US 5,955,431 AGrant
Mast Cell Protease Peptide Inhibitors
Issue Date:1999-09-21
•21 Claims
Abstract
Compositions and methods for inhibiting a complex containing a mast cell protease are provided. The compositions are useful for treating inflammatory disorders, such as asthma, that are mediated by release of a tryptase-6 protein. Methods for identifying additional specific inhibitors of a complex containing tryptase-6 protein and a serglycin glycosaminoglycan also are provided.
Metadata
Assignee
- Brigham and Women's Hospital, Inc.
Inventors
- Richard L. Stevens
- Chifu Huang
Application Information
Application Number:US 016366&
Filing Date:1998-01-30
Priority Date:1998-01-30
Art Unit:164
Classifications
IPC:
A61K 3804A61K 3805A61K 3808
Field of Search:
530514329-33117-18
Patent Drawings
This patent does not have any drawings.
Description
Field of the Invention
This invention relates to compositions containing a mast cell protease inhibitor and methods for use thereof in the prevention and treatment of inflammatory disorders mediated by mast cell tryptases. Methods utilizing the compositions for identifying additional inhibitors of the mast cell protease also are provided.
Background of the Invention
Mast cells play central roles in varied inflammatory reactions due to their ability to release a diverse array of biologically active factors. During the last decade, the primary focus has been on the role of mast cell-derived histamine, leukotrienes, prostaglandins, cytokines, and chemokines in inflammation. Little attention has been paid to the role of tryptases even though these serine proteases are major constituents of the secretory granules of human, mouse, rat, gerbil, and dog mast cells. Accordingly, the mechanisms by which mast cell tryptases mediate inflammation have not been identified.
All mast cell proteases are targeted to the secretory granule as inactive zymogens but they are rapidly activated at this site. Thus, they are stored in the granule in their mature, enzymatically active forms. Tryptases, the major secretory granule proteases of human mast cells, are glycosylated, heparin-associated tetramers of heterogenous, catalytically active subunits. These enzymes are stored in an enzymatically inactive state in the mast cell's secretory granules and are released from the cell following activation through the high affinity IgE receptor. Tryptases have been implicated in a variety of biological processes including tissue inflammation.
Various attempts have been made to identify inhibitors of tryptase for treating inflammatory disorders. For example, small aromatic molecules have been proposed as tryptase inhibitors for preventing and treating inflammatory diseases associated with the respiratory tract, such as asthma and allergic rhinitis. (See, e.g., U.S. Pat. No. 5,525,623, issued to Spear et al., "Compositions and Methods for the Treatment of Immunomediated Inflammatory Disorders"; and International Application Nos. PCT/US95/11814, WO96/09297, and PCT/US94/02706, WO94/20527, Applicant: Arris Pharmaceutical Corporation.) Unfortunately, such molecules nonspecifically inhibit a variety of serine proteases (including pancreatic trypsin) that are present in vivo and, accordingly, the therapeutic value of such molecules for treating conditions mediated by mast cell tryptase remains questionable.
In view of the demonstrated involvement of mast cells in the initiation of inflammation, a need still exists to understand the mechanisms by which mast cells control such inflammation and to develop new and useful agents that inhibit or prevent inflammation in the first instance. Preferably, such agents would selectively inhibit specific components produced by the mast cell that are responsible for the inflammation, thereby requiring administration of relatively low doses of the agent and minimizing the likelihood of side reactions that may be associated with the administration of a high dosage of the agent.
Summary of the Invention
The present invention overcomes the problems of the prior art by providing a preferred peptide substrate (protease inhibitor) and its derivatives which can be used to selectively inhibit aL mast cell tryptase that induces neutrophilia when administered to mice. The invention involves in one respect the discovery of a peptide sequence (SEQ. ID NO. 1) that is a substrate for a complex containing mouse mast cell protease 6 ("mMCP-6") and heparin glycosaminoglycan. This peptide sequence can be used to selectively inhibit this and related mast cell tryptase complexes in vitro and in vivo. Although not intending to be bound to a particular mechanism of action, it is believed that the human tryptases .alpha., I, .beta./II, and III (GenBank Accession Nos. are shown in the sequence listing ) and rat tryptase (GenBank Accession No. U67909, J. Exp. Med. 1997; 185:13-29) are the homologs of mMCP-6 and that one or more of these human tryptases play a key role in the pathogenesis of mast cell-mediated inflammatory disorders including the emigration of neutrophils.
In view of the foregoing, the protease inhibitors of the invention are useful for treating a variety of inflammatory disorders including asthma, allergic rhinitis, urticaria and antioedema, eczematous dermatitis (atopic dermatitis), and anaphylaxis, as well as hyperproliferative skin disease, peptic ulcers, inflammatory bowel disorder, inflammatory skin conditions, and the like. The protease inhibitors of the invention also are useful in screening assays for identifying additional inhibitors that selectively inhibit tryptase-6 cleavage of a peptide having SEQ. ID NO.1.
It remains to be determined exactly how many tryptases exist in humans. Four human tryptase cDNAs (designated tryptase .alpha., I, .beta./II, and III) were isolated by two groups of investigators using two different cDNA libraries (Miller et al., J. Clin. Invest. 1989; 84:1188-1195; Miller et al., J. Clin. Invest. 1990; 86:64-870; Vanderslice et al., Proc. Natl. Acad. Sci. USA 1990; 87:3811-3815). Since the isolated human cDNAs encode enzymes that are >90% identical in their overall amino acid sequences, since humans are not inbred, and since the genes and the region of the chromosome where the tryptase genes reside have not yet been sequenced, the actual number of human mast cell tryptase genes is still unknown. There may be one gene in the human possessing multiple alleles or there may be four or more tryptase genes, some of which are nearly identical. Nevertheless, most investigators believe that human tryptase .alpha. and .beta. are derived from distinct genes.
