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Plants and microbes as drug targets

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Plants and microbes as drug targets Prof. H.S. Prakash Department of Biotechnology University of Mysore Manasagangotri, Mysore 570 006 Chemistry Compound integrity ... – PowerPoint PPT presentation

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Title: Plants and microbes as drug targets


1
Plants and microbes as drug targets
  • Prof. H.S. Prakash
  • Department of Biotechnology
  • University of Mysore
  • Manasagangotri, Mysore 570 006

2
Higher plants a rich source of novel compounds
  • 400,000 higher plant species
  • 10 characterized chemically
  • One-fourth of pharmaceuticals derived from plants
  • 11 of the 252 basic and essential drugs (WHO)
    are exclusively derived from flowering plants
  • Plant-derived drugs have huge market value US30
    billion in USA (2002)

3
Natural metabolites
  • Secondary metabolites have no recognized role in
    maintaining fundamental life processes but have
    important role in the interaction of the cells
    with its environment
  • Only half the structures elucidated
  • Chemically highly diverse but characteristic of a
    plant

4
Role of secondary metabolites
  • Not clear
  • Important for survival of the plants in its
    ecosystem
  • Antimicrobial, anti-insect, deter potential
    predators
  • Discourage competing plant species
  • Attract pollinators or symbionts
  • Flovours, fragrances, dyes, pesticides and
    pharmaceuticals

5
Major groups
  • Based on biosynthetic origins structurally
    divided into five major groups
  • Polyketides Acetate-mevalonate
  • Isoprenoids Terpenoids and steroids from 5-C
    precursor isopentenyl diphosphate (IPP)
  • Alkaloids via classical mevalonate pathway or
    the novel MEP (non-mevalonate or Rohmer) pathway
  • Phenylpropanoids having C6-C3 units from
    aromatic amino acids phenylalanine or tyrosine
  • Flavonoids combination of phenylpropanoids and
    polyketides

6
Biosynthetic pathways
  • Often long, complex multi-step events catalyzed
    by various enzymes and still largely unknown
  • Alkaloid biosynthesis Best studied, 12,000
    structures are known
  • Production of specific alkaloid Often restricted
    to certain plant families
  • Flavonoids are abundant in many plant species

7
Examples Natural drugs
  • Vinblastine, vincristine Madagaskar periwinkle
    Catharanthus roseus
  • Anticancer Paclitaxel (Taxol), podophyllotoxin,
    camptothecin
  • Analgesic Morphine
  • Semi-synthetic drugs steroidal hormones derived
    from diosgenin Dioscorea corticosteroids,
    contraceptives, sex hormones

8
Plant chemical diversity
  • Much greater than any chemical library
  • Enormous reservoir
  • Omics-based functional genomics screening
  • Limited success of combinatorial chemistry or
    computational drug design

9
Combinatorial chemistry
  • Designed organic chemistry enables optimization
    of molecular structures Not attractive
  • 1981-2002 28 (351) of 1031 new drug entities
    (NDEs) either natural products or derivatives
  • 24 synthesized based on natural resources
  • Major group Anti-microbial (66), Anti-cancer
    (52), Anti-hypertensive, Anti-inflammatory

10
Plant cell culture an alternative production
system
  • Plants are difficult to cultivate or becoming
    endangered
  • Chemical synthesis complex structure/specific
    stereochemical requirements
  • Cell or organ culture attractive alternative
  • But limited commercial success because of
    empirical nature of selecting high-yielding,
    stable cultures and lack of biosynthetic pathway
    and its regulation

11
Cell cultures
  • Production limitation
  • Treatment of undifferentiated cells with
    elicitors such as methyljasmonate, salicylic
    acid, chitosan and heavy metals
  • Organ culture Hairy root (alkaloids) or shooty
    teratoma (tumor-like) cultures monoterpenes

12
Cell suspension cultures
  • Shikonin from Lithospermum erythrorhizon
  • Berberine from Coptis japonica
  • Rosmarinin acid from Coleus blumeii
  • Sanguinarine from Papaver somniferum
  • Paclitaxel from Taxus brevifolia
  • Bottlenecks low productivity, process
    technological issues (Bioreactor design,
    cultivation conditions)
  • Functional genomics Newer opportunities

