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Plant ChemiCal Biology

Plant ChemiCal Biology

edited by

Dominique auDenaertPaul overvoorDe

Copyright © 2014 by John Wiley & Sons, Inc. All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, New JerseyPublished simultaneously in Canada

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Library of Congress Cataloging-in-Publication Data:

Plant chemical biology / edited by Dominique Audenaert, Paul Overvoorde. p. cm. Includes bibliographical references and index. ISBN 978-0-470-94669-5 (hardback)1. Botanical chemistry. I. Audenaert, Dominique, 1976– editor of compilation. II. Overvoorde, Paul, 1968– editor of compilation. [DNLM: 1. Plants–chemistry. 2. Biochemical Phenomena. 3. Plant Physiological Phenomena. QK 861] QK861.P525 2014 572′.2–dc23 2013035521

ISBN: 9780470946695

10 9 8 7 6 5 4 3 2 1

v

Contents

PrefaCe vii

ContriButors ix

Part one introDuCtion 1

1.1 from herbal remedies to Cutting-edge science: a historical Perspective of Plant Chemical Biology 3Michelle Q. Brown, Abel Rosado, and Natasha V. Raikhel

Part two sourCes of small moleCules 19

2.1 Compound Collections 21Reg Richardson

2.2 Combinatorial Chemistry library Design 40Robert Klein and Stephen D. Lindell

2.3 natural Product-Based libraries 64Alan L. Harvey

Part three iDentifiCation of new ChemiCal tools By high-throughPut sCreening 73

3.1 assay Design for high-throughput screening 75Frank W. An and Jose R. Perez

vi CONTeNTS

Part four use of ChemiCal Biology to stuDy Plant Physiology 93

4.1 use of Chemical Biology to understand auxin metabolism, signaling, and Polar transport 95Ken-ichiro Hayashi and Paul Overvoorde

4.2 Brassinosteroids signaling and Biosynthesis 128Takeshi Nakano and Tadao Asami

4.3 Chemical genetic approaches on aBa signal transduction 145Eunjoo Park and Tae-Houn Kim

4.4 Jasmonic acid 160Christian Meesters and Erich Kombrink

4.5 Chemical genetics as a tool to study ethylene Biology in Plants 184Yuming Hu, Filip Vandenbussche, and Dominique Van Der Straeten

Part five use of ChemiCal Biology to stuDy Plant Cellular ProCesses 203

5.1 the use of small molecules to Dissect Cell wall Biosynthesis and manipulate the Cortical Cytoskeleton 205Darby Harris and Seth DeBolt

5.2 the use of Chemical Biology to study Plant Cellular Processes: subcellular trafficking 218Ash Haeger, Malgorzata Łangowska, and Stéphanie Robert

Part six target iDentifiCation 233

6.1 target identification of Biologically active small molecules 235Paul Overvoorde and Dominique Audenaert

Part seven translation of Plant ChemiCal Biology from the laB to the fielD 247

7.1 Prospects and Challenges for translating emerging insights in Plant Chemical Biology into new agrochemicals 249Terence A. Walsh

7.2 In Vitro Propagation 263Hans Motte, Stefaan Werbrouck, and Danny Geelen

inDex 289

vii

PrefaCe

Plant biologists have a long history of using small molecules (e.g., MW < 500 Da) to explore developmental, physiological, and metabolic pathways. Compared to molecular genetic approaches used to study biological processes in plants, the roots of plant chemical biology became well established in the first decades of the twentieth century with the discovery of plant growth regulators or plant hormones. Since this time, the capacity of organic synthetic chemists to create and characterize the effect of small molecules provided the basis for understanding processes such as cell elongation through the action of auxins, the stimulation of cell division via the activity of cytokinins, the control of fruit senescence by gibberellins, and the influence of abscisic acid on seed dormancy. In addition, naturally occurring molecules as well as synthetic analogues formed the basis for genetic screens that looked for resistant or hypersensitive mutant plants. During the past 30 years, this approach led to the discovery of cellular components and pathways that inte-grate environmental and developmental information into regulated phenotypic outcomes.

