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Plants and microbes as drug targets . Prof. H.S. Prakash Department of Biotechnology University of Mysore Manasagangotri, Mysore 570 006 . Higher plants – a rich source of novel compounds. 400,000 higher plant species 10% characterized chemically

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Plants and microbes as drug targets l.jpg
Plants and microbes as drug targets

Prof. H.S. Prakash

Department of Biotechnology

University of Mysore

Manasagangotri, Mysore 570 006


Higher plants a rich source of novel compounds l.jpg
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 US$30 billion in USA (2002)


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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


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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


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Major groups

  • Based on biosynthetic origins structurally divided into five major groups

  • Polyketides: Acetate-mevalonate

  • Isoprenoids:Terpenoids and steroids from 5-C precursor isopentenyldiphosphate (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


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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


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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


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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


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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


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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


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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


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Cell suspension cultures

  • Shikonin from Lithospermumerythrorhizon

  • Berberine from Coptis japonica

  • Rosmarinin acid from Coleus blumeii

  • Sanguinarine from Papaversomniferum

  • Paclitaxel from Taxusbrevifolia

  • Bottlenecks – low productivity, process technological issues (Bioreactor design, cultivation conditions)

  • Functional genomics – Newer opportunities


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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


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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 6β-hydroxylase (H6H) of tropane alkaloid biosynthesis enhanced production of scopolamine in cultivated hairy roots


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Transcriptional regulation

  • Part of terpenoidindole alkaloid biosynthesis in Catharanthusroseusis under the control of ORCA3, a jasmonate-responsive APETALA2 (AP2)-domain transcription factor

  • Constitutive overproduction of ORCA3 – enhanced terpenoidindole alkaloids

  • Maize transcription factors LC and C1 in tomato fruits upregulated the flavonoid pathway – kaempferol

  • ANT1 - Anthocyanin biosynthesis, glycosylation and transport in vacuoles


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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


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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


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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


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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


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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


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Problems in characterizing plant metabolome

  • Highly complex nature and chemical diversity

  • Range of chemical properties

  • Far more complex than metabolite profiling of primary metabolites


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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


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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


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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 – Solanumbrevidensx S.tuberosum, both produce glycoalkaloid precursor teinemine – converted by a hydrogenase to tomatidine in S. brevidens and solanidineglycoalkaloid 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


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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


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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


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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)


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Chemical reactions catalyzed by wild-type yeast whole-cell biocatalysts

  • Baker’s 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


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Main enzymatic reactions performed by wild-type yeast biocatalysts

Reduction of C=O bonds

  • Asymmetric reduction of carbonyl-containing compounds (E.g. Furfural to furfuryl alcohol)

  • Reduces simple aliphatic and aromatic ketonesresulting 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


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Wild type yeast biocatalysts

  • Different strains – different specificities

  • Different dehydrogenaseswith 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 C=C-bonds biocatalysts

  • Flavin-depredox 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 enoatereductaseand alcohol dehydrogenases, both depend on the same nicotinamide cofactor

  • E.g. asymmetric bioreductions of α,β-unsaturated ketones with S. cerevisiaeled to (R)-2,2,6-trimethylcyclohexane-1,4-dione used for 3-hydroxycarotenoid production


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Oxidation and biocatalystsracemization 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 chiralsulfoxides used in organic synthesis as asymmetric auxilliary groups to control the stereochemical outcome


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Hydrolase reactions biocatalysts

  • Hydrolysis reactions – proteinases, lipases, esterases

  • Converted 1-alkyn-3-yl acetates to corresponding alcohols and acceptable enantioselectivities


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Formation of C-C-bonds biocatalysts

  • Acyloin condensation forms (1R)-phenylacetylcarbinol, a chiralsynthon of D-ephedrine

  • Involve pyruvatedecarboxylase

  • Benzaldehyde subjected to acyloid condensations

  • Conversion of α,β-unsaturated aldehydes giving optically active diols and production of the α-14 chromanyl moiety of α-tocopherol (Vit E)


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Industrial application of yeast whole-cell biocatalysts

  • Combining microbiological and chemical synthesis

  • E.g. S. cerevisiae: Acyloin-type condensation of benzaldehyde resulting in (1R)-phenylacetylcarbinol which is subsequently converted to (1R, 2S)-ephedrine and (1R,2S)-pseudoephedrine

  • Candida rugosa enoyl-CoA hydratase catalyzes butyric acid to (R)-β-hydroxy-n-butyric acid

  • Zygosaccharomyces rouxii: enantioselective reduction of 3,4-methylenedioxy-phenylacetone to the corresponding (S)-alcohol (Eli Lilly)


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Industrial application of yeast whole-cell biocatalysts

  • Pichia methanolica (Bristol-Myers Squibb): Reductase for the reduction of ethyl5-oxo-hexanoate 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


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Recombinant yeasts biocatalysts

  • 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


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Engineered yeast platform biocatalysts

  • 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


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Organic single or few-step transformations biocatalysts

  • Optically pure ethyl (R)4-chloro-3-hydroxybutanoate from prochiral β-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 β-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 biocatalysts

  • 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


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Transport limitations and displaying enzymes on the surface biocatalysts

  • 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, β-glucosidases from A. oryzae


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Synthetic pathways in yeasts biocatalysts

  • 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 biocatalysts

  • 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


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Taxol (1), the world’s first billion biocatalysts

dollar anticancer drug


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Microbial factories for recombinant pharmaceuticals biocatalysts

  • 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


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Camptothecin was first isolated from the wood of isolated and characterized from endophytic microbes, especially from fungal endophytesCamptotheca 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.