13
Metabolic engineering strategies
  • Decrease the catabolism of the desired compound
  • Enhance the expression or activity of a
    rate-limiting enzyme
  • Prevent feedback inhibition of a key enzyme
  • Enhance expression or activity of all genes
    involved in the pathway
  • Compartmentalization of the desired compound
  • Conversion of an existing product into a new
    product

14
Metabolic engineering strategies
  • Gain-of-function and loss-of-function of genes
  • Discovery of transcription factors that regulate
    the entire pathway
  • Overexpression of transporters
  • Eg. Overexpression of rate-limiting upstream
    enzyme putrescine N-methyltransferase (PMT) and
    the downstream enzyme hyoscyamine 6ß-hydroxylase
    (H6H) of tropane alkaloid biosynthesis enhanced
    production of scopolamine in cultivated hairy
    roots

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Transcriptional regulation
  • Part of terpenoid indole alkaloid biosynthesis in
    Catharanthus roseus is under the control of
    ORCA3, a jasmonate-responsive APETALA2
    (AP2)-domain transcription factor
  • Constitutive overproduction of ORCA3 enhanced
    terpenoid indole alkaloids
  • Maize transcription factors LC and C1 in tomato
    fruits upregulated the flavonoid pathway
    kaempferol
  • ANT1 - Anthocyanin biosynthesis, glycosylation
    and transport in vacuoles

17
Transporters
  • Nicotine and other alkaloids are exported by
    overexpressed yeast ABC transporter PDR5 in
    transgenic tobacco thus decreasing cellular
    toxicity
  • Multidrug resistance protein cjMDR1 obtained from
    berberine-producing Coptis japonica
    cells functions as an ABC transporter probably
    pumps berberine into xylem cells for
    root-to-rhizome translocation

18
Functional genomics and secondary metabolites
  • Transcriptomics, proteomics, metabolomics
  • Genomics tools for medicinal plants are limited
  • Tools 2-D gel electrophoresis-based proteomics,
    transcript analysis tools such as differential
    display, EST databases, micro-arrays

19
Possible functional genomics approach
  • High-throughput selection and testing of genes
  • Could be used without pre-existing sequence
    knowledge
  • Profiling methods (e.g. micro-arrays) require
    genomic information, hence cannot be used

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Functional genomic approach
  • Elicit the metabolites (e.g. methyl jasmonate)
  • Use Genome-wide transcript profiling methods to
    identify expressed genes
  • Combining cDNA-amplified fragment length
    polymorphism, transcript profiling and targeted
    metabolic profiling in a time course experiment
    following elicitation, 591 genes out of 20,000
    visualized genes were identified

22
Functional genomic approach
  • Homology searches 58 genes had known function
  • Include Nicotine biosynthesis genes ornithine
    decarboxylase (ODC), arginine decarboxylase
    (ADC), quinolinate acid phosphoribosyl
    transferase (QPT) and many novel genes alkaloid
    biosynthesis
  • Also putative proteins of unknown function (15),
    signal transduction proteins such as receptors,
    kinases, phosphatases and transcription factors

23
High-throughput functional analysis
  • Isolation of full-length cDNAs.
  • High-throughput construction of expression
    vectors
  • Developing a rapid transformation system
  • Downscaling the plant cell cultures
  • Rapid targeted metabolite profiling
  • Quantitative analysis of desired compounds

24
Problems in characterizing plant metabolome
  • Highly complex nature and chemical diversity
  • Range of chemical properties
  • Far more complex than metabolite profiling of
    primary metabolites

25
New lead molecules through combinatorial
biochemistry
  • Major classes of secondary metabolites
    (Polyketides, isoprenoids, alkaloids,
    phenylproponoids and flavonoids) subdivided into
    several subclasses
  • Eg. 12,000 known alkaloids subdivided into 15
    subclasses proto-, piperdine-, pyrrolidine-,
    pyridine-, quinolizidine-, trophane-,
    pyrrolizidine-, imidazole-, purine-, quinoline-,
    isoquinoline-, quinazoline-, indole-, terpenoid-
    and steroidal-alkaloids

