Antagonists of the C5a Receptor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1973
(6) Bubeck, P.; Grotzinger, J .; Winkler, M.; Kohl, J .; Wollmer, A.;
Klos, A.; Bautsch, W. Site-specific mutagenesis of residues in
the human C5a anaphylatoxin which are involved in possible
interaction with the C5a receptor. Eur. J . Biochem. 1994, 219,
897-904.
(7) Federwisch, M.; Wollmer, A.; Emde, M.; Stuhmer, T.; Melcher,
T.; Klos, A.; Kohl, J .; Bautsch, W. Tryptophan mutants of human
C5a anaphylatoxin: a fluorescence anisotropy decay and energy
transfer study. Biophys. Chem. 1993, 46, 237-48.
(8) Toth, M. J .; Huwyler, L.; Boyar, W. C.; Braunwalder, A. F.;
Yarwood, D.; Hadala, J .; Haston, W. O.; Sills, M. A.; Seligmann,
B.; Galakatos, N. The pharmacophore of the human C5a ana-
phylatoxin. Protein Sci. 1994, 3, 1159-68.
(9) Chenoweth, D. E.; Hugli, T. E. Human C5a and C5a analogues
as probes of the neutrophil C5a receptor. Mol. Immunol. 1980,
17, 151-61.
(10) Siciliano, S. J .; Rollins, T. E.; DeMartino, J .; Konteatis, Z.;
Malkowitz, L.; VanRiper, G.; Bondy, S.; Rosen, H.; Springer, M.
S. Two-site binding of C5a by its receptor: an alternative binding
paradigm for G protein-coupled receptors. Proc. Natl. Acad. Sci.
U.S.A. 1994, 91, 1214-8.
(11) DeMartino, J . A.; Van Riper, G.; Siciliano, S. J .; Molineaux, C.
J .; Konteatis, Z. D.; Rosen, H.; Springer, M. S. The amino
terminus of the human C5a receptor is required for high affinity
C5a binding and for receptor activation by C5a but not C5a
analogues. J . Biol. Chem. 1994, 269, 14446-50.
(12) Kawai, M.; Quincy, D. A.; Lane, B.; Mollison, K. W.; Luly, J . R.;
Carter, G. W. Identification and synthesis of a receptor binding
site of human anaphylatoxin C5a. J . Med. Chem. 1991, 34,
2068-71.
(13) Kawai, M.; Quincy, D. A.; Lane, B.; Mollison, K. W.; Or, Y.-S.;
Luly, J . R.; Carter, G. W. Structure-function studies in a series
of carboxyl-terminal octapeptide analogues of anaphylatoxin
C5a. J . Med. Chem. 1992, 35, 220-3.
simulated annealing protocol consisted of 20 ps of high-
temperature molecular dynamics (1000 K) with a low weight-
ing on the repel force constant and NOE restraints. This was
followed for a further 10 ps with an increased force constant
on the experimental NOE restraints. The dihedral force
constant was increased prior to cooling the system to 300 K
and increasing the repel force constant over 15 ps of dynamics.
Refinement of these structures was achieved using the con-
jugate gradient Powell algorithm with 1000 cycles of energy
minimization32 and a refined force field based on the program
CHARM.33 Structures were displayed using INSIGHT (Biosym
Technologies, San Diego, CA). All structural analysis described
in the text was done using structures calculated without
explicit H-bond restraints. The final structures were examined
to obtain pairwise rms differences over the backbone heavy
atoms (N, CR, and C).
Recep tor Bin d in g Assa y. Assays were performed with
fresh human PMNs, isolated as previously described,18 and a
buffer of 50 mM HEPES, 1 mM CaCl2, 5 mM MgCl2, 0.5%
bovine serum albumin, 0.1% bacitracin, and 100 µM PMSF
(phenylmethanesulfonyl fluoride). Buffer, unlabeled human
recombinant C5a (Sigma) or test peptide, [125I]C5a (∼50 pM)
(New England Nuclear, MA), and PMNs (0.2 × 106) were
added sequentially to a Millipore Multiscreen assay plate.
After incubation for 60 min at 4 °C, the samples were filtered
and the plate was washed once with buffer (100 µL). Filters
were dried, punched, and counted in an LKB γ-counter.
Nonspecific binding was assessed by the inclusion of 1 mM
peptide or 100 nM C5a which typically resulted in 10-15%
total binding.
(14) Mollison, K. W.; Krause, R. A.; Fey, T. A.; Miller, L.; Wiedeman,
P. E.; Kawai, M.; Lane, B.; Luly, J . R.; Carter, G. W. Hexapeptide
analogues of C5a anaphylatoxin reveal heterogeneous neutrophil
agonism/antagonism. FASEB J . 1992, 6, A2058.
(15) Ember, J . A.; Sanderson, S. D.; Taylor, S. M.; Kawahara, M.;
Hugli, T. E. Biologic activity of synthetic analogues of C5a
anaphylatoxin. J . Immunol. 1992, 148, 3165-73.
(16) Sanderson, S. D.; Ember, J . A.; Kirnarsky, L.; Sherman, S. A.;
Finch, A. M.; Taylor, S. M. Decapeptide agonists of human C5a:
the relationship between conformation and spasmogenic and
platelet aggregatory activities. J . Med. Chem. 1994, 37, 3171-
80.
(17) Sanderson, S. D.; Kirnarsky, L.; Sherman, S. A.; Vogen, S. M.;
Prakesh, O.; Ember, J . A.; Finch, A. M.; Taylor, S. M. Decapep-
tide agonists of human C5a: the relationship between conforma-
tion and neutrophil response. J . Med. Chem. 1995, 38, 3669-
75.
