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J . Med. Chem. 1992, 35, 402-406
Improvements in the Minimum Binding Sequence of CSa: Examination of His-67l C5a is a biologically active glycoprotein fragment released during the adivation of serum complement proteins. This glycoprotein, representing the N-terminus of the a-chain of complement protein C5, is enzymatically released from the parent molecule by the actions of the complement-derived C5 convertase. C5a shares with C3a and C4a, two other complement-derived anaphylatoxins, the ability to produce a variety of inflammatory respmses.3 However, C5a is believed to play a major role as a potent inflammatory mediator by ita additional activities in recruiting and stimulating inflammatory leukocytes. Both C5a and ita des Arg-74 derivative exist in vivo as biologically active forms and are both inflammatory mediators. Once it is liberated from ita parent molecule, C5a interacts with specific membrane receptors present on white blood cells including polymorphonuclear leukocytes (PMNL), monocytes, basophils, and eosinophils, as well as with tissue resident cells such as macrophages and mast cells. The interaction of C5a with ita receptor in target cells stimulates the directed migration (chemotaxis) of leukocytss.4 C5a induces PMNL shape changes and increased adhesion to other cells and surface^.^ C5a stimulates the oxidative metabolism of inflammatory leukocytes as indicated by enhanced superoxide anion generation.6 The activation of neutrophils and other target cells by C5a also increases the release of granular enzymes,’ histamine: and other vasoactive inflammatory substance^.^ These substances promote the destruction of tissues, the increase of vascular permeability, the contraction of smooth muscle, and an influx into the tissues of inflammatory cells such as neutrophils, eosinophils, and basophils. C5a may also be important in mediating inflammatory effects of phagocytic mononuclear cells that accumulate at sites of (1) (2)
(3)
(4)
chronic inflammation.1° Recent studies show that C5a may also exert immunomodulatory effects at sites of inflammation by enhancing antibody production.” Thus, the search for a specific C5a receptor antagonist to inhibit the involvement of C5a in inflammation has been an active and ongoing process in recent years because of ita therapeutic implications. Human anaphylatoxin C5a is a single-chain cationic glycoprotein consisting of 74 amino acid residues and an N-linked carbohydrate moiety of approximately 3 kd attached at asparagine 64.12 Six of the seven cysteines in C5a are linked via intramolecular disulfide bonds to stabilize the folded peptide chain. Based on circular dichroism (CD) spectra, as many as 50% of the residues in C5a are estimated to be in a-helical conformation. The primary structures of human C3a, C4a, and C5a have been elucidated by chemical analysis. The sequence comparison between human C3a and C5a indicates 34% homology, while homology between C4a and C5a is 39%.3c The three-dimensional structure of the major portion of C5a has been modeled from the homologous C3a crystal structure by comparative modeling technique^.'^ The “active site”, or the minimum active sequence of C3a and C4a, has been identified by synthetic peptide mapping to be the C-terminal penta- and ~ctapeptides.’~J~ Unlike the
(5) O’Flaherty, J. T.; Ward, P. A. Leukocyte Aggregation Induced by Chemotactic Factors. Inflammation 1979,2, 177-194. (6) Goldstein, I. M.; Feit, F.; Peit, F.; Weissmann, G. Enhancement of Nitroblue Tetrazolium Dye Reduction by Leukocytes Exposed to a Component of Complement in the Absence of Phagocytosis. J. Zmmunol. 1975,114, 516518. (7) Henson, P. M.; Zanolari, B.; Schwartzman, N. A.; Hong, S. R. Intracellular Control of Human Neutrophil Secretion. I. C5a-Induced Stimulus-specificDwnsitization and the Effects of Cytochalasin B. J. Zmmunol. 1978,121,851-855. (8) (a) Grant, J. A.; Dupree, E.; Goldman, A. S.; Schultz, D. R.; Jackson, A. L. Complement-mediated Release of Histamine from Human Leukocytes. J. Zmmunol. 