Vol. 80, No. 2, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
January 30, 1978
ANTIBIOTICS
Pages
AND PEPTIDES WITH AGONIST AND ANTAGONIST CHEMOTACTIC ACTIVITY
S. Aswanikumar, E. Schiffmann and B. A. Corcoran Connective Tissue Section, Laboratory of Developmental and Anomalies, National Institute of Dental Research C. B. Pert, Section on Biochemistry, National Institute of Mental Health
Adult
Biology
Psychiatry
Branch
J. L. Morel1 and E. Gross Section on Molecular Structure, Reproduction Research Branch National Institute of Child Health and Human Development National Bethesda,
Institutes Maryland
Received
of Health 20014
December
2,1977 ABSTRACT
It has been found that the polypeptide antibiotics gramicidin S, tyrocidin and bacitracin,containing Leu-Phe or Ile-Phe sequences,are chemoattractants for neutrophils. Related synthetic pentapeptides containing the sequence Leu-Phe have stronger biological activities, provided the N-terminal residue is acylated. The formylat -5 d peptide fL-Phe-D-Leu-L-Phe-D-Leu-L-Phe is a potent agonist (4 x 10 -7M) whereas the t-butyloxycarbonyl analog is a good antagonist (8 x 10 M). INTRODUCTION N-formylation residue with
with
an aromatic
chemotactic
structure
Methionine
that
there
potency
the sequence hydrophobic
leukocytes
L-Met-X-(X
side
(1).
resulted (10 -"M).
is not
may be replaced
activity
for
relationships
of high
was found
with
or other
activity
activity
attractants
chains)
A detailed
requirement
study
by norleucine
with
(2)
little
loss
of
of chemo-
investigations for
acid
gave products
in the synthesis In subsequent
an absolute
= amino
it
methionine
(3).
in chemotactic
(3). Since
found peptides whether
of peptides
partially
characterized
to be leukoattractants contain fully
a defined
Leu-Phe
(4)
peptides
and the synthetic
sequence
bacterial
(2,3),
products
~00~6-291x/78fU802-0464$01.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproductioti in any form reserved.
of bacterial
464
it
potent
origin
chemotactic
was of interest
such as gramicidin
were
to determine S, tyrocidin
464-471
Vol. 80, No. 2, 1978
and bacitracin
BIOCHEMICAL
which
also
have the sequence
attractants.
The related
(Illustration
2, R = H) and its
butyloxycarbonylated were
also
-a
for
their
here
the
leukoattractants
demonstrated
Leu-Phe
or
Ile-Phe
were
L-Phe-D-Leu-L-Phe-D-Leu-L-Phe-OH N-formylated, (2;
N-acetylated,
R = HCO -,
chemotactic
H3C CO-,
and N-tert(CH3)3C 0 CO-)
potential.
R-L-Phe-D-Leu-L-Phe-D-Leu-L-PheOH
We report indeed
peptide
derivatives
examined
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
for
synthetic
finding
that
and interact peptides MATERIALS
certain with
polypeptide the same cell
antibiotics receptor
are previously
(5). AND METHODS
Fo~yl-L-norleucyl-L-leucyl-L-p-[3H]-phenylalanine from formyl-L-norleucyl-L-leucyl-L-p-chloro-phenylalanine catalytic replacement technique (3).
