Small molecular probes for G-protein-coupled C5a receptors: Conformationally constrained antagonists derived from the C terminus of the human plasma protein C5a

Article abstract of DOI:10.1021/jm9800651

Activation of the human complement system of plasma proteins in response to infection or injury produces a 4-helix bundle glycoprotein (74 amino acids) known as C5a. C5a binds to G-protein-coupled receptors on cell surfaces triggering receptor-ligand inte

Full text of DOI:10.1021/jm9800651

J . Med. Chem. 1998, 41, 3417-3425
3417
Sm a ll Molecu la r P r obes for G-P r otein -Cou p led C5a Recep tor s:
Con for m a tion a lly Con str a in ed An ta gon ists Der ived fr om th e C Ter m in u s of th e
Hu m a n P la sm a P r otein C5a
†
‡
§
†
‡
Allan K. Wong, Angela M. Finch, Gregory K. Pierens, David J . Craik, Stephen M. Taylor, and
,
†
David P. Fairlie*
Centre for Drug Design and Development and Department of Physiology and Pharmacology, University of Queensland,
Brisbane, Qld 4072, Australia, and School of Science, Griffith University, Nathan Campus, Brisbane, Qld 4111, Australia
Received J anuary 28, 1998
Activation of the human complement system of plasma proteins in response to infection or
injury produces a 4-helix bundle glycoprotein (74 amino acids) known as C5a. C5a binds to
G-protein-coupled receptors on cell surfaces triggering receptor-ligand internalization, signal
transduction, and powerful inflammatory responses. Since excessive levels of C5a are associated
with autoimmune and chronic inflammatory disorders, inhibitors of receptor activation may
have therapeutic potential. We now report solution structures and receptor-binding and
antagonist activities for some of the first small molecule antagonists of C5a derived from its
2
hexapeptide C terminus. The antagonist NMe-Phe-Lys-Pro-D-Cha-Trp-D-Arg-CO H (1) surpris-
1
ingly shows an unusually well-defined solution structure as determined by H NMR spectros-
copy. This is one of the smallest acyclic peptides found to possess a defined solution
conformation, which can be explained by the constraining role of intramolecular hydrogen
bonding. NOE and coupling constant data, slow deuterium exchange, and a low dependence
on temperature for the chemical shift of the D-Cha-NH strongly indicate an inverse γ turn
stabilized by a D-Cha-NH‚‚‚OC-Lys hydrogen bond. Smaller conformational populations are
associated with a hydrogen bond between Trp-NH‚‚‚OC-Lys, defining a type II â turn distorted
by the inverse γ turn incorporated within it. An excellent correlation between receptor-affinity
and antagonist activity is indicated for a limited set of synthetic peptides. Conversion of the
C-terminal carboxylate of 1 to an amide decreases antagonist potency 5-fold, but potency is
increased up to 10-fold over 1 if the amide bond is made between the C-terminal carboxylate
and a Lys/Orn side chain to form a cyclic analogue. The solution structure of cycle 6 also
shows γ and â turns; however, the latter occurs in a different position, and there are clear
conformational changes in 6 vs 1 that result in enhanced activity. These results indicate that
potent C5a antagonists can be developed by targeting site 2 alone of the C5a receptor and
define a novel pharmacophore for developing powerful receptor probes or drug candidates.
In tr od u ction
Activation of the human complement system,1,2
a
cascade of plasma proteins involved in immunological
defense against infection and injury, contributes sig-
nificantly to the pathogenesis of numerous acute and
chronic diseases.3 Although broad features of comple-
ment activation are known, mechanistic details remain
poorly understood. A very potent inflammatory media-
tor generated during complement activation is a 74-
4
residue glycoprotein known as C5a. This peptide binds
to a G-protein-coupled receptor (C5aR)4 of the rhodop-
,5
sin superfamily of GTP-linked binding proteins, with
the characteristic seven transmembrane helices (Figure
5
1
). C5a receptors are found on a variety of tissues and
cells including mast cells, neutrophils, monocytes, mac-
rophages, and vascular endothelial cells. C5a recruits
4
F igu r e 1. Two-site model for binding of C5a to its G-protein-
coupled receptor C5aR.
*
Corresponding author. Fax: +61-7-33651990. E-mail: d.fairlie@
neutrophils and macrophages to sites of injury and
alters their morphology, induces degranulation, in-
creases calcium mobilization, vascular permeability
mailbox.uq.edu.au.
†
Centre for Drug Design and Development, University of Queens-
land.
‡
Department of Physiology and Pharmacology, University of Queens-
(edema), and neutrophil adhesiveness, contracts smooth
land.
§
Griffith University.
muscle, stimulates release of inflammatory mediators
S0022-2623(98)00065-X CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/08/1998

3
418 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Wong et al.
