Drawing on Nature

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Drawing on Natures Complexities

• Feasibility and Future Directions

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• Progress is impeded less by ignorance than by the
  illusion of knowledge.

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• Harnessing nature to benefit humans.

Whole plants or derived plant components
(metabolites)

• New drugs
• Botanical Dietary Supplements (Phytomedicines)
• Nutrition

Three major components to this effort
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• Chemicals in plants (or microbes or animals)

• Their effects in humans

• Supply to develop and use

Goal is to use plant chemistry and biology to
benefit humans (not necessarily to get as many
good compounds as possible).
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Drug Discovery From Natural Sources
Sample acquisition Biological evaluation Isolation
structure elucidation Synthesis, SAR Mechanism
of action Bulk supply Preclinical
develop. Clinical trials Launch
Botanicals (Phytomedicines) Multiple
components Standardized material for biological
studies
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• How can we more successfully achieve the goal?

Accept and work within two abiding truths

• Theres a reason for everything.

• Nothing is simple.

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OBJECTIVE

• Challenge us to rethink the drug and
  phytomedicine discovery and development paradigm
  in light of knowledge acquired and lessons
  learned from studies in diverse disciplines.

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What do we know?

• What lessons have we learned?

What big questions do we have?
What would we do if we could?
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• What we know

• Plants produce chemicals for protection and
  survival.

• A single plant produce multiple metabolites
  (structurally related and structurally unrelated)
  at the same time.

• Were beginning to understand how genes are
  organized and regulated, cloned or exogenously
  expressed.

• There can be spatial and temporal differences in
  the metabolic profile of a single plant.

• Correct annotation is more than a function of
  knowing the gene sequence.

• Elicitors can trigger metabolite production.

• Biotic factors such as herbivores and pathogens
  influence the evolution of secondary metabolism

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• What we know

• Plant metabolites have effects on other organisms.

• A single compound has multiple mechanisms of
  action, multiple biological targets in an
  organism, therefore, multiple effects.

• A single molecular target can be relevant to
  multiple diseases/conditions.

• The genetic diversity inherent in humans will
  influence the positive/negative outcomes of
  exposure to plant chemicals.

• Plants have been successfully engineered for
  specific traits, including metabolite content.

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Plants produce chemicals for protection and
survival
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Ernst Stahl (1888). Pflanzen und Schnecken,
Biologische Studie über die Schutzmittel der
Pflanzen gegen Schnecken fraß. Jenaische
Zeitschrift f. Naturwissenschaften 22, 557-684.

• Plant chemistry is protection against snails and
  other herbivores.

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A single plant produces multiple metabolites at
the same time

• Whole tobacco plants make a series of diterpene
  glycosides and caffeoyl putrescine in response to
  MeJA (Keinänen et al. 2001)
• Tobacco cells make anabasine, anatabine,
  anatallines 1 2 and free and soluble methyl
  putrescine in response to MeJA (Goosens et al.
  2003)

At least one explanation is synergy - of both
related and unrelated compounds
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Synergy - structurally related compounds

• Berenbaum and Zangerl (1993) Oecologia 95370-375
• Type A and B streptogramins - alone
  bacteriostatic but together bacteriocidal

Cytochrome p450 metabolism
Growth rate
Papilio polyxenes
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Synergy - structurally unrelated compounds

• Percent oviposition response of Papilio xuthus

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Mechanisms of synergy

• Inhibition of phase 1 2 detoxification enzymes
• Complexation that facilitates transport
• Phosphatidylcholine and other phospholipids
• Lecithin
• Proteolytic enzymes that facilitate transport
• Inhibition of efflux pumps

From Gilbert and Alves (2003) Synergy in Plant
Medicines, Current Medicinal Chemistry 10 13-20
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Understanding how genes are organized and
regulated, and recognizing spatial and temporal
differences
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S. Aubourg, A. Lecharny and J. Bohlmann. 2002.
Genomic analysis of the terpenoid synthase
(AtTPS) gene family of Arabidopsis thaliana.
Molecular Genetics and Genomics 267 730-745.
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Regulation of biosynthetic genes

• Elicitor triggers Jasmonate signalling
• Transcription factors
• ORCA (Octadecanoid-Responsive Catharanthus
  AP2/ERF-domain) transcription factors - Terpenoid
  indole alkaloids
• MYB and bHLH proteins - Anthocyanin biosynthesis

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Elicitors or transcription factors can be used to
search for genes

• Microarray data with elicitors

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• Correct annotation is more than a function of
  knowing the gene sequence

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Elicitors can trigger metabolite production

• Cell wall oligosaccharides
• Pathogen elicitors
• elicitin
• chitosan
• Insect elicitors
• ß-glucosidase
• Fatty acid amino acid conjugates (FACs)
• Other salivary constituents

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Biotic factors such as herbivores and pathogens
influence the evolution of secondary metabolism
Duplication
Polyploidy
Epigenetic effects - Functional
diploidization - Gene silencing
Rearrangements - Diversification -
Psuedogenation
Environmental Interaction
Speciation chemical evolution
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Plant metabolites have effects on other organisms

• Animals
• Taste/odor receptors that govern behavior
• Detoxification enzymes
• Target site modification
• Plants
• Biosynthetic enzymes
• Inhibitors of proteinases
• Signalling pathways

ouabain
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• A single compound has multiple mechanisms of
  action, multiple biological targets, therefore,
  multiple effects in an organism

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Repair protect - multiple mechanisms