In terms of their overall amino acid sequences, mature mMCP-7 and mMCP-6 are 71% identical. Mature mMCP-7 exhibits homologies with human tryptases .alpha., I, .beta./II, and III of 74%, 76%, 76% and 78%, respectively, whereas mature mMCP-6 exhibits homologies of 73%, 78%, 78% and 78%, respectively. Thus, it is difficult to conclude from their overall amino acid sequences which tryptase is the human homolog of mMCP-6. However, a comparison of the pro-peptides of mMCP-6 (see below) with those of human tryptases .alpha., I, .beta./II, and III indicate that human tryptase a probably is not the human homolog of mMCP-6. Comparative analysis of the seven loops that Dr. Sali predicts form the substrate binding pocket of each tryptase also indicates that human tryptase a probably is not the human homolog of mMCP-6. However, at present it is not possible to definitively conclude whether the pocket of mMCP-6 is more similar to that in tryptase I, .beta./II, or III.
As used herein, "tryptase-6", and "mast cell tryptase" are used interchangeably to refer to an enzymatically active serine protease that selectively cleaves a peptide sequence having SEQ. ID NO.1. The preferred tryptase-6 for use in the screening assays of the invention is the mature mMCP-6 tryptase or the corresponding mature human tryptase. The nucleic acid and encoded protein sequence of the mMCP-6 zymogen from BALB/c mice are provided as SEQ. ID NOS.13, 14 and 15, and have been accorded GenBank Accession Nos. M57625 and M57626 (see also Reynolds, et al., J. Biol. Chem. 1991, 266:3847-3853). The GenBank accession numbers and reference citations for these and related mast cell protease nucleic acids and/or proteins are provided in the Sequence Listing. In particular, the Sequence Listing identifies the nucleic acid and encoded protein sequence of the potential human homolog(s) of the mMCP-6 zymogen (SEQ. ID NOS. 16-23). These protein sequences include the sequence of the "mature" tryptase-6 proteins. By "mature", it is meant that the sequence represents the serine protease which is the enzymatically active form of the protein (i.e., the form that associates with heparin glycosaminoglycan to form the tryptase-6 complex that selectively cleaves SEQ. ID NO. 1).
In general, the enzymatically active serine proteases of the invention are associated with a mast cell specific glycosaminoglycan such as heparin in a complex that can be formed in vitro and is also known to exist in vivo. Surprisingly, association of a glycosaminoglycan, such as heparin glycosaminoglycan, with the tryptase-6 appears to be essential for the peptide substrate specificity of the cleavage reaction. The Examples demonstrate the extraordinary specificity of an mMCP-6 tryptase/heparin glycosaminoglycan complex for cleaving SEQ. ID NO. 1 and the lack of specificity for mMCP-6 in the absence of this glycosaminoglycan. Prior to this discovery, the dependence of mMCP-6 cleavage specificity on an association with heparin glycosaminoglycan was unknown and could not have been predicted in view of the reported nonspecific cleavage properties of this tryptase or its homologs in other species.
According to one aspect of the invention, a mast cell tryptase-6 inhibitor that competitively inhibits cleavage of a peptide having SEQ. ID NO. 1 by a mast cell protease is provided. Preferably, the mast cell tryptase-6 is mMCP-6 or human tryptase that is complexed with a mast cell specific glycosaminoglycan (e.g., heparin or ChS-E glycosaminoglycan). In a particularly preferred embodiment, the mast cell tryptase-6 inhibitor is a peptide having the amino acid sequence: Arg-Asn-Arg-Gln-Lys-Thr (SEQ. ID NO.1). The invention also includes functionally equivalent peptides of SEQ. ID NO. 1, namely, (1) fragments (2) chemically modified forms of the peptide, and (3) homologs of SEQ I.D. No. 1 that can be used in accordance with the methods of the invention to selectively inhibit a mast cell tryptase-6 complex in vitro or in vivo. Functionally equivalent peptides contain from three to twelve amino acids and are capable of inhibiting the specific cleavage of SEQ. ID NO. 1 by a mast cell tryptase-6 complex, i.e., tryptase-6 associated with a serglycin proteoglycan.
According to one aspect of the invention, a method for inhibiting a mast cell tryptase-6 complex that selectively cleaves SEQ. ID NO. 1 is provided. The method involves contacting the mast cell tryptase-6 complex with one or more protease inhibitors of the invention for a time sufficient to permit the protease inhibitor to enter the substrate binding site of the enzyme.
According to still another aspect of the invention, a method for selecting a mast cell tryptase-6 complex inhibitor is provided. The method involves determining whether a mast cell tryptase-6 complex cleaves a peptide having SEQ. ID NO. 1 in the presence of a putative protease inhibitor. In a particularly preferred embodiment, the putative protease inhibitor is contained in a phage display library. These methods (also referred to herein as "screening assays") are useful for identifying the above-mentioned functionally equivalent peptides of SEQ. ID NO. 1. Such screening assays rely upon biochemical measurements, physical measurements or functional activity tests to determine whether cleavage of SEQ. ID NO. 1 has occurred.
Exemplary functionally equivalent peptide fragments of SEQ. ID NO. 1 are provided in SEQ. ID NOS. 2-11. Exemplary functionally equivalent homologs of SEQ. ID NO. 1 are derived from the naturally-occurring proteins that contain SEQ. ID NO. 1 or a sequence that is substantially identical to SEQ. ID NO. 1. Functionally equivalent peptides of SEQ. ID NO. 1 optionally contain from one to six conservative amino acid substitutions.
The protease inhibitors of the invention competitively inhibit cleavage by a mast cell tryptase-6 of SEQ. ID NO. 1. The preferred protease inhibitors of the invention are irreversible competitive inhibitors. Such irreversible protease inhibitors include, for example, a derivatizing, agent that reacts with an amino acid in the substrate binding site of the mast cell protease to form a covalent bond. Preferably, such derivatizing agents can reside anywhere in the protease inhibitor. In general, such irreversible protease inhibitors have a structure that mimics the transition state of the enzyme-substrate complex formed during reaction of the mast cell protease with SEQ. ID NO. 1. According to yet other aspects of the invention, pharmaceutical compositions containing the above-described protease inhibitors and methods for making the pharmaceutical compositions are provided. The methods involve placing the protease inhibitors of the invention in a pharmaceutically acceptable carrier.
