Development of Natural Products and Their ... - ACS Publications

Chapter 1

Development of Natural Products and Their Derivatives for Pest Control in the 21st Century Downloaded by UNIV OF NEBRASKA - LINCOLN on April 3, 2015 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0551.ch001

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Paul A. Hedin , Julius J. Menn , and Robert M. Hollingworth 1

Crop Science Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Mississippi State, MS 39762 Plant Sciences Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705 Pesticide Research Center, Michigan State University, East Lansing, MI 48824

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This overview chapter and those that follow explore the emerging technologies for crop protection that are accelerated by the development of new natural pest management agents and materials derived from natural models. We are moving into a new era in which crops w i l l be protected from a broad spectrum of pests including weeds, insects, and diseases by these new biotechnologies. The development of these agents, along with genetically altered pest resistant crops, w i l l provide new means by which crops w i l l be protected from pests i n future years. Also addressed here i s the evolving approach to pesticide discovery, such as computer-aided design and strategies based on understanding of the underlying biochemical target systems. Finally, the monitoring of these products i n the environment, and as a related process, the devising of strategies to regulate them with an appropriate balance between safety and the encouragement of innovation are discussed. Over the past 25 years, there has been much activity directed to chemical work on the isolation and identification of a wide array of biologically active natural products that in some way affect the behavior, development and/or reproduction of pests such as insects, diseases and weeds. However, with regard to crop plants, agronomists, entomologists, and other agricultural scientists have generally depended on traditional methods for selecting resistant varieties with adequate yield properties. Even though various biologically active compounds have been identified, only in a limited percentage of situations was chemical guidance the leading factor i n screening for these properties. Agents such as pheromones, antifeedants, and insect and plant growth regulators, to name a few, have found limited commercial application. In 0097-6156/94/0551-0002$06.00/0 © 1994 American Chemical Society In Natural and Engineered Pest Management Agents; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

Downloaded by UNIV OF NEBRASKA - LINCOLN on April 3, 2015 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0551.ch001

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contrast, synthetic analogues based on the activity of some natural products such as the avermectins have found a wider market. However, the day of biologically active natural products entering the mainstream of crop protection may have cone. Environmental concerns are constricting synthetic chemical pesticide usage. Growing resistance of pests to present pesticides has given added urgency to the search for better, safer cxxrpcunds, and improved delivery systems. The need to treat crops precisely has also provided additional opportunities for the use of natural products. It should be stated, however, that resistance can be expected to develop rapidly to toxic natural products that act on a single target site. Natural Product Pesticides Some biologically active natural products can be considered as bioregulators, the name modeled in part on the plant growth hormones that were shewn to regulate various plant growth and development processes. The levels required for activity of these compounds are often very lew. For example, pheromones are active at picogram levels and below. Insect neurohoromones have been found to be just as active physiologically, and these are just two examples of endogenous bioactive coipounds that may be further exploited in control strategies. The elucidation of the often complex structure and stereochemistry of natural products becomes more and more feasible as access to powerful instrumental techniques rapidly increases. It i s t h r i l l i n g to realize that so much power and capability has been developed, particularly in the past decade. With regard to the pest species that need to be controlled, they can most broadly be classified as those attacking animals, plants, and their products. Those pests that attack animals are often arthropods, usually insects. Other pests of animals are microbes and nematodes. Ihe protection of plants from pests can be divided into at least five categories; control of (1) mammals and other vertebrates, (2) insects and other arthropods, (3) nematodes, (4) diseases, and (5) weeds. While pest control has traditionally been accomplished over the past 50 years with pesticides of synthetic origin, this was not the case prior to the development of synthetic pesticides such as DDT. In recent years, a number of natural products from fermentation such as the avermectins have been finding a niche in the market place, because of lower costs of production and increasing environmental concerns about synthetic chemical pesticides. An effort has also been made to identify those natural products such as azadirachtin, which have been found useful for the control of a broad diversity of pests. Natural products provide leads for the synthesis of pesticides with desirable properties. Often, from an economic standpoint, only synthesis w i l l provide adequate quantities to justify the development of a product. There are a few examples of identification of a natural product that has led to a marketable product. They include the pyrethroids, azadirachtin, the β -methoxyacrylate fungicides, insect pheromones and hormones, and the avermectins.