Ta ble 1. 1H NMR Assignmentsa for 1 in d6-DMSO
residue
HNb
8.83
HR
Hâ
Hγ
1.32
1.88, 1.78
0.76
others
c
d
g
MePhe
Lys
Pro
D-Cha
Trp
D-Arg
4.06
4.54
4.30
4.35
4.65
4.20
3.09, 3.06
1.74, 1.55
2.04, 1.74
1.19, 1.06
3.11, 2.94
1.73, 1.58
7.17, 7.29; 2.46; 8.98
e
f
g
1.51; 2.74, 7.76 (NH2)
3.61, 3.48e
e
f
7.91
8.01
8.44
1.43, 1.08; 1.61, 1.58; 0.73
6.97, 7.06, 7.13, 7.32, 7.65; 10.80
c
g
e
g
1.42
3.08; 7.60
a
b
3
Referenced to residual d5-DMSO at 2.50 ppm. Amide NHs, J NH-CRH values (Hz): 7.91 (Lys), 7.77 (D-Arg), 8.34 (Trp), 8.53 (D-Cha).
c
Aromatics. N-Me. Hδ. f Hꢀ. NH/NH2 amine.
d
e
g
(histamine, TNF-R, IL-1, IL-6, IL-8, prostaglandins, and
leukotrienes) and lysosomal enzymes, promotes forma-
tion of oxygen radicals, and enhances antibody produc-
tion.3 Overexpression or underregulation of C5a is
implicated in the pathogenesis of immunoinflammatory
diseases such as rheumatoid arthritis, adult respiratory
distress syndrome (ARDS), systemic lupus erythema-
tosus, tissue graft rejection, ischemic heart disease,
reperfusion injury, septic shock, psoriasis, gingivitis,
atherosclerosis, lung injury, and extracorporeal post-
dialysis syndrome.3 One strategy for developing thera-
peutics for such diseases is to interfere with the action
of C5a, and we now describe chemical and structural
studies aimed at the design of small molecule probes
that inhibit the interactions of C5a with its receptor.
The NMR solution structure of the protein C5a has
6
been determined, and although it is essentially a
4
-helix bundle, the C-terminal end was found to be
unstructured. Indeed, this conformational flexibility in
the C-terminus has made structure-function studies
extremely difficult to interpret. C5a is thought to
1
F igu r e 2. Stacked plots of H NMR spectra showing time-
dependent decay of amide-NH resonances for Trp (8.1 ppm)
and D-Cha (7.9 ppm), as well as Lys-NH
of 1 in d -DMSO containing D
+
7
,8
3
(8.07 ppm), residues
interact with its receptor at two sites (Figure 1).
An
6
2
O after 10 min (bottom) and
N-terminal interhelical positively charged region of C5a
binds to a negatively charged extracellular domain of
C5aR (site 1), while the C-terminal “effector” region of
C5a is thought to bind with an interhelical domain of
the receptor (site 2) and is responsible for receptor
activation leading to signal transduction. Numerous
then 25, 40, 55, 70, 130, 190, 250, 385, and 520 min.
resonances for amide-NH protons (Table 1). To estab-
lish their possible involvement in intramolecular hy-
drogen bonds, a deuterium exchange experiment was
performed by adding a 10-fold excess of D2O to the
solution. Two of the amide-NH doublets disappeared
immediately along with the resonance attributable to
the N-terminal methylamine-NH protons. However, the
other two amide-NH resonances, as well as a broad
resonance at 8.07 ppm, persisted for up to 6.5 h (Figure
short peptide derivatives of the C terminus of C5a have
been found to be agonists9
-20
of C5a, while only one
small molecule (1, MeFKP-D-Cha-Wr) is reported to be
a full antagonist with no agonist activity2 in isolated
cells and membranes.
0
We now report the surprising observation that the
small hexapeptide 1 and a new constrained cyclic
analogue 6 have well-defined solution structures. These
compounds and several analogues have been examined
for their receptor-binding affinities and antagonist
activities using intact human polymorphonuclear (PMN)
cells. The results demonstrate for the first time that
certain turn conformations are responsible for antago-
nist activity, providing new insight to the binding of C5a
to its receptor. This information should be useful for
the design and development of even more potent C5a
antagonists or agonists as molecular probes of the
complement system or as potential drug candidates. The
results may also be valuable for the design of small
molecules that bind to other G-protein-coupled receptors
which are important drug targets due to their crucial
roles in signal transduction.21
2
). These three slowly exchanging protons are assigned
(Table 1) to the amide NHs of Trp and D-Cha and to
the side-chain amine of Lys. The amine assignment was
established from the TOCSY spectrum where cross-
peaks were observed between the protonated amine and
the ꢀ, δ, and γ CH2 protons. The slow exchange
behavior for the three NH protons indicates a degree of
protection from solvent that is indicative of intramo-
lecular hydrogen bonding.
The temperature independence of the amide chemical
22
shifts (<3 ppb/deg) is another useful marker of hy-
drogen bonding. A temperature dependence (20-60 °C)
study of the amide-NH chemical shifts (∆δ/T ) 2.5 ppb/
deg, D-Cha-NH; 6.0 ppb/deg, Trp-NH; 6.5 ppb/deg, Lys-
NH; 8.7 ppb/deg, Arg-NH; Figure 3, top) unambiguously
confirms the involvement of only the D-Cha-NH in
intramolecular hydrogen bonding. Thus the D-Cha-NH
hydrogen bond appears to be present in a majority
population of conformers. On the other hand, the slow
exchange behavior but high-temperature coefficient (∆δ/
T) of the amide Trp-NH most likely reflects solvent
Resu lts
1
NMR Str u ctu r e of 1. The 1D H NMR spectrum of
peptide 1 in d6-DMSO at 24 °C shows four distinct

Probes for G-Protein-Coupled C5a Receptors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3419
F igu r e 4. Backbone and proline heavy atoms of twenty lowest
energy minimized NMR structures of 1 in d -DMSO (24 °C).
