i. Field of the Invention
This invention relates to an assay to identify compounds useful as C5a antagonists. The invention also comprises C5a antagonists and uses thereof as anti-inflammatory agents and as immunoregulants.
ii. Background
The importance of the C5a receptor finds its origin in its relationship with complement derived C5a and its role in the overall immune response. The complement system is a complex group of proteins present in body fluids that, working together with antibodies or other factors, plays an important role in mediation of immune, allergic, immunochemical and immunopathological reactions. Activation of the complement system can result in a wide range of reactions such as lysis of various kinds of cells, bacteria, protozoa, inactivation of viruses, and the direct mediation of inflammatory processes. Through the hormone-like activity of several of its components, the complement system recruits and enlists the participation of other humoral and cellular effector systems. These in turn induce directed migration of leukocytes, trigger histamine release from mast cells, and stimulate the release of lysosomal constituents from phagocytes.
The complement system consists of at least twenty distinct plasma proteins capable of interacting with each other, with antibodies, and with cell membranes. In a sequential series of activation steps (the "complement cascade"), proteins are activated and then combine with additional proteins to form complexes that cleave and activate still other proteins in the system. The sequential activation of these proteins follows two main pathways, the classical pathway and the alternative pathway. Both pathways use a common terminal trunk that leads to cell lysis or virus inactivation.
The classical pathway can be activated by antigen-antibody complexes, aggregated immuno-globulins and non-immunological substances such as DNA and trypsin-like enzymes. The classical pathway includes activation of C.sub.1, C.sub.4, C.sub.2 and C.sub.3 molecules. These components can be grouped into two functional units: C.sub.1 or recognition unit; and C.sub.4, C.sub.2 and C.sub.3 or activation unit. Five additional components denominated C.sub.5, C.sub.6, C.sub.7, C.sub.8, and C.sub.9 define the membrane attack unit forming the terminal trunk common to both pathways.
The acute inflammatory response depends on the attraction of phagocytic polymorphonuclear leukocytes (PMN's) to the site of microbial invasion by a chemotactic stimulus. The C5a component is pivotal to the effectuation of this response in humans.
C5a is a 74 amino acid protein derived from the fifth component of serum complement during complement activation. C5a is known to mediate various pathological conditions, chronic inflammation, acute pulmonary disorders and even the metastatic spread of cancerous tumors. C5a is chemotactic for neutrophils, monocytes, macrophages, eosinophils and basophils. Thus, this serum factor is an important attractant to leukocytes and is crucial to their accumulation in vivo at sites of immunologic injury.
Leukocytes accumulated at a site of inflammation release granular contents, various hydrolytic enzymes and other cytotoxic components into the extracellular spaces in a process referred to as degranulation, as a result of which the surrounding tissue is damaged.
Numerous chronic inflammatory diseases are thought to involve the aberrant presence of C5a in tissue. Rheumatoid arthritis, osteoarthritis and psoriasis are a few examples. The lung is particularly vulnerable; excess C5a in circulation or in the lung can result in aggregation and migration of leukocytes into this organ. This can lead to microvascular occlusion, endothelial damage and subsequent edema. In J. Amer. Med. Soc., 244, 199 (1980), Hammerschmidt suggested that noncardiac pulmonary edema associated with blood transfusion and hemodialysis depend on an aberrant increase in circulating C5a.
In view of the above, and as generally supported in the literature, it is believed that compounds which could antagonize the binding of C5a to its receptor on effector cells would be useful in the treatment of a wide variety of C5a mediated diseases such as acute inflammatory response, including acute respiratory distress syndrome (ARDS), and anaphylactic shock; chronic inflammation including rheumatoid arthritis, osteoarthritis and psoriasis and metastatic spread of cancerous tumor. See Yancy, K.B., Review--Biological Properties of Human C5a: selected in vitro and in vivo studies, Clin. Exp. Immunol. (1988) 71, 207-210, and references cited therein.
Previous attempts to develop antagonists of C5a utilized an assay to screen for inhibitors of the binding of the intact C5a molecule. The rational design of a useful antagonist requires detailed knowledge of the important receptor-ligand interactions. The binding subunit of the C5a receptor has been cloned and shown to be a member of G protein-coupled receptor superfamily (Gerard, N. P. and Gerard, C. (1991 ) Nature 349, 614-617; Boulay, F., Mery, L., Tardif, M., Brouchon, L. and Vignais, P. (1991) Biochemistry 30, 2993-2999). The region of this molecule which interacts with C5a consists of two subsites. The first is in the binding subunit's N-terminus, and although the second subsite has not been definitely localized, it appears to lie between the transmembrane spanning helices.
