- Email: [email protected]
Translational Opportunities in Plant Biochemistry
Recent Advances in Phytochemistry, vol. 40 John T. Romeo (Editor) 9 2006 Elsevier Ltd. All rights reserved.
Chapter Thirteen
TRANSLATIONAL OPPORTUNITIES IN PLANT BIOCHEMISTRY Cecilia A. Mclntosh
Department of Biological Sciences East Tennessee State University Box 70703 Johnson City, Tennessee 3 7614
Email: [email protected]
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . International Natural Products Repository Network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inflammatory Pathway Platforms and Plant Natural Products . . . . . . . . . . . . . . . . . . Transcription Factor Over-Expression for Metabolite Manipulation ........... Heterologous Sesquiterpene Production Platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . New Technologies for Metabolomics Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SumInary o . . . . ,
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INTRODUCTION Great strides have been made in plant science research in recent years, and some of these relate directly to efforts using genomic, proteomic, and metabolomic approaches. While these approaches have provided the ability to address increasingly complex questions, many challenges remain. These challenges include (but are not limited to) concerns related to: high throughput techniques and the requisite issues of processing large numbers of samples and minimizing sample preparation; developing analytical methods that mesh accuracy, reliability, and high sensitivity; natural product profiling and development of complex analytical software; development of in vivo test systems; and availability of identified source plant material and reference compounds. Some of these challenges are long-standing and others are emerging as inroads in technology used to ask questions of increasing complexity. Added to the above hurdles are those that result from fostering interdisciplinary approaches. Several agencies, including the U.S. National Science Foundation, actively promote and support imegrative research addressing these questions. The make-up of the NSF divisions that support the integrative programs, as well as a degree of imeragency cooperation, reflects the imerdisciplinary requirements of the research itself. 1-5 The following is a review of invited talks given at the La Jolla symposium that focused on resources and techniques. The symposium provided information on developing opportunities and/or methodologies to meet challenges in proteomic/metabolomic research. Speakers included: Troy SmiUie (University of Mississippi), Bryan Greenhagen (Allylix, Inc.), Sekhar Boddupalli (Galileo Pharmaceuticals), Fabricio Medina-Bolivar (Arkansas Biosciences Institute at Arkansas State University), and David Weil (Agilem Technologies). Their purpose was to present new technologies, applications, and/or opportunities with relevance to phytochemistry and plant biochemistry research. Two of the topics refer to platforms or production platforms. In this sense, a platform refers to research and development and/or production and manufacturing requirements and may include the: research and development strategy, requisite hardware and software, biotechnology, production plan, etc. Due to the diversity of topics, it is not possible to do a thorough literature review of each area to accompany presentation of new approaches, but citations of representative articles, reviews, or books have been included. INTERNATIONAL NATURAL PRODUCTS REPOSITORY NETWORK There are thousands of natural products made throughout the plant kingdom. 6 Research addressing questions or testing hypotheses relating to chemistry,
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309
biochemistry, metabolism, pharmacology, pharmacognosy, physiology, nutrition, etc. depends upon the continued isolation and structural identification of novel compounds. Obtaining reliable information on the identification of source materials and the availability of reference compounds is not always straightforward. Only a relative handful of compounds are available from commercial sources. Information on methods for extraction, purification, and identification of natural products are scattered throughout the literature. One research problem for phytochemists that has been under discussion for several years is the issue of how to sift through all of the sources to obtain information on any particular compound and related compounds. Dr. Troy Smillie presented on-going efforts by the University of Mississippi to address these challenges. The University of Mississippi established their Research Institute of Pharmaceutical Sciences in 1964 which led to the launch of the Thad Cochran Research Center housing the National Center for Natural Products Research in the 1990's. This is a large center comprised of several research faculty and staff from various academic departments in the university as well as U.S. Department of Agriculture scientists. The center has several research and development programs for natural products discovery and development (including both terrestrial plant and marine products) as well as medicinal plant research. The Natural Product Repository currently has more than 14,000 samples of extracts, derived fractions, and pure compounds in its inventory. Ongoing collection efforts yield over 1000 additional samples per year with geographic representation that includes the Americas, Africa, Papau New Guinea, and India. Collaborations with other institutions provide access to further samples for bioassay screening. Natural products chemists at the Center continue to isolate compounds based on their own current projects. Plant physiologists and researchers in genomics, proteomics, and metabolomics have an increasing need for natural products for use as substrates or molecular probes, and these are often difficult to obtain. To assist in these efforts, the university is in the process of establishing an International Natural Products Repository Network (INPRN). 7 The goal of the INPRN is to expand research interactions within the scientific community, and it was conceived to serve as a research tool for investigators in various fields by improving access to natural products that are critical reagents but may be unavailable from a commercial source or, if available, may be prohibitively expensive. An international steering committee is guiding network development. 7 There are two components of the INPRN. There will be both a physical and a virtual repository of natural products that will be available to investigators via a webpage interface. The organizational chart is shown in Figure 13.1. One major requirement of such a large-scale effort is the development of a database to serve the program. Requirements of the database are that it be internet ready, secure, easy to use, robust and scalable, and able to handle multiple relations while remaining
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Figure 13.1: Organization of INPRN (provided by T. Smillie).
