Habibah A Wahab, PhD
Malaysian Institute of Pharmaceuticals and Nutraceuticals,
Ministry of Science, Technology and Innovation
SAINS@USM, 11900, Penang
Pharmaceutical Design and Simulation Laboratory,
School of Pharmaceutical Sciences, USM
Shaharum Shamsuddin D. Phil (Oxon.)
Advance Molecular and Cellular Biology Laboratory
Deputy Dean
School of Health Sciences, USM
Leveraging Malaysia’s
Biodiversity towards Value
Creations using Bioinformatics
World’s Megadiversity
Taxonomic Group Species Endemic Species Percent Endemism
Plants 25,000 15,000 60.0
Mammals 380 172 45.3
Birds 769 142 18.5
Reptiles 452 243 53.8
Amphibians 244 196 80.3
Freshwater Fishes 950 350 36.8
Biodiversity hotspot: Sundaland
http://www.biodiversityhotspots.org/xp/hotspots/sundaland/Pages/biodiversity.aspx
Country Mammals Birds Plants
Malaysia 337 746 15500
Malaysia Biodiversity and National Biotechnology Policy
► Innovation to create wealth by utilising and advancing
biotechnology for socio-economic benefits of the nation in
accordance with established social and ethical norms
Mission
Statement
► Position biotechnology as the new economic engine to enhance
prosperity and wellness of the nation by 2020
► Through Healthcare, Industrial and Agriculture
► Enhance R&D and Tech Acquisition, Human Capital Development,
Legal and Financial Structure
Vision
To optimise economic benefits from
sustainable utilisation of the components
of biological diversity;
• Identification and
Development of Bioactive
Compounds
• Bioprocessing
• Pre-formulation for Product
Development
• Screening of Bioactive
Compounds
• Advanced Drug Delivery
Systems
IPHARM
Wealth from plant biodiversity
• Food (through agriculture and harvest of natural population)
– Crops, livestock, forestry and fish
– Food products: fruits, vegetables, nuts
– Food Additives: colourings, flavourings, preservatives
• Medicine
– ~ 119 pure chemicals are extracted from < 90 species of higher plants and used as medicines
throughout the world, for example caffeine, quinine, vincristine, vinblastin.
• Industry
– Fibers for clothing; wood for shelter and warmth
– Source of energy (biomass);
– Industrial products: oils, lubricants, perfumes, fragrances, dyes, paper, waxes, rubber,
latexes, resins, poisons
– All these can be derived from various plant species.
• Tourism & Recreation
– Biodiversity is a source of economical wealth for many regions of the world
– Nature reserves, parks and forests
– Ecotourism, National park, wetland resort
Natural Products Discovery System (NADI) & Value creations
• Natural products
• Value creations
– Pharmaceuticals
– Nutraceuticals
– Cosmeceuticals
– Specialty chemicals
Natural product based pharmaceutical discovery
• The global pharmaceutical markets worth $770 billion in 2008 and expected to grow 4 to 6% in 2010 exceeding $825 billion following stable demand in 2009 (IMS report, June 2009).
• Natural products are established and long source of drug. 61% of the 877 small molecule
new chemical entities introduced as drugs worldwide during 1981-2006 can be traced to or
were inspired by natural products.
David J. Newman; Gordon M. Cragg; J. Nat. Prod. 2007, 70, 461-477.
Natural Products: Source of Drugs (& medications)
Natural Product Drug Discovery: Opportunity
• ~250,000 flowering plant known , ~ 125K found in the tropical forests.
• Only ~1% of tropical species have been studied for their pharmaceutical potential.
• 2002-2006, 26 are plant derived.
BioMalaysia2009, 18th Nov. 2009, KLCC, Kuala Luampur
Natural Product Discovery: Opportunity
• Malaysia has > 15000 flowering species, 2000 with medicinal values
• Many compounds (vincristine, vinblastine, reserpine) in Malaysian natural products are
current drugs in the market though not discovered in Malaysia.
• Herbal industry in Malaysia, RM8 billion/year – growing at 10% per year.
