Gregory Petrossian1,
Dahai Zhang2 Ph.D.,
Neal Goebel2 M.S.,
and Mark Zabriskie2* Ph.D.
Department of Bioresource Research
2 Department of Pharmaceutical Sciences
Oregon State University
1
 Drug
discovery:
• Synthetic library vs. Natural Products
 Natural
Products evolved specifically
for biology
• Growing number of resistant strains
 Unique
environments
• Diverse isolates
• Untapped sources
1)
Screening of actinomycete strains from
Indonesia for Staphylococcus aureus
inhibition
2)
Purification of enduracidin from
Enradin®
3)
Enradin® fraction ; bioactive compound
isolation/purification
 Order
Actinomycetals: Gram (+), aerobic,
chemoorganotrophic
• Fungus-like characteristics: metabolic diversity
and mycelia producing
 Actinobacteria
natural products
responsible for multitudes of antibiotics
 “Cosmopolitan” genus Streptomyces
• Antibacterials, antifungals, anticancer,
antiparisitic, statin drugs, immunosuppressents
 Rare
non-Streptomycete actinomycetes
 Exclusive
Black Water ecosystem in southern
Indonesia
•
•
•
•
Dissolved organic matter
Horizontal ground water flow
Acidic
Tea/cola appearance
 Indonesian
Center for Biodiversity and
Biotechnology Culture Collection of
Microorganisms (ICB-CC)
 Transfer
simple media culture of strain to
complex medium
 Six
unique complex mediums
 Promote secondary metabolite
production
 Ferment, harvest, 96-well
plate bioassay
8 mL complex
Media + strain
6-well plate
 Improvised
method by Smith et. al. (2006)
 Employed
high concentration (>50 mg)
and low concentration (<5 mg) assay
 Bioactivity
 Positive
= growth of <50% S.aureus
control: kanamycin serial dilution
 Negative control: S.aureus and media
 Blank: media (BHI broth)
A
B
C
D
1
2
3
256
ug/mL
-15%
1C - 8340
128
ug/mL
-15%
1C
64
ug/ml
-15%
1C
30 mg/mL
31%
1C
45%
53%
1C
1C
3 mg/mL
90%
5C – 8340
96%
94%
5C
2 mg/mL
126%
98%
7
8
9
32 ug/mL 16 ug/mL 8 ug/mL
-10%
-16%
-15%
4 ug/mL
24%
2 ug/mL
41%
1 ug/mL
92%
2C - 8340
2C
2C
3C – 8340
3C
3C
1.1 mg/mL
102%
90%
110%
.7 mg/mL
101%
109%
112%
5C
6C – 8340
6C
6C
1C – 8352
1C
98%
5 mg/mL
89%
6C
80%
74%
6C
6C
5 mg/mL
92%
1C
.5 mg/mL
90%
4C – 8352
33%
95%
4C
4C
5 mg/mL
109%
102%
82%
E
F
3C – 8352
3C
3C
1.8 mg/mL
122%
125%
127%
4
5
6
10
11
12
 (+)
Control
4C – 8340
4C
4C
.7 mg/mL
109%
132%
137%
1C
2C – 8352
2C
2C
111%
92%
1.1 mg/mL
103%
103%
139%
1C
1C
.5 mg/mL
98%
5C – 8352
97%
90%
5C
5C
6C – 8352
6C
6C
1.5 mg/mL
110%
104%
103%
50 mg/mL
16%
6C
15%
15%
6C
6C
5 mg/mL
81%
Blank
0.15%
99%
105%
Blank
0.15%
Blank
0.15%
G
H
(-)
Control
(-)
Control
(-)
Control
Centrifuged (20 min 3000 rpm)
250 mL
Culture
broth
8 mL complex
Media + strain
6-well plate
mycelia
ICBB8340 in M2 media
• Pastel pink culture broth
No
bioactivity at any concentration
Strains
8340 and 8352 given to Dr.
