ASystems Biology

Approach toNatural Products

Research

ASystems Biology

Approach toNatural Products

Research

P l a n t B i o t e c h n o l o g y 2 0 th A p r i l 2 0 1 5

Ritesh Bhagea, Rouksaar Buctowar, Christabelle Cécile, Keshavi Ghoorbin, Huda Nazeer

Contents

● Introduction

● Overview

● Reductionist Approach

● Holistic Approach

● What is Systems Biology?

○ Advantages of Systems Biology

● Tools of Holistic Approach - Systems Biology

○ Proteomics, Transcriptomics and Metabolomics

● Conclusion

● References

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Introduction

● Medicine is the most important health care; to cure diseases (antibiotics),

relieve symptoms (analgesics), for preventive measures (antihypertensive

drugs) or for the substitution of endogenous compounds (insulin).

● Many modern drugs have been developed thanks to traditional medicines

with receptors and mechanisms of action.

● These consequently lead to the screening for novel bioactive compounds,

and the design and synthesis of similar structures.

● Nevertheless, with the advent of technology, society moved from broad

screening and testing on humans towards molecular-level of screening

(Holistic approach -> reductionist approach)

3

Overview

4

Reductionist approachAn approach to understanding the nature of complex things by reducing to a

simpler level - Introduced by Descartes in Part V of his “Discourses” of 1637.

Also known to be the basis for many of the well-developed areas of modern

science, including physics, chemistry and cell biology.

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Holistic approach

A way to analyze all components

and interactions within and among

organisms unlike the reductionist

approach.

The collective data gives a broad

picture how:

● an organism operates

● affects systems

● other biological processes

respond to an organism

Better understanding and

interpretation of data, thereby

making more accurate predictions

about the human immune system’s

response.

Approach can be done in two

different ways,

● via clinical trials

● systems biology

● (or both)6

Systems Biology - What is it?

“Systems biology is an approach in biomedical research to understanding the

larger picture - be it at the level of the organism, tissue, or cell - by putting its

pieces together. It is in stark contrast to decades of reductionist biology,

which involves taking the pieces apart.” (NIH, 2015)

Measurements

Genetic level: genome,

transcriptome, proteome,

metabolome

Physiological level: pulse,

blood pressure, pain, fever

“Systems biology aims at understanding

biological complexity by unbiased

measurements of as many as possible

parameters without having any hypothesis.”

(Verpoorte et al., 2006)

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Systems Biology - What is it?● It thus explains the biological

phenomenon, not on a gene basis,

but through the interaction of all

cellular and biochemical

components in a cell or an

organism.

● It identifies the elements (genes,

molecules, cells), ascertains their

relationships and involves new

dynamic computer modeling

programs which integrate the info

and allow to simulate entire

organisms based on their biological

components.

The Systems Biology Triangle

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The different levels of measurement in a systems approach

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Advantages of the Systems Biology Approach

● Decrease development time of drugs by half.

● Decrease the cost of development by 70%.

○ Can further reduce costs if coupled with traditional medicines.

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Tools of Holistic approach● The present-day reductionist approach for drug development is not

sufficient to prove efficacy of natural products (medicinal plants).

● Thus, Holistic approaches are employed because:

○ Activity might be due to pro-drugs which are activated upon

administration of the medicine,

○ To synergy among compounds.

● Important tools of Holistic approaches:

1. Transcriptomics

2. Proteomics

3. Metabolomics

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1. Transcriptomics in Systems Biology● The genome is a store of biological

information.

● Biochemical reactions involving enzymes

and proteins w.r.t the genome is genome

expression.

● Transcriptome is the initial product of

genome expression.

● It is a collection of RNA molecules derived

from protein-coding genes in a cell at a

point in time.

● Transcriptomes are maintained by

transcription.

● Transcriptomes are then translated into

proteomes. 12

1. Transcriptomics in Systems Biology

● Transcriptomics aims to comprehensively profile all information that

appears in the RNA pool within a system.

● Cellular functions are mediated by mRNA via gene expression.

● RNA profiling provides clues to:

○ Expressed sequences and genes of a sequence.

○ Gene regulation and regulatory sequences.

○ Function and interactions between genes.

○ Functional differences between tissues and cell types.

○ Identification of candidate genes for any given disease.

● There are various types of RNA molecules:

○ rRNA, snoRNA, tRNA, snRNA, miRNA

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1. Transcriptomics in Systems Biology● There are also non-coding RNA molecules, with activities like:

○ Binding to target sequence by base pairing,

○ Functioning as an enzyme by folding on itself,

○ Binding to protein and modulating its activity.

