Potent Cyclic Antagonists of the Complement C5a Receptor on Human Polymorphonuclear Leukocytes. Relationships between Structures and Activity

Article abstract of DOI:10.1124/mol.65.4.868

Human C5a is a plasma protein with potent chemoattractant and pro-inflammatory properties, and its overexpression correlates with severity of inflammatory diseases. C5a binds to its G protein-coupled receptor (C5aR) on polymorphonuclear leukocytes (PMNLs) through a high-affinity helical bundle and a low-affinity C terminus, the latter being solely responsible for receptor activation. Potent and selective C5a antagonists are predicted to be effective anti-inflammatory drugs, but no pharmacophore for small molecule antagonists has yet been developed, and it would significantly aid drug design. We have hypothesized that a turn conformation is important for activity of the C terminus of C5a and herein report small cyclic peptides that are stable turn mimics with potent antagonism at C5aR on human PMNLs. A comparison of solution structures for the C terminus of C5a, small acyclic peptide ligands, and cyclic antagonists supports the importance of a turn for receptor binding. Competition between a cyclic antagonist and either C5a or an acyclic agonist for C5aR on PMNLs supports a common or overlapping binding site on the C5aR. Structure-activity relationships for 60 cyclic analogs were evaluated by competitive radioligand binding with C5a (affinity) and myeloperoxidase release (antagonist potency) from human PMNLs, with 20 compounds having high antagonist potencies (IC₅₀, 20 nM-1 μM). Computer modeling comparisons reveal that potent antagonists share a common cyclic backbone shape, with affinity-determining side chains of defined volume projecting from the cyclic scaffold. These results define a new pharmacophore for C5a antagonist development and advance our understanding of ligand recognition and receptor activation of this G protein-coupled receptor.

Full text of DOI:10.1124/mol.65.4.868

0026-895X/04/6504-868–879$20.00
MOLECULAR PHARMACOLOGY
Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 65:868–879, 2004
Vol. 65, No. 4
2983/1140113
Printed in U.S.A.
Potent Cyclic Antagonists of the Complement C5a Receptor
on Human Polymorphonuclear Leukocytes. Relationships
between Structures and Activity
Darren R. March, Lavinia M. Proctor, Martin J. Stoermer, Robert Sbaglia,
Giovanni Abbenante, Robert C. Reid, Trent M. Woodruff, Khemar Wadi,
Natalii Paczkowski, Joel D. A. Tyndall, Stephen M. Taylor, and David P. Fairlie
Institute for Molecular Bioscience (D.R.M., M.J.S., R.S., G.A., R.C.R., J.D.A.T., D.P.F.) and School of Biomedical Sciences
(L.M.P., T.M.W., K.W., N.P., S.M.T.), University of Queensland, Brisbane, Queensland, Australia
Received September 22, 2003; accepted January 7, 2004
This article is available online at http://molpharm.aspetjournals.org
ABSTRACT
Human C5a is a plasma protein with potent chemoattractant tagonists supports the importance of a turn for receptor bind-
and pro-inflammatory properties, and its overexpression corre- ing. Competition between a cyclic antagonist and either C5a or
lates with severity of inflammatory diseases. C5a binds to its G an acyclic agonist for C5aR on PMNLs supports a common or
protein-coupled receptor (C5aR) on polymorphonuclear leuko- overlapping binding site on the C5aR. Structure-activity rela-
cytes (PMNLs) through a high-affinity helical bundle and a tionships for 60 cyclic analogs were evaluated by competitive
low-affinity C terminus, the latter being solely responsible for radioligand binding with C5a (affinity) and myeloperoxidase
receptor activation. Potent and selective C5a antagonists are release (antagonist potency) from human PMNLs, with 20 com-
predicted to be effective anti-inflammatory drugs, but no phar- pounds having high antagonist potencies (IC50, 20 nM-1 ␮M).
macophore for small molecule antagonists has yet been devel- Computer modeling comparisons reveal that potent antago-
oped, and it would significantly aid drug design. We have nists share a common cyclic backbone shape, with affinity-
hypothesized that a turn conformation is important for activity determining side chains of defined volume projecting from the
of the C terminus of C5a and herein report small cyclic peptides cyclic scaffold. These results define a new pharmacophore for
that are stable turn mimics with potent antagonism at C5aR on C5a antagonist development and advance our understanding
human PMNLs. A comparison of solution structures for the C of ligand recognition and receptor activation of this G protein-
terminus of C5a, small acyclic peptide ligands, and cyclic an- coupled receptor.
Activation of the complement system, a cascading network smooth muscle, neurons, glial cells, astrocytes, and mast
of plasma proteins, is crucial in host defense to infection and
injury (Abbas et al., 1997). The ultimate product of comple-
ment activation is the membrane attack complex that is
responsible for bacterial cell lysis. Along the way, 70- to
80-residue pro-inflammatory anaphylatoxins C3a, C4a, and
C5a are also produced. C5a is a very potent pro-inflammatory
agent that interacts with a G protein-coupled C5a receptor
(C5aR) on the surface of myeloid (e.g., neutrophils, eosino-
phils, monocytes, T lymphocytes, and macrophages) and non-
myeloid cells (e.g., vascular endothelial cells, vascular
cells) (Wetsel, 1995; Zwirner et al., 1999; Barnum, 2002). C5a
is a potent chemotaxin for inflammatory cells and causes
smooth muscle spasmogenesis as well as release of lysozomal
enzymes, reactive oxygen species, and inflammatory cyto-
kines (e.g., TNF-␣, IL-1, -6, -8) (Okusawa et al., 1988; Ember
et al., 1994, 1998), all components of the host response to
injury or infection.
Sustained or aberrant complement activation is associated
with tissue damage and inflammatory disease. Elevated lev-
els of C5a correlate with immune and inflammatory condi-
tions (Kohl, 2001) and there is growing evidence that C5a is
pathogenic in numerous inflammatory diseases (Abbas et al.,
1997; Kohl, 2001; Barnum, 2002), including rheumatoid ar-
This work was supported in part by the Australian National Health and
Medical Research Council and the Australian Research Council.
D.R.M. and L.M.P. should be considered joint first authors.
ABBREVIATIONS: TNF-␣, tumor necrosis factor-␣; IL, interleukin; TFA, trifluoroacetic acid; DIEA, N,N-diisopropylethylamine; DMF, N,N-
dimethylformamide; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; rpHPLC, reversed-phase high-perfor-
mance liquid chromatography; DMF, dimethylformamide; C5a, complement factor 5a; C5aR, C5a receptor; Boc, N-tert-butoxycarbonyl; Fmoc,
fluoromethyloxycarbonyl; TOCSY, total correlation spectroscopy; ECOSY, exclusive correlation spectroscopy; NOESY, nuclear Overhauser effect
(or enhancement) spectroscopy; 1D, one-dimensional; Cha, cyclohexylalanine; MPO, myeloperoxidase; PMNL, polymorphonuclear leukocyte.
868

Potent Cyclic Antagonists of Human C5a Receptor
869
thritis, immune complex disease, inflammatory bowel syn- synthesis grade; Auspep, Parkville, Victoria, Australia), HBTU and
BOP (Richelieu Biotechnologies, St Hyacinthe, QC, Canada) were
obtained from commercial sources. All other materials were reagent
grade. Preparative scale reversed-phase HPLC (rpHPLC) separa-
tions were performed on a Vydac C18 column (2.2 ϫ 25 cm); analyt-
ical rpHPLC on VYDAC or Phenomenex C18 column (250 ϫ 4.6 mm);
using gradient mixtures of solvent A ϭ water/0.1% TFA and solvent
B ϭ water 10%/acetonitrile 90%, 0.09% TFA. The molecular weight
was determined by electrospray mass spectrometry (LCT MICRO-
MASS). All compounds were analyzed for purity and molecular
weight by rpHPLC and mass spectrometry.
dromes, ischemia/reperfusion injury, myocardial infarct,
pancreatitis, cystic fibrosis, multiple sclerosis, atherosclero-
sis, fibrosis, allergy, diabetes type I, Alzheimer’s disease,
graft rejection, gingivitis, and psoriasis.