6
Residues are labeled at CR positions. Distances between heavy
atoms discussed in text: D-Cha-NH‚‚‚OC-Lys (3.16 Å) and Trp-
NH‚‚‚OC-Lys (4.97 Å).
any minor populations containing a cis form. This lack
of evidence further supports the conclusion that peptide
1
exists in a well-defined conformation in solution and
that there are not multiple slowly exchanging conform-
ers present.
The remaining NOEs detected in 1 also supported the
existence of a well-defined conformer and were used to
derive a set of 55 interproton distance restraints. These
data, combined with the dihedral angle restraints for
the D-Cha and Trp φ angles based on the observed
coupling constants shown in Table 1, were used to
generate a set of 50 structures, using a simulated
annealing algorithm within the program X-PLOR. It
is remarkable that, in the absence of any explicit
hydrogen bonding restraints, the majority of these
structures showed a good convergence to a folded
conformation. This conformation is represented in
Figure 4 by a superposition of 20 of the lowest energy
structures. The pairwise rmsd is 1.01 Å for all of the
backbone CR, N, and C atoms in this family of struc-
tures, but this reduces to 0.30 Å for the turn region Lys
to Trp. This difference reflects the mobility of the
terminal residues Phe and Arg, as seen in Figure 4. In
these structures, the D-Cha-NH and Lys-CO are within
hydrogen bonding distance, consistent with the slow
exchange and temperature coefficient data for this
amide proton.
F igu r e 3. Temperature dependence (20-65 °C) of chemical
6
shifts for amide-NH protons of 1 (top) and 6 (bottom) in d -
DMSO. Legend: (9) D-Arg, (0) D-Arg (side chain), (2) Trp, (b)
D-Cha, ([) Lys, ([) Orn, (O) Orn-(CH -NH, (4) Phe.
2 3
)
protection/intramolecular hydrogen bonding in only a
small population of solution conformers.
The 3J NH-HR coupling constants for the amide-NH
protons were measured from the 1D spectrum and are
reported in Table 1. In small linear peptides it is
unusual for backbone coupling constants to differ from
2
3
the conformationally averaged value of ∼7 Hz, and
thus it is significant that the two constants for D-Cha
and Trp exceed this value (Table 1). This observation
strongly indicates the presence of a well-defined local
structure near these residues, and the existence of this
structure is supported by the involvement of the D-Cha
residue in hydrogen bonding interactions.
Medium range NOEs often provide the strongest
evidence for the presence of folded conformers of pep-
2
3
1
tides in solution, and thus a series of 2D H NMR
spectra were recorded to assign all resonances and to
determine if such NOE interactions are present. TOC-
SY and DQ-COSY experiments were used to identify
residue types, while sequential assignments were made
from the analysis of NOESY data. Among the identified
NOEs for this small peptide are several that indicate
the presence of a folded conformer. These include i, i
The hydrogen bond suggested by the NOE, the
coupling constant data, the temperature-independent
chemical shift, and the slow exchange behavior defines
a seven-membered ring or an inverse γ turn formed by
the Lys-Pro-D-Cha residues. The D-Cha-NH‚‚‚OC heavy
atom distance is 3.16 Å and the N-H‚‚‚O angle is 142°.
2
4
+
2 NOEs for D-Cha to Lys and Pro to Trp, and a D-Cha-
Hydrogen bonds in γ turns are often weak, and thus
it is remarkable that such a bond is detected in this
small peptide. The other NH proton, Trp-NH, that
showed evidence for partial involvement in hydrogen
bonding is relatively well aligned with a suitable ac-
ceptor group, Lys-CO, in the family of structures in
Figure 4. However, it is not within hydrogen bonding
distance (4.97 Å). Trial calculations in which hydrogen
Trp NH-NH NOE which suggest a turn in the peptide
centered on the Pro-D-Cha residues.
Other significant NOEs were observed between Lys-
RH and the two Pro-δH protons. These confirm that
the Lys-Pro bond exists in the trans conformation.
Although the presence of cis Pro bonds is not uncommon
in small peptides, in this case there is no evidence of

3
420 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Wong et al.
Ta ble 2. Receptor Binding Affinities and Antagonist Activities
in Human PMNsa
IC50, µM
receptor antagonist agonist
affinityb
potencyc activity
d
compd
1
2
3
4
5
6
7
8
MeFKP(D-Cha)Wr
MeFKP(D-Cha)Wr-CONH2 14 (5)
1.8 (15)
0.085 (9)
0.5 (3)
0.7 (3)
>1000 (3)
0.090 (5)
0.012 (4)
no
no
no
nd
no
no
MeFKP(D-Cha)WR
MeFKPLWR
11 (5)
144 (1)
3.2 (4)
0.28 (6)
>1000f
0.0008 (9)
AcF[KP-D-Cha-Wr]e
AcF[ÃP-D-Cha-Wr]e
C5a65-74, ISHKDMQLGR
C5a
yes
a
Number of experiments in parentheses. Corrected for amino
b
acid content. nd ) not determined. Fifty percent reduction in
binding of 1 I-C5a to intact human PMNs. c Fifty percent reduc-
tion in myeloperoxidase secretion from human PMNs mediated
by 100 nM C5a. Agonist activity in dose range 0.1 nM to 1 mM.
Cyclic compounds (Figure 5). O is symbol for ornithine. Ref 10.
