Download Marine Natural Products Drug Discovery

Marine Natural Products
Drug Discovery
Amy Wright Ph.D.
Center for Marine Biomedical and Biotechnology Research
HARBOR BRANCH Oceanographic Institute at
Florida Atlantic University
Definition 1: 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 2: Secondary Metabolites
(aka 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
O
H
HH
H
H
OH
H
N
H
O
O
O
H
HO
O
HO
O
H
O
H
O
NH
OH
O
O
O
OH
O
O
OH
O
H H
N
O
O
O
O
H
H
H
O
H
OH
Why does nature make natural products?
Defense
Reproduction
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.
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)
A Historical Perspective: Florida as a
Source of Important Natural Products
Florida Historical Perspective
O
O
HN
HO
HN
N
O
CH3
HO
O
N
O
O
HO
HO
OH
OH
spongouridine
• 1940’s and 1950’s
• spongouridine and
spongothymidine reported
from Cryptotethia crypta
collected near Elliott Key
• Bergmann JOC 1951 15: 981
• Bergmann JOC 1955 20:1502
spongothymidine
NH2
N
HO
N
O
O
HO
OH
Nucleoside antiviral and
Anticancer agents
Ara-C AZT etc.
Florida Historical Perspective
O
• 1969 Siegel and co-workers at
the University of Miami
• Discover potent antitumor and
immunomodulatory properties
of the mangrove tunicate
Ecteinascidia turbinata
• Approved for use against soft
tissue sarcoma in Europe
2007
Siegel et. al. Food Drugs from the Sea 1969
Wright et. al. JOC 1990
Rinehart et. al. JOC 1990
O
HO
O
H HH
S
O
O
O
N
N
H
OH
O
O
NH
HO
Ecteinascidin 743
The first marine derived drug approved by the FDA is:
Prialt™
• 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
An Excellent View
A Variety of Manipulator Tools
Unique Work Platforms
How do we choose dive sites?
Multi-Beam Echo Sounding produces a 3-D image
of the seafloor with a width of about 4 times
water depth
600–800m
Resolution 20-50m
From: Kongsberg Simrad
Reconnaissance Mapping Tool : EM 1002
Depth range
585-750m
1 km
Slide Courtesy of Grasmueck et al. CSL U. Miami 2006
Highres Mapping Tool : AUV C-Surveyor II
AUV: Autonomous Underwater Vehicle
Free swimming- No tether- no cables
Pre-programmed to perform set tasks
Sensors
200 kHz Multibeam Echo Sounder
120 kHz Side Scan Sonar
2-8 kHz Sub-Bottom Profiler
Acoustic Doppler Current Profiler
Temperature, Salinity, Methane
40m
Resolution 1-3 m
Picture courtesy of Reson Inc. and Bluefin Robotics Inc.
Highres Mapping Tool : AUV C-Surveyor II
Depth range
585-750m
Grasmueck et al. CSL U. Miami 2006
1 km
Reconnaissance
Mapping Tool:
EM 1002
High resolution
Mapping Tool:
AUV C-Surveyor II
Grasmueck et al. CSL U. Miami 2006
Depth range
585-750m
40 m
Ground-truthing GBB5z
900 m
0.5 km
Correa et al., 2006 CSL Meeting
Collection Strategies
Biological Diversity = Chemical Diversity
Habitat Diversity = Chemical Diversity
Myrmekioderma metabolites are depth dependent
OH
H OH
O
O
< 33 m,
Anti-tumor Activity
H
O
33 m < X < 66 m
Anti-tumor Activity
> 66 m
Antiviral Activity (HSV-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
O
H H
H
N
O
O
H
H
H
OH
HO
H
HO
NH
H
H
HO
H H
H
NH
O
H
H
O
OH
H
N
O
O
Alteramide
Alteramide
Ikarugamycin
Discodermide
O
Ikarugamycin
O
O
OH
Discodermide
Alteromonas associated
with Halichondria
Terrestrial
Streptomycete
Discodermia dissoluta
H
Biosynthetic Gene clusters localized to
bacterial symbionts
O
O
N
S
N
H
Patellamide A produced by the tunicate symbiont
Prochloron didemni
N
O
HN
NH
O
N
S
N
H
N
Gene cluster expressed in E. coli
Schmidt et. al. PNAS 2005 102: 7315
O
O
O
N
H
Onnamide from sponge Theonella
swinhoeii
Gene cluster sequenced- associated
with bacterium
Piel et. Al. PNAS 2004, 101: 16222
COOH
HO
O
O
OH
O
H2N
O
H
N
O
O
NH
NH
O
The challenge remains to culture these microbes!