was prepared employing
a
Gramicidin S, TPCK* and TLCK** were purchased from Calbiochem, bacitracin from Sigma, tyrocidin from Serva Chemicals, New York, New York. t-Boc-L-phenylalanyl-D-leucyl-L-phenylalanyl-D-leucyl-L-phenylanine a, R = (H3C)3C-OCO-] was synthesized by an active ester method beginning with Boc-L-phenylalanine (I). The N-hydroxysuccinimide ester was prepared by allowing I to react with N-hydroxysuccinimide and dicyclohexylcarbodiimide in ethyl acetate (1 h, O'C; 1 h, 25"). After filtration of the urea and the evaporation of ethyl acetate the product was dissolved in tetrahydrofuran and added to an equal volume of a water suspension of the next amino acid to be coupled and 2 equivalents of NaHC03. After 1 h of reaction at room temperature the tetrahydrofuran was evaporated, the solution was acidified to pH 2 by the addition of hydrochloric acid and extracted with ethyl acetate. The theoretical weight gain occurred at each step and amino acid analysis indicated quantitative coupling. The final product showed one major spot and minor contaminants in thin layer chromatography (TLC:*** benzene:acetic acid = 7:l). Crystallization from ethyl acetate/petroleum ether gave a material pure by amino acid Both the formylated analysis and TLC in the solvent system given above. and the acetylated pentapeptides were prepared by an analogous procedure utilizing the respective esters of N-hydroxysuccinimide. In order to obtain the free pentapeptide, the Boc-derivitive (80 mg) was allowed to stand in 10 ml of trifluoroacetic acid at room temperature for 60 min. Following the removal of trifluoroacetic acid under vacuum, Upon addition of ammonium the salt was dissolved in 30% ethanol-water. After adjustment hydroxide to neutrality the free peptide precipitated. *L-(1-Tosylamido-2-phenyl)ethylchloromethyl ** N-o-p-Tosyl-L-lysylchloromethyl *** thin-layer chromatography
ketone ketone
465
BIOCHEMICAL
Vol. 80, No. 2, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of its pH to 2.0 with hydrochloric acid, the free peptide was extracted with ethyl acetate. The peptide obtained after solvent evaporation showed the correct amino acid analysis and weight recovery of amino acid. Activated rabbit neutrophils were obtained 12 to 14 hours after the intraperitoneal injection of 0.1% glycogen in phosphate buffered saline (6). Chemotactic activities were measured using a Boyden chamber (7) by counting the cells which had migrated through and adhered to the underside of a micropore filter which separated two chambers, the upper one containing the cells, the lower the attractant. In the standard binding assay (5) neutrophils (4.4 x 1A6 cells) briefly treated with 0.1 mM TPCK, were incubateg at 0°C for 1 h in 2 ml of Gey's solution (8) containing 50,000 cpm of [ H]fNorleu-ku-Phe and the ligand bound to the cells retained after filtration on the glass fiber filter and subsequent washing was counted in a liquid scintillation counter with an efficiency of 40%. RESULTS Chemotactic
properties
The polypeptide
antibiotics
of 10 -6 - 1o-5
range displace
the
closely
related
they fNorleu
to their
the potency
(5).
of magnitude,
(Table
l),
(1).
The Boc-protected
the concentration
curve
is
agreement
shows the
previous
findings
peptide
50% inhibition
is
8 x 10 -7 M (Table
pentapeptide
and its
N-protected
of the labeled
attractant
binding is that
ligand. with
shown in Fig. the peptides The calculated
their
behavioral
2.
I).
interact
at the
ID50 values activities
466
of chemotaxis;
A typical
[3H]
The curves
activity
> of chemotactic
EDSO are
of the free
two
on the specificity
1 and the calculated
in Fig.
agonist;
lowest
an inhibitor
(ID5o
is that
by approximately
given
the leukocyte
labeled
effect
peptide
to
and
is a potent
is
specific
indicating
to
as do fMet
lower
a
1) indicating
to an agonist
The effect the
with
is
within
to neutrophils
(Table
pentapeptide
derivative
peptides
antibiotics
attractant
potencies
The formylated
in accord
active
the same receptor
and nonacylated
a result
of acylation
response
through
synthetic
of these
of a labelled
of the acetylated
orders
The ability
chemotactic
chemotaxis
peptides
1).
binding
and related
are chemotactically
M (Table
specific
modulate
of antibiotics
dose-response
shown in Table derivatives
same receptor for (Table
on
fNorleu-Leu-Phe
are essentially
these 1).
I.
to
parallel site
compounds
as the are
in
BIOCHEMICAL
Vol. 80, No. 2, 1978
01
-LOG
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
02
fM1
-LOG
[M]
Figure 1. Chemotactic activity of neutrophils in response to H-L-Phe-DLeu-L-Phe-D-Leu-L-PheOH (0) HCO-L-Phe-D-Leu-L-Phe-D-Leu-L-PheOH (o), H CCO-L-Phe-D-Leu-L-Phe-D-Leu-L-Phe-OH (A) and (H3C)3COCO-L-Phe-D-Leu-LThe results are expressed as the average number of neutrophils PaeOH (A). The SEM in 10 fields at a magnification of X970 for triplicate samples. did not exceed 10% for values above 10. Figure 2. Inhibition of specific binding of (3H)f-Norleu-Leu-Phe to neutrophils by H-L-Phe-D-Leu-L-Phe-D-Leu-L-PheOH (m), HCO-L-Phe-D-Leu-LPhe-D-Leu-L-PheOH (o), H CCO-L-Phe-D-Leu-L-Phe-D-L-Leu-PheOH (A) and (H C)3 COCO-L-Phe-D-Leu-I!-Phe-D-Leu-L-PheOH (A). IDS0 for binding was es 2.imated from these plots of specific binding versus concentration of peptides. The values are means of triplicate samples varying less than
Effect
of proteolytic
From results been invoked
inhibitor
inhibitors,
with
inhibitors
in the
effects
similar
tested
were
was more effective
for
aromatic
(9)
have
to peptide
of the chemotactically
were
suggesting
proteases
response
on neutrophils
observations
of trypsin, specificity
specific
chemotactic
and the antibiotics
of chymotrypsin,
TLCK, an inhibitor peptidase
proteolytic
When the
pentapeptide
protease
inhibitors
as participants
attractants. active
with
in the presence
made (Table
II).
