Download Anatoxin-a Irreversibly Binds Acetylcholine Receptors and Inhibits

Bioactive Compounds from Marine
Microbes
Bioactive Compounds from Algae (Part 2)
Cyanobacteria (“Blue-Green Algae”)
Bioactive Compounds from (Other) Marine Bacteria
Bioactive Compounds from Marine Fungi
Algae
Monera
(“Prokaryotes”)
Protista
Plantae
Fungi
Animalia
Algae
Prokaryotic
Kingdom Monera (Bacteria)
Cyanobacteria (“Blue-Green Algae”)
Eukaryotic
Kingdom Protista
“Microalgae”
Dinophyta (“Dinoflagellates”)
Raphidophyta (“Raphidophytes”)
Bacillariophyta (“Diatoms”)
Chrysophyta (“Golden Algae”)
Chlorophyta (“Green Algae”)
Phaeophyta (“Brown Algae”)
“Macroalgae”
Rhodophyta (“Red Algae”)
“Harmful Algal Blooms” (HABs)
Eukaryotic HAB Toxins
CH3
H
COOH
Domoic Acid
CH 2COOH
H 3C
COOH
Syndrome
“Amnesic Shellfish
Poisoning” (ASP)
Source Species
Pseudonitzschia spp.
Target
Glutamate
Receptors
“Paralytic Shellfish
Poisoning” (ASP)
Alexandrium tamarense
Inhibits
Sodium
Channels
N
H
R4
H
H
N
R1
Saxitoxin
N
NH2+
N
H OH
+H 2N
OH
HO
R2
R3
Brevetoxin
O
O
OHC
CH3
CH3
CH2
CH3
O
O
CH3
O
O
O
“Neurotoxic Shellfish Karenia brevis
Poisoning” (ASP)
(and others)
“Florida Red Tide”
Activates
Sodium
Channels
“Ciguatera Fish
Poisoning” (CFP)
Activates
Sodium
Channels
O
O
CH3
O
O
O
CH3
H3C
Ciguatoxin
(and others)
CH3
H
H
HO
H
H
O
H
H
H
H
H
OH
O
H
H
H
H
O
O
O
O
O
H
H
H
H 3C
O
O
CH3
H
9
Gambierdiscus toxicus
OH
O
H
O
O
H
H
H
H
OH
Pectenotoxin
(and others)
HO
OH
O
“Diarrhetic Shellfish Dinophysis spp.
Poisoning” (CFP)
O
CH3
H 3C
O
O
O
O
O
OH
OH
H3 C
OH
H3 C
O
OH
O
O
O
CH 3
CH3
?
Polyketides from Dinoflagellates
“Polyether Ladders”
HO
O
O
e.g. PbTx-1
OHC
CH3
CH3
CH2
CH3
O
O
CH3
O
O
O
O
O
CH3
O
O
O
CH3
Macrolides
Linear Polyethers
O
CH3
O
CH3
H 3C
O
e.g. PTX-1
OH
O
H
O
O
O
O
O
O
OH
OH
H3 C
OH
H3 C
R1
R4
H3C
O
OH
OH
O
OR3
O
O
CH3
O
CH 3
CH3
H
OH
O
O
O
H
O
e.g. Okadaic Acid
CH3
R2
Polyketide Synthases (PKSs) are
Modular Enzymes
H
Sc
E
NADPH
b-ketoacyl
synthase (KS)
O
Sc
Sc
R
E
H
COOH
O
CO2
NADP+
b-ketoacyl
reductase (KR)
Sc
H
Dehydrase
(DH)
R
Sa
Sc
R
Sa
R
Sa
H2O
O
O
O
OH
O
H
E
E
E
Sa
Enoyl
reductase (ER)
R
Sa
O
Polyketide Synthase (PKS)
Acyl
Carrier
Protein Ketosynthase
(KS)
(ACP)
Acyl
Transferase
(AT)
S
CoAS
CoAS
O
R1
O
2
AT
S
O
R1
R2
S
O
R1
O
ACP
S
O
R1
O
HO
PKSI
Primers
S
O
R
R1
R2
KS
AT
S
O
Enoyl
Dehydratase
Ketoreductase
Reductase
(DH)
(KR)
(ER)
O
R1
OH
R2
Organism
Prorocentrum
lima
P. hoffmanianum
Karenia brevis
Symbiodinium sp.