affordable. The cemer is nearing completion of this phase (T. Smillie, personal communication). Information that will be available within the database includes: source material, common name and synonyms, IUPAC nomenclature, class of compound, molecular form/weight, structure, available quantity, spectroscopic information, stability information, toxicity/hazard warnings, storage conditions, and isolation techniques. Samples with no known intellectual property issues are accepted; donations are actively encouraged, and some have already occurred. This is an excellent altruistic opportunity for individual scientists to make significant contributions to research by outreach on a larger scale. All samples donated will have the donor's identity associated with the sample in the database. Contributions to the physical repository have specific requirements as to the minimum amount needed and the
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nature of the requisite storage conditions; contributions to the virtual repository, of course, are not required to adhere to those same guidelines. Multiple submissions of the same material from different investigators are encouraged by the INPRN. It is important to note that the 1NPRN is not a business, and it will be run as a non-profit entity. This fosters altruistic collaborations for the advancement of science in general. Participation is open to educational, governmental, and other non-profit research groups; direct interactions with commercial partners will be discouraged. Contact information and updates are available through the INPRN homepage. 7 I N F L A M M A T O R Y P A T H W A Y P L A T F O R M S AND P L A N T NATURAL PRODUCTS Many challenges are shared by the fields of rational medicinal chemistry and natural products chemistry. Limited chemical diversity and limited emphasis on functional biology versus target-based optimization for drug discovery are coupled to on-going efforts to discover new natural products. Expectations of chemical diversity as a corollary to biodiversity are not always borne out, and reproducing natural chemistry in a laboratory setting can be difficult. Obtaining a renewable supply of source material as well as controlling the expenses incurred in tooling up to identify potential drugs adds to the challenge. Dr. Sekhar Boddupalli of Galileo Pharmaceuticals (www.galileopharm.com) shared information on new innovations to address such issues. Some approaches are geared toward meshing functional chemistry with functional biology to identify novel starting points. Advantages of using nature-derived chemistry from food sources include a lower risk of toxicity, a higher probability of efficacy, and the proprietary advantages to "first-in-class" drugs. First-in-class drugs are those that possess novel characteristics leading to new approaches/targets for treatment of a condition. Disadvantages of using naturederived chemistry from food sources include: the chemical complexity of foods, which leads to the problem that not all of the chemical content of food is known or "mapped;" the potential of off-target effects; and the possibility of poor pharmacokinetics. The specific example presented is related to discovery and development of first-in-class inhibitors against inflammation and metabolic disease targets, specifically those that are inhibitors of lipoxygenases (LO). These could be applied to the treatment of diseases mediated by inflammation such as asthma, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD), cardiovascular disease, and osteoporosis. Galileo Pharmaceuticals has a proprietary Conserved Inflammatory Pathway (CIP) modulator technology platform that enables rapid identification of novel small molecules acting against a broad range of inflammatory targets. The approach may be applicable to other biological problems.
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Information from genomics as well as other data indicate that plants and humans have conserved inflammatory pathways, e.g., pathogen and lipoxygenaseinduced signaling pathways. In mammalian systems, signal transduction leads to production of isoprostanes and prosta~landins, while jasmonates and phytoprostanes are produced in plants (Fig. 13.2). 8-1 It is well established that treating plant cell cultures with various stressors can lead to production of protective phytochemicals. 15 Many of these compounds have activity in mammalian systems, likely due to pathway conservation. Galileo took advantage of the ability to elicit production of potential anti-inflammatory compounds by the systematic exposure of a broad range of plant species to a variety of stress conditions. They then screened the small molecule libraries in functional mammalian cell- and target-based assays, and they have identified novel, drug-like small molecules that modulate therapeutically relevant targets such as lipoxygenases, kinases, and nuclear receptors. By using this approach, they have established a proprietary set of active compounds, new chemical entities, acting as CIP modulators (Fig. 13.3).16
Figure 13.2: Bioactive Lipids: Conserved Inflammatory Pathways (used by permission of Galileo Pharmaceuticals).
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Figure 13.3: Galileo Discovery Platform: Strategy for Development (used by permission of Galileo Pharmaceuticals).
The development of platforms for rational investigation of chemicals may also prove beneficial for compounds with biological activities other than mammalian drugs. Adaptation of this methodology may find application in studies of plantplant, plant-microbe, or plant-insect interactions as well as in studies of plant metabolism and physiology. 172~ TRANSCRIPTION FACTOR OVER-EXPRESSION FOR METABOLITE MANIPULATION
Dr. Fabricio Medina-Bolivar of the Arkansas Biosciences Institute and the Dept. of Biological Sciences at Arkansas State University presented recent results from investigations into use of transcription factors to manipulate metabolite biosynthesis in hairy root cultures as part of his work on the application of metabolic engineering for the discovery of pharmaceuticals from plants. One of his research interests is the identification of specialized metabolites produced in tobacco that
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could lead to the treatment of Parkinson's disease. This work started as collaborative research with Dr. Neal Castagnoli Jr. of the Harvey W. Peters Center for the Study of Parkinson's Diseases and Disorders of the Nervous System at Virginia Tech. Epidemiology data have shown that tobacco smokers have lower incidents of Parkinson's disease, and it has been postulated that this may be related to the lower levels of monoamine oxidase (MAO) activity in the brain of smokers. 21 Dr. MedinaBolivar is using elicitation in tobacco hairy roots as means to produce chemicals found in tobacco smoke and discover novel MAO inhibitors. Hairy roots are useful as bioproduction systems for specialized metabolites as well as recombinant proteins, providing genetic stability and containment. 22"23 Dr. Medina-Bolivar's research group is over-ex~pressing transcription factors that may regulate specialized metabolic pathways. 2 These are being expressed with promoters that have been studied in his laboratory and shown to be highly active and inducible in tobacco hairy roots (Fig. 13.4). Ultimately, genetically engineered hairy roots will be analyzed for production of novel metabolites and tested in bioassays. This novel approach may lead to the discovery of pharmaceutically important therapeutic drugs.
