New Herbicide Target Sites from Natural Compounds - American

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New Herbicide Target Sites from Natural Compounds Stephen O. Duke, Franck Ε. Dayan, Isabelle A. Kagan, and Scott R. Baerson NPURU, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 8048, University, MS 38677

Introduction

The relatively weak effort to use natural compounds as pesticide leads has led to several commercial herbicides, including glufosinate, bialaphos, the triketones, and pelargonic acid. Furthermore, natural phytotoxins have often been found to have molecular target sites that were previously unknown as viable sites for herbicides. Commercially available herbicides target only about 20 enzymes or energy transfer processes. Even if the natural compound is not a competitive herbicide, the knowledge of a new target site can be used to develop new synthetic herbicides with novel modes of action (1). Discovery of new herbicide target sites is even more important since the U.S. Food Quality Protection Act combined food tolerance levels of pesticides with the same molecular target sites. Natural products have not been an area of focus for discovery efforts because of the relatively high cost of bioassay-directed isolation, the probability of rediscovery, the often prohibitively complex structure of the active molecule, and the often more uncertain legal aspects of a patent. Furthermore, the approaches for discovery of natural product-based pesticide discovery are different from those for synthetic compounds, requiring a different laboratory infrastructure, including emphasis on natural product chemistry, rather than

U.S. government woik. Published 2005 American Chemical Society In New Discoveries in Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

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152 synthetic chemistry. The time and effort required for natural product-based herbicide discovery programs have been reduced by new methods, including semi-automated bioassays, automated chemical extraction and analysis, and molecular biology methods to probe biosynthesis and mode of action. We will focus on our own work in this area in this short review.

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New Target Sites Inhibition of the fructose-1,6-bisphosphate aldolase (FBPase; EC 4.1.2.13) step of glycolysis is known to have long-ranging repercussions on the physiology of plants, leading to reduced photosynthetic efficiency, impaired sugar and starch metabolism, and ultimately to reduced growth (2). Efforts have been made to identify inhibitors of animal FBPase to develop novel pharmaceutical compounds. Although this enzyme will probably not be developed as a potential herbicide target site because it is too ubiquitous, our studies have shown that some natural phytotoxins may target this enzyme. The fructose analogue 2,5-anhydro-D-glucitol (AhG) produced by the plant pathogen Fusarium solani (Mart.) Sacc. NRRL 18883 is phytotoxic (3) inhibiting lettuce root growth with a / of 1.6 mM. The mechanism of action of this sugar analog is relatively complex, involving its sequential bisphosphorylation by hexokinase and phosphofructokinase to yield AhG-1,6bisphosphate (Figure 1). This phosphorylated sugar analogue is a competitive inhibitor of FBPase (7 o of 570 μΜ and value of 103 μΜ) (4). The catalytic mechanism of FBPase involves the formation of a covalent bond between the anomeric hydroxyl group of FPB and the gamma amino functionality of a lysine residue of the enzyme. AhG (and AhG-1,6-bisphosphate) do not have this anomeric hydroxyl group, preventing die normal catalytic function of aldolase. Ceramide synthase biosynthesis is another potential target site. Ceramides are essential lipids in plants and animals. They are particularly important to the integrity of nervous system cells and tissues of animals. Their function in plants is not well understood, although they are significant constituents of the plasma membrane, and inhibition of their synthesis causes rapid cell death. The fumonisins, potent inhibitors of ceramide synthase, are highly phytotoxic (5,d), causing rapid cellular leakage and cell death at low (submicromolar) concentrations. AAL-toxin, a plant pathogen-produced phytotoxin and structural analogue of the fumonisins, produces physiological effects on plants similar to those of the fumonisins (7). Fumonisins and AAL-toxin, both structural 9

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In New Discoveries in Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

153 analogues of sphinganine, cause rapid and dramatic increases in phytosphingosine and sphinganine in treated plants (8). These ceramide precursors accumulate when ceramide synthase is inhibited (Figure 2). AhG