Amphidinium
operculatum
R2
R2
PKSI
PKSII
+
+
+
+
+
-
+
-
Snyder et al. (2003) Mar. Biotechnol., 5 (1):1-12.
Prokaryotic Algae
Monera
(“Prokaryotes”)
Cyanobacteria
(“Blue-Green Algae”)
Protista
Plantae
Fungi
Animalia
Cyanobacteria (“Blue-Green Algae”)
Photosynthetic Bacteria
Oldest Organisms on the Earth
(Fossil Record - 3.5 Billions Years!)
Marine, Freshwater and Terrestrial
Stromatolites
Symbiosis - e.g. Lichen
Pigments and Algae
PBs4
Chl a1
Cyanobacteria
x
Dinoflagellata
Bacillariophyta
Chrysophyta
Chlorophyta
Phaeophyta
Rhodophyta
x
x
x
x
x
x
1Chl
b
c
d
Car. 2
Xanth.3 PC5
x
x
x
x
x
x
x
x
x
x
x
x
x
PE6
x
x
x
x
x
= Chlorophyll; 2Car. = Carotenoids; 3Xanth. = Xanthophylls; 4PB =
Phycobilins (Phycobiloproteins); 5PC = Phyocyanin; 6PE =
Phycoerythrin
x
Cyanobacteria as HABs
Cyanobacteria as HABs
First Scientific Report of Toxic Cyanobacteria:
George Francis (1878) Nature
Lake Alexandria, Murray River, Australia
“thick scum like green oil paint, some two to six inches thick,
and as thick and pasty as porridge”
“Unwholesome” for cattle and other livestock that drink at the water
Nodularia spumigens
Cases of Acute Poisoning by Toxic Cyanobacteria in
Drinking Water
1878
L. Alexandria, Australia
Livestock Poisoning
Nodularia spumigens
1931
Charleston, WV
(Ohio River)
Acute Gastroenteritis
Unknown
(9,000 cases/60,000 pop.)
1966
Harare, Zimbabwe
Gastroenteritis (children)
Microcystis aeruginosa
1975
Sewickley, PA
Acute Gastroenteritis
(62% of 8,000 pop.)
Schizothrix calcicola
1983
Armidale, Australia
Liver damage (elevated
g-glutamyltransferase)
M. aeruginosa
1983
Palm Island, Australia
Hepatoenteritis
(139 children)
C. raciborskii
(cylindrospermopsin)
1993
Itaparica Dam, Brazil
Gastroenteritis
(88 deaths, children)
Anabaena, Microcystis
1996
Caruaru, Brazil
Liver failure
(63 deaths)
Aphan., Oscillatoria
(microcystins)
Toxins from Cyanobacterial HABs
Neurotoxins
Hepatotoxins
Dermatotoxins
Neurotoxins from Cyanobacteria:
Anatoxin-a
1950s-1960s Paul Gorham and Colleagues
Cultured Anabaena flos-aquae
Isolated “Very Fast Death Factor”
HN
H3C
O
Anatoxin-a
O
CH3
CH3
H3C
O
N
H
CH3
Acetylcholine (in vesicles)
Synapse
Anatoxin-a Irreversibly Binds
Acetylcholine Receptors and Inhibits
Acetylcholinesterases
Acetylcholine
Receptor
(AChR)
Anatoxin-a Irreversibly Binds
Acetylcholine Receptors and Inhibits
Acetylcholinesterases
+ + + + + + + +
- - - - - - ++ -K+
Na+
Ca2+
Anatoxin-a Irreversibly Binds
Acetylcholine Receptors and Inhibits
Acetylcholinesterases
+ + + + + + + +
- - - - - - ++ -K+