• Sulforaphane - anticarcinogenic properties
• Inducer of phase II enzymes
• Inhibitor of phase I enzymes
• Inducer of apoptosis and cell cycle arrest
• Resveratrol
• Activates p53 (triggers apoptosis)
• Inhibits NF-?B (tumor promoter)
• Inhibits COX-2
• Lycopene
• Excellent antioxidant
• Anticarcinogen
• Protection against cardiovascular disease

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Immune system

• Polysaccharides elicit immune response
• Many plant species inhibit immune system
• Some plants contains both immunostimulant and
  immunodepressant components

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• A single molecular target can be relevant to
  multiple diseases/conditions

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HIV and the CCR5-?32 allele

• CCR5 receptor binds endogenous cytokines, highly
  expressed in macrophages and CD4 T cells and
  important in immune system
• HIV virus uses the CCR5 receptor to enter
  macrophages during the first stage of infection
• Mutated allele has a 32 base pair deletion
• Individuals homozygous for this deletion are
  resistant to HIV
• Individuals heterozygous for this allele have a
  delayed onset of AIDS of 2-3 years
• New direction of HIV therapies centered around
  blocking this receptor, especially since
  individuals homozygous for this allele are healthy

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Effects of CCR5-?32 on resistance to other
diseases
Disease Yes No Ambiguous
Breast cancer 1
Hypertension 1
Childhood asthma 2 1
Brucellosis 1
Hepatitis C 1
Adult cytomegalovirus 1
Homozygous sickle cell disease 1
Multiple sclerosis 2
Chagas disease 1
Coronary artery disease 1
Crohns disease 1
Modified from de Silva and Stumpf (2004) FEMS
Microbiology Letters 2411-12
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Molecular targets are gene products, and
therefore reflective of human genetic diversity

• This is important for development of therapeutic
  targets
• But therapies must address genetic differences in
  populations

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Assay design should consider

• Synergistic effects
• Possible multiple mechanisms of action for a
  single compound
• Improvement of the in vitro to in vivo
  correlation
• And account for genetic diversity in target
  populations

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Plants have been successfully engineered for
specific traits, including metabolite content

• Successful engineering has been done in primarily
  crop plants to confer resistance to pesticides,
  or growth properties, ripening fruit, drought or
  temperature resistance
• Sato et al. recently reported successful
  engineering for alkaloid production in cells.
• It has also been shown that secondary metabolites
  can be deleted via several methods (redirect the
  precursor, antisense gene, RNA interference,
  etc.).

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• Lessons learned.

• Everything interrelates

• Recognizing the impact of genetic diversity is
  critical

• Single vs. multiple - compounds, mechanisms,
  effects

• Knowing the genome is not enough - understanding
  the function of the genes and proteins is vital

• The approach/process for drug discovery has to
  take into account the environment of anticipated
  use

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Some of the big questions

• Why do plants produce specific metabolites?
• What metabolites are produced by various plants?
• Why does a single plant produce multiple
  metabolites (often structurally unrelated) at the
  same time?
• How do the compounds within a suite interact with
  each other and/or enhance the desired outcome?
• How, where, and when are the metabolites
  produced?
• What triggers their production and what triggers
  changes in their production (evolution)?

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• Some of the big questions
• What effects do/could these metabolites have on
  humans (in light of the reasons for their
  production)?
• What are their mechanisms of action and targets
  in humans and animals?
• What other consequences/effects are likely as a
  result of the mechanism/target of the compounds?
• How can plant production of the metabolic profile
  be reliably reproduced?
• How can the information generated from multiple
  disciplines be maximally shared and inform other
  disciplines?

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• Some proposed major areas of future research and
  development

• Synergism - what, why, how to utilize, etc.
• Systems - better understanding of the systems in
  which potential new drugs are produced and used
  (stimuli to production, multiple
  functions/activities, synergism with other
  compounds, drug effectiveness, etc.)
• Plant metabolic profiles - databases, techniques,
  references
• Comparative genomics - what is it about some
  people, animals that make them less susceptible
  to serious human diseases

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• Some proposed major areas of future research and
  development

• Engineered plants - to reliably understand
  effects of phytomedicines to alter the yield of
  specific plant metabolites to obtain
  reproducible whole plants and plant products
• Increase understanding production of secondary
  metabolites - elicitors, gene organization and
  regulation, annotation, evolution
• Advancing/creating the technology and techniques
  to examine these questions in a cost-effective
  but reliable and rapid manner (bioinformatics,
  assays, structure and function determination,
  etc.)

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What would you do, if money and technology were
not an issue?

• Eliminate the challenge of dereplication (have
  good databases of metabolic profiles)
• Be able to rapidly identify both function and
  structure of secondary metabolites
• Have in vitro models that truly predict in vivo
  effects
• Produce engineered plants that differ only in the
  presence/absence/concentration of specific
  secondary metabolites
• Produce engineered plants that localize desirable
  (or undesirable) metabolites in specific tissues
• Know with certainty that a proposed drug target
  is important in the context of the system in
  question

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• What would you do, if money and technology were
  not an issue?
• Know the multiple effects of a single compound
• Know how compounds interact within the context of
  their potential use in humans
• Be able to predictably, reliably produce mass
  quantities of important secondary metabolites in
  a ecologically responsible manner
• Understand the absorption, distribution,
  metabolism, and pharmacology of multicomponent
  botanicals
• Understand why some people who have HIV have not
  developed symptoms of AIDS, or why sharks dont
  get cancer and mice dont get AIDS

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• Acknowledgments
• Dr. Cheryl Frankfater
• Dr. Norman Lewis, organizers, and staff
• National Science Foundation