● Every cell in an organism is, at any given time point, transcribing thousands

of its genes in various quantities.

● Both from basic science and clinical perspectives, it is vital to accurately

quantify the levels of different transcripts and identify the proteins they

code for.

● Various methods are available for quantification and analysis of messenger

RNA levels:

○ Gene by gene methods

○ Global methods14

1. Transcriptomics in Systems BiologyGene by gene method:

● Northern blot

● qRT-PCR

Global methods:

● Expressed sequence tag (EST) sequencing - generates random, 200-900

bp single-pass sequences of cDNA clones.

● Serial analysis of gene expression (SAGE) - was the first approach to

provide large-scale absolute estimates of transcript frequencies. Tags are

sequenced using standard sequencing technology to derive a digital

representation of the transcript frequencies.

● Cap analysis of gene expression (CAP) - uses 5’ cap-trapping methods to

selectively isolate full-length cDNAs and generates 20-bp tags from these.

Tags are cloned in vectors and sequenced.

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1. Transcriptomics in Systems Biology● Massive parallel signature sequencing (MPSS). 3’ sequences of each

transcript are isolated using biotinylated primer in the cDNA synthesis. 3’

signature sequences are ligated into specifically designed plasmid vectors

and amplified using PCR . Tags coupled with microbeads, for mapping

and sequencing.

● Gene identification signature (GIS)- relies on sequencing of

concatenated 3’ and 5’ paired-end ditags.

Mostly applied technology in transcriptomics is the use of Microarrays.

● Microarrays are used to measure levels

of mRNA transcripts, miRNAs, and proteins

but also to analyse characteristics of genomes.

● Fundamental underlying advantage is analysis

of all known protein-coding transcripts.

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Applications of transcriptomics● Microarray platforms are used for genome-wide analysis of expression

changes in diverse experiments investigating mechanisms of neural injury

and repair (Neurosciences).

● Toxicogenomics determines global transcriptomic responses to chemical

exposures and can predict their effects. (E.g: aquatic toxicology)

● Information on how individual cells respond to signals and other

environmental cues at critical stages of cell-fate determination.

● Assess the gene regulatory network -physiological functions, behavior

and phenotype during development in multicellular organisms.

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Applications of transcriptomics● Plant extracts have been used as flavour, fragrances and medicines for

millennia, which are complex molecules and are difficult to produce

synthetically. Much interest is given to natural products.

○ Research was carried out on Salvia plant for aromatic and

pharmacological attributes and is known for tanshinone synthesis.

○ Sequence analysis were carried out before and after hairy root

cultures were established. Combined metabolomic and

transcriptomic approach towards elucidating tanshinone synthesis

was used.

○ Data has revealed a distinct expression pattern correlated with

tanshinone production, providing a firm foundation for further

investigation of the biosynthesis of these medically important

natural products.

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2. Proteomics in Systems Biology● Proteomics aims to detect and quantify a system’s entire protein content,

hence understanding the expression and function of proteins on a global

level (Weston and Hood, 2004).

● More than simply cataloguing the proteome-a quantitative assessment of

the full complement of proteins within a cells the field of proteomics strives

to characterize:

- protein structure and function - enzyme-substrate interactions

- protein-protein - post-translational modifications

- protein-nucleic acid - protein processing and folding

- protein-lipid - protein turnover and synthesis rates

- cellular and subcellular localization - Protein activation

Current Proteomic Technologies:

● high-resolution, two-dimensional electrophoresis (2DE)

● stable isotope tagging

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2. Proteomics in Systems Biology● Detection of known nonribosomal peptide synthetases /polyketide

synthases systems in members of the genera Bacillus and Streptomyces which are known to synthesize natural products with antibiotic, anticancer and antifungal properties (Bumpus et al., 2009)

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3. Metabolomics in Systems Biology

● Metabolomics is the study of

global metabolite profiles in a

system (cell, tissue or organism)

under a given set of condition.

● Integration of genomics,

proteomics, transcriptomics and

metabolomics is a goal of systems

biology.

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Techniques used in metabolomics

MS

NMRGC

MicroarrayOptical spectroscopy

Fourier transformed infrared spectroscopy

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Main applications of metabolomics

● Drug assessment

● Clinical toxicology

● Nutrigenomics

● Functional genomics

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Application of metabolomics

Metabolomics for natural products research

● The use of metabolomic approach, with appropriate statistical analysis,

allows rapid analysis of these complex data.