The binding site for C5a on its receptor and structural
features of both receptor and ligands that influence agonism
and antagonism are poorly defined. The solution structure of
C5a (Zuiderweg et al., 1989; Zhang et al., 1997) consists of a
64-residue, high-affinity ‘receptor-binding’ N-terminal helix
bundle attached to a 10-residue low affinity ‘receptor-activat-
ing’ C-terminal domain thought to bind in a transmembrane
pore region of the receptor (Siciliano et al., 1994; Huber-Lang
et al., 2003). Much effort has been devoted to developing
short peptides derived from the C terminus of C5a as ago-
nists/antagonists (Drapeau et al., 1993; Luly et al., 1993;
Konteatis et al., 1994; Sanderson et al., 1994, 1995; Finch et
al., 1997). Most were partial agonists, but the hexapeptide
MeFKPdChaWr was a full antagonist with Ͻ1 ␮M affinity
for the C5aR (Drapeau et al., 1993; Konteatis et al., 1994).
Peptide Synthesis. His6-tagged rhC5a was synthesized (Pacz-
kowski et al., 1999) with incorporation of a tobacco etch virus (TEV)
cleavage site between the His6 tag and C5a, allowing tag removal
with
retention
of
an
N-terminal
tripeptide
(GGS).
AcF[3H][OP[3H2]dChaWR] was synthesized (Multiple Peptide Sys-
tems, San Diego, CA) and purified to Ͼ99% by rpHPLC (25–70% B in
20 min). The specific activity was 60 Ci/mmol. Short peptides were
assembled by manual step-wise solid phase peptide synthesis using
Boc or Fmoc chemistry (Wong et al., 1998; Finch et al., 1999).
Cyclic Peptide Synthesis. In general, linear peptides (1 Eq) and
BOP (5 Eq) were dissolved in DMF (10 mM peptide concentration)
More recently, we demonstrated that MeFKPdChaWr and before adding DIEA (15 Eq) and stirred at room temperature over-
night. DMF was removed in vacuo at 30°C and compounds purified
by rpHPLC. For cyclic peptides containing a free N terminus, an
Fmoc group was used as the N-terminal–protecting group during
cyclization, after which the peptide was treated with 30% piperidine/
DMF and worked up as above. One example is given below for each
Boc and Fmoc syntheses of cyclic compounds, together with charac-
terization by mass spectrometry and rpHPLC retention times (Rt; in
minutes) under specified elution conditions.
other hexapeptides adopt a turn conformation in solution
(Wong et al., 1998) and the cyclic molecule AcF-[OPdChaWR]
(1) (Fig. 1) was a potent and selective antagonist of C5aR on
human immune cells (Finch et al., 1999; Haynes et al., 2000)
with potent anti-inflammatory activity in vivo (Short et al.,
1999; Strachan et al., 2000, 2001; Arumugam et al., 2002,
2003; Woodruff et al., 2002, 2003).
Herein, we investigate structure-potency relationships for
60 cyclic antagonists like 1, report solution structures that
Synthesis of Cycle AcF-[OPdChaWR] (1). The peptide Ac-Phe-
Orn-Pro-dCha-Trp-Arg was synthesized by Boc chemistry on a 0.20-
define cyclic backbone shape and side chain projections mmol scale using HBTU/DIEA activation and in situ neutralization
on a Boc-L-Arg(Tos)-phenylacetamidomethyl] resin (338 mg, substi-
tution value ϭ 0.591 mmol/g). Cleavage and deprotection of the resin
(457 mg) was achieved by treating the resin with HF (10 ml) and
p-cresol (1 ml) at Ϫ5 to 0°C for 1 to 2 h, to give crude peptide (160 mg,
90%). Cyclization involved stirring the crude peptide (41 mg, 45
␮mol), BOP (126 mg, 0.28 mmol), and DIEA (158 ␮l, 0.9 mmol) in
DMF (57 ml) for 15 h. Solvent was removed in vacuo, and the cyclic
peptide was purified by rpHPLC (18.8 mg, 47%) Rt ϭ 10.8 min
(gradient, 70% A/30% B to 0% A/100% B over 30 min). Mass spec-
trometry: [MϩH]ϩ (calc.) ϭ 896.5, (found) ϭ 896.5.
Synthesis of cycle AcF-[OPdCha(N-Me-Phe)R] (43). Linear
peptide Ac-Phe-Orn-Pro-dCha-(N-Me-Phe)-Arg-OH was synthesized
by Fmoc chemistry using HBTU/DIEA activation and in situ neu-
tralization on Fmoc-L-Arg(pbf)-Wang resin (0.35 mmol/g; Novabio-
chem) using Fmoc-N-methyl-L-Phe-OH (281 mg, 2 Eq), Fmoc-dCha
(275 mg, 2 Eq), Fmoc-Pro (472 mg, 4 Eq), Fmoc-Orn(Boc) (477 mg, 3
Eq), Fmoc-Phe (542 mg, 4 Eq), and Ac2O (4 Eq). Cleavage and
deprotection of resin with 95% TFA (15 ml) for 1 h gave crude peptide
(150 mg) after precipitation with diethyl ether. Cyclization involved
stirring the rpHPLC purified peptide (100 mg), BOP (200 mg), and
DIEA (222 ␮l) in DMF (2 ml) for 4 h. Solvent was removed in vacuo
and cyclic peptide was purified by rpHPLC (50 mg) Rt ϭ 33 min
(gradient: 70% A/30% B to 0% A/100% B over 30 min). Mass spec-
trometry: [MϩH]ϩ (calc.) ϭ 871.5, (found) ϭ 871.5.
(through C␣-C␤ and C␤-C␥ vectors) for three cyclic antago-
nists, and compare structures and receptor binding proper-
ties with C5a and small acyclic peptide ligands. This infor-
mation reveals how the cyclic scaffold directs important side
chains of Phe, dCha, Trp, and Arg in 1 for receptor binding
and antagonism, information that is valuable for our under-
standing of the mechanism of antagonism, the activation of
C5aR, for refining the antagonist pharmacophore and for
developing potent and selective nonpeptidic C5aR antago-
nists into drugs.
Materials and Methods
Chemical Synthesis
General Methods. Protected amino acids and resins (Novabio-
chem, Laufelfingen, Switzerland), TFA, DIEA, and DMF (peptide
Structural Analysis
NMR Spectra. 1H NMR spectra were recorded on 4 mM peptides
in DMSO-d6 (550 ␮l) on Bruker ARX 500 and Avance DRX 500
spectrometers at 293, 298, and 308 K. Proton assignments were
made by TOCSY (80 ms mixing time), double quantum-filtered cor-
relation spectroscopy, ECOSY, and NOESY (40-, 120-, and 350-ms
mixing time) spectra using a sequential assignment method (Wuth-
rich, 1986). Variable-temperature 1D 1H and TOCSY spectra were
typically collected at 5-K increments from 293 to 338 K. For identi-
Fig. 1. Chemical structure of AcF-[OPdChaWR].

870
March et al.
fication of slowly exchanging amides, a series of 1D and TOCSY
spectra were run after diluting samples with D2O (100 ␮l).
Pad Software, San Diego, CA), inhibition curves were produced, and
IC50 values were determined. Receptor affinities and antagonist
Structure Calculations. Backbone dihedral angle restraints potencies (Ϫlog IC50) were compared statistically with compound 1
3
were derived from JH,NH coupling constants measured from high- using a one-way analysis of variance coupled with a Tukey post test,
resolution 1D 1H NMR spectra; angles were restrained to Ϫ120° Ϯ and statistical significance was determined (P Յ 0.05).