in inhibiting
a role
in
of the present
study
chemotaxis
chemotaxis
for
residues.
show that
467
certain
of
TPCK, an
DISCUSSION The results
10%.
polypeptide
a
than
BIOCHEMICAL
Vol. 80, No. 2, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Chemotactic
Activities
1
of Synthetic
Peptides
Peptide
Chemotaxis ED50 W a
HCO-L-Met-L-Leu-L-PheGH
7
x lo-l1
H-L-Phe-D-Leu-L-Phe 'D-Leu-L-PheOH
4
H CCO-L-Phe-D-Leu 5L-Phe-D-Leu-L-PheOH HCO-L-Phe-D-Leu-L Phe-D-Leu-L-PheOH
and Peptide Specific Bindingb ID5* U-0
S
Inhibition Chemotaxfs ID5* 00
x lo-lo
-
x 10-6
1 x 10-5
-
5
x 1o-7
4
x 10
4
x 10
2
x 10
7
x 1o-7
3.3
-9
t-Boc-L-Phe-D-LeuL-Phe-D-Leu-L-PheQH Gramicidin
-8
8
5
x 10
Tyrocidin
4
x lo+
2
x 10
Bacitracin
2
x 10-5
9 x 10 -5
-5
were employed as described in Methods for chemotaxis The values are means of triplicate samples varying
aED5. is defined as that concentration maximal chemotactic response.
of attractant
giving
b
is defined as that3concentration of peptide causing ID50 of specific binding of [ HI-fNorku-Leu-Phe on neutrophils.
'ID is defined as that concentration cauzkg 50% inhibition of leucotactic attractant fMet-Leu-PheOH.
are
receptor factor
chemotactic
on these (5).
peptides potent
having defined
cells
for
activities sequences
peptide
leukocytes
to identify
of
attractants
and interact peptides
active
of these Leu-Phe
a half50% inhibition
of peptide, not itself response to the standard
as do synthetic
In efforts
to the chemotactic
x 1O-7
-5
x 1o-6
antibiotics
of
-7
3
Standard conditions and binding assays. less than 10%.
Antibiotics
were
as well
468
and a crude
groupings
antibiotics,
with
which
the
an attractant potent
same
bacterial may contribute
some synthetic
selected
since
as the
antibiotics
the most contained
Vol. 80, No. 2, 1978
BIOCHEMICAL
AND BIOPHYSICAL
Table Effect
of Specific Chemotactic
Peptide
II
Protease Response
Addition
RESEARCH COMMUNICATIONS
Inhibitors to Peptides
on the Percent Inhibition Chemotaxisa
to cells
of
Bacitracin,
O.lmM
TPCK, O.lmM
Bacitracin,
O.lmM
TICK,
O.lmM
49
Gramicidin O.OlmM
S
TPCK, O.lmM
100
Gramicidin O.OlmM
S,
TLCK, O.lmM
62
Tyrocidin,
O.OlmM
TPCK, O.lmM
100
Tyrocidin,
O.OlmM
TLCK,O.lmM
0
HCO-L-Phe-D-Leu-LPhe-D-Leu-L-PheOH 1OnM
TPCK, O.lmM
84
HCO-L-Phe-D-LeuL-Phe-D-Leu-LPheOH, 1OnM
TLCK, O.lmM
0
HCO-L-Phe-D-LeuL-Phe-D-Leu-L PheOH, 1OnM
BTEEb O.lmM
100
HCO-L-Phe-IkLeuL-Phe-D-Leu-LPheOH, 1OnM
TAME=O.lmM
aAssays
were
b
for
chemotaxis
BTEE, Benzoyl-L-tyrosine
'TAME,
These
N-formylation observations Boc results
of certain here
is
studies
The formylated
out as described
ester
also
confirm
the finding
for that
the great
chemotaxis replacing
of potent
pentapeptide,
in Methods.
ester
methyl
peptides
in the production
0
carried
ethyl
Tosyl-L-arginine
such a sequence.
10-7M).
