664
J. J. Chen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 662–664
16. Ji, H.; Ohmura, K.; Mahmood, U.; Lee, D. M.; Hofhuis, F. M. A.; Boackle, S. A.;
Table 3
Takahashi, K.; Holers, V. M.; Walport, M.; Gerard, C.; Ezekowitz, A.; Carroll, M.
C.; Brenner, M.; Weissleder, R.; Verbeek, J. S.; Duchatelle, V.; Degott, C.; Benoist,
C.; Mathis, D. Immunity 2002, 16, 157.
Functional activity of lead compounds in various cell lines
Assay
5
7
17
17. Wenderfer, S. E.; Ke, B.; Hollmann, T. J.; Wetsel, R. A.; Lan, H. Y.; Braun, M. C. J.
Am. Soc. Nephrol. 2005, 16, 3572.
18. Bao, L.; Osawe, I.; Puri, T.; Lambris, J. D.; Haas, M.; Quigg, R. J. Eur. J. Immunol.
2005, 35, 2496.
19. Girardi, G.; Berman, J.; Redecha, P.; Spruce, L.; Thurman, J. M.; Kraus, D.;
Hollmann, T. J.; Casali, P.; Caroll, M. C.; Wetsel, R. A.; Lambris, J. D.; Holers, V.
M.; Salmon, J. E. J. Clin. Invest. 2003, 112, 1644.
20. Finch, A. M.; Wong, A. K.; Paczkowski, N. J.; Wadi, S. K.; Craik, D. J.; Fairlie, D. P.;
Taylor, S. M. J. Med. Chem. 1999, 42, 1965.
27
PMN FLIPR IC50
(
l
M)
0.067
5.7
27
>60
>60
>60
>60
0.059
2.5
0.4
Human PMN chemotaxis27
(
lM)
>60
>60
3.1
>60
>60
>60
Human PMN Shape Change, whole blood28
(l
M)
29
RAW cell FLIPR IC50
CXCR3 FLIPR IC50
CXCR4 FLIPR IC50
(
M)
M)
l
M)
>60
>60
>60
>60
(l
l
(
P2Y2 FLIPR IC50 (lM)
21. Sumichika, H.; Sakata, K.; Sato, N.; Takeshita, S.; Ishibuchi, S.; Nakamura, M.;
Kamahori, T.; Ehara, S.; Itoh, K.; Ohtsuka, T.; Ohbora, T.; Mishina, T.; Komatsu,
H.; Naka, Y. J. Biol. Chem. 2002, 277, 49403.
22. Brodbeck, R. M.; Cortright, D. N.; Kieltyka, A. P.; Yu, J.; Baltazar, C. O.; Buck, M.
E.; Meade, R.; Maynard, G. D.; Thurkauf, A.; Chien, D.-S.; Hutchison, A. J.;
Krause, J. E. J. Pharmacol. Exp. Ther. 2008, 327, 898.
in human PMN. Furthermore, compound 5 also inhibited C5a-med-
iated human PMN shape change in whole blood. The diminished
potency of the compounds in these assays may be due to longer
incubation time in the chemotaxis assay and non-specific binding
of compounds to serum proteins in the whole blood assay. In con-
trast to the lack of rodent cross-reactivity reported for other C5aR
small molecule antagonists,20–23 compound 17 showed antagonis-
tic activity for the mouse receptor (RAW cells) which would enable
in vivo studies. All three compounds were founded inactive to-
wards other GPCRs, such as human lymphocyte CXCR3 and CXCR4
in human T cells, and P2Y2 in CHO cells, suggesting that these com-
pounds are selective C5aR antagonists.
In summary, a new class of small molecular C5a antagonists has
been identified through HTS and a hit-to-lead process. The lead
molecules exhibit promising binding and functional activities with
human and mouse cell lines. Further optimization of this series will
be the subject of future communication.