Plant Chemical Biology provides tools that overcome the pleiotropic effects of plant hormones. A single hormone makes defining the role of specific components or signaling pathways a challenge. The capacity to identify novel compounds that stimulate or inhibit a subset of a signaling cascade holds tremendous promise for refining our understanding of these pathways. The coupling of this potential with advances in combinatorial chemistry offers exciting opportunities to develop new tools for basic and applied applications. In recent years, the querying of large collec-tions of diverse compounds through the use of robust assays has led to an impressive array of new discoveries in the field of plant biology.

viii PReFACe

This book intends to provide advanced students and professionals in plant biology—molecular biologists, physiologists, biochemists, or those active in agriculture, horticulture, or agronomy—a summary of key aspects of plant chemical biology. even as the pace of progress in this subfield accelerates, we hope that you will discover in the following chapters a summary of key findings to date, as well as an overview of the available compound collections, principles of assay design, and the translatability of these new tools to applied settings. In this day and age, no volume provides an encyclopedic review. We hope that our selection of topics reveals one of the ways that the quest for understanding biological processes drives the innovation of new technologies, which in turn propel our understanding of the biology.

We thank the authors of this collection. Their expertise and experience allows each topic to be handled with insight and depth. Without their contributions and patience, this book would not have been possible. We thank our institutions, Macalester College and VIB, for their support during the preparation of this book. Finally, we acknowledge the steadfast encouragement of our spouses, Lynn Overvoorde and eva Vermeulen, and our children, Mia, Anika, Vigdis, Sigun, and eldrid, during this process.

Dominique AudenaertPaul Overvoorde

ix

ContriButors

frank w. an Center for the Science of Therapeutics, The Broad Institute of Harvard and MIT, Cambridge, MA, USA

tadao asami Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan, and CReST, Japan Science and Technology, Chiyoda-ku, Tokyo, Japan

Dominique audenaert Compound Screening Facility, VIB, Ghent University, Ghent, Belgium

michelle q. Brown Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California, Riverside, CA, USA

seth DeBolt Department of Horticulture, Agricultural Science Center North, University of Kentucky, Lexington, KY, USA

Danny geelen Department of Plant Production, Faculty of Bioscience engineering, Ghent University, Ghent, Belgium

ash haeger Department of Forest Genetics and Plant Physiology, SLU/Umeå Plant Science Centre, Umeå, Sweden

Darby harris Department of Horticulture, Agricultural Science Center North, University of Kentucky, Lexington, KY, USA

alan l. harvey Strathclyde Institute of Pharmacy and Biomedical Science, University of Strathclyde, Glasgow, UK

Ken-ichiro hayashi Department of Biochemistry, Okayama University of Science, Ridai-cho, Okayama, Japan

x CONTRIBUTORS

yuming hu Department of Physiology, Laboratory for Functional Plant Biology, Faculty of Sciences, Ghent University, Ghent, Belgium

tae-houn Kim Department of Prepharm-Med/Health Functional Biomaterials, Duksung Women’s University, Seoul, Korea

robert Klein Bayer CropScience AG, Frankfurt am Main, Germany

erich Kombrink Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Köln, Germany

malgorzata Łangowska Department of Forest Genetics and Plant Physiology, SLU/Umeå Plant Science Centre, Umeå, Sweden

stephen D. lindell Bayer CropScience AG, Frankfurt am Main, Germany

Christian meesters Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Köln, Germany

hans motte Department of Plant Production, Faculty of Applied Bioscience engineering, University College Ghent, Ghent, Belgium (Member of the Ghent University Association), and Department of Plant Production, Faculty of Bioscience engineering, Ghent University, Ghent, Belgium

takeshi nakano RIKeN Advanced Science Institute, Hirosawa, Wako, Japan, and CReST, Japan Science and Technology, Chiyoda-ku, Tokyo, Japan

Paul overvoorde Department of Biology, Macalester College, St. Paul, MN, USA

eunjoo Park Department of Prepharm-Med/Health Functional Biomaterials, Duksung Women’s University, Seoul, Korea

Jose r. Perez Center for the Science of Therapeutics, The Broad Institute of Harvard and MIT, Cambridge, MA, USA

natasha v. raikhel Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California, Riverside, CA, USA

reg richardson ChemBridge Corporation, San Diego, CA, USA

stéphanie robert Department of Forest Genetics and Plant Physiology, SLU/Umeå Plant Science Centre, Umeå, Sweden

abel rosado Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California, Riverside, CA, USA

Dominique van Der straeten Department of Physiology, Laboratory for Functional Plant Biology, Faculty of Sciences, Ghent University, Ghent, Belgium

filip vandenbussche Department of Physiology, Laboratory for Functional Plant Biology, Faculty of Sciences, Ghent University, Ghent, Belgium

CONTRIBUTORS xi

terence a. walsh Dow AgroSciences LLC, Indianapolis, IN, USA

stefaan werbrouck Department of Plant Production, Faculty of Applied Bioscience engineering, University College Ghent, Belgium, and Department of Plant Production, Faculty of Bioscience engineering, Ghent University, Ghent, Belgium

Part one

introDuCtion

Plant Chemical Biology, First Edition. Edited by Dominique Audenaert and Paul Overvoorde. © 2014 John Wiley & Sons, Inc. Published 2014 by John Wiley & Sons, Inc.

