Marine Natural Products

(Produk Semulajadi
Marin)

PROF. DR. NORAZNAWATI ISMAIL

(PhD)
ROOM NO: 3
Ground Floor, IMB
EMAIL:noraznawati@umt.edu.my
 Marine natural products
• Study for their unusual and unique
chemical structure
• Secondary metabolites, not essential
for growth and reproduction but give
selective advantage to the organisms
• Advantages: medical usefulness and
exhibit several types of activities:
anti-tumor, anti-viral etc
Definition : Primary
metabolites
A primary metabolite is a chemical substance
which is required for cells to survive and replicate
and is therefore required for survival of the
organism
Examples are:
– Proteins
– Carbohydrates
– Lipids
– Nucleic acids
Definition: Secondary Metabolites
(@ Natural products)
• A secondary metabolite is a chemical compound
produced by an organism which is not required for
survival of the organism but presumably confers
an evolutionary advantage
H OH
O N H H
H O O

HH O O HO
O H
HH
H HO
O
O H H
NH O OH O
O H
O H
OH OH
OH H O
O N H
O
O
O O O
Why can marine natural products be used
as medicines?
• Barnacles use a protein with homology to a
histamine/adrenergic receptor to analyze surfaces prior
to settlement

• In humans adrenergic receptors regulate vasodilation,

muscle contraction, etc.

• Active sites of proteins are often conserved

between species, but
can have very different functions.
Why natural products?

• Provide an opportunity for public sector in drug discovery

• New technologies and experts may overcome the ‘problems’
• Chemical diversity of natural products still remains the
driving force
Why does nature make natural products?

Defense

Reproduction
 Nature As a Source of Medicines

• Over 62% of small molecule agents approved

for use as drugs can be traced back to natural
products
• Some examples:
• aspirin (willow/birch )
• morphine (Poppy)
• penicillin (fungus)
• Lovastatin (fungus)
• Adriamycin/dauxorubicin (bacterium)
• Taxol™ (yew tree)
The sustainability of research in drug discovery

• Emerging of new diseases

• Emerging of new drug-resistant pathogens (diseases)
• Identification and validation of new targets for existing diseases
• New innovative assays

• Private vs Public
• Profit vs Social-responsibility

• Continuous search for new sources of drug-like compounds

• Synthetic compounds
• Natural products
The sustainability of research in drug discovery

• Emerging of new diseases

• Emerging of new drug-resistant pathogens (diseases)
• Identification and validation of new targets for existing diseases
• New innovative assays

• Private vs Public
• Profit vs Social-responsibility

• Continuous search for new sources of drug-like compounds

• Synthetic compounds
• Natural products
Current Drugs

• 37% of small molecule NCEs (1981-2006) are synthetic molecules

• 63% of small molecule NCEs can be traced back to natural products
• 1981-2002, natural products and related drugs were used to treat 87%
of all categorised human diseases
• 1982-2002, ~79% of approved drugs worldwide can be tracked back to
natural products

• The pool of natural products is huge and largely untapped

OPPORTUNITY!
Why marine biodiversity?

• Marine organisms diversity is higher than tropical forest

• Of the 36 phyla, 34 are found in marine environment and only 17
in terrestrial area
• Of the 33 phyla of animals, only 1 is exclusive to land whilst 20 are
exclusive to the oceans
Megadiverse Countries
 Why are new drugs needed?

• unmet medical need;

new diseases (AIDS, Alzheimer’s; obesity);
low efficacy (dementia, cancer);
side effects (antidepressants, antipsychotics)
• cost of therapy; (Interleukins)
• costs to individual/country; (Alzheimer’s; spinal injury,
depression)
• sustain industrial activity; pharmaceutical industry
employs thousands and makes a massive contribution to
overseas earnings); patent expiry
 Steps in New Drug Development

1. Idea or hypothesis
2. New drug discovery
3. Screening
4. Preclinical studies
5. Formulation development
6. Clinical studies
7. Official license / Regulations/Marketing
A Historical Perspective: Source of
Important Natural Products
 Historical Perspective

O O • 1940’s and 1950’s

CH3 • spongouridine and
HN HN
spongothymidine reported
HO O N HO O N from Cryptotethia crypta
O O collected near Elliott Key
HO HO
• Bergmann JOC 1951 15:
OH OH 981
• Bergmann JOC 1955
spongouridine spongothymidine
20:1502
NH2

HO O N
O
HO
Nucleoside antiviral
and Anticancer agents
OH Ara-C AZT etc.
 O
• 1969 Siegel and co-workers at O HO
the University of Miami O H HH
N
• Discover potent antitumor and S
N
immunomodulatory properties O
H OH
O
of the mangrove tunicate O
Ecteinascidia turbinata O O
NH