25
d
e
f
observed for intact cells. Thus, although the hexapep-
tide 1 has previously been reported to compete with C5a
binding to receptors on isolated PMN membranes (IC50
1
9
F igu r e 5. Possible H bonding in 1 (top) and 6 (bottom) from
NMR spectra in d -DMSO.
70 nM), we decided to reexamine this competition
using intact PMN cells since this is physiologically more
relevant. Under these conditions we establish that 1
binds with a much lower receptor affinity of IC50 1.8
µM (Figure 6a, Table 2) but confirm that it is indeed a
full antagonist with no agonist properties. The relative
affinity of 1 for the C5aR in intact PMNs was, however,
similar to that previously reported for isolated PMN
6
bonding restraints were included showed that a Trp-
NH‚‚‚OC-Lys hydrogen bond can be readily accom-
modated within the experimental restraint set. This is
consistent with the evidence presented earlier that such
a hydrogen bond is present in a small population of
solution conformers. Hydrogen bonding involving the
Lys-amine side chain was also implicated from the H/D
exchange experiment, but a potential acceptor group
could not be distinguished from the structures. The
hydrogen bonds for the proposed â and γ turns are
indicated in Figure 5.
1
9
membranes.
We find that 1 shows antagonist activity against both
C5a (Figure 6b) and a C-terminal peptide agonist
decapeptide analogue YSFKPMPLaR (receptor affinity
1
7
IC50 6 µM) of the C terminus of C5a65-74 (antagonism
not shown), suggesting that it acts on site 2 of the
receptor (Figure 1). These two small molecules have
similar micromolar affinities for the receptor C5aR on
intact polymorphonuclear leukocytes despite their op-
posing modes of action. A striking feature of the data
in Table 2 is the excellent linear correlation (r ) 0.93)
between the log of the binding affinities and the log of
the antagonist potencies for this limited set of site 2
antagonists (compounds 1-6, Table 2). Since receptor
affinity and antagonist activity are directly proportional
here, the experimentally simpler approach of measuring
receptor binding can also be used to quickly estimate
the antagonist activity, provided that there is no
evidence of agonist activity. We have also tested some
compounds in human umbilical artery, and our prelimi-
nary results indicate that they are also antagonists in
this preparation (Finch and Taylor, unpublished).
It has been proposed that the C-terminal carboxylate
of C5a and of agonist peptides is essential for activity
due to its interaction with a positively charged Arg206
The fact that the Trp-NH and Lys-NH3+ hydrogen
bonds were not formally detected in the structures may
reflect the well-known difficulty of defining solution
conformers of small peptides because of the relative
2
5
weakness of NOEs due to correlation time effects or
may reflect a degree of conformational averaging. In
any case, a hydrogen bond is not required to define a â
turn, and analysis of the CR-CR distances (∼4.6 Å) in
the structures in Figure 4 confirms the fact that the
geometry of the four residues Lys-Pro-D-Cha-Trp does
satisfy the criteria26 for a â turn.
The i + 1 φ and ψ angles for 1 (φ2 ) -86°, ψ2 ) 33°)
are consistent with those reported for inverse γ turns
(
φ2 ) -70 to -85°, ψ2 ) 60-70°)27 and both i + 1 and
i + 2 φ and ψ angles for 1 (Table 4) most closely match
a type II â turn,28 the differences being attributed to
distortion by the presence of the γ turn wholly within
the â turn (Figure 5). To our knowledge, this is the first
example of an intramolecular hydrogen bond between
residues within a â turn, although there are many
examples of hydrogen bonds between a residue within
the “10-membered ring” of a â turn and a residue
outside of it.28
2
0
of the receptor.
We confirm here (Table 2) that the
C-terminal carboxylate is indeed important for affinity,
since the amide 2 shows a 5-fold reduction in both
receptor affinity and antagonist activity (2 vs 1, Table
2). Changing chirality of the Arg-CR (3 vs 1) causes a
similar reduction in activities, and replacing D-Cha with
the L-Leu residue (4) is also detrimental to receptor
binding (Table 2). Clearly the hydrophobic D-cyclohexyl-
alanine residue is not only important in defining the γ
turn through hydrogen bonding but also directs its
Str u ctu r e-Activity Rela tion sh ip s. Others9-13,19,20
have previously claimed nanomolar affinities of C5a
agonist peptides for the PMN C5aR, but their assays
used membranes, isolated from PMN cells, for which
affinities of both C5a and peptide analogues are
9
-13,30
typically
2 orders of magnitude greater than those

Probes for G-Protein-Coupled C5a Receptors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3421
Ta ble 3. 1H NMR Assignmentsa for 6 in d6-DMSO
residue
HNb
HR
Hâ
Hγ
others
AcPhe
Orn
Pro
D-Cha
Trp
D-Arg
8.06
8.23
4.55
4.47
4.55
4.50
4.30
4.02
2.71, 2.94
1.56, 1.70
1.71, 2.26
1.41, 1.43
3.0
1.76 (Me); 7.20, 7.29c
d
1.42
1.88
1.06
2.85, 3.27; 7.37 (NHꢀ)
3.48, 3.59d
d
7.38
8.50
8.51
0.78, 0.84; 1.62, 1.65, 1.48, 1.58
c
6.98, 7.05, 7.33, 7.58; 10.83 (NH)
d
1.34, 1.81
1.20
2.97, 2.97; 7.39 (NHꢀ)
a
Referenced to residual d5-DMSO at 2.50 ppm. Amide NHs, 3J NH-CRH (Hz): 8.41 (Phe), 6.99 (Orn), 8.02 (D-Cha), 4.81 (Trp), 8.28
b
c
d
(
D-Arg). Aromatic CHs. Hδ.