According to a related aspect of the invention, a method for treating a mast cell-mediated inflammatory disorder is provided. Exemplary mast cell-mediated inflammatory disorders include asthma, allergic rhinitis, urticaria and antioedema, and eczematous dermatitis (atopic dermatitis), and anaphylaxis, as well as hyperproliferative skin disease, peptic ulcers, inflammatory bowel disorder, inflammatory skin conditions, and the like. Such mast cell-mediated inflammatory disorders are believed by the Applicants to be mediated by a tryptase-6. Accordingly, the method of the invention involves administering to a subject in need of such treatment one or more protease inhibitors of the invention in a pharmaceutically acceptable carrier. The protease inhibitor is administered to the subject in an amount effective to inhibit activity of a mast cell tryptase-6 complex in said subject.
These and other aspects of the invention as well as various advantages and utilities will be more apparent with reference to the detailed description of the preferred embodiments and in the accompanying drawings. All patents, patent publications and references identified in this document are incorporated in their entirety herein by reference.
Detailed Description of the Invention
The present invention in one aspect involves the discovery that a macromolecular complex containing mouse mast cell tryptase-6 ("mMCP-6") associated with heparin glycosaminoglycan selectively cleaves a peptide having the sequence of SEQ. ID NO. 1 and that this and other structurally-related peptides can be used to selectively inhibit the enzymatic activity of mMCP-6 and its homologs (e.g., human tryptase) in vitro and in vivo. Although not intending to be bound to any particular mechanism or theory, it is believed that the naturally-occurring ("physiological") substrate(s) of tryptase in vivo contains a peptide sequence that is substantially identical to SEQ. ID NO. 1 and that cleavage by a tryptase-6 in vivo of its physiological substrate represents a fundamental step in the pathogenesis of mast cell mediated-inflammatory disorders. By "substantially identical" it is meant that the peptide cleavage site sequence of the physiological substrate of tryptase-6 differs from SEQ. ID NO. 1 by, at most, one amino acid.
As used herein, a "tryptase-6" protein refers to the enzymatically active "mature" mMCP-6 protein, its naturally occurring alleles, and homologs of the foregoing proteins in other species. The tryptase-6 proteins, like other serine proteases, are synthesized in cells as zymogens (i.e., in an enzymatically inactive precursor form) which include a hydrophobic "pre" peptide sequence (also referred to as a "signal sequence" or "signal peptide") and a "pro" sequence (also referred to as a "pro-peptide sequence") attached to the N-terminal portion of the mature protein. The nucleic acid and encoded protein sequence of the mMCP-6 zymogen from BALB/c mice are provided as SEQ ID NOS. 13, 14 and 15, and have been accorded GenBank Accession Nos. M57625 and M57626, (see also Reynolds, et al., J. Biol. Chem. 1991, 266:3847-3853). The GenBank accession numbers and reference citations for these and other mast cell protease nucleic acids and/or proteins are provided in the Sequence Listing. In particular, the Sequence Listing identifies the nucleic acid and encoded protein sequence of the potential human homologs of the mMCP-6 zymogen (SEQ ID NOS. 16-23), including the protein sequences for the "mature" tryptase-6 proteins for these proteins. By "mature", it is meant that the sequence represents the serine protease which is the enzymatically active form of the protein.
The tryptase-6 proteins that are inhibited by the protease inhibitors of the invention are members of the serine protease superfamily. In particular, the tryptase-6 proteins are members of the trypsin-like serine protease family of proteins that are the major constituents of the secretory granules of mouse, rat, gerbil, dog, and human mast cells. Lung, heart, and skin mast cells in the BALB/c mouse express at least two tryptases designated mouse mast cell protease 6 ("mMCP-6") and 7 ("mMCP-7")! which are 71% identical in terms of their overall amino acid sequences. This tryptase family of mast cell proteases has been implicated in the pathobiology of Fc.epsilon.RI-elicited responses in airways. Linkage analysis has implicated the region of chromosome 17 where the mMCP-6 and mMCP-7 genes reside as one of the candidate loci for the inheritance of intrinsic airway hyper responsiveness. A physiological substrate for the mMCP-7 protein recently has been identified as fibrinogen (see, U.S. Ser. No. 60/032,354 filed Dec. 4, 1996, now U.S. Ser. No.08/978,404 filed Nov. 25, 1997 by R. Stevens). To date, the inability to definitively identify the physiological substrate for mMCP-6 has prevented the development of therapeutic agents that mediate conditions attributable to an under- or over-abundance of the mMCP-6 protein or its physiological substrate. Accordingly, the identification of the specific cleavage sequence disclosed herein for the mMCP-6 protein permits the development of therapeutic agents for treating conditions that are mediated by this tryptase.
The mMCP-6 protein is stored in acidic granules of the cell as a complex containing the mature, enzymatically active form of the enzyme ionically bound to the glycosaminoglycan side chains of serglycin proteoglycans (Ghildyal, et al., J. Exp. Med. 1996; 184:1061-1073, whose content is incorporated herein by reference in its entirety). As used herein, a "tryptase-6 complex" refers to a mature mMCP-6 tryptase (its alleles, homologs) in association with a serglycin proteoglycan (containing heparin or another mast cell- specific chondroitin). Although mMCP-6 and mMCP-7 are negatively charged at neutral pH and are associated with serglycin proteoglycans at neutral pH, the two tryptases differ in their ability to dissociate from the proteoglycans following their exocytosis from the mast cell. As a result, these proteases exhibit different substrate specificities and are metabolized quite differently in mice undergoing passive systemic anaphylaxis.