Figure 13.4: Hairy roots elicited with cooper sulfate. The dark area produced in the root tips upon elicitation is used to determine the efficacy of the elicitor. (used by permission of F. Medina-Bolivar).
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H E T E R O L O G O U S S E S Q U I T E R P E N E PRODUCTION P L A T F O R M S Dr. Bryan Greenhagen of Allylix, Inc. shared general strategies that are bein~ used to develop platforms for production of heterologous sesquiterpenes. 25"2 Intellectual property issues preclude presentation of specific details. However, the overall scheme for developing model systems for pathways and for producing compounds is dependent upon understanding structure(s) of the biocatalyst(s) involved, obtaining the genes for the enzymes/biocatalysts, inserting the gene into a production microbe, producing the compound through fermentation, and optimizing activity of the compound through final chemical modification. The production host must be well suited to production of the compound of interest. For example, production of sesquiterpenes would require a host with metabolism that results in a high flux of carbon into famesyl pyrophosphate to enable reconstruction of the pathway. A key to success is to evolve the biosynthetic potential by mapping enzyme active sites by mutational analysis and identification of change-in-function mutations. This has the potential to lead to enantio-specific product engineering. 28 Once structural elements controlling specificity are known, combinatorial engineering of the proteins can be initiated. Advances in the development of biosynthetic production platforms are dependent upon thorough kinetic analyses of enzymes and consideration of the complexities of cellular biosynthesis, as well as development of the actual production process. 29 NEW TECHNOLOGIES FOR METABOLOMIC PROFILING
One of the most important current hurdles facing metabolomics research is relieving the bottlenecks of sample preparation and bioinformatics. Samples for metabolomic analysis are usually complex and may contain thousands of compounds. Many compounds share chemical properties, which presents problems for manual extraction of compounds of interest. 3o Dr. David Weil of Agilent Technologies presented information on new technology developed to address these issues. The approach used is to leverage or adapt advances in proteomics technology (e.g., high throughput capacity, sensitivity, reduced experimental variation, robust bioinformatics) for use in metabolomics. 30 "31 Important factors to consider in developing technology are sample preparation, separation, sensitivity of the method, reduction of experimental variation, and integration of informatics. Capillary HPLC can provide high resolution separation of similar compounds. Current solutions developed by Agilent Technologies involve coupling an Agilent Capillary HPLC system with an Agilent LC/MSD TOF mass spectrometer for nano-scale analyses. 3~ 32-33 The system includes software ("Mass Hunter" and "Mass Profiler") to expedite the informatics
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aspects. 32"33 This system is capable of 32 attamole resolution although routine analysis is at the femtomole range. Details of the system along with sample data are available at the company's web site. 32"33 SUMMARY
Genomic approaches and related technologies have widened the spectra of experimental questions for which the potential of obtaining answers exists. 3435 These questions include those important for basic sciences as well as applied sciences such as pharmaceutical or food science. Proteomic and metabolomic investigations have brought additional requirements for new and/or improved technology. Continued progress in development of biological systems that can be manipulated, production of custom enzymes, development of screening platforms and strategies, and innovations in analytical technology will be critical to on-going success in the field. Additional contributions to scientific advancement in the "omics" related fields will be fostered by sharing of samples of and/or information on phytochemicals through the International Natural Products Repository Network. ACKNOWLEDGEMENTS
Thanks to all speakers for comributing to an exciting symposium session. Special thanks to Dr. Medina-Bolivar, Dr. Boddupalli, and Dr. Smillie for providing materials to aid in writing this symposium summary. REFERENCES
1. National Science Foundation Emerging Frontiers home page http://www.nsf.gov/ div/index.jsp?org=EF 2. National Science Foundation NSF-NIST (Interaction in Chemistry, Materials Research, Molecular Biosciences, Bioengineering, and Chemical Engineering) home page http://www.nsf.gov/funding/pgm_summ.jsp?pims_id=5665&org=MCB 3. National Science Foundation Biochemical Engineering and Biotechnology home page http://www.nsf.gov/funding/pgm_summ.jsp?pims_id= 13368&org=BES&from=home 4. National Science Foundation" Interagency Opportunities in Metabolic Engineering http://www.nsf,gov/pubs/2005/nsf05502/nsf05502.htm 5. National Science Foundation Quantitative Systems Biotechnology and Post Genomic Engineering http://www.nsf.gov/eng/bes/biochemdetail.jsp 6. BARTON, D., NAKANISHI, K. (eds-in-chief), METH-COHN, O. (exec. ed.), Comprehensive Natural Products Chemistry. Elsevier, 1999, vol. 1-9. 7. InternationalNatural Products Repository Network home page http://inprn.org/ 8. MUSIEK, E.S., YIN, H., MILNE, G.L., MORROW, J.D., Recent advances in the biochemistry and clinical relevance of the isoprostane pathway. Lipids, 2005, 40, 987994.