Na+
Ca2+
Anatoxin-a Irreversibly Binds
Acetylcholine Receptors and Inhibits
Acetylcholinesterases
Ca2+
Na+
+ + + + + + + +
- - - - - - ++ --
SR
Ca2+
K+
PKC
Na+
Ca2+
Anatoxin-a Irreversibly Binds
Acetylcholine Receptors and Inhibits
Acetylcholinesterases
Anatoxin-a Irreversibly Binds
Acetylcholine Receptors and Inhibits
Acetylcholinesterases
Ca2+
Na+
+ + + + + + + +
- - - - - - ++ --
SR
Ca2+
K+
PKC
Na+
Ca2+
Anatoxin-a Irreversibly Binds
Acetylcholine Receptors and Inhibits
Acetylcholinesterases
Na+
+ + + + + + + +
- - - - - - ++ -Acetylcholinesterase
K+
Na+
Ca2+
Anatoxin-a Irreversibly Binds
Acetylcholine Receptors and Inhibits
Acetylcholinesterases
Anatoxin-a
Ca2+
Na+
+ + + + + + + +
- - - - - - ++ --
SR
Ca2+
K+
PKC
Na+
Ca2+
Anatoxin-a Irreversibly Binds
Acetylcholine Receptors and Inhibits
Acetylcholinesterases
Neurotoxins from Cyanobacteria:
Anatoxin-a(s)
CH3
N
CH3
HN
N
O
O
P
NH
-O
O
Anatoxin-a(s)
CH3
Anatoxin-a(s) Inhibits
Cholinesterases
LD50 = 20 µg/kg (in mice)
vs.
LD50 of Anatoxin-a = 200 µg/kg (in mice)
Neurotoxins from Cyanobacteria:
Saxitoxin
R4
O
NH 2
O
NHSO3
R4:
H
O
H
N
R1
N
NH2+
N
H OH
+H 2N
OH
R2
R3
Saxitoxin (STX) and
Other “PSP Toxins”
R1
H
H
H
OH
OH
OH
R2
H
H
OSO3H
H
OSO3-
R3
H
OSO3H
H
OSO3H
STX
GTX2
GTX3
NeoSTX
GTX1
GTX4
O
GTX5
C1
C2
GTX6
C3
C4
STX Binds and Blocks Voltage-Gated
Sodium Channels
= STX (or TTX)
Hepatotoxins from Cyanobacteria:
Microcystins
CO2H
CH3
O
N
HN
NH
H3C
O
OCH 3
CH2
H3C
O
O
NH
CH3
CH3
H
N
CH3
HN
H
N
CH 3
O
O
e.g. Microcystin-LR
HN
HN
NH2
CO2 H
CH 3
O
Microcystin Structural Diversity
COOR4
R3
O
N
HN
NH
H3C
O
OR1
CH2
O
H3C
R2
O
X
N
H
R1, R2 , R3 and R4 = -CH3 or -H
X and Z = variable amino acid
CH3
NH
Z
CH3
O
COOH
Over 70 Variants of MIcrocystins!
Microcystin Diversity
CO2H
CH3
O
N
HN
NH
H3C
O
OCH 3
CH2
H3C
O
O
NH
CH3
CH3
H
N
CH3
HN
H
N
CH 3
O
O
Microcystin-LR
HN
HN
NH2
CO2 H
CH 3
O
Hepatotoxins from Cyanobacteria:
Nodularin
O
H
COOH
H3C
H
CH3
CH3
OCH3
O
NH
H3C
H
O
H
CH3
H
N
CH3
NH
O
H
Nodularin
NH
HN
NH2
O
COOH
Microcystins (and Nodularin)
Accumulate in Liver
Approximately 50% of Microcystin in Liver
ATP-Dependent Carrier-Mediated Transport
Microcystins and Primary Liver Cancer
(PLC)
Nandong District, Jiangsu Province, China
Pond/ditch vs. Well as Drinking Water Supply
~ 24-Fold Higher Rate of PLC with Pond/Ditch Water
100.13 cases per 100,000 (pond/ditch)
4.28 cases per 100,000 (well)
Microcystin Levels:
Pond/Ditch: 60% samples positive, avg. 160 pg/mL
Well: None detected
Calculated 0.19 pg/day Seasonal Intake (4 mo./yr.)
