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VOL.
56
NO. 2, FEB.
2003
THE
JOURNAL
OF
ANTIBIOTICS
pp. 55-61
FR252921, a Novel Immunosuppressive
Isolated from Pseudomonas fluorescens
I.
Taxonomy,
and
Fermentation,
Biological
Activities
Isolation,
of FR252921,
Agent
No. 408813
Physico-chemical
FR252922
Properties
and
FR256523
KIYOTAKA FUJINE*, MIHO TANAKA, KEISUKE OHSUMI, MICHIZANE HASHIMOTO, SHIGEHIRO TAKASE,
HIROTSUGU UEDA, MOTOHIRO HINO and TAKASHI FUJII
Exploratory Research Laboratories, FujisawaPharmaceutical Co., Ltd.,
5-2-3 Tokodai, Tsukuba, Ibaraki 300-2698, Japan
(Received for publication October 18, 2002)
Novel immunosuppressive agents, FR252921, FR252922 and FR256523 were isolated
from the cultured broth of a bacterial strain No. 408813. The strain was identified Pseudomonas
fluorescens from morphological and physiological characteristics.
FR252921, FR252922 and FR256523, novel compounds containing macrolactone ring,
showed immunosuppressive activity against murine splenocyte proliferation stimulated with
lipopolysaccharide (LPS) or anti-CD3 mAb in vitro.
In recent years, many new immunosuppressive drugs
have been discovered and developed for clinical use in
organ transplantation1). Almost all of patients are treated
with calcineurin (CN) inhibitors based therapy, using
cyclosporine A (CsA) or FK506, as part of dual, triple, or
sequential therapy2). However, we have some problems to
be resolved, including chronic rejection and side effect. To
establish therapy with more efficacy and safety, we need
another novel immunosuppressant that has different target
from that of immunosuppressants used in clinical,
especially CN inhibitors.
FK506 and CsA act by interaction with their cognate
intracellular receptors, cyclophilin and FKBP, respectively.
The Cat+/calmodulin-regulated phosphatase CN is a major
target of drug-isomerase complexes. They prevent from
transport of NF-AT into nucleus, leading to inability to
rejection and suggested that APC will be important target
cell population for transplantation therapy7). However, CN
inhibitors are insufficient to suppress APC function. For
example, LPS-mediated activation of APC, such as B cell
and dendritic cell, is less sensitive to FK506 than T cell
activation8,9). In order to find a novel drug that inhibits APC
functions, we considered LPS-activated proliferation as
useful in vitro assay.
In the course of searching for immunosuppressants, from
soil microorganisms, FR252921 (1), FR252922 (2) and
FR256523 (3) (Fig. 1) were isolated from the cultured broth
of Pseudomonas fluorescens No. 408813. They inhibited
describes
the taxonomy,
produce IL-23,4). So, FK506 dramatically inhibit T cell
meditated immune response, such as mixed lymphocyte
reaction and splenocyte proliferation stimulated anti-CD3
mAb5,6). On the other hand, many reports mentioned
involvement of antigen presenting cell (APC) with graft
chemical
properties
* Corresponding author: kiyotaka_fujine@po.fujisawa.co.jp
proliferation of splenocyte stimulated with LPS and antiCD3 mAb in contrast to their little toxic effect on nonspecific
proliferation
compounds.
of
and
EL-4,
lymphoma.
fermentation,
biological
This
isolation,
activities
paper
physicoof
these
56
THE
Fig. 1. Structures
FR256523.
of FR252921,
JOURNAL
FR252922
OF
ANTIBIOTICS
FEB.
LPS or anti-CD3 mAb proliferate and produce various
cytokines12-15). So, immunosuppressive activity was
determined with those proliferation assay. Splenocytes was
and
prepared from female C57BL/6 mice (Charles River Japan
Inc.) and suspended in 0.1ml RPMI-1640 medium (Sigma,
St. Louis, MO) supplemented with 10% fetal bovine serum
FR252921 (1)
(Moregate, Bulimba, Australia), 50mM 2-mercaptoethanol
(Nakarai Chemical, Japan), 100units/ml penicillin and
0.1mg/ml streptomycin (Invitrogen, Rockville, MD).
Proliferation
assay
microtiter
FR252922 (2)
in
or
were
of
10μg
to the each
wells,
described
in
(Vol.