Modeling and site-directed mutagenesis analysis of recombinant pro-mMCP-7 (i.e., the expressed protein with its normal "pro-peptide" sequence) suggest that this mature tryptase readily dissociates from serglycin proteoglycans when the protease/proteoglycan macromolecular complex is exocytosed into a pH 7.0 environment because the glycosaminoglycan-binding domain on the surface of mMCP-7 consists primarily of a cluster of His residues. In contrast, the mMCP-6 protein does not readily dissociate from serglycin proteoglycans because its glycosaminoglycan-binding domain consists primarily of a cluster of strongly basic Lys or Arg residues, as found in all mast cell chymases. Although not intending to be limited to a particular mechanism of action, the prolonged retention of exocytosed mMCP-6 complex in the extracellular matrix around activated tissue mast cells is believed by us to be associated with a local activity for this tryptase, whereas the rapid dissipation of mMCP-7 from tissues and its poor ability to be inactivated by circulating protease inhibitors suggests that this distinct, but homologous, tryptase cleaves proteins at more distal sites.
More than 25 genes have been cloned that encode the peptide cores of different proteoglycans. mMCP-6 is preferentially bound to the glycosaminoglycan (GAG) side chains of the serglycin family of proteoglycans. Those mast cells that express mMCP-6 generally have serglycin proteoglycans that have covalently bound heparin chains but sometimes these proteoglycans have highly charged chondroitin sulfate (ChS) chains (e.g., ChS-diB and ChS-E) Human lung mast cells also can express serglycin proteoglycans that can have either heparin or ChS-E chains (Stevens et al., Proc. Natl. Acad. Sci. USA 1988; 85:2284-2287). Although small amounts of serglycin proteoglycan containing ChS-E chains have been identified in cultured human eosinophils (Rothenberg et al., J. Biol. Chem. 1988; 263:13901-13908), mast cells are the only mammalian cell type which can produce relatively large amounts of ChS-E.
It is not known why mast cells synthesize very different types of GAG onto a serglycin peptide core. Since more than 30 enzymes are involved in the differential biosynthesis of heparin and ChS-E, the switch in GAG expression in the mast cell probably is biologically relevant. It is possible that ChS-E influences the substrate specificity of mMCP-6 differently than heparin. Accordingly, other highly charged GAG, such as ChS-E, also may regulate the substrate specificity of mMCP-6.
The specificity of the mMCP-6 complex for cleaving SEQ. ID NO. 1 was discovered during experiments designed to elucidate the preferred amino acid sequences that are cleaved by this mast cell protease. Surprisingly, heparin glycosaminoglycan was found to alter the substrate specificity of mMCP-6 for cleaving peptides in a tryptase-specific bacteriophage display library. We believe that heparin glycosaminoglycan may sterically restrict the substrate-binding cleft of mMCP-6 by directly influencing one of the seven loops that form this pocket. The present invention is based upon the discovery that the mMCP-6 complex selectively cleaves a peptide containing SEQ. ID NO. 1 but that this enzymatic activity is not shared with mMCP-6 (in the absence of a serglycin proteoglycan) or with mMCP-7.
A "mast cell protease inhibitor" or a "protease inhibitor", as used herein, refers to a peptide which competitively inhibits cleavage by a tryptase-6 complex of SEQ. ID NO. 1. The protease inhibitors of the invention are peptides that are or contain SEQ. ID NO. 1 or its functionally equivalent peptides. Protease inhibitors which are functionally equivalent peptides of SEQ. ID NO. 1 are identified in screening assays which measure the ability of a putative protease inhibitor to prevent cleavage by a tryptase-6 complex (e.g., a mMCP-6 or human tryptase-6 complex) of SEQ. ID NO. 1 or its functional equivalents.
As used herein, "functionally equivalent peptides" of SEQ. ID NO.1 refer to (1) fragments, (2) chemicallly modified derivatives, and (3) homologs of SEQ. ID NO. 1, that can be used in accordance with the methods of the invention to inhibit cleavage by a tryptase-6 complex of SEQ. ID NO. 1. Functionally equivalent peptides contain from three to twelve amino acids and competitively inhibit cleavage by a tryptase-6 complex of a peptide that is or that includes SEQ. ID NO.1.
Functionally equivalent peptides of SEQ. ID NO. 1 are identified in one or more "screening assays". In general, such screening assays are of two types: (1) binding assays which detect a complex containing the putative protease inhibitors associated with a tryptase-6 complex (e.g., mMCP-6/heparin glycosaminoglycan) and (2) enzymatic activity assays which measure the ability of a putative protease inhibitor to inhibit cleavage by a tryptase-6 complex of SEQ. ID NO. 1 or a functionally equivalent peptide of SEQ. ID NO. 1. In general, the binding assays (preferably, irreversible binding) involve the detection of a labeled inhibitor (e.g., a fluorescent or radioactive tag) associated with the tryptase-6 complex; enzymatic assays measure the ability of the putative protease inhibitor to competitively inhibit cleavage by the tryptase-6 complex of SEQ. ID NO. 1.
In a particularly preferred embodiment, the protease inhibitor of the invention has SEQ. ID NO. 1. This amino acid sequence was identified in a tryptase-specific bacteriophage peptide display library that was screened with mMCP-6 to determine its preferred substrate peptide sequence (see Example). No particular peptide sequence was favored when the library was screened with mMCP-6 alone; however, a phage clone was preferentially obtained when the library was screened with an mMCP-6/heparin complex. Analysis of this clone revealed a sequence (SEQ. ID NO. 1) that was susceptible to cleavage by the mMCP-6/heparin complex. A search of GenBank indicated that a sequence that is substantially identical to SEQ. ID No. 1 is present in human fibronectin (SEQ. ID NO. 12, amino acid nos. 1351-1356). Although not intending to be bound to any particular theory or mechanism, it is believed that this protein represents a physiological substrate of human tryptase-6 and that tryptase-6 mediates the pathogenesis of inflammatory disorders by selectively cleaving fibronectin at an amino acid sequence that is substantially identical to SEQ. ID NO. 1. (See Example for a more detailed discussion of the role played by fibronectin in integrin-binding and the implications of this discovery with respect to the role played by tryptase in mast-cell mediated inflammation by controlling integrin-dependent signaling pathways.)
The amino acid sequence of SEQ. ID NO. 1 is:
A generic formula that embraces SEQ. ID NO. 1 is:
where R/K represents an Arg or Lys (basic amino acids)and X represents a neutral amino acid. It is believed that the highly charged basic character of SEQ. ID NO. 1 plays an important role in the localization of the peptide to substrate binding site of the mast cell tryptase-6.