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9. MONTUSCHI, P., BARNES, P.J., ROBERTS, L.J. II., Isoprostanes: Markers and mediators of oxidative stress. FASEB J., 2004, 18, 1971-1800. 10. GERSHENSON, J., 2002. Secondary metabolites and plant defense. In: Plant Physiology (L. Taiz and E. Zeiger, eds,), Sinauer Associates, Inc., Sunderland, Massachusetts. 2002. pp 283-308. 11. CREELMAN, R.A., MULLET, J.E., Biosynthesis and action of jasmonates in plants. Annu. Rev. Plant Phys. Plant Mol. Biol., 1997, 48, 355-381. 12. SALZMAN, R.A., BRADY, J.A., F1NLAYSON, S.A., BUCHANAN, C.D., SUMMER, E.J., SUN, F., KLEIN, P.E., KLEIN, R.R., PRATT, L.H., CORDONNIER-PRATT, M.M., MULLET, J.E., Transcriptional profiling of sorghum induced by methyl jasmonate, salicylic acid and aminocyclopropane carboxylic acid reveals cooperative regulation and novel gene responses. Plant Physiol., 2005, 138, 352-368. 13. LOEFFLER, C., BERGER, S., GUY, A., DURAND, T., BR1NGMANN, G., DREYER, M., VON RAD, U., DURNER, J. MUELLER, M.J., Bl-phytoprostanes trigger plant defense and detoxification responses. Plant Physiol., 2004, 137, 328-340. 14. LI, X., SCHULER, M.A., BERENBAUM, M.R., Jasmonate and salicylate induce expression of herbivore cytochrome P450 genes. Nature, 2002, 419, 712-715. 15. GUNDLACH, H., MULLER, M.J., KUTHCEN, T.M., ZENK, M.H., Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures. Proc. Natl. Acad. Sci. USA, 1992, 89, 23 89-2393. 16. BODDUPALLI, S., Conserved inflammatory pathway (CIP) modulator platform (nonconfidential summary). PSNA symposium summary provided by Galileo Pharmaceuticals. 2005, pp 1-3. 17. ROMEO, J.T. (ed.), Chemical Ecology and Phytochemistry of Forest Ecosystems. Rec. Adv. Phytochem., vol. 39, Elsevier, 2005, 307 p. 18. ROMEO, J.T., DOWNUM, K.R., VERPOORTE, R. (eds.), Phytochemical Signals and Plant-Microbe Interactions. Rec. Adv. Phytochem., vol. 32, Plenum Press, 1998, 254 p. 19 ROMEO, J.T. (ed), Integrative Phytochemistry: From Ethnobotany to Molecular Ecology. Rec. Adv. Phytochem., vol. 37, Pergamon (Elsevier), 2003, 329 p. 20. ROMEO, J.T. (ed), Phytochemicals in Human Health, Protction, Nutrition, and Plant Defense. Rec. Adv. Phytochem., vol. 33, Kluwer (Plenum), 1999, 432 p. 21. CASTAGNOLI K., MURUGESAN, T., Tobacco leaf, smoke and smoking, MAO inhibitors, Parkinson's disease and neuroprotection; are there links? Neurotoxicology, 2004, 25, 279-291. 22. MEDINA-BOLIVAR, F., CRAMER, C., Production of recombinant proteins in hairy roots cultured in plastic sleeve bioreactors. In: Recombinant Gene Expression: Reviews and Protocols. P. Balbas and A. Lorence, (eds.). Humana Press, Totowa. 2004, pp. 351363. 23. MEDINA-BOLIVAR, F., FLORES, H., Root culture and natural products: "Unearthing" the hidden half of plant metabolism. Plant Tissue Culture and Biotechnology, 1995, 1, 59-74. 24. LORENCE, A., WOFFENDEN, B.J., SMITH, M., NESSLER, C.L., MEDINABOLIVAR, F., Over-expression of transcription factors to manipulate specialized metabolite biosynthesis. (http://www.psna-online.org/PSNAabst05.pdf; page 96) 25. Commerce Lexington press release. (http;//www.lexicc.com/companies/3350-
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18307%20eBus%20ad-Allylix.pdf) 26. Life Sciences and High-Tech Financial Forum 2005. http://www.connect.org/programs/ lifesciencesff/presenterpr0files.htm 27. Allylix home page. http://Www.allylix.com 28. GREENHAGEN, B., CHAPPELL, J., Molecular scaffolds for chemical wizardry: learning nature's rules for terpene cyclases. Proc. Natl. Acad. Sci. USA, 2001, 98, 13479-13481. 29. TAKAHASHI, S., ZHAO, Y., O'MAILLE, P.E., GREENHAGEN, B.T., NOEL, J.T., COATES, R.M., CHAPPELL, J., Kinetic and molecular analysis of 5-epiaristolochene 1,3-dihydroxylase, a cytochrome P450 enzyme catalyzing successive hydroxylations of sesquiterpenes. J. Biol. Chem., 2005, 280, 3686-3696. 30. MILLER, B., LI, X., FJESDSTED, J., KINCAID, R., CHAKEL, J., WELL, D., Differential detection of metabolites using Mass Hunter and Mass Profiler. (http://www.psna-online.org~SNAabst05.pdf; page 87) 31. WELLS, D.A., WELL, D.A., Directions in automated sample preparation of proteins. PharmaGenomics, 2003, Nov-Dec., 42-54. (http://www.forumsci.co.il/HPLC/ Protein_sample__prep.pdf ) 32. http:/!www.agilent,com. 33. Agilent Technologies product note. Turning samples into answers. http://www.chem.agilent.com/temp/radD793C/00054960.pdf 34. ROMEO, J.T. (ed), Secondary Metabolism in Model Systems. Rec. Adv. Phytochem., vol. 38, Elsevier. 2004, 270 p. 35. ROMEO, J.T., DIXON, R.A. (eds), Phytochemistry in the Genomics and Post-Genomics Eras. Rec. Adv. Phytochem., vol. 36, Pergamon (Elsevier). 2002, 258 p.