3

from herBal remeDies to Cutting-eDge sCienCe: a historiCal PersPeCtive of Plant ChemiCal Biology

Michelle Q. Brown, Abel Rosado, and Natasha V. Raikhel

1.1.1 herBal remeDies anD PharmaCology in the anCient worlD

Herbal medicine is the oldest form of pharmacology known to mankind, and the evolution of its applications has depended upon the availability of plants to emerging cultures over time. Many of the medicinal uses for plants have developed through observations of wild animals and by trial-and-error approaches. As cultures evolved, the acquired knowledge of the medicinal herbs became systematized in order to increase the health and productivity of these civilizations [1]. As early as 2735 b.c., the Chinese emperor Shennong wrote a herbal treatise recommending the use of Ma Huang (Ephedra sinica, which contains the decongestant ephedrine) against respiratory distress. The records of King Hammurabi of Babylon (1800 b.c.) pre-scribed the use of mint (genus Mentha) for digestive disorders, and multiple texts surviving from the ancient cultures of India, Mesopotamia, and egypt illustrate the varied application of medicinal plant products, including white poppies (Papaver somniferum), linseed (Linum usitatissimum), and castor (Ricinus communis) oils, to treat diseases [2].

Deriving knowledge from the medical treatises and methods of those ancient civilizations and other conquered cultures such as Greeks, etruscans, and Persians

1.1

4 FROM HeRBAL ReMeDIeS TO CUTTING-eDGe SCIeNCe

allowed the Roman civilization to devise one of the best and most sophisticated medical systems of the ancient world. Roman contributions to the development of  chemical biology include the cultivation of medicinal herbs for commercial purposes along with the introduction of a variety of painkillers and sedatives including extracts of opium poppies (morphine) and of henbane seeds (Hyoscyamus niger, scopolamine), which Roman physicians tested on soldiers wounded in battle [3]. The Roman physician Galen of Pergamon (130–200 a.d.) outlined the basic prin-ciples of pharmacology by developing a systematic approach to drugs based on reasoning “logos.” For each extract, a defined system of Galenic degrees of potency was used to treat varying levels of dysfunction. This approach enabled physicians and pharmacists to more precisely deliver and monitor the effects of a medicinal substance in a dosage-dependent manner [4]. For almost a thousand years, the drugs and other methods of treatment popularized by Galen were used as standard protocol in european medicine [5].

With the advent and flourishing of the Islamic culture in europe, the Persian text Qanun (Ibn Sina, also known as Avicenna, 1025), translated into Latin as Canon medicinae, displaced the works of Galen and became the textbook for medical education in european universities up to 1650 [6]. The Canon established the basis of modern clinical pharmacology by introducing concepts such as clinical trials, randomized controlled trials, and efficacy tests laying out rules and principles for testing the effectiveness of new drugs and medications. The Canon was also used for medieval medical practitioners as a drug encyclo-pedia. It listed, with commentary, more than 700 preparations, including plant and mineral substances [7–11].

1.1.2 alChemy, Chemistry, anD the isolation of the BioaCtive metaBolites

By the seventeenth century, the knowledge of herbal medicine was widely dissemi-nated throughout europe, and many natural remedies were dependent on plant-based substances. In that period, the study of alchemy and the quest for a “panacea,” a treatment to cure all diseases as well as to prolong life indefinitely, was sought by such renowned scientists as Sir Isaac Newton, Roger Bacon, and Tycho Brahe [12]. Although the final objective was never achieved, alchemy established the experi-mental basis of modern chemistry and was a very important milestone for the development of extractive techniques to isolating plant compounds for medicines, dyes, and perfumes preparation [13]. Thus, as the science of chemistry matured, chemists began extracting and purifying the biologically active molecules from the natural substances that had been used as remedies for millennia. Included in this list of purifications were the isolations of the cardiac glycoside, digoxin from foxglove (Digitalis lanata), which was commonly used as a treatment for edema or dropsy [14], the addictive analgesic morphine from the seed capsules of poppy flowers [15], and the anti-inflammatory salicin from bark of willow (genus Salix) trees [16] (Fig. 1.1.1 Left Panel).