39%
6%
27% 5%
23%
61%
Natural Products
NatProd Deriv ativ es
Sy nthetic compounds w ith NatProd deriv ed pharmacophore
NatProd mimic
Antibacterials 78%
Anticancer 74%
Used to treat 87%
(48/55)
of human diseases
David J. Newman* and
Gordon M. Cragg J. Nat.
Prod., 70 (3), 461 -477,
2007”
Challenges • Approaches to natural discovery:
– random selection followed by chemical screening,
– random selection followed by one or more biological assays, follow-up of biological
activity reports,
– follow-up of ethnomedical (traditional medicine) use of plants
• Challenges:
– Tedious isolation and characterization of natural products and heavy reliance on good
screening and bioassays
– The discovery of compounds that are cytotoxic or have other unsuitable properties.
– Reinventing the wheels
• Problems:
– not enough availability. can be overcome by semi-synthesis/synthesis or using tissue-
culture techniques
– Isolated bioactive compounds are known compounds
NADI: Bioinformatic approach to complementing Natural Product Research
• NADI applications:
– One stop centre for information
– Plant selections • Ethnopharmological basis & Follow-on research
• Systematic evaluation
• Modelling?
– Assay selections • Ethnomedical basis
• Random/systematic screening
• Virtual Screening?
BioMalaysia2009, 18th Nov. 2009, KLCC, Kuala Luampur
NADI:
Bioinformatic databases: small
molecules, receptor databases,
plant monographs
Bioinformatic tools: Blast, Protein
Structure Prediction, Molecular
Docking etc.
NADI: Information Centre for Malaysian Herbs
BioMalaysia2009, 18th Nov. 2009, KLCC, Kuala Luampur
Ethnomedical/follow-on basis
Keyword Search:
Symptom Keyword Search: Plant’s name
Plant’s name
Plant’s part used
Introduction about the plant
BioMalaysia2009, 18th Nov. 2009, KLCC, Kuala Luampur
Traditional Uses of the plant
Scientific Studies on Bioactivities
References
BioMalaysia2009, 18th Nov. 2009, KLCC, Kuala Luampur
Live search on the PubMed on
the related references
From the monographs:
Tongkat Ali has been used:
•Aphrodisiac
•Fever
•Wound healing
•Headache
•And many more
•Scientific studies:
•Increase testosterone
•Anti-malaria
• Antileucaemiac.
•Antitumor
•Antipyretic.
• Antiulcer
BioMalaysia2009, 18th Nov. 2009, KLCC, Kuala Luampur
Systematic Evaluation
Who’s doing what?
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• What has been done? • Do we need to reinvent the wheel?
No of compounds in a plant
0
20
40
60
80
100
120
140
Aca
lyph
a indica
Alpinia raffle
sia
And
rograph
is pan
iculata
Artoc
arpu
s he
tero
phyllus
Bau
hinia malab
arica
Blumea
luce
ra
Cae
salpinia pulch
errim
a
Cas
sia alata
Cen
tella
asiatica
Citrullus vu
lgar
is
Citrus
aur
antiu
m
Citrus
clemen
tina
Citrus
limon
Claus
ena exc
avat
e
Cur
cum
a long
a
Cur
cum
a ze
doa
ria
Cyp
erus ro
tund
us
Eug
enia cum
ini
Eug
enia m
iche
lii
Garcinia atro
virid
is
Har
risonia pe
rfora
te
Jasm
inum
sam
bac
Miche
lia A
lba
Mitragy
na spe
cios
a
Mor
inda
citr
ofolia
Myristic
a fra
grans
Orth
osiph
on stamineu
s
Pan
danu
s am
aryllifolius
Piper
betle
Piper
retro
frac
tum
Pre
mna
tomen
tosa
Ros
a da
mas
cena
Sm
ilax myo
sotiflora
Tab
erna
emontan
a diva
ricata
Trig
onella foe
num
Zingibe
r ca
ssum
unar
Zingibe
r ze
rum
bet
Plant
No
of
co
mp
ou
nd
s
Series1
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BioMalaysia2009, 18th Nov. 2009, KLCC, Kuala Luampur
Rational Selection – Assays
• When a novel compound identified, how do I know it has potential as a drug?
• NADI provides link to USM’s Reverse Docking for prediction of potential receptor(s) to target….