Chapter Thirteen
TRANSLATIONAL OPPORTUNITIES IN PLANT BIOCHEMISTRY Cecilia A. Mclntosh
Department of Biological Sciences East Tennessee State University Box 70703 Johnson City, Tennessee 3 7614
Email: [email protected]
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . International Natural Products Repository Network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inflammatory Pathway Platforms and Plant Natural Products . . . . . . . . . . . . . . . . . . Transcription Factor Over-Expression for Metabolite Manipulation ........... Heterologous Sesquiterpene Production Platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . New Technologies for Metabolomics Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SumInary o . . . . ,
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INTRODUCTION Great strides have been made in plant science research in recent years, and some of these relate directly to efforts using genomic, proteomic, and metabolomic approaches. While these approaches have provided the ability to address increasingly complex questions, many challenges remain. These challenges include (but are not limited to) concerns related to: high throughput techniques and the requisite issues of processing large numbers of samples and minimizing sample preparation; developing analytical methods that mesh accuracy, reliability, and high sensitivity; natural product profiling and development of complex analytical software; development of in vivo test systems; and availability of identified source plant material and reference compounds. Some of these challenges are long-standing and others are emerging as inroads in technology used to ask questions of increasing complexity. Added to the above hurdles are those that result from fostering interdisciplinary approaches. Several agencies, including the U.S. National Science Foundation, actively promote and support imegrative research addressing these questions. The make-up of the NSF divisions that support the integrative programs, as well as a degree of imeragency cooperation, reflects the imerdisciplinary requirements of the research itself. 1-5 The following is a review of invited talks given at the La Jolla symposium that focused on resources and techniques. The symposium provided information on developing opportunities and/or methodologies to meet challenges in proteomic/metabolomic research. Speakers included: Troy SmiUie (University of Mississippi), Bryan Greenhagen (Allylix, Inc.), Sekhar Boddupalli (Galileo Pharmaceuticals), Fabricio Medina-Bolivar (Arkansas Biosciences Institute at Arkansas State University), and David Weil (Agilem Technologies). Their purpose was to present new technologies, applications, and/or opportunities with relevance to phytochemistry and plant biochemistry research. Two of the topics refer to platforms or production platforms. In this sense, a platform refers to research and development and/or production and manufacturing requirements and may include the: research and development strategy, requisite hardware and software, biotechnology, production plan, etc. Due to the diversity of topics, it is not possible to do a thorough literature review of each area to accompany presentation of new approaches, but citations of representative articles, reviews, or books have been included. INTERNATIONAL NATURAL PRODUCTS REPOSITORY NETWORK There are thousands of natural products made throughout the plant kingdom. 6 Research addressing questions or testing hypotheses relating to chemistry,
TRANSLATIONAL OPPORTUNITIES IN PLANT BIOCHEMISTRY
309
biochemistry, metabolism, pharmacology, pharmacognosy, physiology, nutrition, etc. depends upon the continued isolation and structural identification of novel compounds. Obtaining reliable information on the identification of source materials and the availability of reference compounds is not always straightforward. Only a relative handful of compounds are available from commercial sources. Information on methods for extraction, purification, and identification of natural products are scattered throughout the literature. One research problem for phytochemists that has been under discussion for several years is the issue of how to sift through all of the sources to obtain information on any particular compound and related compounds. Dr. Troy Smillie presented on-going efforts by the University of Mississippi to address these challenges. The University of Mississippi established their Research Institute of Pharmaceutical Sciences in 1964 which led to the launch of the Thad Cochran Research Center housing the National Center for Natural Products Research in the 1990's. This is a large center comprised of several research faculty and staff from various academic departments in the university as well as U.S. Department of Agriculture scientists. The center has several research and development programs for natural products discovery and development (including both terrestrial plant and marine products) as well as medicinal plant research. The Natural Product Repository currently has more than 14,000 samples of extracts, derived fractions, and pure compounds in its inventory. Ongoing collection efforts yield over 1000 additional samples per year with geographic representation that includes the Americas, Africa, Papau New Guinea, and India. Collaborations with other institutions provide access to further samples for bioassay screening. Natural products chemists at the Center continue to isolate compounds based on their own current projects. Plant physiologists and researchers in genomics, proteomics, and metabolomics have an increasing need for natural products for use as substrates or molecular probes, and these are often difficult to obtain. To assist in these efforts, the university is in the process of establishing an International Natural Products Repository Network (INPRN). 7 The goal of the INPRN is to expand research interactions within the scientific community, and it was conceived to serve as a research tool for investigators in various fields by improving access to natural products that are critical reagents but may be unavailable from a commercial source or, if available, may be prohibitively expensive. An international steering committee is guiding network development. 7 There are two components of the INPRN. There will be both a physical and a virtual repository of natural products that will be available to investigators via a webpage interface. The organizational chart is shown in Figure 13.1. One major requirement of such a large-scale effort is the development of a database to serve the program. Requirements of the database are that it be internet ready, secure, easy to use, robust and scalable, and able to handle multiple relations while remaining
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Figure 13.1: Organization of INPRN (provided by T. Smillie).