26
Combinatorial biochemistry
  • Secondary metabolites belonging to the same
    subclass are not always synthesized from the same
    primary metabolites but their chemical structures
    share the same basic skeleton
  • Because of activity of enzymes with different
    substrate- and stereo-specificity, the chemical
    diversity and biological activity of the
    molecules belonging to the same subclass can be
    enormous
  • Some subclasses are found only in a few plant
    familites (e.g. tropane alkaloids found in only
    Solanaceae and Erythroxylaceae), whereas
    flavonoids are widely distributed

27
Combinatorial biochemistry
  • Different plants synthesize structurally similar
    but nevertheless diverse molecules
  • An enzyme isolated from one plant might encounter
    new but related substrates when introduced into
    another plant
  • Hybrid new secondary metabolites
  • Somatic hybrids Solanum brevidens x
    S.tuberosum, both produce glycoalkaloid precursor
    teinemine converted by a hydrogenase to
    tomatidine in S. brevidens and solanidine
    glycoalkaloid in S. tuberosum
  • Hybrid produced tomatidine and solanidine, also
    novel glycoalkaloid called demissidine

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Yeast cell factories
  • Biocatalysts biotransformation organic
    synthesis
  • Major synthetic technologies based on
    biocatalytic reactions
  • Fermentation A biological method resulting in
    products which are the result of the complex
    metabolism of microorganisms starting with
    inexpensive simple C and N sources
  • Enzymation (microbial transformation, microbial
    conversion, biotransformation, bioconversion)
    Living cells not necessary but only act as
    simple bag of enzymes or catalysts one or
    few-step

30
Alternative yeast
  • Candida, Cryptococcus, Geotrichum, Issatchenkia,
    Kloeckera, Kluyveromyces, Pichia (including
    Hansenula polymorpha P. angusta), Rhodotorula,
    Rhodosporidium, Schizosaccharomyces pombe,
    Torulopsis, Trichosporon, Trigonopsis variabilis,
    Yarrowia lipolytica and Zygosaccharomyces rouxii

31
Generation of designed microorganisms
  • Increasing number of sequenced genes and whole
    genomes
  • New bioinformatic tools for analyzing the wealth
    of information
  • Biochemically well-characterized biosynthetic
    pathways
  • Well established genetic engineering techniques

32
Designed microorganisms(Genetic engineering
approaches)
  • Construction of synthetic pathways for the
    production of structurally complex, natural
    products like isoprenoids or polyketides
  • Delete harmful genes
  • E. coli, B. subtilis, Schizosaccharomyces pombe
    (fission yeast)

33
Chemical reactions catalyzed by wild-type yeast
whole-cell biocatalysts
  • Bakers yeast (S. cerevisiae) Ideal as
    stereo-selective biocatalysts chiral
    intermediates in the synthesis of
    enantiomerically pure compounds
  • Non-pathogenic, inexpensive, simple to grow at
    large scale, cells can be stored indefinitely in
    dried form

34
Main enzymatic reactions performed by wild-type
yeast
  • Reduction of CO bonds
  • Asymmetric reduction of carbonyl-containing
    compounds (E.g. Furfural to furfuryl alcohol)
  • Reduces simple aliphatic and aromatic ketones
    resulting in (S)-alcohols
  • Whole-cell (redox)-biocatalysts contain necessary
    cofactors and metabolic pathways
  • Cheap C sources (glucose, saccharose)
  • Biocatalysts and cofactors are well protected
    more stable
  • Substrates non-natural toxic (0.3 per
    volume)
  • Large amounts of biomass and by-products impede
    product recovery
  • Transport phenomenon

35
Wild type yeast
  • Different strains different specificities
  • Different dehydrogenases with overlapping
    substrate specificities but opposite
    stereoselectivities
  • Variety of oxidoreductases
  • Improved selectivity substrate modification,
    changes in cultivation conditions, application of
    different C-sources, use of inhibitors, two-phase
    systems, water immiscible ionic liquids,
    biocompatible solvents to provide substrate
    reservoir and in situ extracting agent

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Reduction of CC-bonds
  • Flavin-dep redox enzyme in yeast (Warburg and
    Christian, 1933)
  • Known as old yellow enzymes (Flavin cofactor)
  • Typical substrates are alkenes activated by
    electron-withdrawing substituents
  • Reduced at the expense of NAD(P)H leading to
    enantiomerically pure alkanes creating two chiral
    carbon centers
  • Excellent stereoselectivities
  • Cells provide both enoate reductase and alcohol
    dehydrogenases, both depend on the same
    nicotinamide cofactor
  • E.g. asymmetric bioreductions of a,ß-unsaturated
    ketones with S. cerevisiae led to
    (R)-2,2,6-trimethylcyclohexane-1,4-dione used for
    3-hydroxycarotenoid production