In Natural and Engineered Pest Management Agents; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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NATURAL AND ENGINEERED PEST MANAGEMENT AGENTS


Peptides and Neuropeptides Among the newer categories of potential pest control agents are the biologically active peptides and neuropeptides that appear to have great promise for control of insects, weeds, and diseases because of their very potent hormone-like activities. Their structures and the genes encoding them are already known i n several instances (1). Early work i n this century suggested a functional relationship between the neural and endocrine systems i n insects. This led to the concept of neurosecretion. Subsequent discoveries made using vertebrate systems illustrated the central role of peptides i n regulating behavior and maintaining homeostasis (2). Breakthroughs in technology, and the continuing discovery of proteinaceous factors in insect neural tissue, and most recently, i n plants which have important physiological activities, stimulated numerous efforts to isolate and identify insect (and plant) peptides (1). Coupled with these elements i s the need to develop alternative insect control and crop protection methods which are environmentally acceptable and economically viable. A l l of these forces have generated great interest i n peptides as leads to insect control agents. Efforts are underway to isolate peptides and their genes, analyze sequences and stnic^ure-function relationships, explore the use of natural vectors and the insertion of constructs and foreign genes into these vectors (particularly viruses). Studies are also i n progress to increase virulence, understand the role of plant protective peptides, to identify and understand the molecular biology of peptide (especially neuropeptide) biogenesis, action and clearance, and to improve delivery. The physiological processes regulated by neuropeptides are extensive i n insects. These neuropeptide-regulated processes have been discovered at an ever-increasing pace and are now being followed by the purification and sequencing of the regulatory agents and their genes (3). Since many neuropeptides control essential l i f e processes, mechanisms for blocking their synthesis or action, or enhancement of their degradation may be necessary for developing pest control agents based on this technology. However, the danger of non-target effects may be high. Investigations into sites of synthesis and release continue to reveal complexity i n the insect muroendocrine system. The rapidly increasing knowledge i n this area suggests that useful prototypes for the design of selective pest control agents w i l l emerge i n the near future. Insects respond to the presence of bacteria i n their body cavity with a combination of hemccyte-mediated sequestration processes and the induced synthesis of several families of bacteria-elicited, hemolymph proteins and peptides (1). Among the antibacterial peptides synthesized by various insect species are the cecropins, diptericins, and defensin-like peptides. The structures of these peptides and genes encoding them, and the specificity and mode of action of the peptides are known (Dunn, P., Purdue University, Personal Communication, 1992). Soluble fragments of bacterial c e l l wall peptidoglycan have been shown to e l i c i t the synthesis of cecropin-like peptides by body fat, other tissues and a c e l l line derived from the tobacco hornworm, Manduca sexta. Antibacterial peptides are also synthesized and released



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into the lumen by the metamorphosing midgut of non-infected M. sexta. This has been interpreted as a prophylactic mechanism protecting the insect from infection during gut restructuring. Venoms of insect predators serve as a vast reservoir of toxins which attack key proteins in excitable membranes. Targets for venom toxins include voltage-gated ion channels and components of the neurotrarismitter excitation-secretion process. Using venom toxins as pharmacological probes, i t has become apparent that pronounced phylogenetic differences exist in these target proteins. Such differences indicate that a high potential exists for development of engineered insect-specific toxins (Adams, Μ., University of California, Riverside, Personal Coirmunication, 1992). Tentoxin i s a cyclic tetrapeptide derived from the fungus Alternaria alternata that causes disruption of chloroplast development i n most of the major weed species in soybean and of johnsongrass in corn while having no effect on either of these crop species. Ihe major diipediment to the development of tentoxin as a herbicide i s i t s limited availability because of low biosynthetic yields. Chemical synthesis i s possible and the production of analogues has led to meaningful structure/activity relationships. To date, no analog has provided greater activity or specificity than the parent molecule. Molecular genetic manipulation i s considered the best strategy for the increase in cost effective production. The success of cloning of the biosynthetic genes may determine the eventual deployment of these alternative biorational pesticides (Lax, A., USDA, New Orleans, LA, Personal Communication, 1992). Some antifungal peptides have sufficiently broad spectrum activity against opportunistic plant pathogens to provide resistance against the soft rot diseases that damage most major crops. Research has focused on peptides that were sufficiently small to be amenable to chemical synthesis of both the peptide and the œrresponding coding sequence for the gene. This attribute permitted the research to start with naturally occurring peptides and then, using principles of rational design, move on to unique peptides (4). Natural and Enaineered Viral Agents Although insects are infected by a number of viruses, members of the family Baculoviridae, the nuclear polyhedrosis viruses, and the granulosis viruses are the most promising candidates for use i n insect population management because of their many desirable characteristics. They are found only in Arthropods, frequently cause devastating epizootics i n natural populations, and infect only the larval feeding stage in lepidoptera normally with lethal effect (5). In addition, the infectious virion i s embedded in a protein body that protects i t to some extent from solar inactivation. The alteration of the viruses through recombinant ΕΝΑ techniques to increase their effectiveness i s a key goal of this line of work. Nuclear polyhedrosis viruses have been extensively researched and are used more often for microbial control of insect pests than any of the other viruses. Their generally high degree of host