affordable. The cemer is nearing completion of this phase (T. Smillie, personal communication). Information that will be available within the database includes: source material, common name and synonyms, IUPAC nomenclature, class of compound, molecular form/weight, structure, available quantity, spectroscopic information, stability information, toxicity/hazard warnings, storage conditions, and isolation techniques. Samples with no known intellectual property issues are accepted; donations are actively encouraged, and some have already occurred. This is an excellent altruistic opportunity for individual scientists to make significant contributions to research by outreach on a larger scale. All samples donated will have the donor's identity associated with the sample in the database. Contributions to the physical repository have specific requirements as to the minimum amount needed and the
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nature of the requisite storage conditions; contributions to the virtual repository, of course, are not required to adhere to those same guidelines. Multiple submissions of the same material from different investigators are encouraged by the INPRN. It is important to note that the 1NPRN is not a business, and it will be run as a non-profit entity. This fosters altruistic collaborations for the advancement of science in general. Participation is open to educational, governmental, and other non-profit research groups; direct interactions with commercial partners will be discouraged. Contact information and updates are available through the INPRN homepage. 7 I N F L A M M A T O R Y P A T H W A Y P L A T F O R M S AND P L A N T NATURAL PRODUCTS Many challenges are shared by the fields of rational medicinal chemistry and natural products chemistry. Limited chemical diversity and limited emphasis on functional biology versus target-based optimization for drug discovery are coupled to on-going efforts to discover new natural products. Expectations of chemical diversity as a corollary to biodiversity are not always borne out, and reproducing natural chemistry in a laboratory setting can be difficult. Obtaining a renewable supply of source material as well as controlling the expenses incurred in tooling up to identify potential drugs adds to the challenge. Dr. Sekhar Boddupalli of Galileo Pharmaceuticals (www.galileopharm.com) shared information on new innovations to address such issues. Some approaches are geared toward meshing functional chemistry with functional biology to identify novel starting points. Advantages of using nature-derived chemistry from food sources include a lower risk of toxicity, a higher probability of efficacy, and the proprietary advantages to "first-in-class" drugs. First-in-class drugs are those that possess novel characteristics leading to new approaches/targets for treatment of a condition. Disadvantages of using naturederived chemistry from food sources include: the chemical complexity of foods, which leads to the problem that not all of the chemical content of food is known or "mapped;" the potential of off-target effects; and the possibility of poor pharmacokinetics. The specific example presented is related to discovery and development of first-in-class inhibitors against inflammation and metabolic disease targets, specifically those that are inhibitors of lipoxygenases (LO). These could be applied to the treatment of diseases mediated by inflammation such as asthma, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD), cardiovascular disease, and osteoporosis. Galileo Pharmaceuticals has a proprietary Conserved Inflammatory Pathway (CIP) modulator technology platform that enables rapid identification of novel small molecules acting against a broad range of inflammatory targets. The approach may be applicable to other biological problems.
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Information from genomics as well as other data indicate that plants and humans have conserved inflammatory pathways, e.g., pathogen and lipoxygenaseinduced signaling pathways. In mammalian systems, signal transduction leads to production of isoprostanes and prosta~landins, while jasmonates and phytoprostanes are produced in plants (Fig. 13.2). 8-1 It is well established that treating plant cell cultures with various stressors can lead to production of protective phytochemicals. 15 Many of these compounds have activity in mammalian systems, likely due to pathway conservation. Galileo took advantage of the ability to elicit production of potential anti-inflammatory compounds by the systematic exposure of a broad range of plant species to a variety of stress conditions. They then screened the small molecule libraries in functional mammalian cell- and target-based assays, and they have identified novel, drug-like small molecules that modulate therapeutically relevant targets such as lipoxygenases, kinases, and nuclear receptors. By using this approach, they have established a proprietary set of active compounds, new chemical entities, acting as CIP modulators (Fig. 13.3).16
Figure 13.2: Bioactive Lipids: Conserved Inflammatory Pathways (used by permission of Galileo Pharmaceuticals).
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Figure 13.3: Galileo Discovery Platform: Strategy for Development (used by permission of Galileo Pharmaceuticals).
The development of platforms for rational investigation of chemicals may also prove beneficial for compounds with biological activities other than mammalian drugs. Adaptation of this methodology may find application in studies of plantplant, plant-microbe, or plant-insect interactions as well as in studies of plant metabolism and physiology. 172~ TRANSCRIPTION FACTOR OVER-EXPRESSION FOR METABOLITE MANIPULATION
Dr. Fabricio Medina-Bolivar of the Arkansas Biosciences Institute and the Dept. of Biological Sciences at Arkansas State University presented recent results from investigations into use of transcription factors to manipulate metabolite biosynthesis in hairy root cultures as part of his work on the application of metabolic engineering for the discovery of pharmaceuticals from plants. One of his research interests is the identification of specialized metabolites produced in tobacco that
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could lead to the treatment of Parkinson's disease. This work started as collaborative research with Dr. Neal Castagnoli Jr. of the Harvey W. Peters Center for the Study of Parkinson's Diseases and Disorders of the Nervous System at Virginia Tech. Epidemiology data have shown that tobacco smokers have lower incidents of Parkinson's disease, and it has been postulated that this may be related to the lower levels of monoamine oxidase (MAO) activity in the brain of smokers. 21 Dr. MedinaBolivar is using elicitation in tobacco hairy roots as means to produce chemicals found in tobacco smoke and discover novel MAO inhibitors. Hairy roots are useful as bioproduction systems for specialized metabolites as well as recombinant proteins, providing genetic stability and containment. 22"23 Dr. Medina-Bolivar's research group is over-ex~pressing transcription factors that may regulate specialized metabolic pathways. 2 These are being expressed with promoters that have been studied in his laboratory and shown to be highly active and inducible in tobacco hairy roots (Fig. 13.4). Ultimately, genetically engineered hairy roots will be analyzed for production of novel metabolites and tested in bioassays. This novel approach may lead to the discovery of pharmaceutically important therapeutic drugs.