Glucose

ATP HEXOKINASE

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ADP

«A Glucose-6-phosphate

AhG-P

PHOSPHOGLUCOSE ISOMERASE

Fructose-6-phosphate

ATP I

PHOSPHOFRUCTOKINASE

ΔΠΡ ADP



Fructose-1,6-bisphosphate x

ALDOLASE G-3-P

+ DHAP

Figure 1. Schematic of the mode of action of AhG with the normal glycolytic pathway on the right (solid arrows) and the consecutive bioactivation of AhG into AhG-BP and the ultimate inhibition of the target site on the left (dashed arrows). Symptoms of ceramide synthase inhibition could be caused by depletion of ceramide and ceramide derivatives or by toxic increases in sphinganine and its derivatives. The effects of these phytotoxins are so rapid, that it is unlikely that depletion of ceramides is responsible for cellular death. Others have invoked induction of apoptosis in the mode of action of these compounds (e.g., 9); however, treatment of plant tissues with phytosphingosine and sphinganine causes symptoms very similar to those caused by inhibition of ceramide synthase (10). The very rapid plasma membrane damage at one micromolar and higher

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concentrations makes it unlikely that apotosis is involved in the main phytotoxic effect. The ceramide synthase pathway is clearly a viable site for herbicides, provided an inhibitor can be found that is plant specific. We have been unsuccessful in finding a ceramide synthase inhibitor with high phytotoxicity, but low mammalian toxicity (e.g., 11). Even inhibitors with little structural similarity to the fumonisins, such as australifungin (12), have relatively little difference between mammalian and plant toxicity. This topic is considered in more detail in a recent review (13). Palmitoyl-CoA + Serine

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Apoptosis?

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fumonisins AAL-toxin australifungin

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sphinganine'

phytosphingosine

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ceramide

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ceramide derivatives

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Figure 2. Mechanism of action of ceramide synthase inhibitors in plants. Thick lines indicate increased level effects, whereas thin lines represent decreased level effects. We have found that the key enzyme in asparagine synthesis, asparagine synthetase (AS; EC6.3.5.4), is yet another potential herbicide target site. AS was hypothesized as a potential herbicide target site when it was discovered that the inhibition of growth caused by natural phytotoxin 1,4-cineole (or its synthetic analogue cinmethylin) could be reversed with exogenous supply of asparagine (14). Root uptake of asparagine increased in the presence of the inhibitors, suggesting that the supply of asparagine was low in the treated seedlings. Uptake of aspartate was not affected by the treatments. The in vitro AS activity was inhibited by 1,4-cineole (Figure 3A). The synthetic analogue cinmethylin did not inhibit AS, suggesting that it must be metabolized into a 1,4-cineole analogue, ostensibly 2-hydroxy- 1,4-cineole, prior to inhibiting AS activity (Figure 3B).

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155 This hypothesis was tested by measuring the inhibitory activity of cis- and trans2-hydroxy- 1,4-cineole. The cis-form was more effective against AS than 1,4cineole by more than an order of magnitde (Figure 3). The addition of the hydroxyl group to the molecule renders it less volatile, therefore probably allowing more of the compound to react with the site of inhibition. The transform of 2-hydroxy-1,4-cineole was less active than either the cis-diastereomer or 1,4-cineole (not shown). Greenhouse studies had demonstrated that the cis-form of cinmethylin was more active than its trans-diastereomer, further supporting the current hypothesis that the orientation of the hydroxyl group may play a key role in the potency of this metabolic intermediate.

Figure 3. A. Effect of 1,4-cineole (circles), cis-2-hydroxy-1,4-cineole (triangles) and the commercial herbicide cinmethylin (squares) on the activity of asparagine synthetase from lupin. The dotted line represents 50% inhibition of enzyme activity. B. Scheme for proposed mode of action of cinmethylin. This hypothesis was tested by measuring the inhibitory activity of cis- and trans2-hydroxy-1,4-cineole. The cw-form was more effective against AS than 1,4cineole by more than an order of magnitde (Figure 3). The addition of the hydroxyl group to the molecule renders it less volatile, therefore probably allowing more of the compound to react with the site of inhibition. The transform of 2-hydroxy-1,4-cineole was less active than either the cis-diastereomer or 1,4-cineole (not shown). Greenhouse studies had demonstrated that the cis-form of cinmethylin was more active than its trans-diastereomer, further supporting the current hypothesis that the orientation of the hydroxyl group may play a key role in the potency of this metabolic intermediate.