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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





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A new anti-inflammatory compound, 5, 7-dimethoxy- 4- Anti-bacterialp- ethoxylphenylcoumarin (118), and 5, 7-dimethoxy-4-phenylcoumarin (119) were isolated from endophytic Streptomyces aureofaciens CMUAc130


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Anti-viral metabolites: Four new cyclohexadepsipeptides, pullularins A–D (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


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Molecular farming/’pharming’ pullularins A–D (120-123), were isolated from the endophytic fungus

  • 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


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Molecular farming of proteins pullularins A–D (120-123), were isolated from the endophytic fungus

  • 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


Examples of enzymes produced in plants l.jpg
Examples of enzymes produced in plants pullularins A–D (120-123), were isolated from the endophytic fungus

  • Avidin: diagnostic kits

  • β-glucuronidase: diagnostics kits

  • Trypsis: wound care

  • Cellulase: Ethanol production

  • Thermostable xylanase: Biomass processing

  • Phytase: food processing

  • (1-3)(1-4)β-glucanase: Brewing

  • Lignin peroxidase: Paper manufacture


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Biopharmaceutical recombinant proteins pullularins A–D (120-123), were isolated from the endophytic fungus

  • Protein C: Anticoagulant

  • Hirudin: Anticoagulant

  • Somatotrophin: Growth hormone

  • β-interferon: Hepatitis B + C

  • Serum albumin: Burns/fluid replacement

  • Haemoglobin-α and – β Blood substitute

  • Homotrimeric collagen: Collagen

  • α1 – antitrypsin: cystic fibrosis, haemorrhages

  • Aprotinin (trypsin inhibitor): Transplant surgery

  • Lactoferrin: Antimicrobial

  • ACE: Hypertensis

  • Trichosanthin- α : HIV therapy, cancer


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List of plants with anti-diabetic activity pullularins A–D (120-123), were isolated from the endophytic fungus

Aegle marmelos

Syzygiumcumini

Gymnema sylvestre

Salacia petenensis


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List of plants with anti-inflammatory activity pullularins A–D (120-123), were isolated from the endophytic fungus

Curcuma longa

Boswellia serrata

Ricinus communis 


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List of plants with anti-cancer activity pullularins A–D (120-123), were isolated from the endophytic fungus

Nothapodytes nimmoniana

Aphanamixis polystachya

Dioscorea bulbifera

Taxus brevifolia


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List of plants with pullularins A–D (120-123), were isolated from the endophytic fungus cardiotonic activity

Terminaliaarjuna

Boerhaviadiffusa

Sida cordifolia

Rhamnus purshiana


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List of plants with anti viral activity pullularins A–D (120-123), were isolated from the endophytic fungus

Morinda charantia

Terminaliaarjuna

Calophyllum

inophyllum

Andrographis panniculata

Phyllanthus niruri


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BIOCHEMICAL ASSAYS pullularins A–D (120-123), were isolated from the endophytic fungus


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TLC Visualization Reagents pullularins A–D (120-123), were isolated from the endophytic fungus


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TLC PROFILES pullularins A–D (120-123), were isolated from the endophytic fungus

TLC profile of Piperine


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Drug target assays pullularins A–D (120-123), were isolated from the endophytic fungus

  • Selectivity profiling

  • Target validation

  • Polymerases

  • HT screening (HTS)

  • Nuclear Receptors

  • Kinases

  • Phosphodiesterases

  • GPCRs

  • Transporters

  • Ion channels

  • Nitric oxide synthases

  • Dehydrogenases

  • Proteases


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High throughput screening pullularins A–D (120-123), were isolated from the endophytic fungus

  • 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


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Systems biology in drug discovery pullularins A–D (120-123), were isolated from the endophytic fungus

  • Encompasses many different approaches and models for probing and understanding biological complexity

  • Develop predictive models of human disease

  • Principal approaches:

    Informatic integration of ‘omics’data 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)


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Thank you pullularins A–D (120-123), were isolated from the endophytic fungus


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Ion channels pullularins A–D (120-123), were isolated from the endophytic fungus

  • Fluorometry

  • Electrophysiology

  • Radiometric binding assays


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GPCRs pullularins A–D (120-123), were isolated from the endophytic fungus

  • 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


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Selectivity profiling pullularins A–D (120-123), were isolated from the endophytic fungus

  • Determine mechanism of action and inhibitory profile of compounds

  • Protein kinase and phosphatase targets and disease specialized panels


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Nuclear receptors pullularins A–D (120-123), were isolated from the endophytic fungus

  • HTP cellular reporter gene assay

  • Disease specific nuclear receptor panel screening

  • Biochemical FRET assays


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Target validation pullularins A–D (120-123), were isolated from the endophytic fungus

  • Eliminate non-disease relevant targets

  • Functional assays and histology

  • Disease-specific assays

  • Eliminate non-relevant compounds

  • Angiogenesis, cancer models, nervous system assays


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Proteases pullularins A–D (120-123), were isolated from the endophytic fungus

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

  • Protease inhibitory activity


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Transporters pullularins A–D (120-123), were isolated from the endophytic fungus

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


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Dehydrogenases pullularins A–D (120-123), were isolated from the endophytic fungus

  • Targets for human parasites such as malaria, metabolic disorders, hormone imbalance and anti-angiogenesis

  • Measure oxidation of NADH via dehydrogenase activity


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Other assays pullularins A–D (120-123), were isolated from the endophytic fungus

  • 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


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Toxicological assays pullularins A–D (120-123), were isolated from the endophytic fungus

  • 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


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Chemistry pullularins A–D (120-123), were isolated from the endophytic fungus

  • 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


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Groups of natural compounds pullularins A–D (120-123), were isolated from the endophytic fungus

  • 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 pullularins A–D (120-123), were isolated from the endophytic fungus

  • 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 chemistry)

  • 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 chemistry)

  • 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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