As used herein, functionally equivalent "peptide fragments" of SEQ. ID NO. 1 refer to fragments of SEQ. ID NO. 1 that contain from three to five amino acids (SEQ. ID NOS. 2 through 10). Peptide fragments can be synthesized without undue experimentation using standard procedures known to those of ordinary skill in the art. Each of SEQ. ID NOS. 2-10 contains at least one basic amino acid that can serve as a P1 amino acid for cleavage by the mast cell serine protease.
In general, the term "homolog" refers to a molecule that shares a common structural feature with the molecule to which it is deemed to be an homolog. As used herein in reference to the protease inhibitors of the invention, a "functionally equivalent peptide" that is a "homolog" of SEQ. ID NO.1 is a peptide which shares a common structural feature (amino acid sequence homology) and a common functional activity (inhibiting tryptase-6 complex cleavage of SEQ. ID NO.1) with SEQ. ID NO.1. Functionally equivalent peptide homologs of SEQ. ID NO.1 are derived from naturally-occurring proteins that contain an amino acid sequence having sequence homology to SEQ. ID NO.1. Preferably, such homologs contain at least four and, preferably, five of the amino acid residues in the same order as SEQ. ID NO.1 and, optionally, contain from zero to five amino acids that are derived from the naturally-occurring amino acid sequence. Exemplary functionally equivalent peptide homologs of SEQ. ID NO. 1 include amino acids 1351-1356, 1350-1356, 1349-1356, 1348-1356, 1347-1356, 1346-1356, 1351-1357, 1351-1358, 1351-1359, 1351-1360, 1351-1361- and 1346-1361 of fibronectin (SEQ. ID No. 12).
A computer search of a protein database with SEQ. ID NO.1 revealed a substantially identical sequence in fibronectin. Athough not intending to be bound to any particular theory, it is believed that the physiological substrate for mMCP-6 complex is fibronectin and that tryptase-6 complex is capable of selectively cleaving this protein in vitro or in vivo. Thus, fibronectin represents a "protein homolog" of SEQ. ID NO.1 from which functionally equivalent peptide homologs of SEQ. ID NO. 1 can be derived.
Fibronectin contains the sequence, Arg-Gly-Arg-Gln-Lys-Thr (SEQ. ID NO.11), which differs from SEQ. ID NO.1 in a single amino acid. This sequence is found in fibronectin at amino acids 1351-1356 and is believed to be a cleavage site for the mast cell serine protease in vivo. Functionally equivalent peptide homologs of SEQ. ID NO.1 that are derived from fibronectin include from zero to five amino acids that are N-terminal and/or C-terminal to SEQ. ID NO.11 in the this protein homolog. Additional SEQ. ID NO.1 protein homologs having sequence homology with SEQ. ID NO.1 can be identified using art-recognized methods, e.g., searching data bases such as GENBANK for homologous peptides and/or proteins, as new sequences are added to these databases.
Functionally equivalent peptides of SEQ. ID NO.1 optionally contain conservative amino acid substitutions, provided that the peptides which contain the conservative substitutions competitively inhibit SEQ. ID NO.1 binding to, or cleavage by, a mast cell tryptase-6 complex in the above-mentioned screening assays. As used herein, "conservative amino acid substitution" refers to an amino acid substitution which does not alter the relative charge or size characteristics of the peptide in which the amino acid substitution is made. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M,I,L,V; (b) F,Y,W; (c) K,R,H; (d) A,G; (e) S,T; (f) Q,N; and (g) E,D. In the particularly preferred embodiments, the functionally equivalent peptides of SEQ. ID NO. 1 include one or more conservative amino acid substitution in which arginine and lysine are substituted for one another. It is believed that one, two, or three conservative amino acid substitutions can be made in SEQ. ID NO. 1 without adversely affecting the ability of the peptide to competitively bind to/inhibit a tryptase-6 complex.
Preferably, the protease inhibitors of the invention are peptides that include one or more inter-amino acid bonds that are non-hydrolyzable in vivo. For example, the peptide may contain one or more D-amino acids, thereby rendering the peptide less susceptible to non-specific proteolytic cleavage in vivo. Alternatively, or additionally, the peptide may contain a non-hydrolyzable peptide bond. Such non-hydrolyzable peptide bonds and methods for preparing peptides containing same are known in the art. Exemplary non-hydrolyzable bonds include -psiCH.sub.2 NH!-reduced amide peptide bonds, -psiCOCH.sub.2 !- ketomethylene peptide bonds, -psiCH(CN)NH!-(cyanomethylene)amino peptide bonds, -psiCH.sub.2 CH(OH)!- hydroxyethylene peptide bonds, -psiCH.sub.2 O!- peptide bonds, and -psiCH.sub.2 S!- thiomethylene peptide bonds. Additional non-hydrolyzable peptide bonds can be identified using no more than routine experimentation.
In the preferred embodiments, a derivatizing agent (X) is covalently coupled to the peptide substrate (protease inhibitor) to form an irreversible protease inhibitor (X-P P). Preferably, the derivatizing agent is covalently attached to the N-terminal or the C-terminal amino acid of the protease inhibitor in accordance with standard procedures for derivatizing an amino acid. In general, the derivatizing agent is a reactive group that reacts with an amino acid in the substrate binding site of the mast cell tryptase-6 complex. Preferably, the chemically modified derivative of the peptide substrate (protease inhibitor) possesses a reactive group that functions as an irreversible inhibitor of a tryptase-6 (e.g., mMCP-6). For example, numerous low molecular weight inhibitors of serine proteases have been synthesized that contain a .alpha.-fluorinated ketone or .alpha.-keto ester derivative of a critical amino acid in the preferred peptide substrate (Angelastro et al., J. Med. Chem. 1990; 33:13-16). Additional exemplary derivatizing agents for conferring upon a peptide substrate the ability to irreversibly bind to the substrate binding site are described in U.S. Pat. No. 5,543,396, issued to Powers, et al., "Proline Phosphonate Derivative"; and U.S. Pat. No. 5,187,157 and U.S. Pat. No. 5,242,904, issued to Kettner, et al., "Peptide Boronic Acid Inhibitors of Trypsin-Like Proteases".