ALCHeMY, CHeMISTRY, AND THe ISOLATION OF THe BIOACTIVe MeTABOLITeS 5

With increased purity and knowledge of the active plant extracts and the advances in pharmacology, the first purely synthetic drugs based on natural products were elaborated in the middle of the nineteenth century. For example, in 1839, studies of the willow bark extract showed that salicylic acid was the active ingredient respon-sible for its analgesic properties [17]. In 1853, chemist Charles Frédéric Gerhardt mixed acetyl chloride with sodium salicylate to produce synthetic acetylsalicylic acid for the first time [18]. Academic chemists established the compound’s chemical structure and devised more efficient methods of synthesis that finally led to Felix Hoffman’s formulation of acetylsalicylic acid named Aspirin®, which helped to found the pharmaceutical industry in 1899 and remains the most widely used synthetic drug today [19].

In parallel with the isolation and synthesis of bioactive metabolites derived from plants, the search for antibiotics began in the late 1800s, with the growing acceptance of the germ theory of disease, a theory that linked microbes to the causation of a variety of illnesses [20]. Initially, antibiotics were identified as natural substances

Foxglove

Poppy

Willow

Morphine (analgesic)

Digoxin (cardiac glycoside)

Salicin (analgesic)

Taxol (antitumoral)Western Yew tree

Mayapple Teniposide (antitumoral)

Brefeldin A

Wortmannin

Cycloheximide

Latrunculin B

Endosidin1

Monensin

Cytochalasin D

Concanamycin A

(a)

(c)

(e)

(g) (h)

(f)

(d)

(b)

figure 1.1.1 Many plant metabolites have been utilized as pharmaceuticals, such as morphine, digitalis, and taxol. Several compounds have been utilized in cell biology research. These include compounds that inhibit protein trafficking (a)–(e), protein synthesis inhibitor (f), and cytoskeleton-inhibiting drugs (g)–(h). For color detail, please see color plate section.

6 FROM HeRBAL ReMeDIeS TO CUTTING-eDGe SCIeNCe

released by moss and fungi to inhibit growth of other organisms. Many extracts with antibiotic properties were reported in the literature during the late 1800s. Perhaps the most significant milestone in antibiotic research was the identification and isolation of the antibiotic penicillin from the Penicillium mold [21]. The subsequent development by Sir Howard Florey in 1942 of a process for manufacturing penicillin G has enabled the use of this small molecule to have more positive impact on human health than any other natural substances [22].

The technological innovations that led to the isolation of bioactive compounds toward the end of the nineteenth century impacted more than the area of human health. In fact, these successes set the stage for the development of multiple scientific disciplines, including plant biology. Out of the capacity to identify hormonal and nutritional requirements for plant growth and new ways of applying small molecules to manipulate and interrogate plant systems, the field of plant physiology emerged. Aspects of these advances are highlighted in following sections.

1.1.3 the DisCovery of Phytohormones anD the founDation of moDern Plant ChemiCal Biology

Since antiquity, people have sought to explain plant physiological responses such as gravitropism and touch sensitivity via mechanical or passive physiochemical forces. In this context, Aristotle’s conclusion that plants were insensitive and lack active motion against external stimuli pronounced in the third century b.c. was still prevalent and widely used by eighteenth-century taxonomists to classify and differentiate plants from other organisms within the graduated scale of nature [23]. The 1880 publication of The Power of Movement in Plants by Charles Darwin with assistance from his son Francis was a landmark attempt to integrate plant movements into a biological perspective of behavior by introducing the concepts of plant sensitivity and active perception of external stimuli [24]. The recognition that plants might respond to the environment in much the same way as animals was a paradigm shift for taxonomists and plant physiologists and became the seminal idea that led to the discovery of the first plant growth factor, the auxin phytohormone indole-3-acetic acid (IAA).

The Darwins used experiments on gravi- and phototropism to show that some internal signal or “influence” moved from coleoptile tips of the annual canary grass (Phalaris canariensis) to control cell elongation after stimulation [24]. The suggestion of such signaling molecules led Hans Fitting (1909) to introduce the term “hormone” when referring to substances that stimulate specific plant developmental processes [25]. In  1913, Danish botanist P. Boysen Jensen suggested that the phototropic signal described by the Darwins was indeed a chemical messenger moving down from the tip of the coleoptile [26]. During the early 1920s, several botanists provided circumstantial evidence of the presence of such a chemical in different plants, and finally in 1928, the Dutch physiologist Frits Went defined an experimental procedure for isolating this  chemical and quantifying its physiological activity [27]. A next important step concerned the purification of sufficient amounts of this chemical for analytical purposes.