Similarly, site-directed mutagenesis and studies with peptide analogs of C5a have identified two regions on this molecule which are important for binding to the receptor. The first is in the core of the C5a molecule, centered around Arg40 (Mollison, K. W., Mandecki, W., Zuiderweg, E. R. P., Fayer, L., Fey, T. A., Kranuse, R. A., Conway, R. G., Miller, L., Edalji, R. P., Shallcrss, M. A., Lane, B., Fox, J. L., Greer, J. and Carter, G. W. (1989) Proc. Natl. Acad. Sci. USA 86, 292-297), while the second is contained in the C-terminal eight amino acids (Kawai, M., Or, Y. S., Wiedman, P. E., Luly, J. and Moyer, M. (1990) World Intellectual Property Organization, Publication Number WO 90/09162; Kawai, M., Quincy, D., A., Lane, B., Mollison, K., W., Luly, J., R. and Carter, G., W. (1991 ) Journal of Medicinal Chemistry 34, 2068-2071 ). A synthetic peptide consisting of these eight residues inhibits C5a binding with a Ki=300 .mu.M, and has agonist properties. Subsequent structure activity studies have produced analogs with much higher affinities while reducing the size of the peptides from eight to six residues (Mollison, K. W., Krause, R. A., Fey, T. A., Miller, L., Wiedeman, P. e., Kawai, M., Lane, B., Luly, J. R. and Carter, G. W. (1992) FASEB J. 6, A2058; Kawai, M., Lane, B., Mollison, K. W., Luly, J. R. and Carter, G. W. (1991) 12th Amer. Peptide Symposium, poster #525). However, efforts at generating antagonists of C5a have so far been unsuccessful. The one partial exception is the peptide NMeF--K--P-dCha-F-dR (NMeF is N-methyl phenylalanine, and Cha is cyclohexylalanine), which while not an antagonist, appears to have reduced agonist properties. For example, Mollison et. al. (Mollison, K. W., Krause, R. A., Fey, T. A., Miller, L., Wiedeman, P. e., Kawai, M., Lane, B., Luly, J. R. and Carter, G. W. (1992) FASEB J. 6, A2058) have reported that this peptide evokes only a small chemokinetic response, and is totally devoid of degranulating activity. Further, Drapeau et. al. (Drapeau, G., Brochu, S., Godin, D., Levesque, L., Rioux, F. and Marceau, F. (1993) Biochem. Pharm. 45, 1289-1299) found that the peptide failed to stimulate superoxide release from neutrophils, although it behaved as an agonist in other assays.
The instant inventors have discovered that intact C5a binds to its receptor by interaction at two physically distinct loci. This, at least in part, explains why the existing assays could not distinguish at which locus potentially inhibitory compounds act. In order to develop inhibitors which block the interaction of the C-terminus of C5a with its receptor, we have developed high affinity, labeled peptides to allow our research to focus on this one locus. Prior to the current invention such specificity was not possible.
Peptides from the COOH terminus of C5a have been reported. However, peptides disclosed in these publications have not been used according to the method of the instant invention, and there have not been reports of compounds that exhibit full C5a antagonism at submicromolar concentrations. Thus, see:
Abbott-WO 90/90162; WO 9212168-A1; WO 92/21361 described anaphylatoxin receptor ligands; see also U.S. Pat. Nos. 5,190,922 and 5,223,485, both assigned to Abbott and essentially equivalent to the international publications in content; PA1 Kahn, S. A. et al. Complement 2, 42 (1985) described a 21-residue peptide based on the C5a COOH-terminus; PA1 Kawai, M., et al. J. Med. Chem., 35, 2068 (1991 ) described a C-terminal C5a octapeptide which retained receptor binding activity; Kawai, M. et al. J. Med Chem., 35, 220 (1992) described modified C5a COOH-terminal octapeptide analogs having a Ki for C5a binding of 1-3 .mu.m; PA1 Mollison et al. Proc. Nat. Acad. Sci., 86, 292 (1989) described a Phe for His67 substituted C5a peptide with a Ki of 0.26 .mu.m; PA1 Gerard, C. Get al. J. Reticuloendothel. Soc., 26, 711 (1979) identified the C5a COOH-terminus as critical to activity of C5a as a neutrophil chemotactic agent; PA1 Chenoweth, D. E. Mol Immunol., 17, 151 (1980) identified the C-terminal portion of C5a as important for modulating neutrophil receptor-ligand interaction; PA1 Ember J. A. et al. J. Immuol., 148 3155 (1992) confirmed the increased potency of Phe for His67 substituted C-terminal analogs. PA1 Mollison et al. PNAS USA, 86, 292 (1989) identified receptor binding residues in intact C5a.
A detailed structure/activity relationship for C-terminal peptide analogs of C5a has emerged from studies in a number of laboratories (Kawai, M., Or, Y. S., Wiedman, P. E., Luly, J. and Moyer, M. (1990) World Intellectual Property Organization, Publication Number WO 90/09162; Kawai, M., Quincy, D., A., Lane, B., Mollison, K., W., Luly, J., R. and Carter, G., W. (1991) Journal of Medicinal Chemistry 34, 2068-2071; Mollison, K. W., Krause, R. A., Fey, T. A., Miller, L., Wiedeman, P. e., Kawai, M., Lane, B., Luly, J. R. and Carter, G. W. (1992) FASEB J. 6, A2058; Kawai, M., Lane, B., Mollison, K. W., Luly, J. R. and Carter, G. W. (1991) 12th Amer. Peptide Symposium, poster #525; Ember, J. A., Sanderson, S. D., Taylor, S. M., Kawahara, M. and Hugli, T. E. (1992) J. Immunol. 148, 3165-3173). The minimum size of the peptides has been reduced to 6 residues, as contrasted to 74 in the natural molecule, and analogs with affinities as high as 1-10 nM have been reported. However, all of these molecules retain strong agonist properties. In U.S. Pat. No. 4,692,511, a series of peptides were reported to act as competitive antagonists for the C5a receptor. The peptides were centered around the Asp-Gly-Ala tripeptide at positions 24, 25, and 26 in the naturally occurring C5a molecule. No binding or inhibition data were presented. However, it is manifest from the work we report herein that those purported antagonists were low-affinity molecules with Ki's in the micromolar and higher range. As such, those peptides could not be useful as specific C5a receptor antagonists. As we teach herein, them are two sites for C5a binding to its receptor and peptides disclosed in the 4,692,511 Patent would bind to the external, low-affinity site of the receptor.
Since C5a is an important inflammatory mediator, the development of an antagonist is of considerable therapeutic interest. In this patent disclosure we report that increasing the aromaticity of the residue in position 5 of C5a COOH-terminal hexapeptide analogs leads to a progressive loss of agonism, and we disclose the first full C5a antagonist active at submicromolar concentrations.