BioMalaysia2009, 18th Nov. 2009, KLCC, Kuala Luampur
BioMalaysia2009, 18th Nov. 2009, KLCC, Kuala Luampur
Molecular
Dynamics
Simulation
Molecular
Docking
Virtual Hits
Identified
In vitro
assay
Hit Identified
Literature
review Pharmacop
hore -
Modelling
Lead Identified
Virtual Lead
Identified
Further tests
NADI Drug Design Workflow
Validated target
Identified
*
*
Ensemble Based Docking
AutoDock
Set of docked
complexes, BEs
Post-processing
ranking schemes
Ligand PDBs
Ligands
ZINC
available
NADI NCI
N/A
Org.
synth
Receptor xtal struct
Explicit
MD
Snapshot
10 ps
Receptor ensemble
R. Amaro, JACS, 2008
NADI example: TB
• Caused by Mycobacterium tuberculosis
• One third of the world population is infected
• WHO: 8 million new cases each year
: 30 million people will die of tb this decade
• As much as 30% INH resistant strain TB
• Multi-drug resistant strain in HIV/AIDS patients
Drug Target: InhA (PDB ID: 1ZID)
I47 V78 I95
S94A MT InhA (PDB ID: 1ENZ) I16 I21
Wahab, JCIM, 2009
in-silico screening using NADI
~400 plants,
~4000 compounds
BioMalaysia2009, 18th Nov. 2009, KLCC, Kuala Luampur
INADH-The
ultimate
inhibitor
Virtual Hits Identified
Plants Identified
Table 1: The predicted activities of chemical compounds
reportedly to be isolated from Malaysian plants
Compounds Plant source Predicted Inhibition Constant (Ki)
D Pegaga 5.78E-17
B Akar susun kelapa 4.73E-16
C Sireh
4.13E-16
E Kunyit 1.5E-16
A Bunga tahi ayam
1.05E-16
Isoniazid (INADH) Synthetic molecule 3.795E-13
A B
C
D
E
No.
Plants Plant parts
Extracts MIC
(µg/ml)
1 Lantana camara (Bunga tahi ayam)
leaves Methanol 25
2 Centella asiatica (Pegaga)
whole plant
Methanol 25
3 Psidum guajava (Jambu batu)
leaves Methanol 200
fruits Methanol 50
4 Tabernaemontana coronaria (Jasmin)
leaves Methanol 50
5 Phyllanthus niruri (Dukung anak)
whole plant
Methanol 200
6 Murraya paniculata (Kemuning)
leaves Methanol 50
7
Piper betle (sireh)
whole plant
Ethanol 100
Chloroform 50
Petroleum ether
50
Ethanol:water 50
8 Isoniazid
0.31
CONTROL
Minimum inhibitory concentration (MIC) determinations
NADI example 2 : Avian Influenza
•Major outbreak began 2003.
Affected countries:
•The Republic of Korea, Viet Nam,
Japan, Thailand, Cambodia, The Lao
People’s Democratic Republic,
Indonesia, China, and Malaysia.
•2005, the virus spread to the
Russian Federation, Kazakhstan,
Mongolia, Turkey, Romania,
Croatia, Ukraine and the
Netherland.
•Japan, the Republic of Korea,
and Malaysia have controlled
their outbreaks and are now
considered free of the disease.
Elsewhere in Asia, the virus has
become endemic in several of
the initially affected countries.
•Source:
WHO
viral replication and drug targets
• Influenza virus membranes contain two glycoproteins: haemagglutinin and neuraminidase.
• 2 groups of neuraminidase subtypes: – group-1 contains the subtypes N1, N4, N5 and
N8 (Avian Flu is of N1 subtype). – group-2 contains the subtypes N2, N3, N6, N7
and N9.
• The enzyme facilitates the spread of virus during an infection, thus becomes an attractive target for antiviral drugs. E.g. of drugs inhibit its activity are oseltamivir and zanamivir.
• These inhibitors were originally developed using crystal structures of neuraminidase subtypes N9 and N2 and another neuraminidase from the type B genus of influenza viruses.
• Other drug targets include M2, H2 and RNA polymerase.
