Marine Natural Products Drug Discovery (DM)

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


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


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


Invertebrate Cell Culture


Aquaculture

Polymastia in Rope Culture


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


100nM Taxol


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