Step 2: Screening/Biological
Assays
Screening Approach
Forward Chemical
Genetics Approach
Reverse Chemical
Genetics Approach
CELL
Purified Protein
Treat with small molecule
Treat with small molecule
Detect desired Cell
Phenotype
Detect small molecules
which bind to target
Determine HOW
small molecule
causes phenotype
Define WHAT
EFFECTS the binding
has on the Phenotype
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 HBOITumor 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)
• P388 (murine leukemia)
Cartoon of a Cell-based
Reporter Assay
LIGHT
LIGHT
The Cytoblot Assay
Horse radish peroxidase
LIGHT
LIGHT
LIGHT
O3PO
Secondary Antibody
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, 6:71-83.
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
Enriched fraction
Enriched fraction
Enriched fraction
Enriched fraction
Pure Compound
Spectroscopy is
used to define the Structures
O
HO
HO
HO
O
Cl
O
O
O
O
Cl
N
O
HO
HO
OH
NH2
O
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
O
H
H
N
H
N
H
N
O
OH
H
H
H
O
O
O
HO
H
H
2
3
4
5
6
So, we find a potent drug
candidate…
It comes from a Deep Water
Sponge…
How do we get enough drug to
treat anybody?
(Without destroying the environment)
We have a number of projects focused on
Sustainable Use of Marine Resources
Chemical Synthesis
O
O
OH
OH
OH
OH O
O
NH2
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
Case Studies:
Aphrocallistin
Hexactinellida- “glass sponges”
The Sea Urchin Embryo Assay
1.5 hours post fertilization
Vehicle control
Treated with antimitotic agent
Embryos at 4-8 cell stage
Cell Division arrested
Rapid assay that detects compounds which block cell division
Hexactinellida- Glass sponges
Extraction with Ethanol
n-butanol Partition
C-18 Vacuum Column
Chromatography
Reverse phase MPLC with
Combiflash Companion
Crude extract
Active Compound
PANC-1 cells % Inhibition= 70% at 5
g/ml
M+H+ m/z observed 539.04279
Sea urchin embryo division 100%
Calculated for C20H25O2N6Br2 +2.2 mmu
Inhibition at 100 g/ml
1H
NMR Spectrum
Molecular Formula: C20H24Br2O2N6
Structure of Major Compound
N
H
H2N
H2N
N
N
H3C
Br
O
OH
O
N
Br
CH2
N
NH2
CH3
molokaiamine
Modest activity against cancer cell lines
PANC-1 IC50= 12 g/ml
NCI-ADR-Res IC50= 15 g/ml
CH3
Cell Cycle Analysis—Aphrocallistin
Panc-1 cells; 24 hour treatment
nontreated
methanol
12.5 μg/ml aphrocallistin
Aphrocallistin induces a G1 block in the cell cycle
Flow cytometry on PANC-1
pancreatic carcinoma cells:
Chk1
untreated
Methanol
control
Aphrocallistin
12.5 µg/ml
Example of Flow cytometry on
PANC-1 cells: Chk2
untreated
Methanol
control
Aphrocallistin
12.5 µg/ml
Clear increase in levels of P-Chk2 (thr68)
Aphrocallistin acts via ATM/ATR in
PANC-1 cells
Growth
factors
DNA damage
Stress
Chk1
P-Chk1
Chk2
P-Chk2
P-cyclinD
G1
G0
Cyclin D
CDK4/6
ATM/ATR
CDC25A
M
p16INK4
Chk1/2
p19
p53
p27
CDK7
Cyclin A/B