• Approved for use against soft HO

tissue sarcoma in Europe
Ecteinascidin 743
2007

Siegel et. al. Food Drugs from the Sea 1969

Wright et. al. JOC 1990
Rinehart et. al. JOC 1990
 Marine derived drug approved by FDA

Prialt™
Derived from Conus magus (Cone Snail),

• Prialt (SNX-111) is a synthetic form

of the omega-conotoxin MVIIA

• It is 100-1000x more
potent than morphine

• It has been approved for use by the

FDA for chronic pain
The Process of Marine Drug
Discovery
• Collection
• Biological Screening
• Natural Products Chemistry
• Secondary Testing/Pharmacology
• Production of material for clinical
evaluation
• Clinical investigation
 Marine Natural Product Drug Discovery
Requires a Multidisciplinary Team
• Biologists
– Marine Biologists
– Cell Biologists; Immunologists, Virologists,
Microbiologists
– Pharmacologists/Biochemists
– Molecular Biologists
• Chemists
– Natural Products Chemists
– Spectroscopists
– Synthetic/Medicinal Chemists
• Business Professionals
– Marketing/Technology Transfer/ Patent
Attorneys
Step 1: Collections
Before you collect-Get the right permits
• International Convention on Biological Diversity
– http://www.biodiv.org/convention/articles.asp
• You must have informed consent of host country
– Permits may be issued at a local or country level
– Work with US State Department for Foreign Countries
– Work with State Governments for State waters; National
marine
Fisheries for Federal
– Special Permits for Marine
Sanctuaries
• Often a Memorandum of Understanding is
negotiated prior to collection
– Participation by Host Nation in Project
– Return of Income/Benefit to Host Country
– Reporting requirements
– Sharing of specimens and information on samples
• John E. Fogarty Center (part of NIH) regulates this
– http://www.fic.nih.gov/programs/oecdub.html
 Collection Methods
• Wading
• Scuba
• Trawling
• Submersibles
– AUV (mapping/documentation)
– ROV (mapping/documentation/collections)
– HOV (mapping/documentation/collections)
Harbor Branch Operates the
Johnson-Sea-Link class
subs Depth capability: 3000
ft
 Collection Strategies
Biological Diversity = Chemical Diversity
 Habitat Diversity = Chemical Diversity
Myrmekioderma metabolites are depth dependent
OH O

H OH O
< 33 m,
Anti-tumor
Activity
H

33 m < X < 66 m > 66 m

Anti-tumor Antiviral Activity (HSV-
Activity 1)

Sennett et. al. J. Nat. Prod 1992, 55,1421

Kashman et. al. Tet. Lett. 1987, 28, 546.
 Invertebrate Diversity=Microbial Diversity
• Sponges can have up to
40% of their biomass made
from associated microbes

• The evidence is mounting

that many “sponge derived”
compounds are synthesized
by microbial associates

Thin section of the sponge

Discodermia
There is notable structural similarity between
certain marine natural products and those
produced by microorganisms
H
H

NH NH
H
H
H H
O H
N
O O
H H
O
HH
H H
H O
O H H HO O OH
O H H
H
H H
NH
NH H
N
H
O O
O O O OH

Alteramide Ikarugamycin Discodermide

Alteramide Alteromonas associated

with Halichondria
Ikarugam ycin Terrestrial
Streptomycete
Discodermide Discodermia dissoluta
 Biosynthetic Gene clusters localized to
bacterial symbionts
O
S
N Patellamide A produced by the tunicate
H
O N N symbiont
O
HN
Prochloron didemni
NH
O
Gene cluster expressed in E. coli
N H N Schmidt et. al. PNAS 2005 102: 7315
N O
S
O
O COOH
N
Onnamide from sponge Theonella H
HO
swinhoeii
OH O H2 N NH
O H
O N
Gene cluster sequenced- associated O NH
with bacterium O O O
Piel et. Al. PNAS 2004, 101: 16222

The challenge remains to culture these microbes!