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NATURAL AND ENGINEERED PEST MANAGEMENT AGENTS specificity and ability to produce epizootics are among their most favorable attributes. The broader host ranges of a few NPV's offer hope for using a single virus to control several key pests and expands the potential market for these viruses and i s a challenge for mass production (6). The rather rapid inactivation of NPV's by sunlight and the longer time required to k i l l pests, compared with chemical pesticides, are undesirable attributes. In the future, the addition of feeding stimulants, UV protectants, and natural or chemical enhancers to increase infectivity of viruses may make viruses more competitive with synthetic chemicals for insect control. Many insect species are known to be susceptible to one or more isolates of granulosis viruses (7), however they have been sparingly used for insect control. Nevertheless, many of the susceptible species are eœnomically important Lepidcptera, they are family, genus, or species specific, and they are generally non-hazardous to non-target species. In the case of the codling moth, they have been produced in large quantities using in vivo methods. Their use i s compatible with traditional control methods, and they have been genetically engineered for increased potency. Several approaches have been taken to improve insect baculoviruses as biospecific pesticides. Among these i s the deletion of a v i r a l gene, egt, which encodes an enzyme allowing the virus to block the molting of i t s host by inactivating ecdysteroids, insect molting hormones. Insects infected with viruses lacking functional ecrt cause less crop damage because they attempt to molt, gain less weight, and die sooner than insects infected with the wild-type virus. A second iitprovement involves expression of a neurotoxin gene, Tox34. Viruses expressing Tox34 paralyze insects during infection thus limiting crop damage (8). Viral insecticides must be active in formulations that protect the virus from light degradation. The optimum use of v i r a l insecticides in an insect pest management system requires application systems that deliver the product to the target efficiently and eœnomically. Viruses have typically been applied as sprays by a i r or ground equipment using conventional application equipment. However, this equipment was designed for fast acting contact poisons. Since v i r a l "insecticides" are slow-acting stomach poisons (actually infectious agents), current research i s being centered around spray equipment that w i l l selectively target spray droplets and unique delivery systems (9). Design of Pesticides Ihe development of effective compounds for pest control most often evolves from the identification of a biologically active natural product whose structure i s not conducive for synthesis or microbial production. While the approaches to a synthetic compound vary, they normally u t i l i z e some elements of modeling. Examples consist of various structural modifications of "lower-ordered" lead structures including simple exchanges of bioisosteric groupings, and more drastic skeletal changes such as ring openings and closures. These structural modifications which could apply to starting structures are not necessarily limited within cxxrpound