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Oxidation and racemization reactions
  • Yeast alcohol oxidases oxidation of methanol
    and other primary alcohols
  • Instead of creating a chiral center, it is
    destructed, hence limited synthetic use except
    for regioselective oxidation of polyols
  • Oxidation of sulfides can result in chiral
    sulfoxides used in organic synthesis as
    asymmetric auxilliary groups to control the
    stereochemical outcome

41
Hydrolase reactions
  • Hydrolysis reactions proteinases, lipases,
    esterases
  • Converted 1-alkyn-3-yl acetates to corresponding
    alcohols and acceptable enantioselectivities

42
Formation of C-C-bonds
  • Acyloin condensation forms (1R)-phenylacetyl
    carbinol, a chiral synthon of D-ephedrine
  • Involve pyruvate decarboxylase
  • Benzaldehyde subjected to acyloid condensations
  • Conversion of a,ß-unsaturated aldehydes giving
    optically active diols and production of the a-14
    chromanyl moiety of a-tocopherol (Vit E)

43
Industrial application of yeast whole-cell
  • Combining microbiological and chemical synthesis
  • E.g. S. cerevisiae Acyloin-type condensation of
    benzaldehyde resulting in (1R)-phenylacetylcarbino
    l which is subsequently converted to (1R,
    2S)-ephedrine and (1R,2S)-pseudoephedrine
  • Candida rugosa enoyl-CoA hydratase catalyzes
    butyric acid to (R)-ß-hydroxy-n-butyric acid
  • Zygosaccharomyces rouxii enantioselective
    reduction of 3,4-methylenedioxy-phenylacetone to
    the corresponding (S)-alcohol (Eli Lilly)

44
Industrial application of yeast whole-cell
  • Pichia methanolica (Bristol-Myers Squibb)
    Reductase for the reduction of ethyl5-oxo-hexanoat
    e and 5-oxo-hexanenitrile to the corresponding
    (S)-alcohols
  • Phenylalanine dehydrogenase from
    Thermoactinomyces intermedium used to produce
    Chiral intermediates for the production of
    antihypertensive drug, Omapatrilat

45
Recombinant yeasts
  • Designer yeasts for biocatalytic applications
  • Metabolic engineering for heterologous protein
    production, extension of substrate range,
    pathways leading to new products, pathways for
    the degeneration of xenobiotics, engineering of
    cellular physiology for process improvement,
    elimination/reduction of by-product formation and
    improvement of yield or productivity
  • Production of high value chemicals ethanol,
    glycerol, xylitol, succinic acid and other
    organic acids

46
Engineered yeast platform
  • Mostly for chiral precursors for the
    pharmaceutical, food or feed industry including
    single- and multi-step biocatalytic reactions
  • Pathway engineering leading to structurally
    complex natural products

47
Organic single or few-step transformations
  • Optically pure ethyl (R)4-chloro-3-hydroxybutanoat
    e from prochiral ß-keto ester
  • Altered oxidoreductases combining gene deletion
    and overexpression
  • GRAS status Improved synthesis of the food
    flavoring methyl benzoate by expressing the
    Antirrhinum majus benzoid acid methyltransferase
    (BAMT) under the control of Cu-inducible CUP1
    promoter
  • By encoding ß-glucosidase in yeast elevated the
    resveratrol content
  • Expression of cyclohexanone monooxygenase from
    the Acinetobacter in S. cerevisiae variety of
    substituted cycloalkanones and several sulfides,
    dithianes and dithiolanes

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Cofactor regeneration
  • For asymmetric reductions cofactor-dep enzymes
    are required
  • Considering the cost of NAD(P) and NAD(P)H,
    their stoichiometric application is not
    economically feasible
  • Whole-cell biocatalysts provide the cheapest
    cofactor regeneration system
  • Introduced membrane-bound transhydrogenase from
    E. coli