Ta ble 4. Backbone φ and ψ Anglesa (deg) for 1 and 6
Pro D-Cha Trp
basis for the improved receptor affinity and antagonist
activity of cyclic 6 over acyclic 1, we determined the
solution structure of 6 for comparison with 1.
D-Arg
compd
φ
ψ
φ
ψ
φ
ψ
φ
Ψ
1
NMR Str u ctu r e of 6. The 1D H NMR spectral data
1
6
6
-86
-68
-64
33
-8
-10
97
165
170
-45
-54
-98
-144
-58
-77
124
90
158
111
69
69
for 6 (Table 3) showed three NH protons which have a
chemical shift/temperature coefficient (∆δ/T) < 3 ppb/
deg, characteristic of hydrogen bonding (Figure 3).
These NH protons are assigned to the side chain NH
protons of D-Arg and Orn, as well as to that of the
backbone amide of D-Cha. The latter proton has a
significantly lower temperature coefficient (∆δ/T ) 1
ppb/deg) for 6 than for the open chain derivative 1
a
b
-3
-2
a
For the average structure, restrained by NOE data and
dihedral angles but no hydrogen bond restraints.
(Figure 3), suggesting a strengthening of the hydrogen
bond (Lys-CO‚‚‚HN-D-Cha) which defines a γ turn for
the Orn-Pro-D-Cha residues. Four of the five backbone
3
J NH-HR coupling constants are either >8 Hz or <5 Hz
(Table 3), suggesting a well-defined backbone conforma-
tion and a lack of significant conformational averaging.
2
D TOCSY and NOESY spectra of 6 were recorded
and assigned as described for 1. The NOE data are
consistent with the cyclic derivative adopting a well-
defined solution conformation. In combination with the
coupling constant data, several highly characteristic
medium range NOEs confirm the presence of a γ turn
seen in 1 and also suggest a new â turn involving the
residues D-Cha-Trp-D-Arg-Orn. In particular, the pres-
ence of small and large coupling constants for the two
central residues, Trp and D-Arg, respectively, together
with an NH-NHꢀ NOE between D-Arg and Orn and an
RH-NHꢀ NOE between Trp and Orn suggest a type II
â turn as illustrated for 6 in Figure 5. This fits well
with the observed low-temperature coefficient of the
Orn-Nꢀ proton.
From a set of 89 NOE-based distance restraints and
four dihedral angle (φ) constraints, a family of 50
structures was calculated. The structures converged
with high precision, although there were two distinct
conformational populations which are shown in Figure
7
. For conformer 6a the rmsd over all of the backbone
atoms of the cycle is 0.15 Å, while for conformer 6b it is
.28 Å. These conformers differ mainly in the backbone
F igu r e 6. (a, top) Receptor binding. Inhibition of binding of
1
25
I-C5a to human PMNs by 1 (b), 2 (4), 3 (2), 6 (O). (b, bottom)
C5a antagonist potency as inhibition of myeloperoxidase
MPO) release from human PMNs by 1 (9, n ) 9) and 6 (2, n
4). All data are means ( SEM.
0
conformation of the Trp residue; however, both contain
the γ turn, and this region of the molecule is similar to
that of the open chain analogue 1.
(
)
bulky side chain into specific receptor space that is
critical for antagonism.
Despite the retention of the hydrogen bond that
defines the three-residue γ turn, there are some differ-
ences between the three-dimensional structures of 1 and
6. There are subtle differences in the backbone dihedral
angles associated with the γ turn (Table 4) and some
significant changes in the rest of the molecule. Instead
of the Trp-NH hydrogen bonding with the Orn (Lys in
1) CO, there is a preference for a new hydrogen bond
between D-Cha-CO‚‚‚HꢀN-Orn (Figure 7). The backbone
φ and ψ angles for the Trp and D-Arg residues shown
To further examine the importance of the C-terminal
carboxylate in 1 for receptor binding, a side-chain-to-
main-chain cyclization from the amino acid side-chain
amine of Lys (5) and Orn (6, Figure 5) to the C terminus
was carried out. Despite the fact that both 5 and 6 now
have an amide bond at the C terminus as in 2,
compound 5 is equipotent with 1, while 6 is 1 order of
magnitude more potent. To understand the structural

3
422 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Wong et al.
F igu r e 7. Backbone and proline heavy atoms of twenty lowest energy minimized NMR structures of 6 in d
6
-DMSO (24 °C). Two
sets of conformations 6a (left) and 6b (right) are shown along with the putative H-bond lengths: D-Cha-NH‚‚‚OC-Orn (6a 3.16 Å;
6
b 3.12 Å), Orn-NH‚‚‚OC-D-Cha (6a 3.12 Å; 6b 3.32 Å).
for 6 in Table 4 are qualitatively consistent with typical
values reported27 for type II â turns (φ2 -60°, ψ2 120,
φ3 80°, ψ3 0°). The cyclization and Lys/Orn substitution
in 6 thus induces a new â turn that restricts the
positions of residues in 6 that were relatively disordered
in 1.
amino acids, is the one with the highest propensity for
turn formation. With a Pro at position i + 1 of the â
3
3-35
turn, it would be expected
to be either a type I or a
type II turn, and the latter is discerned from an analysis
of the backbone dihedral angles in the calculated family
of structures. The preference for this form is readily
seen from the NOE data and, in particular, from the
strong RN sequential NOE between residues i + 1 (Pro)
and i + 2 (D-Cha) of the turn relative to a weak δN NOE
Discu ssion
There are very few short acyclic peptides that have
3
3-35
which would be more diagnostic of type I.