1975,114,1101-1106. Improvements in the Minimum Binding Sequence of C5a. Presented at the 200th ACS National Meeting, August 2631, (b) Johnson, A. R.; Hugli, T. E. Muller-Eberhard, H. J. Release 1990, Washington, D.C., Div. Med. Chem., Abstract No. 176. of Histamine from Rat Mast Cells by the Complement Pep(a) Kawai, M.; Quincy, D. A.; Lane, B.; Mollison, K. W.; Luly, tides C3a and C5a. Immunology 1975,28, 1067-1080. J. R.; Carter, G. W. Identification and Synthesis of a Receptor (9) (a) Clancy, R. M.; Dahinden, C. A.; Hugli, T. E. Arichidonate Binding Site of Human Anaphylatoxin C5a. 199th ACS NaMetabolism by Human Polymorphonuclear Leukocytes Stimtional Meeting, April 22-27, 1990, B-ton, MA, Div. Med. ulated by N-formyl-Met-Leu-Pheor Complement Component Chem., Abstract No. 87. (b) Kawai, M.; Quincy, D. Q.;Lane, C5a is Independent of Phospholipase Activation. Proc. Natl. B.; Molliion, K. W.; Luly, J. R.; Carter, G. W. Identification Acad. Sci. U.S.A. 1983, 80, 7200-7204. (b) Stimler, N. P.; Brocklehurst, W. E.; Bloor, C. M.; Hugli, T. E. Complement and Synthesis of a Receptor Binding Site of Human Anaphylatoxin C5a. J. Med. Chem. 1991,34, 2068-2071. Anaphylatoxin C5a Stimulate Release of SRS-A-like Activity (a) Hugli, T. E.; Muller-Eberhard, H. J. Anaphylatoxins: C3a from Guinea-pig Lung Fragments. J. Pharm. Pharmacol. and C5a. Ado. Zmmunol. 1978,26,1-53. (b) Hugli, T. E. The 1980, 32, 804. (c) Stimler, N. P.; Bach, M. K.; Bloor, C. M.; Structural Basis for Anaphylatoxin and Chemotactic FuncHugli, T. E. Anaphylatoxin Mediated Contraction of Guine Pig tions of C3a, C4a, and C5a. Crit. Reo. Zmmunol. 1981, 1, Lung Strips: A Non-histamine Tissue Response. J. Zmmunol. 321-366. (c) Hugli, T. E. Structure and Function of the Ana1982,128,2247-2252. phylatoxin. Springer Semin. Zmmunopathol. 1984,7,193-219. (10) Allison, A. C.; Ferluga, J.; Prydz, H.; Schrolemmer, H. U. The Role of Macrophage Activation in Chronic Inflammation. (a) Shin, H. S.; Synderman, R.; Friedman, E.; Mellors, A.; Mayer, M. M. Chemotactic and Anaphylatoxic Fragment Agents Actions 1978, 8, 27-35. Cleaved from the Fifth Component of Guinea Pig Comple(11) Morgan, E. L.; Weigle, W. 0.; Hugli, T. E. Anaphylatoxinment. Science 1968,162,361-363. (b) Ward, P. A.; Newman, Mediated Regulation of the Immune Responses. I. C3a-MeL. J. A Neutrophil Chemotactic Factor from Human C‘5. J. diated Suppression of Human and Murine Humoral Immune Zmmunol. 1969,102, 93-99. (c) Kay, A. B. Studies on EosiResponses. J. Ezp. Med. 1982,155,1412-1426. Morgan, E. nophil Leukocyte Migration. 11. Factora Specifically ChemoL.; Weigle, W. 0.;Hugli, T. E. Anaphylatoxin-mediated Regtactic for Eosinophile and Neutrophile Generated from Guinulation of Human and Murine Immune Responses. Fed. Proc. ea-pig Serum by Antigen-antibody Complexes. Clin. Ezp. 1984,43, 2543-2547. Zmmunol. 1970,7,723-737. (d) Kay, A. B.; Shin,H. S.; Austin, (12) Fernandez, H. N.; Hugli, T. E. Primary Structural Analysis of K. F. Selective Attraction of Eosinophils and Synergism bethe Polypeptide Portion of Human C5a Anaphylatoxin. J . tween Eoainophil Chemotactic Factor of Anaphylaxis (ECF-A) Biol. Chem. 1978,253,6955-6964; Partial Characterization of and a Fragment cleaved from the Fifth Component of ComHuman C5a Anaphylatoxin. I. Chemical Description of the plement (C5a). Immunology 1973, 24, 96s976. (e) LettCarbohydrate and Polypeptide Portion of Human C5a. J. Brown, M. A.; Boetcher, D. A.; Leonard, E. J. Chemotactic Zmmunol. 1976,117,1688-1694. Responses of Normal Human Basophile to C5a and to Lym(13) Greer, J. Model Structure for the Inflammatory Protein C5a. phocyte-Derived Chemotactic Factor. J.Zmmunol. 1976,117, Science 1985,228,1055-1060. 246-252. (f) Synderman, R.; Shin, H. S.; Hausman, M. H. A (14) Caporale, L. H.; Tippett, P. S.; Erickson, B. W.; Hugli, T. E. Chemotactic Factor for Mononuclear Leukocytes. Proc. SOC. The Active Site of C3a Anaphylatoxin. J. Biol. Chem. 1980, Ezp. Biol. Med. 1971,138,387-390. 255, 10758-10763. 0022-2623/92/1835-Q402$Q3.00f 0 0 1992 American Chemical Society
Journal of Medicinal Chemistry, 1992, Vol. 35, No. 2 403
Communications to the Editor
Table I. Inhibition of '"I-rCBa Binding to PMNL Membranes and Biological Activities of Synthetic Peptides" R-AA67-Lys-AspMet-GIn-Leu-Gly-Arg-OH