51
Transport limitations and displaying enzymes on
the surface
  • Permeabilization protocols include solvent,
    detergent, alkaline treatment, air-drying at 42C
  • Molecular engineering outer membrane structure
    (only for bacteria) or displaying enzymes on the
    cell surface
  • Molecular displaying targeting of heterologous
    proteins to the surface of yeast
  • Hydrolases Rhizopus oryzae lipase,
    ß-glucosidases from A. oryzae

52
Synthetic pathways in yeasts
  • Natural products like isoprenoids, flavonoids or
    polyketides are structurally complex
  • Great commercial potential
  • Carotenoids antioxidants
  • Steroids anti-inflammatory, contraceptive and
    anticancer agents
  • Terpenoids diterpenoid taxol against cancer
  • Flavonoids derived from the phenylpropanoid
    pathway anti-allergenic, anti-inflammatory,
    anti-oxidant
  • Polyketides cancer (adriamycin), cardiovascular
    diseases (lovastatin), immunosuppression
    (rapamycin, tacrolimus) or infectious diseases
    (erythromycin, tetracycline)

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Taxol
  • Isoprenoid (terpenoid)
  • Bark of Pacific yew (Taxus brevifolia)
  • Refractory ovarian and metastatic breast cancer
  • E. coli Taxadiene (a taxol biosynthetic
    intermediate) limitation can not functionally
    express the P450 enzymes
  • S. cerevisiae

58
Taxol (1), the worlds first billion dollar
anticancer drug
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Microbial factories for recombinant
pharmaceuticals
  • 151 recombinant pharmaceuticals approved by FDA
    and/or European Medicines Agency (EMEA)
    bacteria or yeast
  • In spite of lack or unconventional
    post-translational modifications, proteolytic
    instability, poor solubility and activation of
    cell stress responses

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A large number of secondary metabolites have been
extracted, isolated and characterized from
endophytic microbes, especially from fungal
endophytes
65
Camptothecin was first isolated from the wood of
Camptotheca acuminata. Camptothecin has been
obtained from fungal endophytes isolated from the
inner bark of the plant Nothapodytes foetida from
the western coast of India. Some alkaloids
produced by endophytic fungi are also anticancer
agents. Wagenaar et al. isolated three novel
cytochalasins from an endophyte Rhinocladiella
sp. with potential antitumor activity against
selected neoplastic cell lines Rubrofusarin B is
cytotoxic to the colon cancer cell line SW1116
(IC5O 4.5 µg mL-1), and aurasperone A (18) is
inhibitory to xanthine oxidase (XO) (IC50 10.9
µM). Rubrofusarin B fonsecinone A, asperpyrone B
and aurasperone A by fractionation of the extract
of Aspergillus niger IFB-E003, an endophyte of
Cynodon dactylon.
66
Guignardic acid was detected in the culture broth
of an endophytic Guignardia sp. obtained from
Spondias mombin Xanthoviridicatin E and F are
two novel quinine related metabolites produced by
an endophytic Penicillium chrysogenum colonizing
an unidentified plant. These metabolites inhibit
the cleavage reaction of HIV-1 integrase with
IC50 values of 6 and 5 uM, respectively Phomol
is a novel polyketide lactone from Phomopsis sp.,
present in the medicinal plant Erythrina
crista-galli Microcarpalide, a novel
microfilament disrupting agent with weak
cytotoxicity to mammalian cells, was
characterized from an unidentified fungus in
Ficus microcarpa
67
Antimicrobial compounds from fungal Endophytes
Anti-fungal
68
Antimicrobial compounds from fungal Endophytes
Anti-bacterial
69
Antimicrobial compounds from fungal Endophytes
Anti-cancer
70
A new anti-inflammatory compound, 5, 7-dimethoxy-
4-p- ethoxylphenylcoumarin (118), and 5,
7-dimethoxy-4-phenylcoumarin (119) were isolated
from endophytic Streptomyces aureofaciens CMUAc130
71
Anti-viral metabolites Four new
cyclohexadepsipeptides, pullularins AD
(120-123), were isolated from the endophytic
fungus Pullularia sp. BCC 8613. Pullularin A
exhibited activities against the malaria parasite
Plasmodium falciparum K1 (IC50 3.6 µg mL-1) and
herpes simplex virus type 1
72
Molecular farming/pharming
  • Application of molecular biological techniques to
    the synthesis of commercial products in plants
  • CHO Amylose-free starch, high amylose starch,
    cyclodextrins, fructans, trehalose
  • Lipids Medium-chain fatty acids, saturated fatty
    acids, mono-unsaturated fatty acids,
    polyhydroxybutyrate