Type II
been found to have substantial turn structure in
solution.3
3-35
turns are often associated with a Gly residue at position
i + 3 to accommodate the requirement of a positive φ
angle; however, in this case this steric requirement is
met by the presence of a D-amino acid (D-Cha). The
The general consensus is that short
acyclic peptides tend to adopt a myriad of solution
conformations that may include small populations of
turn structures which are frequently responsible for
bioactivity. Here we have presented strong evidence
D-amino acid has implications for the dihedral angle
3
3
defining the J
NH-HR
coupling constant, and in this case
based upon NMR constraint data, J NH-CHR values, and
a large coupling (8.53 Hz) is observed, consistent with
the type II geometry. This contrasts with the small
coupling (5 Hz) expected in a type II turn involving only
L-amino acids. The observed large coupling is particu-
larly significant because coupling constants are suscep-
tible to conformational averaging and thus detection
of a value > 8 Hz strongly suggests a lack of conforma-
tional flexibility.
deuterium exchange and temperature dependence data
for an acyclic hexapeptide 1 in which a high population
of conformers adopt an inverse γ turn stabilized by a
D-Cha-NH‚‚‚OC-Lys hydrogen bond. Structure calcula-
tions suggest that the well-defined γ turn coexists
within a â turn encompassing the residues Lys-Pro-D-
Cha-Trp. Slow exchange and temperature coefficient
data suggest that a hydrogen bond associated with this
â turn, Trp-NH‚‚‚OC-Lys, is populated to a much lesser
extent than that of the γ turn hydrogen bond.
To compare antagonist structures under conditions
in which all compounds are soluble, we performed our
studies using DMSO rather than water as solvent. For
some peptides, DMSO is thought to disrupt local struc-
ture via intermolecular hydrogen bonding interactions,
and therefore, the finding of defined solution conforma-
tions in this structure-destroying solvent makes it
highly likely that an even higher stuctural order is
present under physiological conditions. Antagonist
structures in nonaqueous solvents are also more likely
to be relevant than in water since the antagonists likely
interact with a hydrophobic region (site 2) of the C5a
receptor.
2
3
Cyclic derivatives of 1 were constructed with the aim
of further limiting conformational flexibility of the
hexapeptide residues, particularly the flexible N and C
termini, and increasing the stability of a turn motif.
With a view to ultimately developing bioavailable drug
candidates, cyclization to 5 and 6 was expected to offer
a number of advantages on the basis of the fact that
cyclic peptides in general are known to (i) more ef-
fectively stabilize turn conformations by restricting
conformational freedom, (ii) increase resistance to pro-
teolytic cleavage by degradative enzymes which destroy
peptides, (iii) improve bioavailability, and (iv) increase
3
1-33
target selectivity.
These features tend to make
hydrophobic cyclic peptides more attractive drug can-
didates than their acyclic analogues.
The probability of a turn in 1 is, of course, enhanced
by the presence of the Pro residue, which, of all the
Unfortunately, cycle 5 showed no improvement in
activity over 1, and thus its structure was not investi-

Probes for G-Protein-Coupled C5a Receptors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3423
gated in detail. However, cycle 6 was a much more
potent antagonist than 1 or 5; therefore, its structure
was determined for comparison with that of 1. There
is strong NMR evidence for the presence of a hydrogen
bond across the Orn-Pro-D-Cha segment of the cyclic
derivative 6, thereby defining a γ turn in the identical
position to that in 1. By contrast, the putative type II
â turn that is described above for 1 does not appear to
be prevalent in 6, this cycle being further conforma-
tionally constrained by an additional amide bond be-
tween the Orn side-chain amine and the Arg C termi-
nus. This new amide NH in 6 is strongly implicated in
a hydrogen bond to D-Cha CO. The effect of such a H
bond is to distort the conformation of the Trp-(D-Arg)-
Orn segment, and to some extent also the Orn-Pro-D-
Cha region, of the cycle 6 compared to corresponding
residues of 1. The enhanced antagonist activity for 6
may in part be attributable to the increased stabilization
of a γ turn, but most likely also reflects the part played
by the new type II â turn in positioning the critical
D-Cha, Trp, D-Arg, and Phe residues for receptor bind-
ing.
the hexapeptide by the cycle 6 are responsible for a g104
fold increase in receptor-binding affinity over the con-
formationally flexible decapeptide C terminus of C5a (7,
Table 2). It is interesting to note that, compared with
that of a linear peptide chain as in 7, the turn confor-
mations of both 1 and 6 can bring the hydrophobic Phe,
Trp and D-Cha side chains into closer proximity, creat-
ing a hydrophobic surface “patch”. This may be the key
to developing small molecules with high affinity for site
2 of the C5a receptor. Although this study does not deal
with the locations of the amino acid side chains in a
receptor environment, it does reveal a novel backbone
scaffold (in 6) that leads to potent antagonist activity
and high receptor binding affinity.