30 for 3JH␣,NH Ͼ 8.2 Hz and to Ϫ60° Ϯ 20 for 3J H␣,NH Ͻ 5 Hz (ϩ60°
Ϯ 20 for 3J
Ͻ 5 Hz for D-amino acids). Stereospecific assign-
H␣,NH
Results
ments of ␤-methylene protons and ␹-1 dihedral angles were deter-
mined by examining 3JH␣,H␤ coupling constants in high-resolution
1D 1H NMR and ECOSY spectra in combination with NOE peak
intensities at short mixing times (40, 120 ms). No explicit hydrogen
bonds were used as distance restraints in structure calculations.
NOE distance restraints were derived from spectra at 308 K, but
lower temperature NOESY spectra were also used to resolve ambi-
guities arising from aromatic NH overlap.
Solution structures were calculated using simulated annealing
and energy minimization protocols within XPLOR 3.851 (Bru¨nger,
1992). Standard topology and parameter files for Lys were modified
to accommodate ornithine, and an amide bond patch connected the
Arg C terminus to the ornithine side chain. Cyclohexylalanine (Cha)-
containing peptides were modeled as Leu, as Cha parameters within
topallhdg5.2.pro and parallhdg5.2.pro gave consistently high Ͼ10°
dihedral and violations. Initial structures were generated using ran-
dom ⌽,␺ dihedral angles and energy-minimized. Preliminary struc-
tures were generated by torsion angle simulated annealing involving
a high-temperature (50,000 K) phase comprising 1000 steps of 0.015
ps of torsion angle dynamics, a cooling phase with 1000 steps of 0.015
ps of torsion angle dynamics during which the temperature was
lowered to 0 K, and an energy minimization phase comprising 2000
steps of Powell minimization. Typically, 50 structures were calcu-
lated and the 20 structures of lowest energy were superimposed and
compared using InsightII and Promotif.
Competitive Binding/Antagonism
Compound 1 is a potent antagonist of the human C5aR as
measured by inhibition of the release of myeloperoxidase
from human PMNLs, activated by either human recombi-
nant C5a (Fig. 2A) or a small agonist (Finch et al., 1997)
peptide analog YSFKPMPLaR of the C terminus of C5a (Fig.
2B). Moreover, [3H]Compound 1, which has an affinity of 21
nM (ϪlogIC50 ϭ 7.68 Ϯ 0.01, n ϭ 3), competes with an acyclic
peptide antagonist, MeFKPdChaWr (IC50
ϪlogIC50 ϭ 6.70 Ϯ 0.06, n ϭ 3), and YSFKPMPLaR (IC50
ϭ 201 nM,
ϭ
1180 nM, ϪlogIC50 ϭ 5.93 Ϯ 0.15, n ϭ 4) for binding to
human PMNL C5aRs (Fig. 2C). Importantly, Fig. 2 suggests
that the binding of antagonist 1, agonist YSFPMPLaR, an-
tagonist MeFKPdChaWr and C5a may occur at the same or
overlapping sites on the C5aR. This is supported below by the
finding of a common structural turn motif in all four com-
pounds. Compound 1 has an apparent receptor affinity of 48
nM to human PMNL membranes, with a Kb of 1 nM (pKb ϭ
9.00 Ϯ 0.04, n ϭ 3) or 9 nM (pKb ϭ 8.04 Ϯ 0.35, n ϭ 3) against
MPO release from human PMNLs induced by YSFKPMPLaR
or recombinant C5a, respectively (Fig. 2). By comparison, the
acyclic peptide antagonist MeFKPdChaWr has an apparent
receptor affinity for human PMNL membranes of 370 nM
and Kb of 51 nM against C5a-induced myeloperoxidase re-
lease from human PMNLs (Paczkowski et al., 1999). The
superior receptor affinity and antagonist potency of 1 versus
‘linear’ peptide antagonists such as MeFKPdChaWr is attrib-
uted to preorganization of the constrained cycle in 1 to the
same or similar putative receptor-binding shape adopted nat-
urally by the C terminus of C5a (Fig. 4B). Energy is needed
for solution structures to reorganize to the optimal shape
recognized by a receptor, and an unfavorable position of this
equilibrium leads to low receptor affinity and modest potency
of unconstrained peptides. Elsewhere, we demonstrate that
antagonism exhibited by 1 is insurmountable (Paczkowski et
al., 1999), reflecting a slow off rate from the receptor.
Cellular Assays
Polymorphonuclear Leukocytes. Human PMNLs were iso-
lated from heparinized whole blood by density gradient separation
over a Ficoll-Hypaque solution (Histopaque 1077; Sigma, St. Louis,
MO) as described previously (Finch et al., 1999).
Receptor Binding Assay. PMNLs (2 ϫ 105) or PMNL mem-
branes (0.5–1 ␮g protein) were incubated (4°C, 60 min) in a 96-well
multiscreen filtration plate (GV 0.22 ␮M; Millipore, North Ryde,
New South Wales, Australia) with buffer (50 mM HEPES, 1 mM
CaCl2, 5 mM MgCl2, 0.5% bovine serum albumin, and 0.1% bacitra-
cin), 125I-C5a (50 pM; PerkinElmer Life and Analytical Sciences,
Boston, MA) or [3H3]AcF-[OPdChaWR] (830 pM; Multiple Peptide
Systems) and test compound to a total volume of 200 ␮l. The wells
were then filtered, dried, and the filter-bound radioactivity was
counted. Nonspecific binding was assessed by the addition of 100 nM
C5a, which typically resulted in 5 to 10% total binding for intact
PMNLs and 10 to 20% for PMNL membranes.
Structure of AcF-[OPdChaWR] (1)
Myeloperoxidase Release. PMNLs (1 ϫ 105) were preincubated
(10 min, 37°C) with cytochalasin B (10 ␮g/ml; Sigma) before being
added to a 96-well plate containing buffer (Hanks’ buffered salt
solution ϩ 0.1% gelatin) and test compounds, as described previously
(Paczkowski et al., 1999). The plate was incubated (10 min, 37°C),
C5a was added (MPO release assigned as 100% response), and the
We have reported a preliminary solution structure for the
backbone of 1 (Finch et al., 1999) that did not define side-
chain locations and used inferior topology and parameter
files to those now available for structure calculations. In this
structural refinement of 1, positions for Orn, Pro, Trp, Phe
plate was incubated again (10 min, 37°C). The total final reaction side chains are now defined, the latter two by using ␹ dihe-
1
volume was 125 ␮l. Phosphate buffer, pH 6.8, was added to each well,
a final incubation was performed (10 min, 25°C), and was substrate
dral restraints. These modifications more accurately define
macrocycle shape and locations of side chains responsible for
affinity and antagonism. Importantly, the structure of 1 was
determined under the same conditions as structures of other
cycles herein, allowing direct structural comparisons.
Coupling Constants and Temperature-Independent
Chemical Shifts. 1H NMR spectra and resonance assign-
ments were similar to those previously reported (Finch et al.,
1999), suggesting structure around Phe2, D-Cha5, and Arg7.
added (3,3-dimethoxybenzidine; 2.85 mg/ml; Sigma)
ϩ H2O2
(0.255%; Merck). The absorbance of each well was read at 450 nm on
a TECAN microplate reader, with maximal C5a-induced MPO re-
lease assigned 100% response versus spontaneous MPO release ϳ5%
(subtracted from all values).
Statistical Analysis. Dissociation constants (Kb) for the antago-
nist were measured as detailed elsewhere (Kenakin, 1997; Pacz-
kowski et al., 1999). For both receptor binding and antagonism
assays, nonlinear regression was performed using Prism 3 (Graph- In addition to three ␺ angle restraints derived from coupling

Potent Cyclic Antagonists of Human C5a Receptor
871
constants for these residues, two ␹-1 angle restraints (Phe
and Trp) were obtained from 1D and ECOSY data with NOE
intensities from a short mixing time NOESY. These five
restraints were used in the structure calculations. Variable
temperature data show low temperature coefficients ⌬␦/T for
Arg-␦NH (2.7 ppb/K), Orn-␦NH (1.8 ppb/K) and Arg ␣NH (2.7
ppb/K). In DMSO-d6 containing 15% D2O, both Trp and Arg
␣NHs underwent slower H/D exchange (56% and 62% resid-
ual signal intensities after 11 h) than other NHs (which
exchanged in a few minutes), suggesting some protection
from solvation as expected for H-bonded amide NHs. TOCSY
spectra during the D2O exchange experiment also support
some H-bonding for the Orn ␦NH. In combination these three
observations support the existence of some transannular H-
bonding in a small population of solution structures that
constitute 1.