THe DAWN OF PLANT SYNTHeTIC CHeMISTRY 7

Five years of attempts made by K. Thimann, and J. Haagen-Smit among others, finally succeeded, and the Darwinian “influence” was identified as IAA [28, 29].

The identification of IAA as the growth factor responsible for the gravitropic and phototropic responses crowned a half century of research that revealed the use of chemical signals in plants and opened the door to the development of compounds that could contribute to modern agriculture by stimulating growth or eliminating weeds.

1.1.4 the Dawn of Plant synthetiC Chemistry

The implementation of synthetic and combinatorial chemistry in the agrochemical industry coincided with the development of the modern pharmaceutical industry. Both of these fields were fueled by the diversity and increased availability of organic compounds derived from coal tar, plants, and microorganisms. In this context, pharmacological studies by P. ehrlich (1913) led to the receptor theory of small-molecule action, and receptors were defined as specific body constituents where the active drugs selectively bind [30]. After the identification of IAA as a plant growth factor, the 1940s witnessed the creation of multiple academic and industrial labora-tories in which detailed structure–activity analysis along with combinatorial chemistry led to the development of novel compounds that mimic the activity of the endogenous hormone or that could function as herbicides. As a result, a wide array of IAA analogs and derivatives including 1-naphthaleneacetic acid (1-NAA), 2-methyl-4-chlorophenoxyacetic acid (MCPA), and 2,4-dichlorophenoxyacetic acid (2,4-D) were synthesized. Further research demonstrated that these derivatives elicited the same type of plant responses as IAA such as elongation in aerial tissues and stimulated lateral root formation but with activity that lasted longer and was more potent than the endogenous molecule because of their relatively slower metab-olism by plants [31–33].

With increased concentration, stability, and activity in plant tissues, synthetic auxins gained considerable importance for probing auxin function in basic research as well as for applied civil and military uses. examples of civil uses range from the commercialization of the synthetic auxin 1-NAA marketed as “Hortomone A” to stimulate lateral rooting in crops [34] to the commercial elaboration in 1945 of the first systemic herbicide based on synthetic auxins marketed as “Weedone,” a product that started a new era of weed control in modern agriculture [35]. In parallel, the development of synthetic auxins for military purposes was fast-tracked during World War II, and early investigations regarding the use of synthetic auxins were kept secret because these novel herbicides were deemed putative bioweapons [36]. It was not until the Vietnam Conflict nearly twenty years later, when hormone-based weed killers would become infamous due to the extensive use of the defoliant Agent Orange, an equal mixture of 2,4-D and 2,4,5-trichlorophenoxyacetic acid, as part of the U.S. herbicidal warfare program. The use of Agent Orange in the Vietnam War caused the loss of millions of acres of upland and mangrove forests and crops, and its side effects on humans and the environment awakened interest in the effects of synthetic chemicals on the natural environment [37].

8 FROM HeRBAL ReMeDIeS TO CUTTING-eDGe SCIeNCe

In the post-World War II era, the excitement caused by the synthesis of the novel antibiotic penicillin and its impact for human health, the increased crop production after the use of synthetic herbicides, and the development of affordable synthetic chemicals stimulated the search for more natural and synthetic substances derived from plants and microorganisms. These advances had profound implications in the field of plant chemical biology because multitudes of small molecular probes became available to explore biological processes without the need for stringent pharmacoki-netic or safety profiles required for medicinal applications. Many compounds such as cycloheximide, Brefeldin A, cytochalasins, and latrunculins remain powerful and much used probes that enable answers to basic and applied questions to be found.

1.1.5 serenDiPity versus rational Design of ChemiCal liBraries

By the end of the 1940s, the use of natural and synthetic compounds as probes to modulate biological pathways in plants was extended, and synthetic organic chem-istry had progressed to the point where the large-scale preparation of synthetic analogs of bioactive molecules such as antibiotics and plant hormones was economically feasible. At the same time, the introduction of powerful commercial spectrometers (i.e., nuclear magnetic resonance, 1952) and separation techniques (i.e., high-performance liquid chromatography, 1961) for determining the structures of minute quantities of biologically active natural products boosted the identification and refinement of natural drugs through structure–activity relation analysis. Thus, the resulting “methyl, ethyl, isopropyl” analog model for drug discovery, although empirical, was quite successful for several decades. These collections of natural compound and their synthetic derivatives were, however, limited by two factors. First, the basis of drug discovery relied heavily on the serendipity of finding biolog-ically active products from a relatively small number of plant species, and second, these collections contained a limited number of compounds representing only a minute fraction of the structural diversity possibilities in the chemical space [38].