Yu, 1989, Primary Live Cancer, pp. 30-37; Harada et al., 1996, China Nat. Toxins, 4: 27783; Ueno et al., 1996, Carcinogenesis 17: 1317-21
Microcystins (and Nodularins) are
PP1/2a Ser/Thr Protein
Phosphatase Inhibitors
MacKintosh et al. (1990) FEBS Lett. 264: 187-92.
Non-Ribosomal Peptides are
Characteristic of Cyanobacterial
Toxins
Methyl-dehydro-Ala
(Mdha)
Iso-D-Glu
CO2H
CH3
O
N
Adda
HN
NH
H3C
O
OCH 3
D-Ala
CH2
H3C
O
O
NH
CH3
CH3
H
N
CH3
HN
CH 3
H
N
CH 3
O
O
Arg
Iso-MeAsp
HN
HN
CO2 H
NH2
O
Leu
Non-Ribosomal Peptide
Synthetases (NRPSs) are Modular
Enzymes
A = Adenylation Domain
C = Condensation Domain
T (PCP) = Thiolation
(Peptidyl Carrier) Domain
Mixed NRPS/PKS Pathway for
Synthesis of Microcystins
Dermatotoxins from Cyanobacteria:
Lyngbya Toxins
Lynbyatoxin
Aplysiotoxin
Dermatotoxins from Cyanobacteria:
Lyngbya Toxins
Drugs from Cyanobacteria:
Curacin-A
OCH3
N
Curacin A
S
Curacin A Binds Colchicine Site of
Tubulin and Inhibits TubulinPolymerization
Drugs from NON-MARINE
Cyanobacteria: Cryptophycin
O
O
O
O
HN
Cl
O
O
N
H
O
Cryptophycin 52
(LY355703)
OCH3
Cryptophycin Inhibits Tubulin
Polymerization
Cryptophycin 52 in Clinical Trials
(Phase II)
Clinical Response of Women with Ovarian
Cancer Treated with LY355703 (n=24)
Response
Complete Remission
Partial Remission
Stable Disease
Progressive Disease
No.
0
3
7
14
%
0
12.5
29.2
58.3
D’Agostino et al. (2006) Intl. J. Gyn. Cancer 16: 71-76.
Drugs from Cyanobacteria:
Spirulina?
Bioactive Compounds from
Heterotrophic Marine Bacteria
Monera
(“Prokaryotes”)
Protista
Plantae
Fungi
Animalia
Bacteria: Kingdom Monera
Shape
Cocci = Sphere-Shaped
Bacilli = Rod-Shaped
Spirilla = Spiral-Shaped
“Growth Form”
Staph = Bacteria in Clusters
Strep = Bacteria in Chains
Classification of the Bacteria
(i.e. Kingdom Monera)
Gram-Negative
Gram-Positive
Classification of the Bacteria
(i.e. Kingdom Monera)
Eubacteria
Gram-Positive Bacteria w/ Cell-Walls
Gram-Negative Bacteria w/ Cell-Walls
Bacteria w/o Cell-Walls (Mycoplasma)
Archaebacteria
Cyanobacteria are Eubacteria
Gram-Stained
Cyanobacteria NEGATIVE
Lipopolysaccharides (LPSs) from
Cell-Wall of Gram-Negative
Bacteria are “Endotoxin”
“O-Region”: Repeating tri-,
tetra- or pentasaccharides
(up to 25)
Outside Cell
Polysaccharide “Core”
Cell Wall
HO
OH
O
CH2
P
O
Lipid A
Inside Cell
O
O
CH2
OH
O
O
O
NH
C
O
C
OH
HO
CH2
O
NH
O
OH
CH2
CH
C
O
O
C
O
CH
(CH2) 10
O
O
C
CH3 O
(CH2) 10
CH2
CH2
(CH2) 10
C
HC
OH
HC
CH3
(CH2) 10
CH3
CH3
(CH2) 10
CH3
P
(CH2) 10
CH3
OH
O
Actinomycetes: A Rich Source of
Bioactive Compounds
Approximately Half of Bioactive Metabolites!!
Approximately 60% of Antibiotics
Streptomyces spp.