Figure 13.4: Hairy roots elicited with cooper sulfate. The dark area produced in the root tips upon elicitation is used to determine the efficacy of the elicitor. (used by permission of F. Medina-Bolivar).
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H E T E R O L O G O U S S E S Q U I T E R P E N E PRODUCTION P L A T F O R M S Dr. Bryan Greenhagen of Allylix, Inc. shared general strategies that are bein~ used to develop platforms for production of heterologous sesquiterpenes. 25"2 Intellectual property issues preclude presentation of specific details. However, the overall scheme for developing model systems for pathways and for producing compounds is dependent upon understanding structure(s) of the biocatalyst(s) involved, obtaining the genes for the enzymes/biocatalysts, inserting the gene into a production microbe, producing the compound through fermentation, and optimizing activity of the compound through final chemical modification. The production host must be well suited to production of the compound of interest. For example, production of sesquiterpenes would require a host with metabolism that results in a high flux of carbon into famesyl pyrophosphate to enable reconstruction of the pathway. A key to success is to evolve the biosynthetic potential by mapping enzyme active sites by mutational analysis and identification of change-in-function mutations. This has the potential to lead to enantio-specific product engineering. 28 Once structural elements controlling specificity are known, combinatorial engineering of the proteins can be initiated. Advances in the development of biosynthetic production platforms are dependent upon thorough kinetic analyses of enzymes and consideration of the complexities of cellular biosynthesis, as well as development of the actual production process. 29 NEW TECHNOLOGIES FOR METABOLOMIC PROFILING
One of the most important current hurdles facing metabolomics research is relieving the bottlenecks of sample preparation and bioinformatics. Samples for metabolomic analysis are usually complex and may contain thousands of compounds. Many compounds share chemical properties, which presents problems for manual extraction of compounds of interest. 3o Dr. David Weil of Agilent Technologies presented information on new technology developed to address these issues. The approach used is to leverage or adapt advances in proteomics technology (e.g., high throughput capacity, sensitivity, reduced experimental variation, robust bioinformatics) for use in metabolomics. 30 "31 Important factors to consider in developing technology are sample preparation, separation, sensitivity of the method, reduction of experimental variation, and integration of informatics. Capillary HPLC can provide high resolution separation of similar compounds. Current solutions developed by Agilent Technologies involve coupling an Agilent Capillary HPLC system with an Agilent LC/MSD TOF mass spectrometer for nano-scale analyses. 3~ 32-33 The system includes software ("Mass Hunter" and "Mass Profiler") to expedite the informatics
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aspects. 32"33 This system is capable of 32 attamole resolution although routine analysis is at the femtomole range. Details of the system along with sample data are available at the company's web site. 32"33 SUMMARY
Genomic approaches and related technologies have widened the spectra of experimental questions for which the potential of obtaining answers exists. 3435 These questions include those important for basic sciences as well as applied sciences such as pharmaceutical or food science. Proteomic and metabolomic investigations have brought additional requirements for new and/or improved technology. Continued progress in development of biological systems that can be manipulated, production of custom enzymes, development of screening platforms and strategies, and innovations in analytical technology will be critical to on-going success in the field. Additional contributions to scientific advancement in the "omics" related fields will be fostered by sharing of samples of and/or information on phytochemicals through the International Natural Products Repository Network. ACKNOWLEDGEMENTS
Thanks to all speakers for comributing to an exciting symposium session. Special thanks to Dr. Medina-Bolivar, Dr. Boddupalli, and Dr. Smillie for providing materials to aid in writing this symposium summary. REFERENCES
1. National Science Foundation Emerging Frontiers home page http://www.nsf.gov/ div/index.jsp?org=EF 2. National Science Foundation NSF-NIST (Interaction in Chemistry, Materials Research, Molecular Biosciences, Bioengineering, and Chemical Engineering) home page http://www.nsf.gov/funding/pgm_summ.jsp?pims_id=5665&org=MCB 3. National Science Foundation Biochemical Engineering and Biotechnology home page http://www.nsf.gov/funding/pgm_summ.jsp?pims_id= 13368&org=BES&from=home 4. National Science Foundation" Interagency Opportunities in Metabolic Engineering http://www.nsf,gov/pubs/2005/nsf05502/nsf05502.htm 5. National Science Foundation Quantitative Systems Biotechnology and Post Genomic Engineering http://www.nsf.gov/eng/bes/biochemdetail.jsp 6. BARTON, D., NAKANISHI, K. (eds-in-chief), METH-COHN, O. (exec. ed.), Comprehensive Natural Products Chemistry. Elsevier, 1999, vol. 1-9. 7. InternationalNatural Products Repository Network home page http://inprn.org/ 8. MUSIEK, E.S., YIN, H., MILNE, G.L., MORROW, J.D., Recent advances in the biochemistry and clinical relevance of the isoprostane pathway. Lipids, 2005, 40, 987994.