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Old Target Sites Several natural phytotoxins have been found by us and others to target the same molecular target site as a class of commercial, synthetic herbicides. In one case below, the compound targets two herbicide target sites at low doses, something that no commercial herbicide does. Thus, even when natural products target old sites, they may have utility as new herbicides or herbicide classes for multiple target sites. They could also be used as allelochemicals that could be genetically engineered into crops. The allelochemical sorgoleone (2-hydroxy-5-methoxy-3-[(8\Z,-ll'^- 8 Ί 1 Ί 4 * pentadecatriene]-p-benzoquinone; Figure 4) is exuded in oily droplets from the root hairs of Sorghum species. These droplets are up to 90% sorgoleone and its analogues. It is relatively stable in soil and accumulates in the rhizosphere. This molecule is highly phytotoxic to broadleaf and grass weeds at concentrations as low as 10 μΜ (15), and its presence in the soil surrounding sorghum plants inhibits the growth of weeds. Sorgoleone was initially found to inhibit mitochondrial respiration, but it was later found to be a more potent inhibitor of photosynthetic electron transport of photosystem II (PSII) (15, 16). Sorgoleone is structurally similar to plastoquinone (PQ), a benzoquinone involved in photosynthetic electron transport. Sorgoleone competes for the PQ binding site of the D - l protein in a manner similar to most commercial photosynthetic inhibitors (17). The in vitro PSII inhibiting activity of sorgoleone is similar to some of the commercial herbicides targeting this site (e.g., atrazine and diuron). Sorgoleone and other natural quinones can inhibit the enzyme phydroxyphenylpyruvate dioxygenase (HPPD) (Figure 4), the key enzyme in plastoquinone synthesis (18). In addition to acting as a redox reagent in PSII, plastoquinone is a cofactor for phytoene desaturase, the target of many carotenoid synthesis inhibiting herbicides (19). Without plastoquinone, phytoene desaturase does not function, resulting in cessation of carotenoid synthesis. Most commercial HPPD inhibitors (e.g., sulcotrione (Figure 4) and isoxaflutole) are competitive, time-dependent (tight-binding) inhibitors. As such, these herbicides bind to the enzyme very tightly with t of dissociation ranging from a few hours to several days, as opposed to milliseconds for traditional reversible inhibitors. Sorgoleone does not behave as these herbicides and appears to be a reversible inhibitor of HPPD. This quinone is structurally more planar than the traditional HPPD inhibitors, so it may not form a stable tightlybinding reaction intermediate. Instead, its backbone may resemble the conformation of one of the later intermediate step in the reaction mechanism of HPPD. Usnic acid [2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyl-l,3(2H9pH)dibenzofurandione] (Figure 4) is one of the most common secondary metabolites Vi

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157 found in the lichen genus Usnea. Seedlings grown in the presence of usnic acid develop chlorosis in the cotyledonary tissues. Loss of chlorophylls in response to phytotoxins can be associated with light-dependent destabilization of cellular and sub-cellular membranes, but usnic acid apparently acts differently since it causes membrane leakage in the absence of light, though the loss of membrane integrity is increased in light. There is a strong decrease in β-carotene in plants treated with usnic acid, and total carotenoids decrease with increased usnic acid concentration. Inhibition of carotenoid synthesis is known to be accompanied with the destruction of chlorophyll (bleaching) due to the destabilization of the photosynthetic apparatus.