As discussed above, a computer search of a protein database with SEQ. ID NO.1 revealed that a substantially identical sequence (SEQ. ID NO. 11, fibronectin amino acids 1351-1356) resides in the middle of each subunit of fibronectin. This sequence is conserved from rats to humans. As discussed in detail in the Example, fibronectin possesses numerous conserved domains that enable fibronectin to interact simultaneously with different proteins on the cell's surface and in the extracellular matrix. The mMCP-6 susceptible sequence in fibronectin is located between the collagen and integrin binding domains. Based upon this observation and the results disclosed herein, we believe that specific cleavage at this site has a dramatic effect on fibronectin-mediated adhesion of fibroblasts and inflammation that is mediated by integrin signal transduction.
Described in the Example is an experiment in which mMCP-6 was injected into the peritoneal cavity of a mouse animal model. Surprisingly, the injection of mMCP-6 into the peritoneal cavity of the animal model specifically recruited neutrophils to this site; however, injection of homologous mMCP-7 into the cavity did not have this effect. As discussed in more detail in the Example, we believe that neutrophil emigration in this in vivo assay is mediated, in part, by a generated large-sized fragment of fibronectin that lacks its collagen binding domain. Accordingly, the discovery described herein that mMCP-6 (but not mMCP-7) specifically cuts fibronectin between its collagen- and integrin-binding domains has important implications for mast cell-mediated control of fibrosis and inflammation. More specifically, the animal model results presented herein provide evidence that the protease inhibitors disclosed herein are useful for modulating tryptase-6-mediated inflammation by inhibiting specific cleavage by tryptase of its physiological substrate in vivo. Although mMCP-6 and mMCP-7 (described in U.S. Ser. No. 60/032,354) have different substrate specificities, we believe that both tryptases alter integrin-mediated signaling pathways: mMCP-7 by cleaving fibrinogen and mMCP-6 by cleaving fibronectin. Thus, the results presented herein further suggest that mast cell tryptases play a central role in mast cell-mediated inflammation by controlling different integrin-dependent signaling pathways.
In view of the foregoing, a method for treating a mast cell-mediated inflammatory disorder is provided. The method involves administering to a subject in need of such treatment the tryptase-6 complex inhibitors of the invention in a pharmaceutically acceptable carrier and in an amount effective to inhibit activity of a tryptase-6 complex in said subject.
As used herein, a "mast cell-mediated inflammatory disorder" refers to those diseases associated with mast cell tryptase-6 release and susceptible to treatment with a tryptase-6 inhibitor such as disclosed herein. Examples of such disorders include diseases of immediate type hypersensitivity such as asthma, allergic rhinitis, urticaria and antioedema, and eczematous dermatitis (atopic dermatitis), and anaphylaxis, as well as hyperproliferative skin disease, peptic ulcers, inflammatory bowel disorder, inflammatory skin condiitons, and the like. "Hyperresponsiveness" refers to late phase bronchoconstriction and airway hyperreactivity associated with chronic asthma. Hyperresponsiveness of asthmatic bronchiolar tissue is believed to result from chronic inflammation reactions, which irritate and damage the epithelium lining the airway wall and promote pathological thickening of the underlying tissue. Thus, the protease inhibitors of the invention are useful for the treatment (prevent, delay the onset of, or ameliorate the symptoms) of immunomediated inflammatory disorders, and particularly with those associated with the respiratory tract, e.g., asthma, and hyperresponsiveness.
The protease inhibitors described above are administered in effective amounts. An effective amount is a dosage of the protease inhibitor sufficient to provide a medically desirable result. The effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practioner. For example, an effective amount for treating asthma would be an amount sufficient to lessen or inhibit one or more clinically recognized symptoms of asthma. Thus, it will be understood that the protease inhibitors of the invention can be used to treat mast-cell mediated inflammatory disorders prophylactically in subjects at risk of developing such inflammatory disorders. As used in the claims, "inhibit" embraces all of the foregoing. Likewise, an effective amount for treating any of the above-noted inflammatory disorders is that amount which can slow or halt altogether the particular symptoms of such disorders. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment.
Generally, doses of active compounds will be from about 0.01 mg/kg per day to 1000 mg/kg per day. It is expected that doses in the range of 50-500 mg/kg will be suitable, preferably orally and in one or several administrations per day. Lower doses will result from other forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.
When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptably compositions. Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts. As used herein, tryptase-6 inhibitor or protease inhibitor means the compounds described above as well as salts thereof.
The tryptase-6 inhibitors may be combined, optionally, with a pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration into a human or other animal. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being comingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
The pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the protease inhibitor, which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.
A variety of administration routes are available. The particular mode selected will depend of course, upon the particular drug selected, the severity of the condition being treated and the dosage required for therapeutic efficacy. The methods of the invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, topical, nasal, interdermal, or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intramuscular, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. They could, however, be preferred in emergency situations. Oral administration will be preferred for prophylactic treatment because of the convenience to the patient as well as the dosing schedule.
The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the protease inhibitors into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the protease inhibitors into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
For topical applications, the protease inhibitors can be formulated as ointments or creams. Exemplary pharmaceutically acceptable carriers for peptide drugs, described in U.S. Pat. No. 5,211,657, are useful for containing the protease inhibitors of the invention. Exemplary pharmaceutically acceptable carriers for protease inhibitors that are small molecules and, in particular, for aerosol administration, are described in U.S. Pat. No. 5,525,623. Such preparations also are useful for containing the protease inhibitors of the invention. As used herein, the term "aerosol" refers to a gas-borne suspended phase of the protease inhibitors that is capable of being inhaled into the bronchioles or nasal passages. Such formulations are particularly useful for treating asthma and hyperresponsiveness.
