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 US$30 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 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

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 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

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

16. 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

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

21. 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 – 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

29. 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 • 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

34. Main enzymatic reactions performed by wild-type yeast 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

35. Wild type yeast • 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

37. Reduction of C=C-bonds • 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

40. 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 chiralsulfoxides 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)-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)

43. Industrial application of yeast whole-cell • 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)

44. Industrial application of yeast whole-cell • 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

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-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

50. 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