with the enzyme acetylcholinesterase (AChE), inhibiting its control over the nervous system. For example, tabun, a G-type toxin, was discovered in 1936 by a German scientist involved



8 with developing synthetic insecticides. Later before the end of World War II, Germans developed more powerful nerve agents sarin and soman. A single milligram of nerve agent on the skin is sufficient to cause death; fortunately, the G-agents were not used in World War H. In the 1950s, the V-type nerve gases were developed, such as V-X, that are more toxic and persistent. Thousands of tons of these compounds were stockpiled by major countries over the intervening decades which has created current safety and environmental impact concerns with regard to their detoxification and destruction. In 1995, a terrorist group dispersed sarin in a Tokyo subway station resulting in the death of 3 people and the illness of several thousand commuters. One year earlier, the same sect had murdered one of its own members with the deadly nerve gas, V-X. Five chapters of this book describe the implications of these incidents. An overview of the chemical terrorism attacks in Japan is provided by A. T. Tu (US). Additional chapters by Y. Seto (Japan), Y. Ogawa (Japan), T. Okumura (Japan), and H. Tsuchihashi (Japan) describe the biological effects of both sarin and V-X on human victims. Nerve gases not only may be analyzed directly but, in addition, generate unique metabolites in human blood that can provide conclusive evidence of the specific nerve agent administered.

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SARIN

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V-X

Mild stimulants such as tea and coffee are primarily of social importance in modern times while more potent stimulants such as cocaine and amphetamines are substances of abuse. In primitive cultures, stimulants were more highly prized for their ability to extend endurance and alleviate hunger than for social or mind-altering properties. R.K. Mueller (Germany) discusses state-of-the-art analytical methodology currently employed to monitor modern athletes, some of whom occasionally ingest controlled substances in order to achieve enhanced athletic performance. Literature Cited 1.

Mann, J. Murder, Magic, and Medicine; Oxford University Press: New York, 1994.


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Bioactive Natural Product Database, Szenzor Management Consulting Company, Budapest, Hungary, 1996.

3.

Berressem, P. Chem. Brit. 1999,35,40.

4.

Natural Toxins: Toxicology, Chemistry, and Safety; Keeler, R.F.;Mandava, N.B.; Tu, A.T., Eds.; Alaken, Inc.: Fort Collins CO, 1992

5.

Natural Toxins 2: Structure, Mechanism of Action, and Detection; Singh, B.R.; Tu, A.T., Eds.; Plenum Press: New York NY, 1996


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Structure-Function Relations of Natural Toxins and Nerve Agents: An

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