CDC2
p21
CDK2
Cyclin A/E
Abl
Rb
DP1
E2F
S
DP1
G2
E2F
Total Synthesis of Aphrocallistin
Burnham Institute for Medical Research
Aphrocallistin-Like Library Synthesis
Stepwise Process:
1. Assemble key Building block: 1
2. Install Linker: 4
3. Add Heterocycle: 4 x 4 = 16
4. Deprotect then Add Acyl Groups: 16 x 12 = 192
Example 2:
Leiodermatolide: A Potent Antimitotic
Agent
The cytoblot assay
24 hrs
1. Plate cells and
allow to adhere
48 hrs
2. Treat with HBOI
compounds and
known inhibitor
3. Fix, Permeabilize,
and block cells
ECL
LIGHT
4. Treat with targetspecific antibody
5. Treat with HRPconjugated secondary
antibody
Adapted from a protocol by Stockwell et al Chemistry and Biology 1999 6:71-83
The Phosphonucleolin Cytoblot
TAXOL
“ACTIVE”
EXTRACT
CYTOTOXIC
Nucleolin is
phosphorylated
prior to mitosis
Cells which are
blocked at mitosis
are detected using
an antibody to
phosphonucleolin
and a secondary
antibody tagged
with luciferase
Assay adapted from Stockwell et. al. Chemistry & Biology 1999, 6:71-83.
Leiodermatium sp.
• Extracts shows strong
response in PN
cytoblot assay
• Cytotoxic against
tumor cell lines:
– PANC-1
– A549
– NCI/ADR-Res
Purification
Extraction with Ethanol-Ethyl acetate
Solvent Partitioning Ethyl Acetate-Water
Silica gel Vacuum Column Chromatography
C-18 Reverse Phase HPLC
Pure Compound
Leiodermatolide
H3C H H
CH3
OH
CH3
H
H2N
CH3
O
O
O
H3C
H
HO
O
O
O
H
H3C
H
CH3
Cell Line
A549 (human lung)
IC50 nM
3.3 nM
PANC-1 (pancreatic)
DLD-1 (colon)
NCI-ADR-Res
P388 (murine leukemia)
5.0 nM
8.3 nM
233 nM
3.3 nM
Leiodermatolide blocks cell cycle
progression at the G2/M checkpoint
Methanol
10nM Leiodermatolide
100nM paclitaxel
1nM Leiodermatolide
A549 Stained for Tubulin and with
Propidium Iodide - Adherent
Nontreated
1nM Leiodermatolide
Methanol
10nM Leiodermatolide
100nM Taxol
100nM Leiodermatolide
Leiodermatolide 10 nM
Leiodermatolide does not
directly bind tubulin
Work is on-going to define its molecular target
Microbial Production?
H3C H H
CH3
OH
CH3
H
H2N
CH3
O
O
O
H3C
H
HO
O
O
O
H
H3C
H
CH3
Has O-carbamate
Has signature C-1 alkylation
Probably produced by Type I PKS
This gives us key genes to search for
•Ketosynthase
•O-carbamoyl transferase
•HMG-CO-A synthase
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
Many thanks to:
•
All my colleagues in the CMBBR
•
Mark Grasmuek and Gregor Eberli, RSMAS
•
Greg Roth, Daniela Divlianska, Jennifer Hoffman BIMR Lake Nona
•
Jill Roberts- Leiodermatolide; Diana Pechter –Aphrocallistin
•
Esther Guzmán, Tara Pitts, Pat Linley: Cell Biology
•
Our Funding Sources
– National Institutes of Health, NCI
– Center of Excellence in Biomedical and Marine Biotechnology
– State of Florida, FWRI
– National Science Foundation
– Florida Sea Grant College Program
– NOAA Office of Ocean Exploration
– Gertrude E. Skelly Charitable Foundation
– The Atlantic Foundation