The results presented here for 1 and 6 will assist in
the design and development of even more potent,
conformationally constrained, C5a antagonists that may
find therapeutic applications. Such refined molecules
are expected to be powerful structural probes of the C5a-
binding site(s) on G-protein-coupled receptor(s) for C5a
and of the mechanistic role of C5a in complement-
mediated immune defense.
The constraining γ and â turns proposed in the linear
peptide 1 and cyclic peptide 6 have precedent in other
cyclic peptides. For example, there have been many
other attempts to conformationally fix putative â and γ
turns in bioactive peptides, resulting in notable mimet-
Exp er im en ta l Section
Abbreviations: TFA ) trifluoroacetic acid; DIPEA ) diiso-
propylethylamine; HBTU ) O-benzotriazole N′,N′,N′,N′-tetra-
methyluronium hexafluorophosphate; BOP ) benzotriazole-
1
-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophos-
ics for RGD, somatostatin, and opioid peptides, to name
phate; DMF ) dimethylformamide; TIPS ) triisopropylsilane;
TMSBr ) trimethylsilyl bromide; Mtr ) 4-methoxy-2,3,6-
trimethyl-benzenesulfonyl; O ) L-ornithine; D-Cha ) D-cyclo-
hexylalanine; amino acid symbols “r” and “a” represent D-Arg
and D-Ala, respectively.
a few,3
6-39
derived through structure-activity relation-
ships. Most of these examples preserve some type of
turn structure through cyclization of the peptide. We
have also previously detected overlapping γ and â turns
Gen er a l Meth od s. Protected amino acids and resins were
obtained from Novabiochem. TFA, DIEA, and DMF (peptide
synthesis grade) were purchased from Auspep. All other
materials were reagent grade unless otherwise stated. Pre-
parative scale reverse-phase HPLC (RP-HPLC) separations
were performed on a Vydac C18 reverse-phase column (2.2 ×
25 cm); analytical RP-HPLC on Waters Delta-Pak PrepPak
in the cyclic octapeptide, ascidiacyclamide, found in the
marine organisms ascidians.40 Combinations of a γ and
â turn have also been found in the backbone of cyclic
penta- and hexapeptides, particularly those containing
alternating D- and L-amino acids.3
6-39
For example, a
type II â turn and an inverse γ turn have been identified
in cyclic antagonists 9, c-[D-Glu-Ala-L-allo-Ile-Leu-L-
C
18 reverse-phase column (0.8 × 10 cm); using gradient
mixtures of solvent A ) water/0.1% TFA and solvent B ) 10%
water/90% acetonitrile, 0.09% TFA. The molecular weight of
the peptides was determined by electrospray mass spectrom-
etry recorded on a triple quadrupole mass spectrometer (PE
SCIEX API III) as described elsewhere.40 H NMR spectra
were recorded on either Bruker ARX 500 MHz or Varian Unity
1
4
00 or 600 spectrometers. Proton assignments were deter-
mined by 2D NMR experiments (DFCOSY, TOCSY, NOESY).
All assay data were analyzed by nonlinear regression and
statistics using Student’s t test (p < 0.05).
P ep tid e Syn th esis. The linear peptides 1-4 were as-
sembled by manual stepwise solid-phase peptide synthesis
using HBTU activation and DIPEA in situ neutralization.
Couplings were monitored by the quantitative ninhydrin test.
Trp]4
1-44
and 10, c-[D-Asp-Pro-D-Val-Leu-D-Trp]44 for
R
endothelin receptors, also members of the rhodopsin
Boc chemistry was employed for temporary N -protection of
family of G-protein-coupled receptors with seven trans-
amino acids with two 1 min treatments with TFA for Boc group
removal. Peptide 2 was synthesized on a Peptide Institute
MBHA resin, with a substitution value of 0.79 mmol/g, to give
a C-terminal amide. The other peptides were synthesized on
a Novabiochem Boc-D-Arg(Tos)-PAM resin, with a substitution
value of 0.51 mmol/g. The peptides were fully deprotected and
cleaved by treatment with liquid HF (10 mL), p-cresol (1 mL)
membrane domains.45 An inverse γ turn also forms in
1
0 (Asp-CO‚‚‚Val-NH), as in 1 and 6 (Lys-CO‚‚‚D-Cha-
NH), through hydrogen bonding between residues that
flank the proline.
The preservation of a γ turn conformation in com-
pounds 1 and 6 suggests that both molecules are
preorganized for binding to the G-protein-coupled C5a
receptor. This common feature, together with the
observed linear relationship between receptor-binding
affinity and antagonist potency, highlights the impor-
tant role of this turn conformation in expression of
antagonism. The conformational limitations placed on
at -5 °C for 1-2 h. Analytical HPLC (gradient; 0 to 75% B
+
over 60 min): 1, t
R
) 32.0 min, MS [M + H] (calcd) ) 900.5,
+
+
[
8
M + H] (exptl) ) 900.7; 2, t
R
) 32.2 min, [M + H] (calcd) )
99.6, [M + H] (exptl) ) 899.7; 3, t ) 30.0 min, MS [M +
) 23.8 min,
+
R
+
+
H] (calcd) ) 900.5, [M + H] (exptl) ) 900.7; 4, t
R
+
+
MS [M + H] (calcd) ) 860.5, [M + H] (exptl) ) 860.5.