Dahai Zhang
Verification of bioactivity by disk
diffusion bioassay
No bioactivity (<10 mg/mL)
 Emphasis
of research put on Enradin®
fraction
 Not
effective at low concentrations thus
not a powerful agent against S.aureus
 Dr. Zhang
carried out screening of
remaining rare non-Streptomyces
actinomycete isolates
 Enradin®
 Dried
biomass from
Streptomyces fungicidicus
 5-8% enduracidin
 Poultry feed additive
O
L-
 Enduracidin
– peptide antibiotic
 No known mechanism of
resistance
 Gram (+), MRSA effective
NH
HO2C
L-Asp1
L-Hpg17
O
NH2
D-Hpg3
O
NH
NH
HO
D-allo-Thr5
O
H
N
O
D-Orn4
O
L-Thr2
O
H
N
N
H
N
H
O
OH
O
O
D-Ala16
O
O
HN
HN
NH
HN
L-End15
H2N
OH
HN
O
N
H
N
H
OH
O
H
N
O
Gly14
H
N
N
H
D-Ser12
Cl
Cl
OH
OH
L-Dpg13
L-Hpg11
Enduracidin A
Increasing Polarity
Decreasing Polarity
 THF
fraction loaded on silica gel column
 eluted with 1% MeOH in DCM (250
mL) and 5% MeOH in DCM (500 mL).
 Utilized Thin Layer Chromatography to
pool fractions
 Indicator dye: Thiazolyl Blue Tetrazolium
 High
Pressure Liquid Chromatography
1200
9: 210 nm , 4 nm
3: 254 nm , 4 nm
8: 278 nm , 4 nm
6: 310 nm , 4 nm
1000
mAU
800
600
400
200
0
30
32
34
36
38
Minutes
40
42
44
A
B
Smith
C
1
2
3
4
5
6
7
8
9
10
256
128
64
32
16
8
4
2
1
ug/mL ug/mL ug/mL ug/mL ug/mL ug/mL ug/mL ug/mL ug/mL ← (+)
-15% -15% -15% -10% -16% -15% 24% 41% 92% Control
11
12
0
1
2
3
4
5
6
7
8
9
10
11
23
22
21
20
19
18
17
16
15
14
13
12
34
35
et al. methods (2006)
D
24
Positive
control: kanamycin serial
25
26
27
28
29
30
31
32
33
dilution
Negative control: S.aureus and media
Blank: media (BHI broth)
E
47
46
45
44
43
42
41
40
39
38
37
36
F
48
49
50
51
52
53
54
55
56
57
58
59
G
71
70
69
68
67
66
65
64
63
62
61
60
Turbidity
H
72
measured at t =0 and t =17 h
(-)
(-)
(-)
Blank Blank Blank
Control Control Control 0.15% 0.15% 0.15%
 Smith
et al. methods (2006)
 Positive
control: kanamycin serial dilution
 Negative control: S.aureus and media
 Blank: media (BHI broth)
 Turbidity
measured at t =0 and t =17 h
1200
9: 210 nm , 4 nm
3: 254 nm , 4 nm
8: 278 nm , 4 nm
D
6: 310 nm , 4 nm
E
1000
800
mAU
C
600
400
A
F
B
200
H
G
0
30
32
34
36
38
Minutes
40
42
44
 Individual
compounds (A-H) given to
Neal Goebel for structure determination
 Compounds
D & E: most interesting
based on UV-absorption
Compound E – Unsaturated Fatty Acid
 Enduracidin
purification maximized
(yield = 16.1 mg) for biosynthetic
modification by Neal Goebel
• Improve solubility and maintain bioactivity
 Bioactive THF
Fraction from Enradin®
contains unsaturated fatty acid
analogues
• Common nuisance, inhibit bacterial growth but
do not make ideal antibiotics
Natural
Products are essential
to battle over drug resistance
New
fermentation mediums
New
high-throughput methods
• Cultivation and screening separated
Subsurface Biosphere Initiative (SBI)
summer undergraduate internship
Intervet/Schering Plough Animal Health
Dr. Mark Zabriskie Lab
Bioresource Research Program
OREGON STATE UNIVERSITY