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CELL AND TISSUE CULTURE TECHNOLOGY

ARTEMISININ

PRESENTED BY: Sagarika, Sajin, Shreya Nahata, Sonal, Tsering Angmo

THE BREAKTHROUGH DISCOVERY

SOURCE: www.nobelprize.org

MALARIA
Malaria is predominantly a disease of the developing
world disease is caused by Plasmodium spp.
WHO World Malaria Report (2014): around 3.4 billion
people at risk.

TREATMENT OF MALARIA
Artemisinin plays a central role in combating the
increasingly drugresistant, malaria-causing parasite Plasmodium
falciparum.
Artemisinin-based combination therapy (ACT) is currently
the most
effective means to treat and reduce the transmission rate

DISCOVERY
1950 Failure to eradicate malaria; the disease rebounded
1967 Research programme Project 523 under leadership
of Tu Youyou
Investigated more than 2,000 Chinese herb preparations
and identified 640 hits that had possible antimalarial
activities.
380 extracts obtained from ~200 Chinese herbs were
evaluated against a mouse model of malaria.
The turning point came when an Artemisia annua leaf
extract showed inhibition against parasite growth.
4 October 1971 a nontoxic, neutral extract that was 100%
effective against parasitemia in mice infected with

INTRODUCTION
Artemisinin (historically as qinghao): SECONDARY
METABOLITE
Obtained from Artemisia annua
(sweet wormwood), an herb
employed inChinese traditional
medicine.
Belongs to Asteraceae family.
Artemisinin is stored in glandular
trichomes on the surface of the
leaves/stems and on the corolla
and receptacles of the florets.

STRUCTURE AND
PROPERTIES
1972
a sesquiterpenoid lactone with
endoperoxide ring.
colorless crystalline substance
molecular weight of 282 Da,
molecular formula of C15H22O5,
and

Structure of
artimisinin

Its
melting
point of 156157 C
major role
Antimalarial property- a new class of antimalarial agents that
rapidly kill the malaria parasites at an early stage of their
development.
Hepatitis B
A range of cancer cell lines

Image source:

MODE OF ACTION

Most artemisinins used today areprodrugs of the


biologically activemetabolite dihydroartemisinin,
which is active during the stage when the parasite is
located insidered blood cells.
Artemisinins exert their antimalarial action by radical
formation that depends on their endoperoxide bridge.

When

the parasite that causes malaria infects a red


blood cell, it consumeshemoglobin within its digestive
vacuole, a process that generates oxidative stress.

In the primary theory of the mechanism of action, the


iron of the heme directly reducestheperoxide bond in
artemisinin, generatinghigh-valent iron-oxo species and
resulting in a cascade of reactions that produce reactive
oxygenradicals which damage the parasite and lead to
its death.
Artemisinin derivatives provide the basis for the most
effective treatments for malaria, particularly in the form
of artemisinin-based combination therapies (ACTs) with
lumefantrine and sulfadoxime pyrimetamine

DERIVATIVES OF ARTEMISININ
The therapeutic value of artemisinin was limited by its low
solubility in both oil and water, and this has lead to the
development of semi-synthetic drugs with pharmacological
properties superior to those of the parent.
The most important such derivatives are artemether, arteether
and artesunate which exhibit greater potency than artemisinin
itself, as well as improved solubility, and favourable metabolic
and hydrolytic stabilities.

BIOSYNTHETIC PATHWAY OF
ARTEMISININ
The Biosynthesis of artemisinin involves:
Phase 1 (isopentenyl pyrophosphate to amorpha4,11-diene)
Phase 2 (amorpha-4,11-diene to
dihydroartemisinic acid)
Phase 3 (dihydroartemisinic acid to artemisinin)

BIOSYNTHETIC
PATHWAY OF
ARTEMISININ

METABOLIC ENGINEERING IN
PLANTS
by overexpressing key genes from this pathway
eg CYP
by overexpression of heterologous genes in A.
annua
By down-regulating enzymatic reactions that
compete with ADS for its use, such as squalene
synthase (SQS) involved in sterol biosynthesis.

Activation of artemisinin-pathway genes

Moran Farhi et al (2013) Metabolic engineering of plants for


artemisinin synthesis, Biotechnology and Genetic

OVEREXPRESSION OF
HETEROLOGOUS GENES IN A.
ANNUA
isopentenyl transferase gene (ipt) from Agrobacterium
tumefaciens
It codes for the enzyme, isopentenyl transferase (IPT)- it catalyzes
the condensation of isopentenyl diphosphate (IPP) to form
isopentenyl AMP (iPMP)
reports have shown that overexpression of IPT increases the
content of chlorophyll and artemisinin content is higher in A. annua
plants with higher chlorophyll
the chlorophyll content increased by 2060%, and artemisinin
content increased by 3070% in the transgenic plants.

REPRESSION OF PATHWAYS COMPETING FOR


ARTEMISININ BIOSYNTHESIS
FPP is at a major metabolic
branch point and is used for the
synthesis of important cellular
components such as sterols,
ubiquinones and hormones like
brassinosteroid
One of the major enzymes that
utilize FDP in eukaryotes is SQS,
which shuttles terpenoids into the
sterol pathway.
In A. annua, its suppression was
accompanied by reduction in the
levels of four major sterols and
an increase in artemisinin
content (Yang et al., 2008; Zhang
et al., 2009)

Benye Liu et al 2011

THE SEMI-SYNTHETIC ARTEMISININ PROJECT


Why is there a need for semi-synthetic artemisinin?
Sharply increased demand for artemisinin.
Urgent need to develop an alternative source of
affordable artemisinin.
To stabilize both the cost and the supply of artemisinin
derivatives.
Aim of the Semi-synthetic Artemisinin Project

To engineer a microorganism to produce an artemisinin


precursor at high titres, rates & yields, followed by
chemical conversion to artemisinin.

STAGES IN THE SYNTHESIS


OF SEMI-SYNTHETIC ARTEMISININ

STAGE 1
Engineering of the yeast strain
by overexpression of genes of
mevalonate pathway &
expression of A. annua
amorphadiene synthase.

STAGE 2
Oxidation of amorphadiene to
artemisinic acid by expression of
CYP71AVI,
CPR1, cytb5 and dehydrogenases.

STAGE 3
Extraction of artemisinic acid
and its conversion to
artemisinin.

METABOLIC ENGINEERING OF E.COLI


TO PRODUCE ARTEMISININ
Why in E.coli?
a) The isoprenoid precursor used in artemisinin biosynthesis are
common to both prokaryotes and eukaryotes.
b) Overall yield is too low in both original and engineered plants.

E.coli uses DXP pathway to produce IPP.


But it contain many internal control so the mevalonate pathway
of
S. cerevisiae is engineered as shown in figure in next slide.

Production of amorphadiene via the DXP or


mevalonate isoprenoid pathways and depiction of the
synthetic operons used in this study.

Meaning of signs used in the figure


are as follows

PLAC promoter

ADS, amorphadiene synthase.

Adapted from

CONCLUSION AND CHALLENGES

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