Concentration (μΜ)

Figure 4. Effect of the jS-triketone (-)-usnic acid (circles), the benzoquinone sorgoleone (triangles) and the commercial herbicide sulcotrione (squares) on the activity of p-hydroxyphenylpyruvate dioxygenase. The dotted line represents 50% inhibition of enzyme activity. Although most herbicides that cause normally green tissues to be white target the enzyme phytoene desaturase that converts phytoene to carotenes, this symptom is also associated with inhibition of HPPD, the enzyme responsible for plastoquinone biosynthesis (20). Usnic acid possesses a 2-keto-cyclohexane1,3-dione substructure common to many triketone HPPD inhibitors (e.g., sulcotrione and mesotrione) (21). When tested on this enzyme, usnic acid is strongly inhibitory, with an / of 70 nM, surpassing the activity obtained with the commercial herbicide sulcotrione (Figure 4) (22). Despite high in vitro activity, usnic acid was poorly active on 3-week-old morningglory, barnyardgrass, velvetleaf, sicklepod, and yellow nutsedge seedlings in a preliminary greenhouse study. The discrepancy between the high in vitro activity on HPPD and the low in vivo activity is most likely due to poor 50

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158 foliar uptake associated with inadequate physicochemical properties of usnic acid.

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New Methods for Target Site Determination Molecular biology is having profound effects on the pesticide industry. In addition to its use in producing herbicide-resistant crops, improved biocontrol agents, and more allelopathic or competitive crops (23), it can be used in both the search for new molecular target sites and the elucidation of modes of action of natural phytotoxins. Using DNA microarray technology for transcriptional profiling is a potentially effective means for elucidating inhibitor modes of action. Theoretically, specific patterns of up- and down-regulated genes might be associated with particular molecular target sites. This could allow the researcher to quickly determine if a new phytotoxin fits a particular transcriptional pattern associated with a target site from a gene transcription library of profiles of compounds with known modes of action. If the results do not fit one of these profiles, the phytotoxin may target a new molecular site, and its effects on gene transcription could provide clues as to what that target is. In addition to genes specifically associated with the molecular target site, stress-related and detoxificationassociated genes are likely to be found associated non-specifically in response to exposure to toxic xenobiotics. Separation of target site-specific effects on transcription from effects of these less specific, general toxicant-related genes will simplify this potentially very complicated tool. Another issue has been determination of proper doses and time points for sampling. The later the data are taken, the more genes unrelated to the primary target site are affected. With plants, an issue is whether to use whole plants or cell cultures. In whole plants, herbicides sometime target only cells of certain tissues, so that whole plant extaction of RNA dilutes the RNA of interest. Uniform and rapid uptake of the phytotoxin is also problematic with whole plants. We are in the early stages of building gene expression profile libraries for both fungicides and herbicides, using whole genome DNA microarrays for Saccharomyces cerevisiae and Arabidopsis thaliana, respectively. When generating data on tens of thousands of genes, ordinary statistics will provide hundreds of false positives and negatives (24). Experiments must be well designed and executed to provide robust transcriptional profile fingerprints. In many cases, microarray results for genes of interest must be verified with quantitative real-time RT-PCR (e.g., 25)

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With Saccharomyces cerevisiae microarrays, we have found good gene transcription fingerprints for certain molecular target sites. Results with Arabidopsis thaliana as a model for studying herbicidal modes of action have thus far been encouraging, although our studies with this organism are less developed than with S. cerevisiae. Current experiments should allow us to distinguish between gene expression changes that occur in direct response to the presence of a specific inhibitor, versus those that occur indirectly or due to stress.

Conclusions Powerful, new tools and approaches to natural product discovery and utilization are rapidly expanding our knowledge of chemical diversity, the biological activity, and the molecular genetics of naturally occurring compounds. Many of these compounds are phytotoxic and have the potential for use in weed management, either as a herbicide or as a genetically engineered allelochemical. But, still, compared to the synthetic herbicide discovery and herbicide-resistant crop development efforts, little has been done with natural products. This knowledge gap and the availability of new capabilities should attract future researchers to focus on discovery and development of natural compounds for weed management.

