Natural Products Research:

Quo Vadis?

Rob Verpoorte

Rob Verpoorte

Natural Products Laboratory

IBL, Leiden University

PO Box 9502, 2300 RA Leiden

Verpoort@CHEM.LeidenUniv.NL

The best way to predict the

future, is to invent it

Alan Kay, 1971

My goal today

• To show why I think that natural products

chemistry has excellent perspectives

– Briefly introduce systems biology

– The role of metabolomics therein

– To show how this is helpful in understanding

complex biological systems

• To give you some food (drinks) for thought

Natural Products Research in

the future?

• Not a simple question to answer

• First we need to define the mission

Mission of Natural Products

Research

• What is the mission of science in

general?

• To make this a better world

Useful plants

• Ca. 30 species for our staple food

• Ca. 100 species for fruits

• Ca. 100 species for vegetables

• 40.000- 70.000 medicinal plants

• Many others (fibers, paper, wood, spices,

ornamental, etc.)

Changing global situation

• Growing population

• Need for increased food production

• Need for novel medicines

– Antibiotics, antiparasitic, anticancer, antiviral

• Polution environment

• Sustainable, renewable production

New products and concepts from nature needed

Natural Products Research

can contribute in many ways

to make this a better world!

5 most important natural products

highlights of the past 5 decades

• ?...............? Please give me your candidates

My five:

• NIH plant screening: taxol, camptothecine

• TCM: artemisinin

• By chance: vinblastine and vincristine

• Metabolic engineering: Golden rice

• MEP terpenoid pathway

Each field in science

goes through a growth curve

Where do we stand as a discipline?

• Plant research suffered enormously from the major

marketing blunder in the introduction of GM food

• In the past 30 years we lost a lot of our capacity due

to the advent of molecular biology

• Molecular biology has also reached stationary phase

• Time is ripe for new technologies and new ideas

Our strength

• Chemistry is the basis for

observing biological

systems and

characterizing phenotypes

• For a set of spectral data

(UV, IR, MS, NMR, etc.)

there is only one chemical

structure possible

• Biology is very complex,

models are often highly

hypothetical, bias is

always a threat

• Chromatography a

science or an art?

Life is chemistry at work!

JPA Ioannidis:

“Why most published Research Findings

are False”PLoSMedicine 2(2005)696-701

(www.plosmedicine.org)

• “For many current scientific research fields,

claimed research findings may often be simple

accurate measures of the prevailing bias”

• “Simulations show that for most study designs and

settings, it is more likely for a research claim to be

false than true”

Our weakness

• We have no standardized

extraction protocols

• Preparative phytochemical

separations are poorly

described

• We have no public data

storage systems

• Structure elucidation is not

like squencing C, H, N and O

but still an intellectual

challenge

• Molecular biology works with

“studentproof” extraction kits

• Biochemists work out protein

purification protocol before

final isolation

• Gene and protein sequence

databases

• Sequencing DNA, RNA and

AA is robotized, fast and low

cost

The Challenges

• Translate chemistry to genes, “omics”

• Elucidate biosynthetic pathways, “omics”

• Metabolic engineering, synthetic biology

• Chemistry biological interactions, systems biology

• Activity medicinal plants, systems biology

• Novel fine chemicals from plants, bioprospecting

Bioprospecting

The systematic search for:

- organisms

- genes

- biomolecules

- other compounds

- designs

that might have a potential use.

Sources of chemodiversity

Estimated numbers of species

• Higher plants 25 x 104

• Lower plants 12 x 103

• Vertebrates 12 x 103

• Insects 30 x 106

• Algae 10 x 106

• Fungi 15 x 105

• Prokaryotes 15 x 105

Total 10-100 million

If every organism contains one

unique compound

there are

10-100 million natural products

Known: 150,000-200,000

Ca. 5000 new ones found per year

Still much to discover!