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series shewing similar biological modes of action, but are also sometimes extended to those for bioactive exxnpeund series of similar structures regardless of the differences in biological modes of action. Successful design of novel herbicides based on specific inhibition of selected enzyme targets requires careful consideration of the choice of the target, mechanism of action, design of potent inhibitors, delivery of the inhibitor to the target, and metabolic fate of the inhibitor. Validated targets can be identified by obtaining chemical leads or by genetic methods. Genetic approaches include studies of conditionally lethal bacterial and plant mutants and use of antisense technology. In the absence of chemical leads with known sites of action, targets for chemical validation may be selected by the following criteria: the target i s essential to plants and, preferably, inhibition leads to multiple deleterious effects; the target i s present in plants, but not in mammals; the target has low intracellular concentration, i.e., has potential for low use rates; and the proposed inhibitors of the target are available. Potent inhibition of the selected target may s t i l l not produce an effective herbicide. Studies of the uptake, translocation, and metabolism of the inhibitor are needed to determine i f the cause of poor in vivo performance i s due to these factors or to an intrinsically poor target. Without f u l l appreciation of each of these aspects of herbicide design, the chances for success with the target-site directed approach are reduced (Rendina, A., DuPont de Nemours and Co., Newark, DE, Personal Communication, 1992). Another approach involves some genetic alteration of the plant. For example, the herbicide glyphosate i s currently used for the control of a wide spectrum of weeds. Because glyphosate i s a non-selective, post-emergent herbicide, one limitation of i t s use in a weed management system i s that i t cannot come into contact with normal crop plants during the growing season. Two general methods are available for the development of herbicide-tolerant crops using genetic modification: target-site modification and metabolic inactivation. Using a combination of mutagenesis, X-ray crystallography, and physical methodologies, the glyphosate/phos^onerKDlpyruvate and shikimate~3 -phosphate binding sites of EPSPS have been identified. Glyphosate-tolerant EPSPS's which maintain a high catalytic efficiency have also been identified. In addition, a glyphosate-degrading enzyme has been used to provide in planta tolerance, thus allowing the illustration of both approaches for imparting glyphosate-tolerance to crops (IQ). Another approach involves elucidating the role of substrate conformation in key biological processes. In a model study, the primary step in the biosynthesis of a l l N-linked glycoproteins was found to be catalyzed by the membrane associated enzyme oligc^ccharyl transferase and involved the co-translational transfer of a complex carbohydrate from a pyrophosphate donor to the side-chain nitrogen of an asparagine. The primary peptide sequence requirements for the process are simple and include a minimum -Asn-Xaa-Ser/Ihr- tripeptide recognition motif. This enzyme-catalyzed transformation i s intriguing in that i t represents



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NATURAL AND ENGINEERED PEST MANAGEMENT AGENTS a rare demonstration of nucleophilic behavior at an amide nitrogen, and furthermore, the selectivity i s even more surprising when one considers the potential for competing functionally i n the complex peptidyl substrates i n which the "reactive" asparagine i s localized (11). To provide a convenient means for selective bacterial marking of engineered micrrorgardsms and for semiquantitative bacterial population determination, reversible, metal-based switching sites were introduced into the enzyme, rat pancreatic trypsin. Carefully chosen metal complexes down-regulate enzymatic activity and act as rKDncompetitive, allosteric enzyme inhibitors. The variant trypsins retain good catalytic activity and possess Kj-values for certain copper complexes in the range 0.1-20 yM. Computer-based methods for designing metal-switching properties into enzymes have been developed (Haymore, Β., Monsanto Co., St. Louis, MD, Personal Cctnraunication, 1992). Registration of Biopesticides It i s realized that unless these natural and derived pest management agents, however developed, can be sold i n the marketplace, the underlying research may be viewed by many as having been of no practical consequence. Therefore, along with work directed to their development, parallel efforts to secure approval for their use must also be pursued. The U.S. Environmental Protection Agency (USERA) has encouraged the development of pest management techniques that offer alternatives to chemical control. Many natural products, engineered microbials and viruses, and biologically-derived pest management agents differ significantly from "conventional" chemical pesticides i n their chemistry, complexity, and mode of action. However, such differences are d i f f i c u l t to categorize i n terms of relative risk and there are few regulatory precedents. This has created difficulties for the regulatory community and the decision-making process has sometimes appeared slow. Progress i s being made as more biopesticides are registered. Because certain uses of semiochemicals present comparatively low risk compared with conventional chemicals owing to their target specificity, unique modes of action, and low volume used, the USEPA has exempted these uses from FIERA (Plimmer, J., ABC Laboratories, Columbia, MD, Personal Ccxnmunication, 1992). The public has become increasingly focused on the issue of pollution prevention. The USEPA i s now shifting i t s perspective from being a neutral evaluator to being an active seeker of ways to promote safer approaches to pest control. EPA has begun to realign i t s regulatory programs to promote the development, registration, marketing, and use of safer pesticides, such as the biologicals. These "environmental-friendly" pesticides can be easily divided into two basic groups: naturally-occurring microbials and engineered viruses, and biochemicals and engineered transgenic plants. The registration process for these two pesticide groupings i s governed by different regulatory schemes and policy issues. The new focus on "lower risk" pesticides as well as the loss of •'minor uses" through the re-registration process w i l l result i n greater