(18) Finch, A. M.; Vogen, S. M.; Sherman, S. A.; Kirnarsky, L.; Taylor,
S. M.; Sanderson, S. D. Biological active conformer of the effector
region of human C5a and modulatory effects of N-terminal
receptor binding determinants on activity. J . Med. Chem. 1997,
40, 877-84.
(19) Drapeau, G.; Brochu, S.; Godin, D.; Levesque, L.; Rioux, F.;
Marceau, F. Synthetic C5a receptor agonists. Pharmacology,
metabolism and in vivo cardiovascular and hematologic effects.
Biochem. Pharmacol. 1993, 45, 1289-99.
An ta gon ism Assa y. Antagonist assays were assessed by
monitoring myeloperoxidase release as follows. Cells were
isolated as previously described18 and incubated with cytocha-
lasin B (10 µg/mL, 10 min, 37 °C). Hank’s balanced salt
solution containing 0.1% gelatin and test peptide was added
onto a 96-well plate (total volume 100 µL/well), followed by
25 µL of cells (4 × 106/mL). To assess the capacity of each
peptide to antagonize C5a, cells were incubated for 10 min at
37 °C with each peptide, followed by addition of C5a (100 nM)
and further incubated for 10 min. Then 50 µL of phosphate
buffer (0.1 M, pH 6.8) was added to each well, followed by the
addition of 25 µL of a fresh 1:1 mixture of dimethoxybenzidine
(5.7 mg/mL) and H2O2 (0.51%). The reaction was stopped at
20 min by addition of 2% sodium azide (25 µL). Absorbances
were measured at 450 nm in a Rainbow plate reader and
corrected for control values (no peptide).
Ack n ow led gm en t. We thank Trudy Bond in the
Centre for amino acid analyses, the Australian Research
Council for a Senior Research Fellowship (D.J .C.), and
the National Health and Medical Research Council for
grant support.
(20) Konteatis, Z. D.; Siciliano, S. J .; Van Riper, G.; Molineaux, C.
J .; Pandya, S.; Fischer, P.; Rosen, H.; Mumford, R. A.; Springer,
M. S. Development of C5a receptor antagonists. Differential loss
of functional responses. J . Immunol. 1994, 153, 4200-4.
(21) Wong, A. K.; Finch, A. M.; Pierens, G. K.; Craik, D.; Taylor, S.
M.; Fairlie, D. P. Molecular probes for G-protein coupled
receptors. Conformationally constrained antagonists derived
from the C-terminus of human plasma protein C5a. J . Med.
Chem. 1998, 41, 3417-22.
(22) Zhang, X.; Boyar, W.; Galakatos, N.; Gonnella, N. C. Solution
structure of a unique C5a semi-synthetic antagonist: implica-
tions in receptor binding. Protein Sci. 1997, 6, 65-72.
(23) Pellas, T. C.; Boyar, W.; van Oostrum, J .; Wasvary, J .; Fryer, L.
R.; Pastor, G.; Sills, M.; Braunwalder, A.; Yarwood, D. R.;
Kramer, R.; Kimble, E.; Hadala, J .; Haston, W.; Moreira-
Ludewig, R.; Uziel-Fusi, S.; Peters, P.; Bill, K.; Wennogle, L. P.
Novel C5a receptor antagonists regulate neutrophil functions
in vitro and in vivo. J . Immunol. 1998, 160, 5616-21.
(24) Kessler, H. Conformation and biological activity of cyclic pep-
tides. Angew Chem., Int. Ed. 1982, 21, 512-23.
Refer en ces
(1) J ose, P. J .; Moss, I. K.; Maini, R. N.; Williams, T. J . Measurement
of the chemotactic complement fragment C5a in rheumatoid
synovial fluids by radioimmunoassay: role of C5a in the acute
inflammatory phase. Ann. Rheum. Dis. 1990, 49, 747-52.
(2) Valazquez, P.; Cribbs, D. H.; Poulos, T. L.; Tenner, A. J .
Aspartate residue 7 in amyloid beta protein is critical for
classical complement pathway activation: implications for alzhe-
imers disease pathogenesis. Nature Med. 1997 3, 77-9.
(3) Engler, R. L.; Roth, D. M.; del Balzo, U.; Ito, B. R. Intracoronary
C5a induces myocardial ischemia by mechanisms independent
of the neutrophil: leukocyte filters desensitize the myocardium
to C5a. FASEB J . 1991, 5, 2983-91.
(4) Hammerschmidt, D. E.; Weaver, L. J .; Hudson, L. D.; Craddock,
P. R.; J acob, H. S. Association of complement activation and
elevated plasma C5a with adult respiratory distress syndrome.
Pathophysiological relevance and possible prognostic value.
Lancet 1980, 1, 947-9.
(5) Mollison, K. W.; Mandecki, W.; Zuiderweg, E. R.; Fayer, L.; Fey,
T. A.; Krause, R. A.; Conway, R. G.; Miller, L.; Edalji, R. P.;
Shallcross, M. A.; et al. Identification of receptor-binding resi-
dues in the inflammatory complement protein C5a by site-
directed mutagenesis. Proc. Natl. Acad. Sci. U.S.A. 1989, 86,
292-6.
(25) Dyson, H. J .; Wright, P. E. Defining solution conformations of
small linear peptides. Annu. Rev. Biophys. Chem. 1991, 20, 519-
38.
(26) Rose, G. D.; Gierasch, L. M.; Smith, J . A. Turns in peptides and
proteins. Adv. Protein Chem. 1985, 37, 1-109.