Some characteristics natural products

Value end

product

Activity

range

amounts

Medicines High nM Kg -tons

Cosmetics High μM-mM Kg - tons

Nutraceuticals,

food additives

Intermediate -

low

mM Tons -

bulk

Agrochemicals low nM- μM Tons -

bulk

Small-molecule approved drugs

1981-2010

Newman and Cragg, J. Nat. Prod. 75(2012)311

Drug development 2012

The good news

• About half of all novel

drugs are natural

products or natural

products derived!

The bad news

• The number of novel

drugs is decreasing

dramatically!

Reductionist approach in studying

(medicinal) plants

• High throughput screening: for some targets

upto 100.000 samples per 24 hrs, i.e. within

three days all plants species can be screened

for that activity

• Bioassay guided fractionation to isolate

active compound

– Chromatographic separation

– Measure activity with simple bioassay

– Repeat until pure active compound

Paradigm of modern drug

development:

Single compound single target

Lock and key model

for drug development,

but the door does not

change!

Is there an other way to

find the needle?

We have to rethink drug

development!

Drug Development

Holistic Approach

Reductionist

Approach

HumansAnimals

OrgansCells

Molecules

presentpast

All “Omics” are tools for

observing living systems on all

possible levels:

SYSTEMS BIOLOGY

Systems Biology

not hypothesis, but observation based

• Organism under different conditions

• Measure as many parameters as possible

– Metabolome

– Proteome

– Transcriptome

– Physiological data

• Use e.g. multivariate analysis to find any differences, correlations, etc.

• Hypothesis based on observations

• Datamining

Systems biology

Measure as many parameters as possible

Systems biology as an unbiased,

observation based approach, is a great

opportunity for studying biological

systems, e.g. processes involving synergy

and prodrugs

Metabolomics is the key to identify

compounds that are correlated with

activity, or identify the mode of action

Remember? Life is chemistry at work!

Comparison metabolomic tools

LC-MS GC-MS TLC MS-MS NMR

Sample prep - -- ++ + +++

Reproducible -- + - + +++

Absolute qnt - - - - +++

Relative qnt + ++ + ++ +++

Identity ++ ++ + ++ ++

Compound No ++ +++ + +++ +

Sensitive ++ ++ + +++ -

Wine as a complex system

• What compounds make a

wine to be a good one?

• NMR-based metabolomics of

150 wines ranked for quality

on scale 1-4 (4 = best)

Classification wines

taste panel – NMR data of EtOAc extracts

Studies of traditional medicines

• Mode of action

• Toxicity

• Markers for activity

• Quality assurance

• Synergism, prodrugs

• Variability, contaminations

• Synergism, prodrugs

• How to define quality

- evidence based use

- novel drugs

What is synergism?

Two or more agents working together to

produce a result not obtainable by any of the

agents independently.

Source: Wikipedia

1+1> 2

the basis of life

IsobologramSynergy: IC50 values (μg/ml) of combination of ginkgolides

A and B in PAF-induced in vitro thrombocyte aggregation

H Wagner, Fitoterapia 82 (2011) 34–37

Synergism: How to prove?

• Loss of activity in bioassay guided

fractionation and recovery of activity after

pooling inactive fractions:

– compounds still unknown.

• Isobolograms showing activities of different

combinations of two compounds:

– which compounds to test?

• Systems biology, correlating compounds

with activity:

– combine activity and metabolomics data.

Metabolomics and identification

active compounds in Galphimia glauca

• Collection of 6 different ecotypes

• Only two have strong sedative effect

• Can we identify metabolites

responsible for activity by NMR based

metabolomics?

Collaboration with AT Cardoso Taketa and ML Villarreal, Mexico

Planta Med. 74(2008)1295

-20

-10

0

10

-20 -10 0 10 20

GM

GM

GM

GM

GM

GM

GMGM

JGJG

JGJG

JG

JGJGMCMC

MC

MC

MCMC

MS

MSMS

MS

MSMS

MT

MT

MT

MT

MT

QJ

QJ

QJ

QJ

QJ

QJQJ

Supervised multivariate data analysis

Galphimia glauca ecotypesPartial least squares regression-discriminant analysis

Obtain maximum separation between two or more classes.

Consider only signals that separate classes, rest is noice.