Structure Calculation. The 20 lowest energy structures
(⌬E Ͻ1.3 kJ/mol) shown in Fig. 3 were calculated (Bru¨nger,
1992) from 129 NOE restraints and 5 dihedral angles. The
␹-1 restraints tightened the structure, with Phe and Trp
rings being well defined. The root-mean-square deviation for
all backbone atoms of the cycle was 0.27 Å. The Arg ␣NH and
Orn ␦NH are directed to the interior of the saddle-shaped
macrocycle. The Arg ␣NH was not within H-bonding distance
to the nearest CO of Pro (CO. . . N 4.2Å), nor to CO of D-Cha
(CO. . . N 4.4Å) as reported (Finch et al., 1999). Analysis with
Promotif suggests the lowest energy structure is a type IV
␤-turn, but the protection from solvent of Trp and Arg ␣NHs
suggests some type I-like ␤-turn in water.
NMR-derived solution structure calculations for small cy-
clic peptides are always compromised by the tendency of
software packages to select out lowest energy structures from
conformational ensembles (Nikiforovich et al., 2000). NMR
parameters are time- and ensemble-averaged quantities, so
NOE and J-couplings correspond to population-weighted av-
eraged values, and single conformations calculated from such
data may not reflect the overall structure, unless that con-
formation is highly populated. In the case of 1, a very weak
NOE between the acetyl methyl and the Arg␣NH biases the
structure (Fig. 3A) so that AcPhe moves beneath the plane of
the macrocycle. A structure calculation without this NOE
resulted in a flatter macrocycle with the Ac-Phe directed
further away (Fig. 3B). We attribute the NOE to a small
population of conformers where the AcPhe is located within
NOE distance of the Arg␣NH. This does not affect the C␣-
C␤vectors of Pro, dCha, Trp, and Arg but does reorient the
Phe side chain. The cyclic backbone structure identified here
for 1 shows reasonably well how it acts as a scaffold in
Fig. 2. (A&B) Antagonist potency in vitro. Myeloperoxidase release from
human PMNLs incubated with C5a (A) or agonist YSFKPMPLaR (B) in
the presence of antagonist 1 at 0 nM (f), 10 nM (E), 30 nM (F), and 100
nM (Ⅺ). Data expressed as percentage maximum C5a or agonist response
(mean Ϯ S.E., n ϭ 3–6). C, ligand competition for C5aR. Competitive
binding (mean percentage of specific binding Ϯ S.E., n ϭ 3–4) for unla-
beled 1 (F), MeFKPdChaWr (E), and YSFKPMPLAR (f) versus [3H]la-
beled 1 for PMNLs.
Fig. 3. Solution structure of 1. A, average backbone structures with
(yellow) and without (blue) a weak NOE (Ac3Arg␣NH) constraint
(AcPhe omitted). B, side view showing different locations for Ac-Phe
substituent. C, overlay of 20 lowest energy structures in d6-DMSO (Phe2,
blue; Pro4, red; dCha5, yellow; Trp6, brown; Arg7, white) showing only
the lowest energy conformer for cyclic backbone. Arg7 is directed away
from dCha, Trp, and Phe.

872
March et al.
projecting the side chains responsible for affinity (Phe, dCha, antagonist potency, but an H-bond donor (tyrosine, 15) re-
Trp, and Arg) and antagonism (Trp) into receptor space.
duced receptor affinity by ϳ20 fold. The Phe side chain is
thus important for ligand binding and interacts stereospecif-
ically with the receptor via the L-enantiomer. Figure 7A
Structural Comparison of 1 with Acyclic
Agonists/Antagonists
Small peptides derived from the C terminus of C5a typi-
cally show partial agonist activity in promoting enzyme re-
lease from human PMNLs (Luly et al., 1993; Konteatis et al.,
1994, 1997; Siciliano et al., 1994; Sanderson et al., 1995;
Finch et al., 1997). Decapeptide YSFKPMPLaR is one of the
most potent examples of a full agonist, with an apparent
receptor affinity for human PMNLs of 5 ␮M (Finch et al.,
1997). Before our work, the only known C5aR antagonist was
a hexapeptide analog of the C terminus of C5a, MeFKPd-
ChaWr (Konteatis et al., 1994). We now show (Fig. 4A) for the
first time that the NMR-derived solution structures of antag-
onist MeFKPdChaWr (Wong et al., 1998) and the small ag-
onist peptide YSFKPMPLaR (Vogen et al., 1999) share a
common ‘turn’ conformation (Fig. 4A) that is structurally
mimicked by cyclic antagonist 1 (Fig. 4A). When this work
was originally conducted, the C terminus of hC5a had been
reported to be unstructured (Zuiderweg et al., 1989), but a
more recent solution structure (Zhang et al., 1997) defined an
elongated helical turn for residues C5a70–74 (Fig. 4B), a turn
that is mimicked by the cyclic component of 1 (Fig. 4C). This
latter comparison suggests the possibility that the C termi-
nus of C5a might bind to its receptor as a turn motif, as
seems to be true for many peptide ligands for GPCRs.
Structure-Activity Relationships
To probe features of 1 responsible for receptor affinity and
antagonism, a series of analogs were constructed from a
cyclic scaffold (Fig. 5) with variable substituents. Effects of
structure on receptor binding and antagonist potency (Fig. 6)
can be interpreted from Tables 1–4, which show mainly
single substitutions at positions A-F (Fig. 5). All compounds
were devoid of agonist activity at 10Ϫ10 to 10Ϫ4 M.
Position A. Substitution of Ac in AcF-[OPdChaWR].
Analogs 2–8 (Table 1), incorporating alkyl, aryl, benzoyl, and
tosyl substitutions for acetyl at position A of AcF-[OPd-
ChaWR], had comparable affinity and antagonist potency to
1 for PMNL C5aRs. Thus, the acetyl group can be replaced
with a wide range of groups of variable size, hydrophobicity
and hydrophilicity without substantially reducing receptor
affinity or antagonist activity. Loss of the substituent alto-
gether from position A still produces comparable receptor
affinity (Finch et al., 1999). This position could therefore be
modified to vary solubility, pharmacokinetic parameters, or
to attach fluorescent or radiotracer labels.
Position B. Substitution of Phe in AcF-[OPdChaWR].
The importance of the interaction between Phe and the re-
ceptor was examined through substitutions that varied spa-
tial and stereochemical constraints at position B (Table 1).
Removal of the aromatic side chain (9) significantly lowered
receptor affinity (97-fold), demonstrating the importance of
this component. dPhe (10) at this position produced a 75-fold
reduction in receptor affinity, whereas phenylglycine (11)
slightly lowered receptor affinity and antagonist potency.
Shifting the aromatic ring further from the backbone (homo-
phenylalanine, 12) reduced affinity and antagonist potency
by ϳ20 fold. Substitution of the aromatic ring by fluorine at
Fig. 4. Structural comparison of 1, acyclic peptide agonist/antagonist,
and C5a. Superimposed average solution structures derived from NMR
spectroscopy for 1 (yellow) superimposed on agonist YSFKPMPLaR (red)
(38) and antagonist MeFKPdChaWr (light blue) (41) (A); C5a (white with
C-terminal residues 64–74 in red) (29) (B); 1 (yellow) superimposed on
ortho- or meta- positions (13–14) did not affect affinity and C-terminal residues 64–74 (red) showing balls for C␣ atoms of each (C).