Total: 208 plants
3500 cpds
in-silico screening using NADI
BioMalaysia2009, 18th Nov. 2009, KLCC, Kuala Luampur
Docking of Oseltamivir onto H5N1 Neuraminidase
• RMSD: 1.3 Å
• Predicted Ki = 0.13 nM (exp Ki ranging from 0.073 – 0.37 nM, AAC, Aug. 2006)
Compound name Final Docked energy,
kcal/mol Inhibition constant, Ki
MSC 986 -11.27 5.35E-09
MSC 1026 -12.54 3.45E-09
MSC 1685 -12.71 6.29E-09
MSC 1880 -15.55 8.58E-10
MSC 1941 -12.22 9.42E-10
Top 5 compounds screened from MD trajectories of neuraminidase
MSC986 MSC1026
MSC 1685
MSC 1880
MSC 1941
Preliminary results of enzyme Inhibition Studies % Inhibitory of Neuraminidase by
methanol extract
0
20
40
60
80
100
120
0 5 10 15
Sample Concentration
% i
nh
ibit
ory
Sample Concentration (µg/ml)
% inhibitory i % inhibitory ii Average
12.00 95.50 95.80 95.65
3.00 83.20 84.90 84.55
1.50 59.80 77.10 68.45
0.75 67.80 72.10 69.95
0.05 47.60 49.70 48.65
0
20
40
60
80
100
120
50 100 500 1000 2000 3000 5000
Concentration (ug/mL)
% I
nh
ibit
ion
THF-pet ether extract
Oxalomalic acid
Oseltamivir
Plaque assay experiment : Testing the compound againts live H1N1 virus
NADI, Herbals, Cosmetics and Specialty Chemicals
Natural Product Discovery: Way Forward
• NADI database and bioinformatics approach important and have values to:
– the rational selection of plants for further studies – survey new chemical structures found in various classes of natural compounds – predict compounds with new pharmacological activity (compounds only have
value if they act differently or on new targets relative to known structures) – utilise natural products derived structures as guiding principle for new
chemical library for synthesis. • Analogues of natural products can be more potent than the parent compounds, or
possess superior drug-like properties. • New biological activities not even seen with the parent molecule. • Intensified search for new natural product derived molecules.
• Collaboration efforts from different disciplines – rapid and cost-effective discovery
• Safeguard our natural treasure (wealth, security)
Acknowledgement
NADI Developers: NAPIMM:
Yam Wai Keat, Nurul Bahiyyah, Suhaini Ahmad
NADI-RA
Nur Hanani Che Mat
NADI-HERBS
Nornisah Mohamed
NADI-CHEM
Suhaini Ahmad, Ezatul E Kamarulzaman, Ros Fatihah Halim, Nur Faezah Abdullah
NADI-VISAGE
• Suhaini Ahmad,
• Junya Seo (PhD,
Osaka U)
• Mitsuru Jikeya (MSc,
Osaka U)
• Masafumi Tominaga
(MSc, Osaka U)
Post Doctoral Fellow Dr. Hassan Abdullah
Dr Muhamad TM Al Dajani
Postgraduates: Mohd Razip Asaruddin
(PhD)
Suhaini Ahmad (MSc)
Nurul Izzah (MSc)
Noor Hamdah Musa (MSc)
Siti Marina Maidin (MSc)
Sharizan Abdullah (MSc)
Fauziah Hanim (MSc)
Fauziahanim Zakaria (MSc)
Internship Students:
Vicky Yang (BSc, UCSD)
Cindy Trans (BSc, UCSD)
Ranmali Varahenage
(BSc, UC San Diego)
BIRD FLU RESEARCH GROUP
Academic PI
•Dr. Aisyah Saad Abdul Rahim
•Dr. Hasnah Osman
•Dr. Shafida Abdul Hamid
•Dr. Nornisah Mohamed
•Dr. Razip Samian
•Dr. Normi Yusuf
•Dr. Rashidah Abdul Rahim
•Dr. Shaharum Shamsuddin
•Dr. Sharifah Syed Hassan
Funding
USM Research University Grant
TB Drug Discovery
•Dr. Shaida Fariza Sulaiman
•Suriyati Muhamad
•Dr. Pazilah Ibrahim
•Dr. Choong Yee Siew
Funding
MOSTI-EScience