100
(1).
on the other
group
of chemotaxis hand,
for
One of the significant
the formyl
inhibitors
469
specificity
with
t-
(8 x
was almost
as
BIOCHEMICAL
Vol. 80, No. 2, 1978
potent
an agonist
as the known most
The pentapeptide
tested
Based upon our all
L isomer
active biological
for
synthetic
peptides
by the chymotryptic inhibitor, aromatic
residues
cells
polymerase
a known chemotaxis
results).
results)
chemotaxis. their
proposed
for
other
to them is
inhibited
than by the tryptic
of a peptidase
specific
for
here
since
is
of the labeled
effect
that
Actinomycin here
through
the receptor
and peptidase
is
probably
affected
attractant
RNA synthesis
presented
RNA of these
to the
previously
470
cells
since
we have
these
implicated
mM),
neither
antibiotics
that
the
(0.1
these
D does not affect suggests
not
valinomycin
sequences,
seem likely
the evidence
to inhibit
a new property
by itself
Leu-Phe
Rather,
to
and tyrocidin).their
This
action
by inhibiting
has been shown
and more recently
leukocytes.
that
sole (gift
affect
antibiotics,
response
bacitracin,
Nor does it
movement
(unpublished
to that
(10)
not having
nor binding
cell
to be similar
are
B and EM 49,
B, do not
such as gramicidin
ionophoric
ionophore
residues
may not be the
polymixin
active
(gramicidin, for
acid
and f- Nle-Leu-Phe
sequence
the cell
more
publication).
L amino
chemotactically
What we are reporting
activity
for
to polymixin
is,
is
process.
as an ionophore
of their
only
the
shows a greater
TPCK, more effectively
antibiotic
products
chemotactic
(submitted
the participation
in this
(11).
bacterial
That
implying
A polypeptide
late
similar
inhibitor,
TLCK,
f-L-Phe-L-Met
since
that
f-L-Met-L-Leu-L-Phe
of a Leu-Phe
activity
appears (9).
to postulate
such as f-Phe-Leu-Phe
a new antibiotic
of action
since
(2)].
D and L residues.
seems reasonable
containing
chemotactic
[f-Met-Leu-Phe
of alternating
D enantiomer
sequence
mechanism
lished
its
in the case of the
However,
compound
and since
But the presence
of Squibb)
result
it
tripeptides
-Leu-Phe
requirement
affect
consisted
results
than
other
chemotactic.
active
be even more active
effect
the
here
f-L-Met-D-Leu-L-Phe
Furthermore, with
recent
would
than
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(unpubstimualso
found
chemotaxis. polypeptides in
leukotaxis
act
Vol. 80, No. 2, 1978
Since
(5,9). conditions,
through
leukocyte it
properties
BIOCHEMICAL
may be prudent
of these their
migration
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
contributes to take
antibiotics
in large
into
account
in efforts
part
to inflammatory
the leukotactic
to modulate
inflammation
use.
References: 1.
Schiffmann, Acad. Sci.,
E.,
Corcoran, B. A. and Wahl, 1059-1062.
72:
S. M. (1975)
2.
Showell, H. J., Freer, R. J., Zigmond, S. H., Aswanikumar, S., Corcoran, B. A., and Becker, Med. 1143: 1154-1169.
3.
Day, A. R., Aswanikumar, FEBS Letters:
4.
Schiffmann, E., Showell, H. J., Corcoran, B. and Becker, E. L. (1975) J. Irmnunol.,
5.
Aswanikumar, S., Corcoran, B., Showell, H. J., Becker, E. L., Commun. 74: 810-817.
6.
Snyderman, 1, 521-525.
7.
Tempel, T. R., J. Peridontal.
8.
Gey,
9.
Aswanikumar, Proc. Natl.
Showell, H. J., Becker, S., Pert, C., Radding, 77: 291-294.
R.,
Phillips,
-
S., Schiffmann, Acad. Sci., 73:
:
P. K., 74,
Jordan, 12180.
B. A., Ward, P. A., 114: 1831-1837.
E.,
H. V.,
Am. J.
B.,
Smith,
Corcoran,
S. E. (1970)
Infect.
Mergenhagen,
S. E. (1970)
Cancer,
27:
Irmaunity
45-76.
B. A. and Wahl,
S. M. (1976)
2439-2442.
in Spores, eds. (Amer. Sot. for
and Paulus,
E., Corcoran, R. J. (1977)
Schiffmann, E., Day, A. R., Freer, R. J., and Pert, C. (1977) Biochem. Biophys. Res.
G. E. and Gey, M. K. (1936)
11. Mukherjee, 74.
R.,
Natl.
Schiffmann, E., E. L., (1976) J. Exp.
Schiffmann, and Freer,
J. and Mergenhagen,
Snyderman, 41: 3171
10. Sadoff, H. L. (1972) and Campbell, L. L.,
E. L., J. A.,
Proc.
H. (1977)
780-784.
471
Haborson, Microbial. Proc.
H. O., Hanson, Wash., D. C.) Natl.
Acad.
Sci.
R., pp.
157-166.