Potent Cyclic Antagonists of Human C5a Receptor
873
shows the relationship between receptor affinity and calcu- for side chain volumes ϳ400 to 600 Å3 because they showed
lated steric volume for each side chain with L-stereochemis-
try; the steric fit of the side chain to receptor space is optimal
highest affinity.
Positions A and B. Exocyclic substituents on AcF-
[OPdChaWR]. Three analogs (16-18) simultaneously varied
substituents at positions A and B on the cyclic scaffold (Table
1). Complete removal of the acetylated Phe (16), leaving only
the cyclic component, diminished receptor affinity to unde-
tectable levels. Attaching a cinnamoyl substitutent (17) to
the macrocycle reduced (26-fold) receptor affinity, but hydro-
cinnamyl (18) did not alter C5aR affinity or antagonist po-
tency. The negligible affinity of the cyclic scaffold [OPd-
ChaWR] reveals how important the exocyclic component is
for high affinity with C5aR. Because the substituent at po-
sition A is not crucial for binding, the Phe side chain contrib-
utes substantially to receptor affinity. Flexibility in the exo-
cyclic appendage is important, the trans double bond of the
cinnamoyl group has low affinity for the receptor, whereas
the more flexible saturated hydrocinnamoyl appendage has
affinity and potency comparable with 1.
Position C. Substitution of Pro in AcF-[OPdChaWR].
Replacing proline in 1 by bulkier amino acids like Phe (20)
and Ile (21) substantially reduced (6 and 8-fold, respectively)
affinity for the cellular receptor (Table 2), but smaller Val
(19), hydroxyproline (22), or ␦-N-methyl-Orn (24) substitu-
ents had little effect on receptor affinity. The negligible ef-
fects of these smaller substituents indicate that they are
merely filling receptor space without making important H-
bonding or other intramolecular interactions. 4-cis-Thiopro-
line (23, 75.5 ␮M) displayed a significant loss in binding
affinity, whereas 4-trans-hydroxyproline (22, 0.45 ␮M) re-
tained comparable affinity compared with 1. These are of
similar size and differ only in the stereochemistry, suggest-
ing a steric fit to the receptor. Molecular replacements that
did not possess the structural constraints and turn-inducing
properties of Pro were not accommodated at this site. Figure
7B shows the relationship between receptor affinity and cal-
culated steric volume for residues with L-stereochemistry at
position C. An optimal minimum volume is not clear from
this graph, although side chains Ͼ300 Å3 cause a decrease in
receptor affinity, presumably because receptor space is rela-
tively limited for this position. The outlying point (␤-thiopro-
line) suggests that steric volume alone determines not only
affinity but also how the steric bulk is oriented.
Fig. 5. Scaffold of C5a receptor antagonist cyclic peptides. Positions A-F
were systematically varied in Tables 1–4.
Position D. Substitution of D-Cha in AcF-[OPd-
ChaWR]. D-Stereochemistry is crucial at this position for
receptor affinity (Table 2), replacement of D-Cha by L-Cha
results in IC50 144 ␮M for receptor affinity (data not shown).
Substitution of dCha with smaller aliphatic amino acids like
D-norleucine (29), glycine (25), D-alanine (26), D-valine (27),
and D-leucine (28) produced analogs with reduced receptor
affinity (Table 2). Replacing cyclohexyl with an aromatic ring
(30) or locating the cyclohexyl further from the scaffold (32)
did not affect receptor affinity or antagonist potency. N-
Methylation of phenylalanine (31) resulted in a 2000-fold loss
in affinity for the C5aR. Tyr (33) at this position caused a
6-fold reduction in C5aR affinity, whereas incorporation of
the positively charged arginine (34) eliminated binding up to
100 ␮M. Inclusion of a larger bicyclic residue (36), aminoin-
dancarboxylic acid (37) or Trp (35) produced 24ϫ, 60ϫ, and
84ϫ reduction in affinity. Antagonist potency for 26, 30, 32,
33, and 36 correlated with receptor affinity (Table 2), the
Fig. 6. Receptor affinity and antagonist potency. Inhibition by C5a (F), 1
(f), 18 (E), and 38 (Ⅺ) of binding of 125I-C5a to human PMNLs (A) and
C5a-induced myeloperoxidase release from human PMNLs (B). Each
point represents the mean Ϯ S.E.M.

874
March et al.
antagonist potencies of 30 and 32 were not significantly necessary for receptor affinity and antagonism. The dPhe
different from 1 (Table 2). substituent retains the affinity and activity of 1 but, given
Figure 7C shows a parabolic relationship between receptor that other aliphatic groups can achieve this, it seems likely
affinity and calculated steric volume of side chains, with one that this is caused by a hydrophobic interaction with the
exception (D-homocyclohexylalanine). Because compounds receptor rather than the ␲-stacking properties of this group.
with a larger or smaller side chains have lower affinity, the The dramatic loss in receptor affinity when the ␣NH at this
size/volume of this substitutent must be very important for position on the backbone is methylated (31) suggests a crit-
receptor affinity. Removal of the dCha side chain dramati- ical role for this group in forming either an intramolecular or
cally reduces receptor affinity (25), neutral or charged polar a receptor-specific hydrogen-bonded interaction.
side chains are contraindicated, whereas larger bicyclic sub-
Position E. Substitution of Trp in AcF-[OPdChaWR].
stituents are not accommodated. A hydrophobic side chain Limitations in the size and orientation of the Trp replace-
extending at least two carbons from the ␣-carbon seems ment and the importance of the indole nitrogen of Trp were
TABLE 1
Structure-activity relationships at positions A and B of a C5a antagonist peptide
Receptor Affinity
Antagonist Activity
a
b
No.
Peptide
n
Ϫlog IC50 Ϯ S.E.
IC50
n
Ϫlog IC50 Ϯ SE
IC50
␮M
nM
1
2
Ac-F-͓OPdChaWR͔
Mes-F-͓OPdChaWR͔
Tos-F-͓OPdChaWR͔
Suc-F-͓OPdChaWR͔
MeSuc-F-͓OPdChaWR͔
Ahx-F-͓OPdChaWR͔
Bn-F-͓OPdChaWR͔
Iso-F-͓OPdChaWR͔
Ac-G-͓OPdChaWR͔
Ac-f-͓OPdChaWR͔
Ac-Phg-͓OPdChaWR͔
Ac-hPhe-͓OPdChaWR͔
Ac-(o-fl)F-͓OPdChaWR͔
Ac-(m-fl)F-͓OPdChaWR͔
Ac-Y-͓OPdChaWR͔
H-͓OPdChaWR͔
22
3
3
3
3
3
3
3
4
3
3
3
3
2
2
6.43 Ϯ 0.07
6.32 Ϯ 0.03
6.01 Ϯ 0.04
6.35 Ϯ 0.08
6.07 Ϯ 0.07
6.60 Ϯ 0.06
5.98 Ϯ 0.08
6.07 Ϯ 0.05
4.43 Ϯ 0.11*
4.54 Ϯ 0.12*
6.12 Ϯ 0.05
5.17 Ϯ 0.15*
6.28 Ϯ 0.07
6.36 Ϯ 0.12
5.05 Ϯ 0.02*
⌽
0.38
0.47
0.96
0.45
0.84
0.25
1.10
0.84
36.8
28.7
0.76
6.8
16
3
7.58 Ϯ 0.06
7.47 Ϯ 0.13
6.54 Ϯ 0.15
7.64 Ϯ 0.17
7.42 Ϯ 0.07
7.21 Ϯ 0.07
N.D.
26
34
291
23
38
62
3
4
4
3
5
3
6
3
7
8
N.D.
9
N.D.
N.D.
10
11
12
13
14
15
16
17
18
3
2
3
6.82 Ϯ 0.24
6.32 Ϯ 0.12*
7.42 Ϯ 0.11
N.D.
151
480
38
0.52
0.44
8.9
N.D.
N.D.
Cin-͓OPdChaWR͔
HCin-͓OPdChaWR͔
3
3
5.00 Ϯ 0.01*
6.65 Ϯ 0.06
9.90
0.22
N.D.