Step 2: Screening/Biological
Assays
 Screening
Approach
Forward Chemical Reverse Chemical
Genetics Genetics
Approach Approach

CELL Purified Protein

Treat with small Treat with small

molecule molecule

Detect desired Cell Detect small

Phenotype molecules which bind
to target
Determine HOW
small molecule Define WHAT
causes EFFECTS the binding
 Current Research Focus
Finding Compounds to treat:
• Cancer
– Pancreatic Cancer
– Multidrug resistant cancers
– Cancer “Specific” Agents (NCDDG)
• Infectious Disease
– Drug resistant Staphylococcus aureus
– Anti-malarial- collaborative UCF, WRAIR

• Neurodegenerative Disease- collaborative

– Alzheimer’s & Parkinson’s Diseases
– Neuroprotection (Stroke)
• Inflammation –collaborative
 Types of Assays Run at HBOI-
Tumor Cell Lines
Tumor Cell Line Panel/MTT
assay
• A549 (lung)
• PANC-1 (pancreatic)
• ASPC-1 (pancreatic)
• Mia PaCa2 (pancreatic)
• BxPC-3 (pancreatic)
• MCF-7 (breast)
• DLD-1 (colon)
• NCI-ADR-Res (ovarian)
 Cartoon of a Cell-based
LIGHT
Reporter Assay
LIGHT
The Cytoblot Assay
Horse radish
peroxidLaIsGeHT

LIGHT

O3PO Secondary Antibody LIGHT

Primary Antibody
Protein of Interest

• The target protein increases or decreases in level after treatment

• The protein target is detected with sequential treatment of

antibodies, one of which is labeled with a horseradish peroxidase

• Differences in light emitted from control values indicate if

something is active
Stockwell et. al. Chemistry & Biology 1999,
 Target directed assays
• MAP Kinase Signaling
– p-ERK, p-MEK
– cell survival, invasion, resistance to apoptosis
• Inhibition of GSK-3
– regulates NF-B in pancreatic and breast cancer- controls cell
proliferation
• Apoptosis -
– Restoring sensitivity to TRAIL- induced apoptosis in resistant
tumor cells
• Cancer and Inflammation
– Regulation of NF-B, STAT3. mast cell migration

• HEDGEHOG signaling
– targeting cancer stem cells through regulation of Gli proteins
Step 3: Natural Products
Chemistry

Purify and Determine the structures of the

Active Natural Products
Extracts are Complex Mixtures of
Natural Products
Natural Products Chemistry
The Traditional Way
Chromatography

Bioassay

Chromatography

Bioassay

Pure Active
Compound
Extracts are Complex Mixtures of
Natural Products

• Mixtures are too complex

for modern high throughput
screening operations
• Solution: make a “Peak
Library”
 HBOI Peak Library Generation
Prepare Extract

Solvent partition

Chromatograph using
COMBIFLASH

Assay/Analyze
Fractions
Deep-water Verongid sponge
 Pure Compound

Spectroscopy is
used to define the
Structures
O OH Cl Cl

HO O N
O
HO HO
HO O
O NH 2
O O O

HO OCH3
600 MHz Nuclear Magnetic Resonance
Spectrometer
Step 4: Determine the Mechanism
of Action

For example:
How does the compound kill cancer cells?
 Confocal Microscopy

Flow Cytometry
 Western Analysis for measuring
responses at the Protein level

Immuno-blots
Affinity Chromatography
MW

Phospho-Akt (Thr 473)

OH H H H
N N N
H H
O O

OH
H O
H H O
O
HO
H H
2 3 4 5 6
 We have a number of projects focused on
Sustainable Use of Marine Resources

Chemical Synthesis

O O

O OH O
H O
O OH N 2
H H
 We have a number of projects focused on
Sustainable Use of Marine Resources
Invertebrate Cell Culture
 We have a number of projects focused on
Sustainable Use of Marine Resources

Aquaculture

Polymastia in Rope Culture

 We have a number of projects focused on
Sustainable Use of Marine Resources

Recombinant
Production
Purification
Extraction with Ethanol-Ethyl acetate

Solvent Partitioning Ethyl Acetate-Water

Silica gel Vacuum Column Chromatography

C-18 Reverse Phase HPLC

Pure Compound
Impact on Natural Products
and drug discovery
• New technologies and innovations
– Reducing cost in R & D
– Previously 75% costs spent on failure
– Increase in productivity
– Using new techniques including microarray
technology and bioinformatics
– Accelerating the drug development
process
– Maintaining high standards in quality
assurance
 A promising future for marine natural
products in drug discovery?

• The marine ecosystem holds a rich

biodiversity with vast potential for
biotechnological exploitation that is yet
to be fully explored.
• Studies of new drug leads from marine
sources will significantly expand.
• The marine sources could be the major
source of new drugs for the next
decade!
 Conclusions
• The marine environment holds a wealth of
natural products
• If the compounds are coupled to the wealth of
molecular targets defined by studies on the
human genome and proteome…
• and biotechnology is harnessed to produce
them….then
• the Oceans can play a major role in improving
Human Health