Active

Inactive

-0.12

-0.10

-0.08

-0.06

-0.04

-0.02

-0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

-0.12 -0.10 -0.08 -0.06 -0.04 -0.02 -0.00 0.02 0.04 0.06 0.08 0.10 0.12

10.00000" 9.96000" "9.92000" "9.88000" "9.84000" "9.80000" "

9.76000" "9.72000" "

9.68000" "9.64000" "

9.60000" "9.56000" "

9.52000" "

9.48000" "

9.44000" " 9.40000" "

9.36000" "

9.32000" "9.28000" "

9.24000" "

9.20000" "

9.16000" "

9.12000" "

9.08000" "9.04000" "

9.00000" "8.96000" "

8.92000" "

8.88000" "

8.84000" "8.80000" "

8.76000" "

8.72000" "

8.68000" "

8.64000" "

8.60000" "

8.56000" "

8.52000" "

8.48000" "

8.44000" "

8.40000" "

8.36000" "

8.32000" "

8.28000" "

8.24000" "

8.20000" "

8.16000" "

8.12000" "

8.08000" "

8.04000" "

8.00000" "

7.96000" "

7.92000" "7.88000" "

7.84000" "

7.80000" "

7.76000" "

7.72000" "7.68000" "7.64000" "

7.60000" "

7.56000" "

7.52000" "

7.48000" "

7.44000" "

7.40000" "

7.36000" "7.32000" " 7.28000" "

7.24000" "

7.20000" "

7.16000" "

7.12000" "

7.08000" "

7.04000" "

7.00000" "

6.96000" "

6.92000" "

6.88000" "

6.84000" "

6.80000" "

6.76000" "

6.72000" "

6.68000" "

6.64000" "

6.60000" "

6.56000" "

6.52000" "

6.48000" "

6.44000" "

6.40000" "

6.36000" "6.32000" "

6.28000" "

6.24000" "

6.20000" "

6.16000" "

6.12000" "

6.08000" "

6.04000" "

6.00000" "

5.96000" "

5.92000" "

5.88000" "

5.84000" "

5.80000" "

5.76000" "

5.72000" "

5.68000" "

5.64000" "

5.60000" "

5.56000" "

5.52000" "

5.48000" "

5.44000" "

5.40000" "

5.36000" "

5.32000" "

5.28000" "

5.24000" "

5.20000" "

5.16000" "

5.12000" "

4.52000" "

4.48000" "

4.44000" "

4.40000" "

4.36000" "

4.32000" "

4.28000" "4.24000" "

4.20000" "

4.16000" "

4.12000" "

4.08000" "

4.04000" "

4.00000" "

3.96000" "

3.92000" "

3.88000" "

3.84000" "

3.80000" "

3.76000" "

3.72000" "

3.68000" "

3.64000" "

3.60000" "

3.56000" "

3.52000" "

3.48000" "

3.44000" "

3.40000" "

3.36000" "

3.32000" "

3.28000" "

3.24000" "

3.20000" "

3.16000" "

3.12000" "

3.08000" "

3.04000" "3.00000" " 2.96000" "

2.92000" "

2.88000" "

2.84000" "

2.80000" "

2.76000" "

2.72000" "

2.68000" "

2.64000" "

2.60000" "

2.56000" "2.52000" "

2.48000" "

2.44000" "

2.40000" "

2.36000" "

2.32000" "

2.28000" "

2.24000" "

2.20000" "

2.16000" "

2.12000" " 2.08000" "

2.04000" "

2.00000" "

1.96000" "

1.92000" "

1.88000" "

1.84000" "

1.80000" "

1.76000" "

1.72000" "1.68000" "

1.64000" "

1.60000" "1.56000" "

1.52000" "

1.48000" "

1.44000" "1.40000" "

1.36000" "

1.32000" "

1.28000" "

1.24000" "

1.20000" "

1.16000" "

1.12000" "

1.08000" "

1.04000" "

1.00000" "

0.96000" "

0.92000" "

0.88000" " 0.84000" "

0.80000" "

0.76000" "

0.72000" "0.68000" "

0.64000" "

0.60000" "0.56000" "0.52000" "0.48000" "0.44000" "

0.40000" "0.36000" "0.32000"

Non active

Samples

Active Samples

(Triterpenoids

and

flavonoids)

Differentiation of Galphimia glauca

ecotypes based on anxiolytic activity

Loadings plot PLS-DA

A 1HNMR vinylic H-1, H-2 diastereomeric galphimine B and F

B MeOH extract Galphimia glauca GM sample

Activity correlated

compounds Galphimia glauca

20

18

29

MeO2C

21

OR2

OH

O

O

R1

HO

My Dream!