3
7.51 Ϯ 0.12
31
a Concentration causing 50% inhibition of maximum binding of 125I-C5a to intact PMNLs.
b Concentration causing 50% inhibition of myeloperoxidase release from PMNLs induced by 100 nM C5a.
Mes, mesyl; Tos, tosyl; Suc, succinyl; MeSuc, methyl succinyl; Ahx, aminohexanoyl; Bn, benzoyl; Iso, isovaleroyl; Phg, phenylglycine; hPhe, homophenylalanine; (o-fl)F,
ortho-fluoro phenylalanine; (m-fl)F, meta-fluoro phenylalanine; Cin, cinnamoyl, Hcin, hydrocinnamoyl; N.D., not determined; n, number of experiments; *, significant at P Յ
0.05 difference from 1; ⌽, no C5aR affinity up to 1 mM.
TABLE 2
Structure-activity relationships at positions C and D of C5a antagonist peptide
Receptor Affinity
Antagonist Activity
a
b
No.
Peptide
n
Ϫlog IC50 Ϯ S.E.
IC50
n
Ϫlog IC50 Ϯ SE
IC50
␮M
nM
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
AcF-͓OVdChaWR͔
AcF-͓OFdChaWR͔
AcF-͓OIdChaWR͔
AcF-͓OHypdChaWR͔
AcF-͓OThpdChaWR͔
AcF-͓(␦-N-Me)OPdChaWR͔
AcF-͓OPGWR͔
AcF-͓OPdAlaWR͔
AcF-͓OPdValWR͔
AcF-͓OPdLeuWR͔
AcF-͓OPdNleWR͔
AcF-͓OPdPheWR͔
AcF-͓OP(N-Me)fWR͔
AcF-͓OPdhChaWR͔
AcF-͓OPdTyrWR͔
AcF-͓OPdArgWR͔
AcF-͓OPdTrpWR͔
AcF-͓OPdTicWR͔
AcF-͓OPdAicWR͔
2
3
3
3
2
3
3
3
3
3
3
5
3
3
3
3
2
3
3
6.08 Ϯ 0.05
5.61 Ϯ 0.0.7
5.53 Ϯ 0.12*
6.57 Ϯ 0.20
4.41 Ϯ 0.20*
6.45 Ϯ 0.07
2.97 Ϯ 0.24*
3.84 Ϯ 0.12*
4.89 Ϯ 0.22*
5.95 Ϯ 0.04
6.27 Ϯ 0.14
6.34 Ϯ 0.12
3.14 Ϯ 0.83*
6.41 Ϯ 0.12
5.64 Ϯ 0.17
0.84
2.43
2.94
0.27
N.D.
N.D.
N.D.
N.D.
N.D.
7.45 Ϯ 0.03
N.D.
4.43 Ϯ 0.07*
N.D.
N.D.
N.D.
7.65 Ϯ 0.18
N.D.
7.39 Ϯ 0.1
6.53 Ϯ 0.31
N.D.
N.D.
4.78 Ϯ 0.22*
N.D.
39
0.35
1082
145
13.0
1.13
0.53
0.46
721
0.39
2.2
Ͼ100
32
9.2
22.7
3
3
35
3700
3
22
3
3
41
300
4.50 Ϯ 0.15*
5.04 Ϯ 0.07*
4.64 Ϯ 0.39*
2
1665
a Concentration causing 50% inhibition of maximum binding of 125I-C5a to intact PMNLs.
b Concentration causing 50% inhibition of myeloperoxidase release from PMNLs induced by 100 nM C5a.
Hyp, 4-trans-hydroxyproline; Thp, 4-cis-thioproline; (␦-N-Me)O, ␦-N-methylated ornithine; (N-Me)f, N-methyl-D-phenylalanine; hCha, homocyclohexylalanine; Tic,
tetrahydroisoquinoline; Aic, aminoindanecarboxylic acid; N.D., not determined; n, number of experiments; *, significant (P Յ 0.05) difference from 1; ⌽, no C5aR affinity up
to 1 mM.

Potent Cyclic Antagonists of Human C5a Receptor
875
investigated (Table 3). Smaller aliphatic residues Leu (38) that any side chain that is Ͼ900 Å3 or Ͻ400 Å3 results in
and Cha (39) reduced receptor affinity 50ϫ and 31ϫ, respec- decreased receptor affinity. We attribute these limitations to
tively. Retaining the aromatic character of the indole moiety the receptor space, into which the side chain fits, being too
while reducing the size of the aromatic ring to an imidazole small to accommodate the ligand in the case of the large side
(40) resulted in a 62-fold loss in receptor affinity. In contrast, chains or too large to be adequately filled in the case of the
the smaller aromatic Phe (41) produced no significant change smaller side chains.
in receptor affinity, although extending the aromatic ring
Position F. Substitution of Arg in AcF-[OPdChaWR].
further from the cycle (42) caused a 30-fold reduction in Three analogs (50-52; Table 3) report on the importance of
affinity. N-Methylated Phe (43) significantly reduced affin- size and charge of the Arg side chain for affinity. Removing
ity, as did substitution with dTrp (44). Incorporating benzo- the positive charge through substitution with citrulline (50)
thiazolealanine (45), which retains the size and aromaticity caused a 15-fold reduction in receptor affinity; moving the
of the indole ring while exchanging sulfur for nitrogen, re- Arg side chain one carbon further from the cycle (51) de-
sulted in no change in receptor affinity. With the exception of creased affinity 3-fold, and a Lys substitution (52) produced
1-naphthyl (46), the incorporation of large bi- or tri-cyclic a 63-fold loss in receptor affinity. A terminal Arg in C5a has
groups (47–49) reduced affinity for the C5aR. Of the three been shown to be critical for its activity (DeMartino et al.,
high-affinity analogs, two (41, 46) were more potent antago- 1995) and is present in all C5a peptide analogs. Interaction
nists than the third (45).
between C5aR and the positively charged Arg side chain also
Figure 7D shows the variation in C5aR affinity for com- contributes significantly to affinity of these cyclic antago-
pounds containing an L-residue at this position, with calcu- nists. A low-affinity interaction can still occur when the pos-
lated steric volume of side chains. Volume was calculated by itive charge is removed, because the urea moiety in citrulline
rotation of all rotatable bonds through all sterically accessi- confers high affinity.
ble positions and therefore represents the maximal volume of
Positions D to F of AcF-[OPdChaWR]. Based on the
all side-chain–accessible space. An unambiguous minimum results for analogs with single substitutions at either dCha,
is not clear from this graph, however it does demonstrate Trp, or Arg (positions D-F), a number of compounds featuring
TABLE 3
Structure-activity relationships at position E or F of C5a antagonist peptides
Receptor Affinity
Antagonist Activity
a
b
No.
Peptide
n
Ϫlog IC50 Ϯ S.E.
IC50
n
Ϫlog IC50 Ϯ SE
IC50
␮M
nM
3000
4500
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
AcF-͓OPdChaLR͔
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4.72 Ϯ 0.13*
4.92 Ϯ 0.17*
4.63 Ϯ 0.10*
6.61 Ϯ 0.15
4.93 Ϯ 0.24*
4.47 Ϯ 0.11*
4.52 Ϯ 0.14*
6.55 Ϯ 0.28
6.15 Ϯ 0.01
5.80 Ϯ 0.06
5.43 Ϯ 0.39*
4.54 Ϯ 0.20*
5.22 Ϯ 0.06*
5.87 Ϯ 0.06
4.62 Ϯ 0.05*
18.9
11.9
23.5
3
3
5.52 Ϯ 0.43*
5.34 Ϯ 0.02*
N.D.
7.49 Ϯ 0.07
N.D.
N.D.
N.D.
6.76 Ϯ 0.27
7.33 Ϯ 0.22
N.D.