23. Gong, Y.; Barbay, J. K.; Buntinx, M.; Li, J.; Wauwe, J. V.; Claes, C.; Lommen, G. V.;
Hornby, P. J.; He, W. Bioorg. Med. Chem. Lett. 2008, 18, 3852.
24. FLIPR assay conditions: CHO-G
well in 384 well black clear bottom TC plates (BD 353962) in media (R1
(invitrogen) + 1% FBS + 1 mM -glutamine) 24 h before the assay. On the day of
a16-hC5aR cells were seeded at 15,000 cells/
L
experiment, cells were washed and loaded with Ca3 dye (molecular devices
R8090) for 2 h (1 h 37 °C followed by 1 h at rt). Compounds were added to the
cells by FLIPR3 (molecular devices). After 10 min incubation with compound,
hC5a was added at the concentration of 2 Â EC50 (0.1 nM). C5a induced
intracellular calcium accumulation was recorded on FLIPR3.
25. Binding assay conditions: CHO-G
a16-C5a cells were seeded in growth media
(R1 + 10% FBS + 1 mM -glutamine) at 50,000/well in 96 well white-clear bottom
L
TC plate (Perkin–Elmer 1450-517) the day before the assay. On the day of
experiment, culture media was replaced by assay buffer (1 Â HANKS + 20 mM
HEPES + 0.1% BSA). Compounds were then added to the cells followed by 10 min
incubation at rt. 125I-hC5a (0.6 nM) (Perkin–Elmer, NEX250) was then added to
the cells. After 1 h incubation at room temp, the cell plate was washed eight
times with wash buffer (PBS + 1 mM CaCl2 + 0.1% BSA) on a harvester (Packard).
Excess buffer was removed by blotting with paper. 40 ml of Microscint 40
(Perkin–Elmer) was added to each well. The plates were then counted on Trilux
(Perkin–Elmer).
Acknowledgements
26. To create the alignment, each compound was expanded into multiple low
conformations using OMEGA (OpenEye Scientific Software, Santa Fe, NM. http://
(5) and the conformations of (17) were constructed using ROCS (OpenEye
Scientific Software). The alignment with the most consistent molecular shape
and 3D chemistry orientation was chosen for analysis.
We thank the CPG team and DAC group at Wyeth for their ana-
lytical supports to this project.
References and notes
27. Neutrophil assay conditions: Human neutrophils (PMN) were purified from
human whole blood using a Ficoll gradient protocol. The cells were then re-
suspended in 1 Â Ca3 dye and seeded at 50,000 cells/well in 384 well black
clear bottom TC plates (BD 353962) in assay buffer (1 Â HANKS + 20 mM
HEPES + 0.1% BSA). After 1 hr incubation at 37 °C, 5% CO2, Compounds were
added to the cells by FLIPR3. 10 nM hC5a was added after 10 min incubation
with compound. C5a induced intracellular calcium accumulation was recorded
on FLIPR3.
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incubated at 37 °C for 10 min. Then 250 ll ice-cold 1% (v/v) paraformaldehyde
was added to fix cells, followed by addition of ice-cold hypotonic buffer and
incubation on ice for 40 min to lyse red blood cells. Neutrophils were identified
and gated by flow cytometry on FACSCalibur (Becton Dickinson) using forward
and side scatter. Increase in neutrophil autofluorescence forward scatter on
FL2-H channel, which reflects shape change of neutrophils due to cytoskeletal
rearrangement stimulated by the C5a chemoattractant, was measured by
Flowjo software.
29. RAW assay conditions: RAW264.7 cells were seeded at 50,000 cells/well in 384
well black clear bottom TC plates (BD 353962) in media (DME (invitrogen) + 1%
FBS + 2 mM L-glutamine) the day before. On the day of experiment, cells were
washed and loaded with Ca3 dye for 1 h at 37 °C. Compounds were added to
the cells by FLIPR3. After 10 min incubation with compound, mC5a was added
at the concentration of 2 Â EC50 (10 nM). C5a induced intracellular calcium
accumulation was recorded on FLIPR3.