73
Molecular farming of proteins
  • Antibodies full Igs and engineered types such
    as scFVs (single chain antibodies)
  • Subunit vaccines vaccines based upon short
    peptide sequences that act as antigens
  • Protein antibiotics

74
Examples of enzymes produced in plants
  • Avidin diagnostic kits
  • ß-glucuronidase diagnostics kits
  • Trypsis wound care
  • Cellulase Ethanol production
  • Thermostable xylanase Biomass processing
  • Phytase food processing
  • (1-3)(1-4)ß-glucanase Brewing
  • Lignin peroxidase Paper manufacture

75
Biopharmaceutical recombinant proteins
  • Protein C Anticoagulant
  • Hirudin Anticoagulant
  • Somatotrophin Growth hormone
  • ß-interferon Hepatitis B C
  • Serum albumin Burns/fluid replacement
  • Haemoglobin-a and ß Blood substitute
  • Homotrimeric collagen Collagen
  • a1 antitrypsin cystic fibrosis, haemorrhages
  • Aprotinin (trypsin inhibitor) Transplant surgery
  • Lactoferrin Antimicrobial
  • ACE Hypertensis
  • Trichosanthin- a HIV therapy, cancer

76
List of plants with anti-diabetic activity
Aegle marmelos
Syzygium cumini 
Gymnema sylvestre
Salacia petenensis
77
List of plants with anti-inflammatory activity
Curcuma longa
Boswellia serrata
Ricinus communis 
78
List of plants with anti-cancer activity
Nothapodytes nimmoniana
Aphanamixis polystachya
Dioscorea bulbifera
Taxus brevifolia
79
List of plants with cardiotonic activity
Terminalia arjuna
Boerhavia diffusa
Sida cordifolia
Rhamnus purshiana
80
List of plants with anti viral activity
Morinda charantia
Terminalia arjuna
Calophyllum inophyllum
Andrographis panniculata
Phyllanthus niruri
81
BIOCHEMICAL ASSAYS
82
TLC Visualization Reagents
83
TLC PROFILES
TLC profile of Piperine
84
Drug target assays
  • Selectivity profiling
  • Target validation
  • Polymerases
  • HT screening (HTS)
  • Nuclear Receptors
  • Kinases
  • Phosphodiesterases
  • GPCRs
  • Transporters
  • Ion channels
  • Nitric oxide synthases
  • Dehydrogenases
  • Proteases

85
High throughput screening
  • Convert primary assays into HTP
  • Large scale reagent preparation
  • Multiple methodologies and dependable results
  • Assays
  • ELISA, RT-PCR, RIA, Particle agglutination,
    cell-based assays, cell culture, HPLC, Genomic
    sequence

86
Systems biology in drug discovery
  • Encompasses many different approaches and models
    for probing and understanding biological
    complexity
  • Develop predictive models of human disease
  • Principal approaches
  • Informatic integration of omicsdata sets
    (bottom up approach)
  • Computer modeling of disease or organ system
    from literature (top-down approach to target
    selection, clinical indication and clinical trial
    design)
  • Use of complex human cell system themselved to
    interpret and predict the biological activities
    of drugs and gene targets (direct experimental
    approach)

87
Thank you
88
Ion channels
  • Fluorometry
  • Electrophysiology
  • Radiometric binding assays

89
GPCRs
  • HTP agonist, antagonist and pathway exploration
  • GPCR expressing cell lines and cDNAs
  • Calcium, cAMP readouts
  • Radio-labeled and label-free assays
  • Assays
  • Ligand binding, receptor dimerization and
    downstream signalling

90
Selectivity profiling
  • Determine mechanism of action and inhibitory
    profile of compounds
  • Protein kinase and phosphatase targets and
    disease specialized panels

91
Nuclear receptors
  • HTP cellular reporter gene assay
  • Disease specific nuclear receptor panel screening
  • Biochemical FRET assays

92
Target validation
  • Eliminate non-disease relevant targets
  • Functional assays and histology
  • Disease-specific assays
  • Eliminate non-relevant compounds
  • Angiogenesis, cancer models, nervous system assays