1)10)
Bacterially.
was
BERGEY'S
and
were
Manual
Manual
out
microscope
24
at 30℃.
hours
on
a
of
of strain
microscope
cells
grown
and
on
methods
a
1×105
anti‑mouse
was
37℃
in
was
CD3
added.
a
The
humidified
measured
by
agar
added
to each
blue
for 3 days
well
the
all
mixed
photometer
at 660nm.
96-well
well
containing
in 0.1ml
medium.
5% CO2. Proliferation
from
The plates
microplate
was tested
at 37℃
added
and
crystals.
wavelength
each
cells
was
of the medium
the dark
activity
with
EL-4
described
in RPMI‑1640
on a two-wavelength
plates,
incubated
408813
scanning
nutrient
at
removal
with a reference
lymphoma
Medical
No.
which
Proliferation
was
to dissolve
microtiter
Bacteriology
Identification
light
with
the
Systematic
observation
using
electron
of
for
Morphological
carried
based
U‑bottomed
containing
(10μg/ml)
days
dissolved
In vitro cytotoxic
studies
3
After
2-propanol
at 550nm
taxonomic
of MTT
were measured
Methods
Study
The
CO2.
well.
thoroughly
Taxonomic
to
(Sigma)
for
96‑well
well
colorimetric MTT (3-(4,5-dimethylthiazolyl-2-yl)-2,5diphenyltetrazolium bromide) assay described by
MOSMANN16). Briefly, 4 hours prior to termination of
culture,
and
LPS
5%
in
each
medium,
incubated
atmosphere
FR256523 (3)
performed
with
0.1ml
mAb(1μg/ml)
cells
was
plates
splenocytes
Materials
2003
1×103
The
in a humidified
was measured
U-bottomed
murine
cells
were
atmosphere
of
by MTT
assay
as
above.
for
Results
Media Used for Seed Culture and Production
The seed medium consisted of Nutrient Broth (Kyokuto
Seiyaku, Japan) 2%. The production medium was composed of glycerin 2.2%, modified starch 2.2%, Nutrient
Broth 3.3%, soybean meal 3.3%, CaCO3 0.2%, Antifoam
No. 8 (Kao Corporation, Japan) 0.125%.
Taxonomic
The bacterial
from
a soil sample
Morphological
carried
Analysis
Detection
broth
by
and
HPLC
of
the
using
at
compounds
fractions
1‑3
under
a reverse
from
purification
phase
column
(AM‑302,150×4.6mm
i.d., YMC
phase
acetonitrile.
was
ml/minute.
80%aqueous
The
detection
wavelength
Co.,
The
was
the
were
fermentation
out
by
The
flow
rate
and
mobile
was
shape
set at 210nm.
As
described
and Cytotoxic
previously,
stimulated
1
was
rod
to 32℃.
for catalase,
with
hydrolysis,
light
and
motile
entire-edged.
summarized
Activity
splenocytes
a
The
with
Fig.
in Nagano
prefecture,
No.
strain
2).
Strain
oxidase,
gelatin
electron
408813
was
a
of No. 408813
in color, circular
not formed.
The cell
of 0.8‑1.0×2.0‑3.0μm.
of strain
in Table 2. The growth
No.
No.
Colonies
were
an
was
agar for 24 hours
pale orange
spores
Japan.
408813
and
on nutrient
characteristics
Strain
isolated
of
bacterium.
a size
was originally
microscope
agar were smooth,
Physiological
1.O
3℃
Immunosuppressive
(Table
on nutrient
YMC‑ODS‑AM
collected
with cells grown
Gram-negative,
monitored
Ltd.).
30℃
No. 408813
observation
microscope
HPLC
strain
Studies
408813
Simmons
liquefaction,
gave
No. 408813
temperature
positive
citrate,
and arginine
was from
results
urease,
were
in test
tween
80
dihydrolase.
VOL.
56
THE
NO. 2
Table
1.
Morphological
characteristic
of
JOURNAL
OF
Table
strain
2.
Scale:
2.
Physiological
characteristics
of
strain
No. 408813.
No. 408813.
Fig.
57
ANTIBIOTICS
Electron
micrograph
of strain
No. 408813.
2μm.