• To find a new

drug from a

plant

My nightmare?

The biologically active

compound I finally found,

cannot be produced in sufficient

amounts.

It is feasible to grow plant cells

in large scale bioreactors

Economy:

- 1500 $/kg at 0.3 g/l/14d

- 430 $/kg at 3 g/l/14 d

Successful industrial plant

biotechnology processes:

• Taxol (US/Germany, Korea, Japan)

• Shikonin (Japan)

• Ginseng roots (Japan, Korea)

However, for most important pharmaceuticals

too low production to compete with the plant

Catharanthus roseus (Apocynaceae)

source of terpenoid indole alkaloids

• Ajmalicine

– improving

cerebral blood

circulation

• Vinblastine,

vincristine

– antitumor

Metabolic engineering or synthetic biology?

STR

Plastid shikimate

pathway

TRYPTOPHAN

Cytosol

Mevalonate

terpenoid

pathway

C15, C30

Plastid

Deoxyxylose

terpenoid

pathway

C10, C20, C40

GERANIOL

STRICTOSIDINE

TRYPTAMINE SECOLOGANIN

AJMALICINE

CATHARANTHINE

VINDOLINE

VINBLASTINE

TDC

G10H

SGD

Transgenic Catharanthus

roseus cell cultures

• Stable cell lines overexpressing TDC- and/or STR-

genes

• TDC-overexpression only increased tryptamine

• STR-overexpression increased alkaloid production

(200-300 mg/l)

• Overexpression ORCA regulatory genes

upregulated a series of enzymes, but not alkaloid

levels

Overexpression ORCA3 in

Catharanthus roseus cells

Johan Memelink and co-workers

Hairy root culture of Weigelia

expressing TDC and STR-genes

• secologanin: below detection

limit

• tryptamine: 20 g/g DW

• ajmalicine: 1.4 g/g DW

• serpentine: 0.2 g/g DW

Feed tryptamine and juice of Symphoricarpos albus

berries (contain sugar and 2% secologanin)

yield 2 g/l alkaloid per 3 days.

EU-project SMARTCELL

Biosynthesis secologanin

Integration of

transcriptomics,

proteomics and

metabolomics data

Successful

expression whole

pathway in plant

Major result genetic engineering

Artemisia annua – artemisinin

poor farmer or big pharma?

Blueprint of the cell factory has many levels,

but do we know the actual process?

Metabolism has 4 dimensions:

3 of space and 1 of time

Metabolomics is at best like a

picture of low resolution, but not like

the high resolution film needed to

see where what is happening,

fluxomics!

Plastid

Vindoline

pathway

Plastid

Trp

pathway

Plastid

MEP

pathway

Vacuole

Secologanin? Loganin?

Vacuole

STR, tryptamine, secologanin,

strictosidine, ajmalicine

Vacuole

Peroxidase, Vindoline, vinblastine

Trp tryptamine

TDC

Idioblast

Vascular cell Epidermal cell

ER

G10H

ER

LAMT, SLS?

ER

Intermediate X

Simplified

model intra-

and intercellular

transport

C. roseus

alkaloid

biosynthesis

Alkaloid intermediate Y

diHOHtabersonine

Uptake

alkaloids

and

secologanin

in C. roseus

vacuoles

(no ATP)

secologanin

ajmalicine

strictosidin

e

THAlstonine

catharanthinevindoline

stemmadenin

e

Alkaloid transport into vacuoles of

Catharanthus roseus

Roytrakul and Verpoorte, Phytochem. Rev. 6(2007)383

Overexpression ABC transporter

• Coptis japonica specific berberine ABC transporter gene of the MDR-type CjMDR1* – Imports the alkaloid into the cell

– ATP dependent

– In cell membrane

– Inhibited by vanadate, and glibenclamide,

• Catharanthus roseus CRPM cell cultures

Many thanks to K. Yazaki giving us CjMDR1!