For the well-resourced biomedical community, the development of computer tools, and concurrent advances in structural biology, chemistry, and protein crystallo-graphy in the 1980s, a shift to a “rational” computational archetype for drug discovery emerged, and computational drug discovery was born [39]. The assumption underpinning this approach was that a continuous stream of new protein structures and associated active sites and binding pockets would provide the basis for the computational-assisted rational drug design. Limitations in the crystallographic techniques such as the difficulty in generating protein crystals, the slow pace of protein structure determi-nation, and the time-consuming experimental validation of the candidate molecules have limited the number of successes obtained by this approach. In addition, for plant biologists, limitations to bioavailability due to the protective nature of the cell wall and rapid xenobiotic metabolism further tempered enthusiasm for this approach and led to a return of largely empirical methods of small-molecule library synthesis and high-throughput screening (HTS). Technological advances in  robotics and

THe DeVeLOPMeNT OF COMBINATORIAL CHeMISTRY 9

biological techniques for simultaneously assaying thousands of compounds in concert during the 1990s fueled this paradigm shift.

1.1.6 the DeveloPment of ComBinatorial Chemistry

After the partial drawback of the rational drug design, chemists and biologists realized that large collections of structurally distinct molecules should be generated to perform efficient pharmacological studies and therefore, the traditional synthesis and purification of one compound at a time for screening was no longer a feasible approach. An important step toward increasing the diversity and the speed in which structurally related compounds were generated was the application of combinatorial chemistry techniques involving systematic mixing and matching of various chemical building blocks to generate libraries of small molecules. The development of these techniques, running in parallel with advances in automation and robotics, enabled organic chemists Furka and Lam to apply Merrifield’s solid-phase synthesis of pep-tides to the combinatorial synthesis of diverse sets of peptides on solid phase using mix and split reactions pioneered by them [40–42]. The success of combinatorial chemistry to generate libraries of peptides and peptidemimetics positively impacted pharmacological studies because this approach involved iterative reactions, was ame-nable to automation, and allowed detritus from the products to be removed by washing the resins.

early combinatorial chemistry techniques successfully generated enormous num-bers of compounds; still their “diversity” was limited to variations in appendages attached to a small number of common skeletons and the compounds frequently lacked chirality. Since most of the small molecules known to disrupt biological systems were structurally complex natural products, the organic chemist Stuart Schreiber reasoned that maximizing the number of structures and scaffolds by increasing the size and number of rigidifying elements (macrocycles, polycycles, olefins, etc.) in small-molecule libraries would be the most efficient way to populate the largest amount of chemical space. Hence, the creation of smaller diversity-oriented library of compounds contain-ing wider spectra of molecular shapes would provide both more hits and a wider range of biological targets than the traditional combinatorial libraries [43].

As the number and diversity of characterized bioactive synthetic compounds increased, the biochemist Chris Lipinski and his colleagues identified the general properties and structural features of molecules that had strong biological activity [44]. What they observed, often referred to as the “Lipinski Rule of Five,” is that molecules that are likely to be orally active drugs in humans meet four criteria—they contain not more than 5 hydrogen bond donors, they contain not more than 10 hydrogen bond acceptors, they are less than 500 daltons, and they have logP

oct/wat less than 5. These

guidelines were quickly adopted by the pharmaceutical industry as an early filter for the development of orally active drugs. For plant biologist, these same rules apply for agrochemicals [45, 46].

By the end of the millennia, an important limitation to expanding and defining the possibilities of combinatorial chemistry for basic plant research was the

10 FROM HeRBAL ReMeDIeS TO CUTTING-eDGe SCIeNCe

closed environment of the biotechnology and pharmaceutical industries in which most of the research projects were carried out. In this context, a milestone for the development of plant chemical biology as a subject for basic science was the creation of publicly available HTS facilities such as the Harvard’s Institute of Chemistry and Cell Biology (1998), whose highly successful Investigator-Initiated Screening Program facilitated the development of small-molecule screening projects for basic research and oriented the pursuit of chemical biology as an academic discipline. These efforts were also supported by the availability of libraries from both academic (e.g., the LATCA library) and commercial libraries that continue to grow in size and complexity.