Strain No. 408813 was O-F
test oxidative and pyoverdin
production positive. Nitrate reduction, esculin hydrolysis,
starch hydrolysis, ONPG test and Dnase were negative.
Acid formation was observed from D-glucose, D-xylose
and D-mannitol. The following compounds were utilized as
a sole carbon source: namely, D-glucose, D-mannose, Dxylose, D-mannitol, D-trehalose, glycerin, sucrose, maltose,
N-acetyl-D-glucosamine,
According
gluconate, caprate and malate.
to BERGEY'S Manual
of Systematic
Bacte-
riology (Vol. 1)11), strain No. 408813 was considered
belong to genus Pseudomonas
to
from those characteristics
described above. Thus, strain No. 408813 was compared
with Pseudomonas
species
result, it was found
resemble Pseudoinonas
described
that the strain
fluorescens.
in literature.
As a
proved
to closely
Therefore,
strain No.
408813 was identified as Pseudomonas fluorescens.
Fermentation
Aslant
into
a 225‑ml
medium.
shaker
a
culture
225‑ml
of
Erlenmeyer
After
incubation
(220rpm),
1.2ml
Erlenmeyer
the
producing
flask
at
of
flask
strain
containing
30℃
the
for
culture
containing
24
was
inoculated
60ml
ofthe
hours
on
was
60ml
rotary
the
same
inoculated
of
seed
a
into
58
THE
seed
medium,
rotary
and
shaker
resultant
days
(220rpm).
seed
fermentor.
incubated
Four
culture
The
under
at
was
of
for
hundreds
was
24
hours
on
milliliters
transferred
fermentation
aeration
30℃
JOURNAL
into
carried
a
out
20liters/minute
of
a
After
the
extracted
few
minutes
with
an
was
The
at 25℃
for
agitation
DAISO
500ml
in
Co.,
of
50ml
of
of
Ltd.,
The
extract
The
oily
layer)
reduced
methanol
Japan)
and
Then
was
was
filtered
was
washed
the
soon
with
residue
three
acetonitrile
The
passed
with acetonitrile,
filtered
and dried up
of 2 as a white powder.
was
times
layer
residue
was
through
Physico-chemical
Properties
As shown in Table 3, compounds 1, 2 and 3 were soluble
in chloroform and dimethyl sulfoxide, slightly soluble in
acetonitrile and methanol but insoluble in n-hexane and
water. They show positive color reactions to iodine vapor
and Dragendorf, though negative against Morish, Ahrlich,
FeCl3 and Ninhydrin. They showed UV absorption at 239
and 304nm. The ES-MS spectrum showed a molecular ion
peak at m/z 501, 529 and 527 (M+H+, 1, 2 and 3
respectively). The 1H and 13CNMR spectra of 1 are shown
in Fig. 3 and Fig. 4.
The determinations of the structures were accomplished
concentrated
SP‑120‑ODS‑B
packed
was
for a
filtrate
This
pressure.
and
broth
stirring
acetate(1:1)
residue.
acetonitrile
Daisogel
by
earth.
(upper
n‑hexane.
under
culture
n‑hexane/ethyl
to an
of
volume
of
extract
pressure
(1 liter)
the
of acetone
temperature.
36 liters
concentrated
completed,
volume
was washed
2003
3
of
and Purification
diatomaceous
solvent
in
equal
dissolved
column
of
reduced
dissolved
was
equal
at room
with
solution.
with
an
aid
extracted
under
culture
with
powder
FEB.
jar
200rpm.
Isolation
ANTIBIOTICS
to give 79mg
the
30‑liter
and
OF
primarily by a series of 2-D NMR techniques. Detailed of
the structure elucidation studies of 1, 2 and 3 will be
described elsewhere.
a
(15/30μm,
40%
aqueous
acetonitrile. The column was eluted with 65% aqueous
acetonitrile (6 liters, 1), 70% aqueous acetonitrile (4 liters,
3 and 2) and 75% aqueous acetonitrile (1.5 liters, 2). The
elution was monitored by analytical HPLC. Fractions
containing 1, 2 and 3 were combined respectively. The
portion corresponding to 1 was extracted with equal
volume of n-hexane/ethyl acetate (1:2) solution. The
solvent extract (upper layer) was concentrated under
reduced pressure to a powder. The powder was washed with
acetonitrile, filtered and dried up to give 175mg of 1 as a
white powder.