Uptake of berberine and selected TIA in

ABC transgenic C. roseus cells

Berb Ajmal Cath Serp 4H-Alst

MDR #7 2,10 32,38* 2,34 1,41 11,02*

pRT #4 2,38 1,24 1,86 0,83 3,43

WT 2,35 8,36 2,65 1,28 4,68

(ng/mg of cells)

Ajmalicine and tetrahydroalstonine

increased uptake in ABC transgenic cells

Metabolome is the sum of micro- and nano-

metabolomes

Metabolites in Jacobaea vulgaris leaves

Phenylpropanoids

Pyrrolizidine alkaloids

Nuringtyas et al. In press

Single cell(-type) analysis required!

Phenylpropanoids Pyrrolizidine alkaloids

Nuringtyas et al. In press

Logistics cell factory

Energy

Storage

Production sites

Raw materials

Regulation

The Cell Factory is

like a car factory,

everything must be at

the right moment on

the right place in the

right quantity

Engineering the cell factory

Metabolic Engineering

• A few genes

• Biosynthetic genes

• Regulatory genes

Original cell or organism

Synthetic biology

• Large number of genes

• Biosynthetic genes

• Transporter genes

• Regulatory genes

• RNAi to block

competitive pathways

“Minimal cell”

Technologies: molecular biology and metabolite analysis

Some questions that over the years

accumulated in my mind

• Up to 30% flavonoids in flowers?

• Why is a plant extract a viscous liquid?

• How are non-water soluble compounds like terpenoids, cellulose biosynthesized?

• How do plants survive in the desert?

• How do organisms survive at low temperatures?

• How does a dry seed gets alive?

1H-NMR plant extract: overall picture

Alkaloids

Flavonoids

Phenylpropanoids

Phenoloics

Tannins

Sugars

Amino acids

GlycosidesOrganic acids

Terpenoids

Steroids

Observations we made during

the years

• NMR-based Metabolomics of all kind of cells (mammalian, microbial, plants, zebrafish) always show high levels of some sugars, organic acids, amino acids and organic bases

• Levels are too high to be explained as intermediates

• Choline is common component of synthetic ionic liquids

2007: The EUREKA Moment

Could it be that the organic bases like

choline, betaine, etc.

together with

organic acids like malic acid, succinic

acid, citric acid, etc.

give ionic liquids?

First experiment: malic acid and choline chloride

1:1

Bingo: a beautiful ionic liquid

Down to the lowest level: the cell content

Our hypothesis:

Everywhere in living systems

Natural Deep Eutectic Solvents (NADES)

occur and form a third liquid phase of

intermediate polarity

• Ionic liquids are formed by mixing an acid and a

base (e.g. choline and malic acid)

• Deep eutectic solvents are formed by mixing two

solids (e.g. glucose and malic acid)

YH Choi et al. Plant Physiol. 2011

Evidence for occurrence of

NADES

• Make various combinations of possible

NADES ingredients

• Measure physical characteristics, e.g. polarity

• Study solubility of various natural compounds

• Measure metabolome of systems where they

may occur

• Proof in-situ presence

YH Choi et al. Plant Physiol. 156(2011)1701

.

Combinations Molar ratio

Citric acid-Choline chloride 1:2, 1:3

Malic acid-Choline chloride 1:1, 1:2, 1:3

Maleic acid-Choline chloride 1:1, 1:2, 1:3

Aconitic acid-Choline

chloride

1:1

Citric acid-Proline 1:1, 1:2, 1:3

List of some natural ionic liquids

Glucose-Choline chloride-

Water

1:1:1

Fructose-Choline chloride-

Water

1:1:1

Sucrose-Choline chloride-

Water

1:1:1

Glucose-Fructose 1:1

Fructose-Sucrose 1:1

Glucose-Sucrose 1:1

Sucrose-Glucose-Fructose 1:1:1

Malic acid-Glucose 1:1

Malic acid-Fructose 1:1

Malic acid-Sucrose 1:1

Citric acid-Glucose 2:1

Citric acid-Trehalose 2:1

Citric acid-Sucrose 1:1

Maleic acid-Glucose 4:1

Maleic acid-Sucrose 1:1

Some examples of deep eutectic solvents

Ingredients and NADES(mole/mole)