receptor agonist efficacyb binding: CK:d MPOe SVP:f R AAB7 Ki,C gM % rC5a (E&, pM) % rC5a (ECW,pM) % rC5a (ECw, pM) O.ooOo3 100 (0.00014) 100 (0.001) 100 (0.03) 129 (110) 3 300 H His inactive" H Ala 88 (110) H (l-Me)His 180 90 (17) H (3-Me)His 58 121 (13) Thia 20 H Thi 142 (0.53) 68 (200) 85 (150% response) could be classified as superagonists. The role of the particular position 67 substitutions, Cha and hPhe, respectively, in conferring this property is not clear. The PMNL responses appear to be attributable to interaction of the peptides with the C5a receptor because potencies of the functional responses agree well with the binding affmities. The chemokinetic responses reached EC5,, levels at concentrations within severalfold of the binding Ki values. Inducing MPO release required consistently higher concentrations of peptides than for stimulating locomotion, which is a well-known property of chemoattractants such as C5a.3 The further ability of members of this series of peptides to trigger biologic responses in vivo is established by their activity in the vascular permeability assay. Because C5a has a broad dose-response curve for inducing vascular leakage (not shown) and requires much higher concentrations to reach a maximum, the peptides were tested at a single high concentration to determine whether or not they retained this undesirable agonist response. Vascular leakage involves additional targets, e.g., mast ~ells,~J* indicating that the C5a receptor interaction and agonist properties of the C-terminal peptide analogs are not confined to PMNL. Conclusions A 1000-foldboost of binding affinity of the C5a C-terminal octapeptide has been realized by a single Phe for Hiss7 substitution. However, extension of the Phee7C5a C-terminal peptides up to 14-mers does not further improve the affinity as compared to the Phea7C-terminal &peptide. These peptides, although representing a small portion of the C5a molecule, are agonists as determined by PMNL chemokinesis, PMNL myeloperoxidase release and guinea pig skin vascular permeability assays. As such, they are interesting small molecule mimics of C5a and serve as potent leads in the development of C5a antagonists. We have recently demonstrated the applicability of this Phe for Hisa7modification in other series of ana-
406 Journal of Medicinal Chemistry, 1992, Vol. 35, No. 2
loguess and feel it will have broad utility in C5a analogue design. (28) (a) Or, Y. S.; Clark, R. F.; Lane, B.; Mollison, K. W.; Luly, J. R.; Carter, G. W. Improvements in the C5a Minimum Binding Sequence with Hydrophobic Interactions. 20lst ACS National Meeting, April 14-19, 1991, Atlanta, GA, Div. Med. Chem., Abstract No. 45. (b) Or, Y. S.; Clark, R. F.; Lane, B.; Mollison, K. W.; Luly, J. R.; Carter, G. W. Potent C-terminal C5a Octapeptide Inhibitors by Modification at Residue 70. 201st ACS National Meeting, April 14-19, 1991, Atlanta, GA, Div. Med. Chem. Abstract No. 46. (c) Moyer, M. P.; Rhoades, T. A.; Kawai, M.; Or, Y. S.; Mollison, K. W.; Lane, B.; Luly, J. R.; Carter, G. W. Contribution of Side Chain Functionality, Chirality and Amide N-H to Binding of a Potent C5a Binding Inhibitor. 201st ACS National Meeting, April 14-19, 1991, Atlanta, GA, Div. Med. Chem., Abstract No. 136. (d) Or, Y. S.; Clark, R. F.; Lane, B.; Mollison, K. W.; Luly, J. R.; Carter, G. W. C5a Carboxyl Terminal Octapeptide Analogues with Reduced Chemokinetic Activity. 201st ACS National Meeting, April 14-19,1991, Atlanta, GA, Div. Med. Chem., Abstract No. 137. (e) Or, Y. S.; Clark, R. F.; Kawai, M.; Lane, B.; Mollison, K. W.; Luly, J. R.; Carter, G. W. Effect of N-terminal Acylation on the Chemokinetic Activity of C5a Carboxyl Terminal Octapeptide Analogues. 202nd ACS National Meeting, August 25-30, 1991, New York, NY, Div. Med. Chem., Abstract No. 168.
Communications to the Editor
Acknowledgment. We thank Mr. Richard Conway, Mr. Thomas Fey, Ms. Ruth Krause, and Ms. Loan Miller for biological testing and the analytical research department of Abbott Laboratories for spectral measurements. Supplementary Material Available: Experimental and characterization data for the compounds discussed in this work (5 pages). Ordering information is given on any current masthead Page.
* T o whom correspondence should be addressed. Yat Sun Or,* Richard F. Clark, Benjamin Lane Karl W. Mollison, George W. Carter, Jay R.Luly Pharmaceutical Products Division Abbot t La bora tories Abbott Park, Illinois 60064 Received October 31, 1991