The portion corresponding to 3 was extracted with equal
volume of n-hexane/ethyl acetate (1:2) solution. The
solvent extract (upper layer) was concentrated under
reduced pressure to a powder. The powder was washed with
acetonitrile, filtered and dried up to give 74mg of 3 as a
white powder.
The portion corresponding to 2 was extracted with equal
volume of n-hexane/ethyl acetate (1:2) solution. The
solvent extract was concentrated under reduced pressure to
a powder. This powder was dissolved in 50ml of methanol
and soon passed through a column (1 liter) of Daisogel SP120-ODS-B packed with 40% aqueous acetonitrile. The
column was eluted with 75% aqueous acetonitrile.
Fractions containing 2 were combined. And then, 2 was
extracted with equal volume of n-hexane/ethyl acetate
(1:2) solution. The solvent extract (upper layer) was
concentrated under reduced pressure to a powder. The
In Vitro Immunosuppressive
and Cytotoxic
Activity
Activity
The in vitro immunosuppressive activities of 1, 2 and 3
were analyzed using lymphocyte growth inhibition assay. In
this study, the immunosuppressive effect was calculated by
reduction in lymphocyte proliferation quantified as strength
of staining by MTT. 1, 2 and 3 inhibited proliferation
stimulated with LPS, comparably insensitive to FK506 or
CsA. Moreover, 1, 2 and 3 also inhibited splenocyte
proliferation stimulated with anti-CD3 mAb with comparable IC50value to CN inhibitors, FK506 or CsA (Table 4).
Similar inhibitory effects of them on mixed lymphocyte
reaction were confirmed (data not shown).
The cytotoxic activities of 1, 2 and 3 against EL-4,
murine lymphoma cell line were analyzed. Their IC50were
641, 146 and 760ng/ml, respectively.
Discussion
Compounds 1, 2 and 3, which exhibited immunosuppressive activities, were isolated from the culture broth of
Pseudomonas fluorescens No. 408813. From the evidence
of physico-chemical data, these compounds were to have
novel structures (Fig. 1). Compounds that have 19-members
macrolactone ring containing two amide bonds are rarely
found in nature. Thus, our screening system with LPSactivation assay and cytotoxic assay is useful to find novel
immunosuppressant different from CN inhibitor.
VOL.
56
NO. 2
THE
Table
3.
JOURNAL
Physico-chemical
OF
ANTIBIOTICS
properties
of compound 1-3.
a Plate: Silica gel 60 F254(E. Merck Co.), CHCl3:CH3OH=10:1.
b Plate: RP-18 WF254(E. Merck Co.), 80% aq. Acetonitrile.
Fig.
3.
400MHz 1H
NMR spectrum
of 1 in CD2Cl2.
59
THE
60
Fig.
4.
JOURNAL
100MHz
OF
FEB.
ANTIBIOTICS
13C NMR spectrum
2003
of 1 in CD2Cl2.
Table 4. IC50values of 1, 2, 3, FK506 and CsA against murine splenic proliferation and cytotoxic activity
in vitro (ng/ml).
Compounds
proliferation
to their
EL-4.
1,
and
stimulated
little toxic
proliferation
little effect
effect
activation.
the
on non-specific
FK506
showed
suggest
from a target of FK506
on
inhibited
lymphocyte
The details
in contrast
proliferation
of
and CsA, T cell specific
strong inhibition
of
these
papers17,18).
of in vivo evaluation
compounds
The
the
are
structural
and the mode of action
described
in
the
following
analysis
will
be
described
elsewhere.
of splenocyte
stimulated with anti-CD3 mAb, in contrast to
on proliferation
stimulated
with LPS. These
strongly
studies
3
with LPS and anti-CD3
On the other hand,
immunosuppressant,
results
2
Acknowledgment
that target of 1, 2 and 3 is different
and CsA, and may facilitate
signaling
pathway
of
further
LPS-mediated
We
wish
spectrometric
to
thank
FUMIE ABE and Ms.
preparation
Mr.
measurements,
MASAAKI
and Ms.
KATSUOKA
SATOKO FURUKAWA for assistance
of this manuscript.
for
mass
MICHIKO USHIODA, Ms.
in the
VOL.
56
NO. 2
THE
JOURNAL
OF
ANTIBIOTICS
61
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