1: sucrose, 2: fructose, 3: glucose, 4:

malic acid, 5: sucrose-fructose-glucose

(1:1:1), 6: sucrose-malic acid (1:1)

1 2 3 4 5 6

H-1

H-2’’

H-2’

H-3’’

Other sugar HOH and water

* *

Sucrose Malic acid

Correlation between molecules

by 1H-1H-NOESY

Sucrose-malic acid-water (1:1:1) = about 5% of water!

Solubility of Rutin in some NADES

Media

W S G F M A

So

lub

lity

of

ruti

n (

g/k

g)

0

20

40

60

80

100

Water (W)

sucrose-choline chloride (S)

glucose-choline Cl (G)

fructose-choline Cl (F) maleic

acid-choline Cl (M)

aconitic acid-choline Cl (A)

NADES

extractions

safflower

87

Dai et al. In press

Solubility macromolecules (mg/ml)

NADES Starch Gluten DNA

LGH - 4.8 286.6

GCH 15.8 0.2 2.5

PCH 11.5 0.3 7.7

PMH 4.3 173.7

Water 1.5 252.1

SoCH - 0.03 2.8

Laccase activity in malic acid-choline Cl (1:1)

0 % water, 2: 25% water, 3: 50% water.

g

3.43.94.44.95.4

sg

g

s+g+f

1H NMR Cleome hassleriana nectar. s: sucrose, g: glucose, f: fructose

Ocurrence in Plants?– Plants secrete non-volatile saps

• To attract insects: nectar

• To catch insects

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ff

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Drosera: liquid for

ever?

day 0 day 10Day

Day 1 Day 10?

Sucrose – choline chloride

1:1 molar

Water

Gently poor water on

NADES

Carthamin dissolved in

either phase

NADES may explain

• Biosynthesis of water insoluble compounds

• High level of accumulation of poorly water

soluble compounds

• How lichen can survive drought

• How cacti and resurrection plants survive

• How a seed can germinate after 30,000

years in the permafrost

• ..........

You see it, when you understand it.

Johan Cruijff

NADES beginning of life?

• Self organizing structures, liquid crystals

• Different chemistry than in water

• Intermediate polarity between water and lipids

• Water miscible, but remain stable upon dehydration

• Strongly retain water

• Liquid in large temperature range, even far below 00 C

After this existential question

back to basics, experiments for at home!

How do you make the best caipirinha?

How do you make

the best caipirinha?

• Sugar is a solvent!

• Sequence of lime extraction is important

• First sugar or cachaça/wodka?

• We measured clear difference of the

caipirinha’s metabolome as measured by

NMR

• But what tastes best?

You may send me the results:

verpoort@chem.leidenuniv.nl

What does my crystal ball tells me?I see you working in

multidisciplinary teams exploring

nature in a systemic way, leading

to understanding and exploiting

nature to our benefit

Dutch government’s view on innovation

Major discoveries

Anecdotic examples• Discovery of penicillin

– Growth inhibition zones with two microorganisms on one plate → Antibiotics

• Discovery of vinblastine and vincristine

– Testing plant for antidiabetes, observing effect on leucocytes → Antitumor medicines

• Discovery Omeprazole (Losec) no 1 best sold drug worldwide for some 15 years

– Not active but pro-drug to treat ulcers

• Viagra, a failed antihypertension drug → k€€€

There are no navigators

for research

You need to be a good

observer with an open

mind

Discovery is by chance

8th International Workshop

“Metabolomics”

April 12-17, 2015

Leiden, The Netherlands

www.plantsandmetabolomics.nl

verpoort@Chem.LeidenUniv.NL