According to another aspect of the invention, the protease inhibitors of the invention are useful as agents for modulating integrin-mediated signal transduction. Thus, the invention advantageously provides mast cell protease inhibitors in a form that can be administered in accordance with art-recognized methods for drug delivery in vivo. For example, the protease inhibitors can be formulated into an aerosol or topical pharmaceutic preparation to deliver to local cells an amount of protease inhibitor sufficient to inhibit mast cell-mediated fibrosis, inflammation, and integrin-related signal transduction pathways such as those involved in cell trafficking and proliferation. Topical application to the skin of a protease inhibitor of the invention is useful for inhibiting cell proliferation associated with conditions such as psoriasis. Aerosol application of a protease inhibitor is useful for inhibiting inflammation associated with asthma and other disorders associated with intrinsic airway hyperresponsiveness.
Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the protease inhibitors. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the protease inhibitors described above, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer based systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the protease inhibitor is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,832,253, and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.
Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. Long-term release, as used herein, means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days. Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
According to yet another aspect of the invention, a method for manufacturing a pharmaceutical composition containing the protease inhibitors of the invention is provided. The method involves placing the above-described protease inhibitor in a pharmaceutically acceptable carrier to form a pharmaceutical composition and administering the pharmaceutical composition containing a therapeutically effective amount of the protease inhibitor to the recipient.
It should be understood that the preceding is merely a detailed description of preferred embodiments. It therefore should be apparent to those of ordinary skill in the art that various modifications and equivalents can be made without departing from the spirit and scope of the invention. All references, patents and patent publications that are identified in this application are incorporated in their entirety herein by reference. The specific example presented below is illustrative only and is not intended to limit the scope of the invention described herein.
Example
Experimental Procedures.sup.1
Expression of pro-mMCP-6 and pro-EK-mMCP-6-FLAG in Insect Cells--The novel bioengineering approach developed recently to obtain a pseudozymogen form of mMCP-7 that could be proteolytically activated after its purification from the conditioned media of insect cells was used to obtain a similar pseudozymogen (pro-EK-mMCP-6-FLAG) form of mMCP-6. Expressed pro-EK-mMCP-6-FLAG has an EK-susceptible peptide (Asp-Asp-Asp-Asp-Lys, SEQ ID NO. 29) in between the domain that encodes the endogenous pro-peptide and the N-terminal Ile residue of the mature tryptase. The recombinant protein also has the 8-residue FLAG peptide attached to its C terminus. In order for a serine protease to have catalytic activity the .alpha.-amino of the N-terminal Ile residue must form a internal ion pair with the carboxyl group of an internal specific Asp residue after the pro-peptide is removed (Freer et al., Biochemistry 1977, 9:1997-2009). Thus, it is critical that mature mMCP-6 have an N-terminal Ile residue. Because EK is a highly specific enzyme that cleaves the Lys-Ile bond in its recognition motif (Light and Janska, Trends Biochem Sci 1989, 14(3):110-112), is a relatively stable enzyme at pH 5.0, and will specifically cleave pro-mMCP-7-FLAG, it was anticipated that pro-mMCP-6-FLAG could be proteolytically activated by EK under conditions where the recombinant tryptase, itself, would have very little enzymatic activity until the pH is raised to 7.0. That the pseudozymogen also has the FLAG peptide at its C-terminus enabled its rapid purification from the insect cell conditioned media by means of an affinity column containing anti-FLAG IgG antibody (Prickett et al., Biotechniques 1989, 7:580-589; Brizzard et al., Biotechniques 1994, 16:730-735).
While, in theory, EK digestion of pro-mMCP-6-FLAG should remove the modified pro-peptide, the resulting recombinant product still will have the FLAG peptide attached to its C-terminus. Nevertheless, it was anticipated that mMCP-6-FLAG would be enzymatically active because the FLAG peptide does not influence the enzymatic activity of mMCP-7.
The relevant cDNA constructs, created using standard polymerase chain reaction approaches, were inserted in the correct orientation into the multiple cloning site of pVL1393 (PharMingen, San Diego, Calif.) downstream of the promoter of the polyhedrin gene. Insect cells were induced to express pro-mMCP-6 and pro-mMCP-6-FLAG, as described previously for pro-mMCP-7 (Matsumoto et al., J. Biol. Chem. 1995, 270:19524-19531) and pro-mMCP-7-FLAG. Briefly, purified plasmid DNA (.about.5 .mu.g) was mixed with 0.5 .mu.g of linearized BaculoGold.TM. DNA (PharMingen) and calcium phosphate, each resulting DNA solution was added to 3.times.10.sup.6 adherent Spodoptera frugiperda 9 insect cells (Invitrogen, San Diego, Calif.) that were in their log phase of growth, and the infected cells were cultured for 7 days at 27.degree. C. in medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (Sigma, St. Louis, Mo.). Recombinant virus particles (.gtoreq.3.times.10.sup.7) from these insect cells were added to a new culture dish containing 6.times.10.sup.6 Trichoplusia ni High Five.TM. insect cells (Invitrogen) in their log phase of growth, and the infected cells were cultured in serum-free, Xpress medium (BioWhittaker, Walkersville, Md.). Generally 4 d later, the conditioned medium was centrifuged at 1500 g for 15-min at room temperature before attempting to purify the secreted recombinant protein.
Purification of pro-mMCP-6 and pro-EK-mMCP-6-FLAG from Insect Cell Conditioned Media and EK Activation of the Recombinant Zymogen--Pro-mMCP-6 and pro-EK-mMCP-6-FLAG were purified by heparin-Sepharose chromatography, as described for pro-mMCP-7 (Matsumoto et al., J. Biol. Chem. 1995, 270:19524-19531). The purification of pro-EK-mMCP-6-FLAG also was carried out using an affinity column containing the mouse anti-FLAG M2 monoclonal antibody (Eastman Kodak/International Biotechnol.). This immunoaffinity column (2 ml) was washed with 0.1 M glycine, pH 3.5, and then with 50 mM Tris-HCl and 150 mM NaCl, pH 7.4. After the application of the insect cell conditioned media, the column was washed briefly with the above pH 7.4 buffer, and then bound pro-EK-mMCP-6-FLAG was eluted with 0.1 M glycine, pH 3.5. The eluate was collected into tubes that contained 0.1 M Tris-HCl, pH 7.0, to minimize acid-mediated denaturation of pro-EK-mMCP-6-FLAG. The protein concentration of the eluate was estimated by measuring the absorbance at 280 nm.