93
Proteases
  • Proteases are one of the largest classes of
    potential drug targets with over 600 human gene
    targets and infectious diseases
  • Protease inhibitory activity

94
Transporters
  • Small molecules that inhibit transporter activity
    in the kidney may improve drug half life

95
Dehydrogenases
  • Targets for human parasites such as malaria,
    metabolic disorders, hormone imbalance and
    anti-angiogenesis
  • Measure oxidation of NADH via dehydrogenase
    activity

96
Other assays
  • Polymerases
  • HERG channel screening assays
  • NAD/NADH assay
  • Neutrophil elastase release assay Cell-based
    screening for anti-inflammatory drug
  • Nitric oxide synthases inflammatory response to
    cancer and bacterial invasion

97
Toxicological assays
  • Mammalian toxicology General, inhalation,
    reproduction, opthamologic, juvenile, dermal,
    phototoxicity, immunotoxicity
  • Cytotoxicity Zebrafish embryos, epidermal cells,
    hematopoietic cells, hepatocytes, tubule
    epithelial cells, endothelial cells,
    cardiomyocytes
  • Genotoxicity Ames test, chromosome aberration,
    comet assay, micronuclus test
  • Cardiotoxicity HERG inhibition, cardiac
    channels, action potential duration, perfused
    heart
  • Safety pharmacology Off-target effects,
    respiratory, cardiovascular, gastrointestinal,
    CAN (behaviour)
  • Drug-drug interaction
  • Clinical pathology
  • Anatomic pathology

98
Chemistry
  • Compound integrity GC-MS, LC/MS, NMR, High
    resolution MS, elemental analysis, melting point,
    UV and IR profiling, Optic rotation
  • Natural products screening, analysis,
    purification, fermenation
  • Chemical properties Thermal analysis, aqueous
    solubility, chemical stability, partition
    coefficient, photostability

99
Groups of natural compounds
  • Natural compounds Vincristine (leukemia)
  • Semisynthetic derivatives Podophyllotoxin
    (cancer)
  • Basic skeleton diosgenin Dioscorea sp.
  • Steroid skeleton corticosteroids,
    contraceptives, sex hormones

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Production of nanoparticles
  • An array of physical, chemical and biological
    methods synthetic nanomaterials
  • Particular shape and size
  • UV-radiation, aerosol techniques, lithography,
    laser ablation, ultrasonic fields, photochemical
    reduction techniques
  • Expertise and need hazardous chemicals

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Production of nanoparticles using organisms
(Green chemistry)
  • Unique potential in environmentally friendly
    production and accumulation of nanoparticles with
    different shapes and size
  • Different applied chemical composition,
    controlled monodispersity, desired morphologies,
    interested particle size
  • Biosynthesis of gold, silver, gold-silver alloy,
    selenium, platinum, palladium, silica, titanium,
    zirconia, quantum dots, magnetite etc.
  • Rate of synthesis slow
  • Optimize microbial cultivation methods,
    extraction techniques, combinatorial appraoch
    photobiological methods

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Comparing a cell to a factory
  • Plasma membrane like shipping/receiving
    department regulates what enters and leaves the
    cell, where cell makes contact with the external
    environment
  • Nucleus is like CEO controls all cell activity,
    determines what proteins will be made
  • Cytoplasm like factory floor contains the
    organelles, site of most cell activity
  • ER like assembly line where ribosomes do their
    work
  • Ribosomes like workers in the assembly line build
    the proteins
  • Golgi apparatus like finishing/packaging
    department prepare proteins for use or export
  • Lysosomes like maintenance crew responsible for
    breaking down and absorbing materials taken in by
    the cell
  • Cytoskeleton like support beams (walls, ceilings,
    floors) maintains cell shape
  • Mitochondria /chloroplasts like power plant
    transforms one form of energy into another

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Examples
  • Sulfite reductase-mediated synthesis of gold
    nanoparticles capped with phytochelatin
  • Nitrate reductase-mediated synthesis of silver
    nanoparticles from silver nitrate
  • Facile biosynthesis, separation and conjugation
    of gold nanoparticles to doxorubicin
  • CdS quantum dots Enzyme mediated in vitro
    synthesis, characterization and conjugation with
    plant lectins
  • Extracellular biosynthesis of CdSe quantum dots
    by Fusarium oxysporum

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