AcF-͓OPdChaChaR͔
AcF-͓OPdChaHR͔
AcF-͓OPdChaFR͔
0.25
3
32
AcF-͓OPdChahFR͔
AcF-͓OPdCha(N-Me)FR͔
AcF-͓OPdChawR͔
11.7
27.2
30.4
AcF-͓OPdChaBtaR͔
AcF-͓OPdCha1NalR͔
AcF-͓OPdCha2NalR͔
AcF-͓OPdChaTicR͔
AcF-͓OPdChaFlaR͔
AcF-͓OPdChaWCit͔
AcF-͓OPdChaWhArg͔
AcF-͓OPdChaWK͔
0.28
0.71
15.8
3.7
28.9
6.0
3
3
172
47
2
3
4.95 Ϯ 0.19*
6.10 Ϯ 0.09*
N.D.
1130
800
1.4
24.1
N.D.
N.D.
a Concentration causing 50% inhibition of maximum binding of 125I-C5a to intact PMNLs.
b Concentration causing 50% inhibition of myeloperoxidase release from PMNLs induced by 100 nM C5a.
hPhe, homophenylalanine; (N-Me)F, N-methyl phenylalanine; Bta, benzothiazolealanine; 1Nal, 1-Napthylalanine; 2-Nal, 2-napthylalanine; Tic, tetrahydroisoquinoline;
Fla, fluorenylalanine; Cit, citrulline; hArg, homoarginine; N.D., not determined; n, number of experiments; *, significant (P Յ 0.05) difference from 1; ⌽, no C5aR affinity
up to 1 mM.
TABLE 4
Structure-activity relationships at positions D and E or E and F of C5a antagonist peptides in combination
Receptor Affinity
Antagonist Activity
a
b
No.
Peptide
n
Ϫlog IC50 Ϯ S.E.
IC50
n
Ϫlog IC50 Ϯ SE
IC50
␮M
5.2
3.1
69.2
3.2
3.5
6.8
Ͼ1 mM
0.93
nM
53
54
55
56
57
58
59
60
AcF-͓OPfFR͔
5
3
3
3
3
3
3
3
5.29 Ϯ 0.22*
5.51 Ϯ 0.04*
4.16 Ϯ 0.13*
5.50 Ϯ 0.09*
5.46 Ϯ 0.10*
5.17 Ϯ 0.15*
3
5.28 Ϯ 0.10*
N.D.
5210
AcF-͓OPf1NalR͔
AcF-͓OPfYR͔
N.D.
AcF-͓OPdChaF(pCl)F͔
AcF-͓OPdChaF(pNH2)F͔
AcF-͓OPdChaFhF͔
AcF-͓OPdArgFR͔
AcF-͓OPGmbTyrR͔
N.D.
N.D.
N.D.
N.D.
6.03 Ϯ 0.08
2
6.17 Ϯ 0.09*
679
a Concentration causing 50% inhibition of maximum binding of 125I-C5a to intact PMNLs.
b Concentration causing 50% inhibition of myeloperoxidase release from PMNLs induced by 100 nM C5a.
(pCl)F, para-chlorophenylalanine; (pNH2)F, para-aminophenylalanine; hPhe, homophenylalanine; mbTyr, metabenzyltyrosine; N.D., not determined; n, number of
experiments; *, significant (P Յ 0.05) difference from 1; ⌽, no C5aR affinity up to 1 mM.

876
March et al.
double changes were synthesized (Table 4). Three analogs Solution Structures of 41 and 53
incorporated dPhe at position D and either Phe (53),
We determined solution structures of active (41) and inac-
1-napthylalanine (54), or Tyr (55) at position E. Analogs
56–58 showed significantly reduced binding affinity. A fourth
compound incorporating dArg (59) at position D and Phe at
position E had no detectable affinity up to 1 mM. Complete
removal of the dCha sidechain via a Gly substitution coupled
with incorporation of a meta-benzyl tyrosine at the Trp posi-
tion (60) did not have a significant effect on receptor affinity.
Analogs 53 and 60 were less potent antagonists.
Attempts to simultaneously change dCha and Trp residues
to the most favorable residues identified from above led to
reduced affinity and activity. Either there is a complex spa-
tial interaction between the groups at these sites or the
receptor responds cooperatively to simultaneous changes on
the ligand. The receptor affinity and antagonist activity of
the double Phe substitution does not correlate with the other
AcF-[OPdChaWR] analogs. The disproportional loss in activ-
ity seen with these substitutions underlines the complexity of
cooperative protein-ligand interactions. Optimal binding at
one location can become unfavorable if changes are made
elsewhere because of the interactions that are induced after
compound binding. Ligand-protein interactions are not rigid
‘lock and key’ contacts that can be readily optimized. Both
the receptor and the antagonist are flexible and changes at
one site can have unintended and unpredictable effects at
another site.
tive (53) compounds to investigate whether cyclic backbone
shape is altered by side-chain variation. This is important
because side-chain bulk can either influence ligand fit to the
receptor directly or indirectly, via changes to macrocycle
shape.
Structure of 41 (Ac-Phe[Orn-Pro-dCha-Phe-Arg]). 1H
NMR spectra for 41 in DMSO-d6 were similar to those for 1
with seven well resolved amide NH resonances. Indicators of
local structure were the 3JH␣,NH coupling constants for Phe2
(8.5 Hz), D-Cha5 (4.6 Hz), and Arg7 (8.3 Hz). Like 1, 41 shows
low temperature coefficients (␦⌬/K) for Arg ␣NH (2.3 ppb/K),
Orn ␦NH (2.4 ppb/K), and Arg ␦NH (2.7 ppb/K). Stereospe-
cific assignments of ␤-protons and ␹-1 dihedral restraints for
Phe2 and Phe6 were determined as for 1. The solution struc-
ture of 41 was calculated from 79 NOE restraints and 5
dihedral angle restraints to produce 20 convergent lowest
energy structures (⌬E Ͻ0.3 kJ/mol) shown in Fig. 8A. The
root-mean-square deviation over all backbone atoms of the
cycle was 0.12 Å. The Ac-Phe portion of the molecule is less
defined than in 1, because of the difficulty in unambiguously
assigning NOEs from aromatic residues to Phe2 or Phe6. The
Pro C␣. . . Arg C␣distance (5.7 Å) was shorter than in 1
(6.9Å), and this may reflect a higher population of Type IIЈ
␤-turn conformers.
Fig. 7. Volume-Affinity Relationships. Calculated steric volume of the side chain versus measured affinity for PMNLs of analogs of 1 with
replacements for Phe (10–17) (A); Pro (19–24) (B); D-Cha (25-37) (C); and Trp (38-49) (D).

Potent Cyclic Antagonists of Human C5a Receptor
877
Structure of 53 (Ac-Phe[Orn-Pro-dPhe-Phe-Arg]). In term rat models of sepsis (Short et al., 1999; Huber-Lang et
3
contrast to 1 and 41, JH␣,NH coupling constant for D-Phe5 al., 2002), reverse passive Arthus (Strachan et al., 2000)
(6.5 Hz) was not indicative of structure, whereas Phe2 (8.3 dermal inflammation (Strachan et al., 2001), arthritis (Woo-
Hz) and Arg7 (8.3 Hz) were similarly high and used to derive druff et al., 2002), gastrointestinal and renal ischemia-reper-
two ␾ dihedral angle restraints. Variable-temperature NMR fusion injury (Arumugam et al., 2002, 2003), and colitis (Woo-
experiments indicated low-temperature coefficients for Arg druff et al., 2003). This anti-inflammatory activity is partly
␣NH (1.7 ppb/K), D-Phe5 (2.4 ppb/K), and Arg ␦NH (2.7 related to indirect inhibition of the release of proinflamma-
ppb/K). All three possible sets of stereospecific H␤-assign- tory cytokines (e.g., TNF␣, IL-6) from PMNLs, monocytes,
ments (Phe2, dPhe5, Phe6 AMX spin systems) and three ␹-1 and other immune cells that we have directly monitored
dihedral restraints could be extracted from 1D 1H and (Haynes et al., 2000; Strachan et al., 2000; Arumugam et al.,
ECOSY data. NMR structure calculations based on 71 NOE 2002).