Syn th esis of Cycle AcF [KP -D-Ch a Wr ] (5). The linear
peptide Ac-Phe-Lys-Pro-D-Cha-Trp-D-Arg was synthesized by

3
424 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Wong et al.
Fmoc chemistry using HBTU/DIPEA activation on a Fmoc-D-
Arg(Mtr)-Wang resin, with a substitution value of 0.32 mmol/
g. Fmoc group removal was effected by two 1 min treatments
with 50% piperidine/DMF. Cleavage and deprotection using
Recep tor -Bin d in g Assa y. Assays were performed with
1
7
fresh human PMNs, isolated as previously described, and a
buffer of 50 mM HEPES, 1 mM CaCl , 5 mM MgCl , 0.5%
2
2
bovine serum albumin, 0.1% bacitracin, and 100 µM PMSF
(phenylmethylsulfonyl fluoride). In assays performed at 4 °C,
buffer, unlabeled human recombinant C5a (Sigma) or test
peptide, Hunter/Bolton labeled 1 I-C5a (∼50 pM) (New Eng-
9
5% TFA/2.5% TIPS/2.5% H
peptide which was purified by RP-HPLC. The linear peptide
15 mg, 13.1 µmol) and BOP (41 mg, 92.7 µmol) were dissolved
2
O for 1 h gave the Mtr-protected
25
(
6
in DMF (12 mL), DIPEA (18 µL) was added, and the mixture
was stirred for 15 h at room temperature. Analysis by RP-
HPLC and IS-MS indicated the completion of the reaction, and
then the solvent was removed in vacuo and the cyclized peptide
fully deprotected by treatment with 1 M TMSBr in TFA for 1
h. A final RP-HPLC gave the desired cyclic peptide (7.8 mg,
land Nuclear, MA), and PMNs (0.2 × 10 ) were added
sequentially to a Millipore Multiscreen assay plate (HV 0.45)
having a final volume of 200 µL/well. After incubation for 60
min at 4 °C, the samples were filtered and the plate washed
once with buffer (100 µL). Filters were dried, punched, and
counted in an LKB gamma counter. Nonspecific binding was
assessed by the inclusion of 1 mM peptide or 100 nM C5a
which typically resulted in 10-15% total binding.
+
+
6
(
5%): t
exptl) ) 910.7.
Syn th esis of Cycle AcF [OP -D-Ch a Wr ] (6). The linear
R
) 44.0 min, MS [M + H] (calcd) ) 910.5, [M + H]
An ta gon ism Assa y. Antagonist activity was assessed by
peptide Ac-Phe-Orn-Pro-D-Cha-Trp-D-Arg was synthesized by
Boc chemistry as per the procedure for peptides 1-4. Cy-
clization of the peptide was carried out by stirring the peptide
monitoring myeloperoxidase release as follows. Cells were
17
isolated as previously described and incubated with cytocha-
lasin B (5 µg/mL, 15 min, 37 °C). Hank’s Balanced Salt
solution containing 0.1% gelatin and test peptide were added
(
52 mg, 57 µmol), BOP (126 mg, 0.28 mmol) and pyridine (460
µL) in DMF (57 mL) for 15 h. The solvent was removed in
vacuo and the cyclic peptide purified by RP-HPLC (t ) 43.7
and 44.7 min). Yield 11.3 mg, 22%: MS [M + H] (calcd) )
onto a 96-well plate (total volume 100 µL/well), followed by
R
6
2
5 µL cells (4 × 10 /mL). To assess the capacity of each peptide
+
to antagonize C5a, cells were incubated for 5 min at 37 °C
with each peptide, followed by addition of C5a (100 nM) or
agonist peptide and further incubation for 5 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
+
8
96.5, [M + H] (exptl) ) 896.5. The peptide was tested as a
mixture of these two diastereomers. For the NMR structure,
the peptide was cyclized using the above procedure, but DIPEA
was used as the base instead of pyridine, resulting in only one
+
of these two compounds (RP-HPLC, t ) 44.7 min; [M + H]
R
dimethoxybenidine (5.7 mg/mL) and H
2 2
O (0.51%). The reac-
)
896.5) which is attributed to the cycle AcF[OP-D-ChaWr]
rather than to AcF[OP-D-ChaWR].
NMR Str u ctu r e Deter m in a tion . 1H NMR spectra were
recorded for compounds 1 and 6 (3 mg in 750 µL d -DMSO, δ
.50) referenced to solvent on a Varian Unity 400 or 600
tion was stopped at 20 min by addition of 2% sodium azide
25 µL). Absorbances were measured at 450 nm in a Bioscan
50-plate reader and corrected for control values (no peptide).
(
4
6
2
Ack n ow led gm en t. We thank Trudy Bond in the
1
spectrometer at 24 °C. Two-dimensional H NMR NOESY
Centre for amino acid analyses and the Australian
Research Council for a Senior Research Fellowship
(D.J .C.).
(
relaxation delay 2.0 s, mix time 50-300 ms), DQF-COSY and
TOCSY (mixing time 75 ms) experiments were acquired and
recorded in phase sensitive mode. Acquisition times ) 0.186
s, spectral width ) 5500 Hz, number of complex points (t
1
dimension) ) 1024 for all experiments. Data were zero-filled
and Fourier-transformed to 1024 real points in both dimen-
sions.
NMR data were processed using TRIAD software (Tripos
Assoc.) on a Silicon Graphics Indy work station. 2D NOE
cross-peaks were integrated and characterized into strong
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