Actinomycetes: A Rich Source of
Bioactive Compounds
Antibiotics from Streptomyces
Antibiotic
Amphotericin B
Erythromycin
Neomycin
Streptomycin
Tetracycline
Vancomycin
Rifamycin
Source
Streptomyces nodosus
S. erythreus
S. fradiae
S. griseus
S. rimosus
S. orientalis
S. mediterranei
Marine Actinomycetes
H
OH
O
Salinospora spp.
HN
O
O
CH3
Cl
Salinosporamide A
(NPI-0052)
Feling et al. (2003) Agnew Chem Int Ed Engl, 42:355-7
Are Salinospora “True” Marine
Bacteria?
Jensen et al. (1991) Appl. Environ. Microbiol., 57: 1102-8.
Salinosporamide A Inhibits the
Proteasome
H
OH
OH
O
O
HN
HN
O
O
O
O
CH3
CH3
H 3C
Cl
Salinsporamide A
Omuralide
Macherla et al. (2005) J. Med. Chem. 48: 3684-7.
Salinosporamide A Inhibits the
Proteasome
Macherla et al. (2005) J. Med. Chem. 48: 3684-7.
The Proteasome Degrades ShortLived and Abnormal Proteins
19S “Caps”
(Regulatory)
26S Proteasome
20S Proteasome
(Proteolytic)
Proteins Marked for Degradation
by Ubiquitinylation
-Helix
b-Strands
Ubiquitin (Ubq)
76 Amino Acids (Mol. Mass 8500)
Two C-Terminal Gly -> Bind to Lys of Proteins
Gly Bind Lys of Other Ubq = Polyubiquitinylation
The Ubiquitin-Proteasome
Pathway
Salinosporamide A Inhibits NF-kB
Activation
Macherla et al. (2005) J. Med. Chem. 48: 3684-7.
Proteasome Degrades IkB and
Activates NF-kB
Bioactive Compounds from
Salinospora and Marine
Actinomycetes
Taken from Jensen et al. (2005)
Bioactive Compounds from Marine
Fungi
Fungi: The First Source of
Antibiotics
1928 - Alexander Fleming Discovers
Inhibition of Bacteria by Penicillum notatum
Penicillin G
Fleming (1929) Br. J. Exp. Pathol. 10: 226
1939 - Florey and Chain Cure Mice of
Bacterial Infection with Penicillin Injection
…by the end of World War II, U.S. companies
were making 650 billion units/month
Antibacterial Compounds from
Marine Fungi: Pestalone
Inhibit methicillin- and
vancomycin-resistant
Enterococcus faecium
(MIC = 37 and 78 ng/mL,
respectively)
OH
O
Cl
HO
O
H3 CO
OH
H
Pestalone
(from Pestalotia sp.)
CH3
Cl
Antiviral Compounds from Marine
Fungi: Halovirs
Anti-HSV Activity
C6
C10 C14 C16
C18
Antimalarial Compounds from
Marine Fungi: Aigialomycin D
HO
O
O
Aigialus parvus
(isolated from mangrove)
Aigialomycin D
CH 3
Antimalarial Compounds from
Marine Fungi: Aigialomycin D
Compound
Aigialomycin A
Aigialomycin B
Aigialomycin C
Aigialomycin D
Aigialomycin E
P. falciparum K1
(IC50, µg/mL)
>20
>20
>20
6
>20
Antimalarial Compounds from Marine
Fungi: Ascosalipyrrolidinone A
H3C
H 3CO
H
N
O
O
CH3
H
Ascochyta salicorniae
(symbiont with green alga, Ulva)
H
H
H3C
H
Ascosalipyrrolidinone A
Culture of Bacteria
(and other microbes)
“Non-Culturability” of Bacteria
Approximately 99% of Heterotrophic Bacteria
are Non-Culturable
Required Nutrients, Conditions and Other
Factors Unknown
Metagenomics – NRPS, PKS
Metagenomic Analysis of Bacteria
from Discodermia dissoluta
PKS
NRPS
Filamentous
Unicellular
Shirmer et al. (2005) Appl. Environ. Microbiol., 71:4840-9.