1.1.7 Plant ChemiCal Biology in the -omiCs era

As chemical biology was gaining prominence as an academic discipline, plant biol-ogists were also gaining a new host of molecular and genetic tools allowing plant genetics to undergo its own development into the age of genomic research. The release of the complete sequence of the model plant Arabidopsis thaliana (2000), together with many unique genetic/genomic tools developed in this plant system such as whole-genome microarrays, a large collection of knockout and activation-tagged mutations, a rich array of mutants, and the ease of cloning a gene by map-based cloning aided by DNA microarrays, allowed the integration of chemical screens at various biological levels ranging from subcellular to the whole organism.

The development of sophisticated imaging techniques such as confocal laser scanning microscopy (CLSM) that allows for greater resolution than traditional fluo-rescence microscopy and that can generate a 3 D representation of the specimen, coupled with the development of plant-specific probes such as engineered green fluorescent protein (GFP), originally isolated from the jellyfish Aequorea victoria, fused to the now sequenced Arabidopsis genes, allowed researchers to view the dynamic, in vivo subcellular localization of proteins instead of the static state of fixed specimens [47]. Thus, rather than have to wait for growth or gross morphological plant phenotypes, plant researchers could now observe subcellular phenotypes caused by small molecules based on the aberrant localization of proteins in real time. The successes of this approach are documented in other chapters in this book. Collectively, the benefits of obtaining novel insights by using chemical tools are reflected in the growing number of publications during the past two decades that use chemical biology approaches for plant research (Fig. 1.1.2).

1.1.8 introDuCtion of BioinformatiCs anD CheminformatiCs

The deluge of information generated by the -omics analyses could only be managed with the help of computers and their programmers, and thus, the field of bioinformatics was born. Facilitating the growth of bioinformatics were the Internet-accessible public

INTRODUCTION OF BIOINFORMATICS AND CHeMINFORMATICS 11

repositories, such as the National Center for Biotechnology Information (NCBI). At first, bioinformatics merely managed the wealth of sequences and structures. Algorithms to analyze homology of gene sequences, such as Basic Local Alignment Search Tool (BLAST), were necessary to navigate the over 100 million sequence records contained within GenBank at NCBI. This helped to identify the members of large gene families and genes similar in sequence to ones with known function in other sequenced organisms, such as yeast. Bioinformatics has helped identify common promoter motifs among genes that promote both brassinosteroid and auxin pathways [48]. Bioinformatics also provides a critical tool in the field of comparative genomics. Now, with a wealth of analysis and modeling algorithms and tools available, bioinformatics represents just a portion of the field of computational biology.

As high-throughput chemical biology screens continue to be performed, the need for new tools and data repositories resulted in the formation of cheminformatics. The formation of the NCBI’s PubChem open-access database in 2004 was initially a cause of concern for the American Chemical Society Chemical Abstract Service (CAS) Registry, which maintained a subscription-based comprehensive database of compounds dating back to the early 1900s. Since the inception of PubChem, however, several publicly accessible compound databases have been established (e.g., ChemDB [49], DrugBank [50], ChemmineR [51]). In these databases, 3 D compounds are described using the simplified molecular input line entry specification (SMILeS).

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2009

2010

figure 1.1.2 evolution of the number of publications using chemical genomic approaches in the plant model system Arabidopsis thaliana. Source: National Center for Biotechnology Information (NCBI).

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These descriptors along with physiochemical descriptors can be mined for structural and activity relationships among compounds.

Future chemical biology approaches will likely involve better and more efficient computational starting points. While a little more than 10 million pure compounds are described in chemical databases, the potential chemical diversity (defined as the number of unique chemical structures) of compounds composed of carbon, hydrogen, nitrogen, oxygen, sulfur, phosphorous, and the halogens (the organic chemist’s periodic table) of molecular weight less than 1000 likely exceeds 1060 [52]. However, in a time when Moore’s Law (1963), which states that computer processing speed doubles every two years, still prevails, it is not utopist to think that in the near future the virtual vastness of chemical spaces could be overcome with enough computing power. The virtual screening of compounds is extremely appealing because it saves the time and expense of physically screening extensive numbers of compounds. In silico screening requires either the identification of the ligand to be bound or the specifications of the docking site for competitive inhibi-tion. Docking programs that generate the possible conformations of interaction between ligand and docking site are of critical importance. Maintaining the open-access example of the databases, many docking programs such as AutoDock [53] and ZINC [54] are publicly accessible. In silico screening allows an enrichment of putatively active compounds based on the ligand or docking site. These compounds then need to be actively assayed to determine actual activity. To demonstrate the utility of this approach, Doman et al. [55] in a screen to identify novel inhibitors of tyrosine phosphatase 1B utilized both in silico chemical genomics and HTS. The in silico program determined 365 putative hits and 127 of these were confirmed, for a hit rate of 34.8%. The HTS screen of over 400,000 compounds produced only 85 hits, for a hit rate of 0.021%. With in silico applications, however, results need to be verified with wet lab experiments when the targets are known. Without a directed chemical screen, compound targets must be identified through mutational analysis and mapping or biochemical means.