References 1. Duke, S.O.; Dayan, F.E.; Rimando, A . M . In Herbicides and Their Mechanisms ofAction; Cobb, A.H.; Kirkwood, R.C. Eds.; Sheffield Academic Press, Ltd., Sheffield, UK, 2000, pp. 105-133. 2. Haake, V.; Zrenner, R.; Sonnewald, U.; Stitt, M. Plant J. 1998, 14, 147-157. 3. Tanaka, T.; Hanato, K.; Watanabe, M; Abbas, H.K. J.Nat. Toxins 1996, 5, 317-329. 4. Dayan, F.E.; Rimando, A . M . ; Tellez, M.R.; Scheffler, B.E.; Roy, T.; Abbas, H.K.; Duke, S.O. Z. Naturforsch. 2002, 57c: 645-653, 5. Abbas, H.K.; Boyette, C.D. Weed Technol. 1992, 6, 548-543. 6. Abbas, H.K.; Paul, R.N.; Boyette, C.D.; Duke, S.O.; Vesonder, R.F. Can. J. Bot. 1992, 70, 1824-1833. 7. Abbas, H.K.; Vesonder, R.F.; Boyette, C.D.; Peterson, S.W. Can. J. Bot. 1993, 71, 155-160.

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160 8. Abbas, H.K.; Tanaka, T.; Duke, S.O.; Porter, J.K.; Wray, E.M.; Hodges, L.; Sessions, A.E.; Merrill, A.H.; Riley, R.T. Plant Physiol. 1994, 106, 10851093. 9. Gilchrist, D.G. Cell Death Differ. 1997, 4, 689-698. 10. Tanaka, T.; Abbas, H.K.; Duke, S.O. Phytochemistry 1993, 33, 779-785. 11. Abbas, H.K.; Tanaka, T.; Shier, W.T. Phytochemistry 1995, 35, 16811689. 12. Abbas, H.K.; Duke, S.O.; Merrill, A.H.; Want, W.; Shier, W.T. Phytochemistry 1998, 47, 1509-1514. 13. Abbas, H.K.; Duke, S.O.; Shier, W.T.; Duke, M.V. In Advances in Microbial Toxin Research and its Biotechnological Exploitation; Upadhyay, R.K., Ed.; Kluwer, Amsterdam, 2002, pp. 211-229. 14. Romagni, J.G.; Duke, S.O.; Dayan, F.E. Plant Physiol., 2000, 123, 725-732. 15. Nimbal, C.I.; Yerkes, C.N.; Weston, L.A.; Weller, S.C. Pestic. Biochem. Physiol. 1996, 54, 73-83. 16. Einhellig, F.A.; Rasmussen, J.A.; Hejl, A . M . ; Souza, I.F. J. Chem. Ecol. 1993, 19, 369-375. 17. Gonzalez, V . M . ; Kazimir, J.; Nimbai, C.I.; Weston, L.A.; Cheniae, G.M. J. Agric. Food Chem. 1997, 45, 1415-1421. 18. Meazza, G.; Scheffler, B.E.; Tellez, M.R.; Rimando, A . M . ; Nanayakkara, N.P.D.; Khan, I.A.; Abourashed, E.A.; Romagni, J.G.; Duke, S.O.; Dayan, F.E. Phytochemistry 2002, 59, 281-288. 19. Devine, M.D.; Duke, S.O.; Fedtke, C. Physiology of Herbicide Action. Prentice Hall, Englewood Cliffs, NJ, 1993, pp. 395-424. 20. Pallett, K.E.; Little, J.P.; Sheekey, M.; Veerasekaran, P. Pestic. Biochem. Physiol. 1998, 62, 113-124. 21. Lee, D.L.; Knudsen, C.G.; Michaely, W.J.; Chin, H.-L.; Nguyen, N.H.; Carter, C.G.; Cromartie, T.H.; Lake, B.H.; Shribbs, J.M.; Fraser, Pestic. Sci. 1998, 54, 377-384. 22. Romagni, J.G.; Meazza, G.; Nanayakkara, N.P.D.; Dayan, F.E. FEBS Lett. 2000, 480, 301-305. 23. Duke, S.O. TrendsBiotechnol.2003, 21, 192-195. 24. Beneš, V.; Muckenthaler, M. Trends Biotechnol. 2003, 28: 244-249. 25. Agarwal, A.K.; Rogers, P.D.; Baerson, S.R.; Jacob, M.R.; Barker, K.S.; Cleary, J.D.; Walker, L.A.; Nagle, D.G.; Clark, A . M . J. Biol. Chem. 2003, In press.

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