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9. MONTUSCHI, P., BARNES, P.J., ROBERTS, L.J. II., Isoprostanes: Markers and mediators of oxidative stress. FASEB J., 2004, 18, 1971-1800. 10. GERSHENSON, J., 2002. Secondary metabolites and plant defense. In: Plant Physiology (L. Taiz and E. Zeiger, eds,), Sinauer Associates, Inc., Sunderland, Massachusetts. 2002. pp 283-308. 11. CREELMAN, R.A., MULLET, J.E., Biosynthesis and action of jasmonates in plants. Annu. Rev. Plant Phys. Plant Mol. Biol., 1997, 48, 355-381. 12. SALZMAN, R.A., BRADY, J.A., F1NLAYSON, S.A., BUCHANAN, C.D., SUMMER, E.J., SUN, F., KLEIN, P.E., KLEIN, R.R., PRATT, L.H., CORDONNIER-PRATT, M.M., MULLET, J.E., Transcriptional profiling of sorghum induced by methyl jasmonate, salicylic acid and aminocyclopropane carboxylic acid reveals cooperative regulation and novel gene responses. Plant Physiol., 2005, 138, 352-368. 13. LOEFFLER, C., BERGER, S., GUY, A., DURAND, T., BR1NGMANN, G., DREYER, M., VON RAD, U., DURNER, J. MUELLER, M.J., Bl-phytoprostanes trigger plant defense and detoxification responses. Plant Physiol., 2004, 137, 328-340. 14. LI, X., SCHULER, M.A., BERENBAUM, M.R., Jasmonate and salicylate induce expression of herbivore cytochrome P450 genes. Nature, 2002, 419, 712-715. 15. GUNDLACH, H., MULLER, M.J., KUTHCEN, T.M., ZENK, M.H., Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures. Proc. Natl. Acad. Sci. USA, 1992, 89, 23 89-2393. 16. BODDUPALLI, S., Conserved inflammatory pathway (CIP) modulator platform (nonconfidential summary). PSNA symposium summary provided by Galileo Pharmaceuticals. 2005, pp 1-3. 17. ROMEO, J.T. (ed.), Chemical Ecology and Phytochemistry of Forest Ecosystems. Rec. Adv. Phytochem., vol. 39, Elsevier, 2005, 307 p. 18. ROMEO, J.T., DOWNUM, K.R., VERPOORTE, R. (eds.), Phytochemical Signals and Plant-Microbe Interactions. Rec. Adv. Phytochem., vol. 32, Plenum Press, 1998, 254 p. 19 ROMEO, J.T. (ed), Integrative Phytochemistry: From Ethnobotany to Molecular Ecology. Rec. Adv. Phytochem., vol. 37, Pergamon (Elsevier), 2003, 329 p. 20. ROMEO, J.T. (ed), Phytochemicals in Human Health, Protction, Nutrition, and Plant Defense. Rec. Adv. Phytochem., vol. 33, Kluwer (Plenum), 1999, 432 p. 21. CASTAGNOLI K., MURUGESAN, T., Tobacco leaf, smoke and smoking, MAO inhibitors, Parkinson's disease and neuroprotection; are there links? Neurotoxicology, 2004, 25, 279-291. 22. MEDINA-BOLIVAR, F., CRAMER, C., Production of recombinant proteins in hairy roots cultured in plastic sleeve bioreactors. In: Recombinant Gene Expression: Reviews and Protocols. P. Balbas and A. Lorence, (eds.). Humana Press, Totowa. 2004, pp. 351363. 23. MEDINA-BOLIVAR, F., FLORES, H., Root culture and natural products: "Unearthing" the hidden half of plant metabolism. Plant Tissue Culture and Biotechnology, 1995, 1, 59-74. 24. LORENCE, A., WOFFENDEN, B.J., SMITH, M., NESSLER, C.L., MEDINABOLIVAR, F., Over-expression of transcription factors to manipulate specialized metabolite biosynthesis. (http://www.psna-online.org/PSNAabst05.pdf; page 96) 25. Commerce Lexington press release. (http;//www.lexicc.com/companies/3350-
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18307%20eBus%20ad-Allylix.pdf) 26. Life Sciences and High-Tech Financial Forum 2005. http://www.connect.org/programs/ lifesciencesff/presenterpr0files.htm 27. Allylix home page. http://Www.allylix.com 28. GREENHAGEN, B., CHAPPELL, J., Molecular scaffolds for chemical wizardry: learning nature's rules for terpene cyclases. Proc. Natl. Acad. Sci. USA, 2001, 98, 13479-13481. 29. TAKAHASHI, S., ZHAO, Y., O'MAILLE, P.E., GREENHAGEN, B.T., NOEL, J.T., COATES, R.M., CHAPPELL, J., Kinetic and molecular analysis of 5-epiaristolochene 1,3-dihydroxylase, a cytochrome P450 enzyme catalyzing successive hydroxylations of sesquiterpenes. J. Biol. Chem., 2005, 280, 3686-3696. 30. MILLER, B., LI, X., FJESDSTED, J., KINCAID, R., CHAKEL, J., WELL, D., Differential detection of metabolites using Mass Hunter and Mass Profiler. (http://www.psna-online.org~SNAabst05.pdf; page 87) 31. WELLS, D.A., WELL, D.A., Directions in automated sample preparation of proteins. PharmaGenomics, 2003, Nov-Dec., 42-54. (http://www.forumsci.co.il/HPLC/ Protein_sample__prep.pdf ) 32. http:/!www.agilent,com. 33. Agilent Technologies product note. Turning samples into answers. http://www.chem.agilent.com/temp/radD793C/00054960.pdf 34. ROMEO, J.T. (ed), Secondary Metabolism in Model Systems. Rec. Adv. Phytochem., vol. 38, Elsevier. 2004, 270 p. 35. ROMEO, J.T., DIXON, R.A. (eds), Phytochemistry in the Genomics and Post-Genomics Eras. Rec. Adv. Phytochem., vol. 36, Pergamon (Elsevier). 2002, 258 p.