1.1.9 unique Challenges in the fielD of ChemiCal genomiC researCh

Plant chemical genomics, defined as the use of small molecules to study the functions of the plant cell at the genome level, is a developing field that provides valuable tools and novel perspectives for plant research. However, the use of small molecules in plant systems is associated with a unique set of challenges. Still unre-solved is the synthesis of enough quantities of compound required for larger-scale studies and structure–activity relation analyses. In some cases, obtaining sufficient supply of a particular bioactive compound can be difficult when dealing with natural metabolite compounds too difficult to synthesize in a laboratory and found in minimal concentrations in nature [56]. Also, the compound isolation from natural sources can provoke intellectual property and environmental conservation

CONCLUDING ReMARKS 13

arguments for regionally endogenous and endangered species. To cope with these challenges, the future chemical genomics will undoubtedly have to incorporate and streamline novel technologies for increased synthesis, utility, and understanding in a sustainable and environmentally friendly setup, as well as to implement efficient intellectual property protocols.

A challenge of a different nature is derived from the plant system itself. Being sessile organisms, plants have adapted to deal with a multitude of environmental and toxicological stresses. These adaptations can help a plant to habituate to a compound over time at the cellular level, as in the case of Brefeldin A [57], and at  the gross morphology level, as evidenced by the regeneration of shoots in non- hormone-containing media from calli of Nicotiana bigelovii habituated to the herbicide 2,4-D [58]. However, sometimes it is not always the plant metabolic path-ways that are altered, but the structure of the compound. Chemical compounds can act as precursor molecules, metabolizing into the smaller bioactive molecule, or be modified by the plant’s metabolism in vivo. Two compounds that are now known to be modified in planta are sirtinol and hypostatin. Sirtinol was originally identified in a yeast screen as an inhibitor of the Sirtuin family of nicotinamide adenine dinucleotide (NAD)-dependent deacetylases [59], was later characterized as an activator of auxin-inducible genes through the degradation of negative auxin regulators, and finally determined to be converted in vivo to an active auxin that is transported in cells through a polar-independent pathway [60, 61]. Hypostatin was originally identified as a cell expansion inhibitor and found to be glycosylated by the UDP-glycosyltransferase HYPOSTATIN ReSISTANCe 1 (HYR1) in vivo. These examples underscore the need to ascertain and verify in vivo the compound responsible for the biological phenotypes observed.

1.1.10 ConCluDing remarKs

Despite the unique challenges in the field of chemical biology, the use of small compounds to circumvent the problems of gene redundancy and lethality encoun-tered by classical genetics has proven to be a useful approach in interrogating biological systems. The discipline has always been an interface between chemistry and biology, maturing as advances in the separate fields occurred and eventually including computer science through cheminformatics, in silico screening, and dock-ing programs. As the future of chemical genomics is pondered, it is easy to imagine the continued incorporation of other scientific disciplines as well.

In contrast to classical biologists, taking a reductionist view of the plant sys-tems and reducing them to basic parts and pathways, the new breed of plant system biologists look for the integration of the whole organism into a complex network of pathways, with the belief that unique properties can only be observed through the study of the whole system. The next natural step would be to incorporate chemical genomics into the systems biology paradigm as it has been done in study with yeast [62]. Thus, chemical genomics will continue its interdisciplinary

14 FROM HeRBAL ReMeDIeS TO CUTTING-eDGe SCIeNCe

journey, including various biological fields such as genomics, epigenomics, metabolomics, and phenomics, with chemistry and computer sciences.

aCKnowleDgments

We would like to thank Dr. Sean Cutler for critical reading of the manuscript. This work was supported by the National Science Foundation (DGe-0504249 to M.Q.B.) and by the DOe/BeS Physical Biosciences program (De–FG02–02eR15295 to A.R. and N.V.R.)

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