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reliance on integrated pest management, which i s USEPA s challenging goal to the pesticide-prOducing community (Lindsay, A., Office of Pesticide Programs, USEPA, Washington, DC, Personal Ocinnunication, 1992). Products developed using the tools of reccmbinant ΕΝΑ technology are subject to special regulations that affect not only the final product approval but a l l phases of f i e l d research. Many products may be subject to regulation by more than one federal agency, and most w i l l be subject to both federal and state controls. Close consulation with regulatory o f f i c i a l s early i n the research and development process i s the key to insuring that research activities are focused not just on product development but also i n developing the data base necessary for i n i t i a l f i e l d testing and for final product approval. Managing the regulatory process involves creating a framework for responding to public concerns raised by individual citizens, public interest groups and the press (Davis, J., Crops Genetics International, Hanover, MD, Personal Communication, 1992). Technologies based on new knowledge of biological ecosystems such as those involving insect pheromones, genetically modified organisms, or suppression of biological populations have been developed by support from the public sector i n response to the demand for alternative pest control methods. They are usually most suited for limited niche markets where commercial sales are limited, or they represent entirely different biological approaches where significant risk must be taken by business before profitable markets are created. These newly developed techniques are regulated by the USEPA under FIERA and USDA's Animal and Plant Health Inspection Service under the Plant Pest Act. However, the regulatory procedures can become an important obstacle which can impede œmmercial development. As these new agents become available, they should be used as part of a management system that w i l l embrace increasingly species-specific agents used very sparingly in an integrated program, and that involves environmental management to minimize the incidence and impact of pests along with the use of a multiplicity of pest control agents. The trend toward species specificity i n pest control agents w i l l select for those that have substantial commercial potential against major economic pests, so that the costs of registration can be recovered through sales. Unless the regulatory process i s relaxed or i s subsidized, the control of moderately severe, sporadic or geographically limited pests w i l l remain the province of broad-spectrum agents. The development of natural and derived pest management agents seems to be a highly desirable strategy for obtaining government approval and public support so that crops, animals, and the public can be protected from pests. In most cases, these new agents have met with much scrutiny, and they hopefully w i l l gain public support because they appear to be environmentally friendly. Literature Cited 1.

Kelly, T. J.; Masler, E. P.; Menn, J . J . In Pesticides and Alternatives: Innovative Chemical and Biological Approaches to


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5. 6. 7. 8. 9. 10.

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NATURAL AND ENGINEERED PEST MANAGEMENT AGENTS Pest Control; Casida, J., Bd.; Elsevier, Amsterdam, 1990, pp 283-297. Thorpe, A.; Duve, H. In Stimulus-Secretion Coupling i n Neuroendocrine Systems. Current Topics i n Neuroendcœinology; Ganten, D.; Pfaff, D., Eds.; Springer-Verlag: New York, NY, 1988, Vol. 9; pp 185-230. Dave, H.; Thorpe, A. In Neurohormones i n Invertebrates; Ihorndyke, M. C.; Goldsworthy, G. J., Eds.; Cambridge University, 1988; pp 115-139. Soravia, E. S.; Martini, G.; Zasloff, M. F.E.B.S. Lett. 1988, 288. 337. Vaughn, J . L.; Dougherty, Ε. M. In Viral Insecticides for Biological Control; Maramorosch, K.; Sherman, Κ. E., Eds.; Academic Press, Inc.: Orlando, FL, 1985, pp 569-633. Harper, J . D. In Epizootiology of Insect Diseases; Fuxa, J. R.; Tanada, Υ., Eds.; John Wiley and Sons, Inc.: New York, NY, 1987, pp 473-496. Crook, Ν. E. In Viruses of Invertebrates; Kurstak, Ε., Ed.; Marcel Dekker, Inc.: New York, NY, 1991, pp 73-110. Tomalski, M. D.; Miller, L. K. Nature. 1991, 352, 82. Groner, A. In Safety of Microbial Insecticides; Laid, M.; Lacey, L. A.; Davidson, E. W., Eds.; CRC Press: Boca Raton, FL, 1989, pp 135-148. Barry, G.; Kishore, G.; Padgette, S.; Taylor, M.; Kblacz, K.; Weldon, M.; Re, D.; Elchholtz, D.; Fincher, K.; Hallas, L. In Biosynthesis and Molecular Regulation of Amino Acids i n Plants; Singh, B. K.; Flores, Η. Ε.; Shannon, J . C., Eds.; American Society of Plant Physiologists: 1992, pp 139-145. Imperiali, B.; Shannon, K. L.; Rickert, K. W. J . Am. Chem. Soc.. 1992, 114, pp 7942-7944.

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