○ E.g., NMR clustering techniques have also been used to cluster the

spectra of crude plant extracts to determine the biochemical

mechanism of action for herbicides (Rochfort, 2005).

■ In this study, 27 herbicidal compounds with 19 different MOAs

were applied to plants for 24h before an aqueous extract of the

plant was taken.

■ Artificial neural network analysis was employed to analyse the

NMR spectroscopic data of these extracts.

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Application of metabolomics■ Treated samples were easily

distinguished from untreated

samples and with the use of

an appropriate training set,

many different MOAs were

distinguished for the

commercially available

compounds tested.

■ This approach has been recently

demonstrated for a herbal medicine.

● A medicinal herb extract

(Anoectochilus formosanus: a popular

folk medicine with anticancer activity)

was compared to a single compound

drug in MCF-17 cells by metabolomic

and transcriptomic analysis, and a

similar level of gene expression

regulation was observed in both,

suggesting a similar MOA.

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Conclusion

For phytochemist and pharmacognosist, metabolomics and systems biology

are great opportunities as they have all the knowledge for analysis of natural

products.

These approaches could be associated with chemometrics and bioinformatics

to make a major contribution in the research of medicinal plants.

“To waste, to destroy, our natural resources, to skin and exhaust the land;

instead of using it so as to increase its usefulness, will result in undermining in

the days of our children, the very prosperity we ought by right to hand down

to them amplified and developed.”

-Theodore Roosevelt

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ReferencesBumpus, S.B., Evans, B.S., Thomas, P.M., Ntai, I. and Kelleher, N.L. (2009). A

Proteomics Approach to Discovery of Natural Products and Their Biosynthetic Pathways., Nat Biotechnol, 27(10), pp 951–956.

Fang, F.C. and Casadevall, A. (2011). Reductionistic and Holistic Science. Infect. Immun. 79 (4), pp. 1401-1404.

Gao, W., Sun, H.-X., Xiao, H., Cui, G., Hillwig, M. L. and Jackson, A. (2014). Combining

metabolomics and transcriptomics to characterize tanshinone biosynthesis in Salvia miltiorrhiza.

BMC Genomics 15(1), 73.

Munro, K. M. and Perreau, V. M. (2009). Current and Future Applications ofTranscriptomics for Discovery in CNS Disease and Injury. Neurosignals 17(4), 311–327.

Rochfort, S. (2005). Metabolomics reviewed: A new “ omics” platform technology for systems biology and implications for natural products research. J. Nat.Prod, 68, pp 1813-1820.

Sumner, L.W., Mendes, P. and Dixon R.A. (2003). Plant metabolomics: large-scale phytochemistry in the functional genomics era. Phytochemistry ,62, pp 817–836.

Tang, F., Lao, K. and Surani, M. A. (2011). Development and applications of single-cell transcriptome analysis. Nature Methods.

Verpoorte, R., Choi, Y.H. and Kim, H.K. (2005). Ethnopharmacology and systems biology: A perfect holistic match. Journal of Ethnopharmacology, 100, pp 53-56.

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Verpoorte, R., Choi, Y.H. and Kim, H.K. (2006). Plants as source of medicine: new perspectives. In: Medicinal and Aromatic plants - Agricultural, Commercial, Ecological, Legal, Pharmacological and Social aspects. Eds: Bogers, R.J. Cracker, L.E. and Lange, D. Springer, Dordrecht, pp 261.

Wang, M., Lamers, R.J.A.N., Korthout, H.A.A.J., Nesselrooij, J.H.J.V., Witkamp, R.F., Heijden, R., Voshol, P.J., Havekes, L.M., Verpoorte, R. and Greefan, J.V. (2005). Metabolomics in the Context of Systems Biology: Bridging Traditional Chinese Medicine and Molecular Pharmacology. Phytother. Res., 19, pp 173–182.

Wei Chen. (2013). Transcriptome profiling--Past, Present and Future. Berlin Institute for Medical Systems Biology. Max-Delbrueck-Center for Molecular Medicine

Weston, A.D. and Hood, L. (2004). Systems Biology, Proteomics, and the Future of Health Care: Toward Predictive, Preventative, and Personalized Medicine. Journal of Proteome Research, 3, pp 179-196.

Wirta, V. (2006). Mining the transcriptome - methods and applications. School of Biotechnology, Royal Institute of Technology, Stockholm.

References

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