restraints and the 5 dihedral restraints yielded a highly
In the absence of detailed structural knowledge of the
converged family of structures (Fig. 8B) with cycle backbone transmembrane protein C5aR and its active site, structure-
pairwise root-mean-square deviation ϭ 0.15 Å. The 20 lowest activity relationships for ligands that bind to the human C5a
energy structures (Fig. 8B) describe an inverse ␥-turn about receptor can provide valuable information for defining the
Orn-Pro-D-Phe and suggest a ␲-␲ interaction between D-Phe5 agonist/antagonist binding site and for the development of
and Phe6. The D-Phe5 ␣NH does not exchange rapidly with potent C5aR agonists and antagonists. We reported herein
D2O; Arg and Trp ␣NHs were also somewhat slow to ex- the refined solution structure (Fig. 3) of our prototype cyclic
change.
antagonist AcF[OPdChaWR] (1) consisting of a cyclic scaffold
Comparision of Structures. Fig. 8C shows a superimpo- that supports affinity-determining and antagonist-defining
sition of the average calculated structures for 1, 41, and 53 side chain moieties. Structural comparisons between cyclic
using all macrocycle backbone heavy atoms. The three struc- antagonist 1, the C terminus of C5a, and a small acyclic
tures show remarkable similarity in the position of their peptide agonist and antagonist (Fig. 4) suggest the impor-
cyclic C␣-C␤ side chain vectors, suggesting that subtle alter- tance of a common turn conformation for receptor binding.
ations to the cyclic backbone may not dramatically disrupt The turn shape in 1 is also displayed by cyclic analogs [e.g.,
the projections of the side chains. Thus, the steric bulk of side 41 and others (M. J. Stoermer and D. P. Fairlie, unpublished
chains will probably directly determine affinity and antago- results)]. Together with this structural similarity, competi-
nism.
tion between cyclic antagonist 1 and either C5a or acyclic
peptide agonist or antagonists for the C5aR on whole PMNLs
(Fig. 2) is consistent with a common bioactive shape for the
antagonist, agonist and C terminus of C5a, and supports the
same or a similar binding site on C5aR.
Discussion
In contrast to reported peptide analogs of the C terminus of
C5a, cyclic antagonist 1 is a potent antagonist of the C5aR on
human PMNLs, and it is devoid of agonist activity, as mon-
itored in sensitive assays for chemotaxis and polarization
(Haynes et al., 2000). It is also highly receptor selective,
failing even at 100 ␮M concentrations to antagonize myelo-
peroxidase release from PMNLs stimulated with fMet-Leu-
Phe, leukotriene B4, platelet-activating factor (Finch et al.,
1999), C3a, or IL-8 (N. Paczkowski, K. Wadi, and S. M.
Taylor, unpublished data). Unlike linear peptides and com-
plement-targeting proteins, cyclic antagonist 1 is chemically
stable to peptidase degradation in blood and gastric fluids
(data not shown) and is both small enough and sufficiently
lipophilic to be orally active at Յ1 mg/kg/day in vivo (Woo-
druff et al., 2002). We have found that 1 has potent and
efficacious anti-inflammatory activity in short- and long-
To understand the influence of each component of 1 on
receptor affinity and antagonist potency, structure-activity
relationships were investigated for 60 cyclic analogs of 1
evaluated against the C5aR on human PMNLs by radioli-
gand binding for receptor affinity and by enzyme release for
antagonist potency. Twenty compounds had receptor affini-
ties IC50 Ͻ 1 ␮M. There was a positive general correlation
between affinity and antagonism for the majority of analogs
examined, allowing insights to size and shape limitations at
each antagonist position. The solution structures for cyclic
analogs of 1 [41, 53, and others not shown (M. J. Stoermer
and D. P. Fairlie, unpublished results)] reveal a common
cyclic backbone shape, supporting the idea that side chain
variations directly affect ligand fit to the receptor rather than
indirectly by altering macrocyclic shape. This is further sup-
ported by relationships between calculated steric volume of
side chains and measured affinity for PMNLs (Fig. 7) that
reveal optimal side chain bulk at each site on the cyclic
antagonist scaffold. Whereas position A (Ac) can be varied
widely without loss in potency, positions B (Phe), C (Pro), D
(D-Cha), and F (Arg) are likely determinants of receptor af-
finity rather than antagonist potency per se, and position E
(Trp) alone seems to mechanistically dictate whether the
cyclic scaffold will be an antagonist or agonist. The latter
observation is similar to what was learned from acyclic pep-
tide ligands for C5a (Konteatis et al., 1994), except that side
chains other than the indole ring of Trp (e.g., Phe) on the
cyclic scaffold also produce potent antagonism herein and the
Arg needs to have L- rather than the D-stereochemistry
(Wong et al., 1998) preferred for acyclic antagonists. Inter-
Fig. 8. Superimposition of Structures. Overlay of 20 lowest energy struc-
tures of 41 (A) and 53 (B) (Phe2, blue; Pro4, red; dCha5, yellow; Phe6,
brown; Arg7, white but not shown for clarity in 53). Putative ␲-␲ inter-
action and inverse ␥-turn defining H-bond around Pro4 are shown for 53.
C, overlay of averaged structures for cyclic backbones of 1 (red), 41
(yellow), and 53 (blue, ␤-carbons for residues 4–7 in cyan).

878
March et al.
Ember JA, Jagels MA, and Hugli TE (1998). Characterization of complement ana-
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estingly the NMR structure of 53 supports a similar cyclic
scaffold to that in 1 but also suggests D-Phe. . . . L-Phe
␲-stacking interaction in DMSO-d6. In a hydrophobic recep-
tor environment, this may interfere with ligand fitting asso-
ciated with antagonism because compound 53 was not a
potent antagonist.
The availability of potent and selective antagonists of the
C5aR can help to establish the pathophysiology of C5a and to
probe the selective pathological responses to this component
of the complement network. C5a formation or action is inhib-
ited in vivo by soluble recombinant complement receptor type
I (sCR1) (Hill et al., 1992), C5 and C5a antibodies (Amster-
dam et al., 1995; Wang et al., 1995), or recombinant C5a
polypeptides (Pellas et al., 1998) but these large molecules
have inherent limitations as drugs, including poor bioavail-
ability, low metabolic stability, potential immunogenicity,
and manufacturing costs. Here we reported a series of novel
small molecules, derived from the solution structure of just a
small portion of the bioactive surface of C5a, 20 of which are
potent (IC50 Ͻ 1 ␮M) and selective antagonists of human
C5aR. This class of cyclic compounds shows promising anti-
inflammatory activity in vivo even when given orally, so
structure-activity relationships reported herein serve to de-
fine a pharmacophore for potential development of C5aR-
directed small molecule anti-inflammatory drugs. In conjunc-
tion with site-directed mutagenesis studies of the receptor,
these data may help to better define the mechanism of ago-
nism versus antagonism, enabling a better understanding of
the origin and control of C5a-mediated disease pathology.
Rational drug design based on protein-protein interactions
has often proven to be frustrating for medicinal chemists,
particularly the development of small nonpeptidic antago-
nists of protein hormones and cytokines. Some important
pathogenic factors, such as TNF␣, can be controlled by an-
tagonist drugs now in the clinic (e.g., Enbrel), but they are
mainly protein-based therapies requiring parenteral admin-
istration. The immediate challenge is to understand surface
protein-protein interactions in sufficient detail to enable the
development of small orally active antagonists. The struc-
ture- and ligand-based approach demonstrated here for a G
protein-coupled receptor, uses constrained cyclic molecules
as intermediates in the development of nonpeptidic drugs.
Such cyclic intermediates can provide valuable insight to the
receptor-binding ligand structure and are sometimes suit-
able in their own right as orally active molecular probes for in
vivo target validation, as in this case.
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Address correspondence to: Professor David Fairlie, Institute for Molecular
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