Purified pro-EK-mMCP-6-FLAG (.about.100 .mu.g in 100 .mu.l) was separately mixed with 100 .mu.l of a pH 5.2 buffer consisting of 50 mM sodium acetate and 5 mM calcium chloride. One .mu.l of a solution containing 550 U of calf intestine EK (Biozyme) was added to each, and the mixture was incubated at 37.degree. C. generally for 3 h to allow EK to activate the zymogen in the absence of heparin. The spectrophotometric method of Svendsen and coworkers (Throm. Res. 1972, 1:267-278) was used to determine whether or not mMCP-6-FLAG is enzymatically active. Generally, 1-.mu.l samples of each activation reaction were placed in 1 ml of a pH 7.4 buffer containing 25 mM sodium phosphate, 1 mM EDTA, and 50 .mu.g of tosyl-Gly-Pro-Lys-p-nitroanilide. The change in optical density at 405 nm was then determined after a 3-min incubation at room temperature. The ability of recombinant mMCP-6-FLAG to cleave the trypsin-susceptible substrates tosyl-Gly-Pro-Arg-p-nitroanilide, benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide, benzoyl-Pro-Phe-Arg-p-nitroanilide, and acetyl-Ile-Glu-Ala-Arg-p-nitroanilide were also evaluated.
SDS-PAGE/Immunoblotting and N-terminal Amino Acid Analysis--Insect conditioned media (.about.20 .mu.l) containing either pro-mMCP-6, pro-EK-mMCP-6-FLAG, or EK-activated mMCP-6-FLAG (.about.1 .mu.l) were diluted in SDS-PAGE buffer (1% SDS, 5% 2-ME, 0.1% bromophenol blue, and 500 mM Tris-HCl, pH 6.8) and boiled for 5 min before being loaded onto 12% polyacrylamide gels. After SDS-PAGE, the resolved proteins were stained with Coomassie Blue or were transferred in 20 mM Tris-HCI, 150 mM glycine, pH 8.3 buffer containing 20% methanol for 2 to 4 h at 200 mA to PVDF membranes (Millipore) using a BIO-RAD (Richmond, Calif.) immunoblotting apparatus. For immunoanalysis of the resulting protein blots, each membrane was sequentially incubated 1 h in 5% non-fat milk, 1 h with a 1:500 dilution of affinity-purified rabbit anti-mMCP-6 Ig (Ghildyal et al., J. Immunol. 1994, 153:2624-2630) in TBST buffer (Tris-buffered saline with 0.01% Tween 20), TBST buffer alone, and then a 1:1,000 dilution of anti-rabbit IgG alkaline phosphatase conjugate (.about.1 ng/ml final concentration) in TBST buffer. Immunoreactive proteins were visualized using nitroblue tetrazolium (0.2 mg/ml) and 5-bromo-4-chloro-3-indolyl phosphate (0.1 mg/ml) as substrates.
For N-terminal amino acid analysis, SDS-PAGE-resolved proteins were electrobloted. unto PVDF membranes, briefly stained with 0.5% Ponceau S red (Sigma), and the relevant protein/peptide bands were subjected to automated Edman degradation by the Harvard Microchemistry Facility (Harvard Biological Laboratories, Cambridge, Mass.).
Screening of a Tryptase-Specific, Bacteriophage Peptide Display Library with mMCP-6--A peptide display library that encodes an altered pIII containing at its N terminus the FLAG peptide followed by an 8-residue hypervariable peptide was screened with recombinant mMCP-6-FLAG. Briefly, phage were obtained that express on their surface a pill fusion protein with an extension peptide consisting of the FLAG peptide and a hypervariable octamer peptide containing a Lys/Arg residue at the PI site. After the varied phages in the library were allowed to bind to the anti-FLAG IgG column, the immuno-affinity column was incubated with recombinant mMCP-6-FLAG in the presence or absence of heparin. Those phage recovered in the column's eluate were amplified, and the selection procedure was repeated one lo three times. By determining the nucleotide sequence of the relevant portion of the geneIII construct in each clone, the amino acid sequence of the mMCP-6-susceptible peptide in the random domain of the pIII fusion protein was deduced. To prepare the phage column used in the screening process, 10 ml of the phage-enriched supernatant was added to 2 ml of 20% polyethylene glycol (8 kDa; Sigma) and 2.5 M NaCl and the mixture incubated at 4.degree. C. for 30 min. After a 30 min centrifugation at 10,000 g, the recombinant phage in the pellet were resuspended in 2 ml of 150 mM NaCl, 1 mM NaCl.sub.2, and 10 mM sodium phosphate, pH 7.0, and applied to a 1-ml affinity column containing the anti-FLAG M1 monoclonal antibody. The column was washed 3 times with 10 ml of the same pH 7.0 buffer to remove unbound phage. EK-activated mMCP-6-FLAG (.about.50 .mu.g in 200 .mu.l buffer) in the absence or presence of heparin glycosaminoglycan (.about.50 .mu.g) was added, and the column was sealed and incubated at room temperature for 90 min. After protease treatment, the column was washed with 2 ml of the pH 7.0 buffer to recover those phage which possessed protease-susceptible pIII fusion proteins. Log-phase E. coli were infected with the obtained phage to produce phagemid. Bacteria were again grown in 2.times.YT medium containing 2% glucose and the phagemid in the bacteria were converted to phage with the addition of helper phage. This screening procedure was repeated up to 4 times to select the phage in the library which are most susceptible to degradation by mMCP-6-FLAG.
E. coli was infected with resulting mMCP-6-FLAG-susceptible phage to generate phagemids. The infected bacteria were seeded onto a plate containing 1.5% agar, 2% Bacto-tryptone, 0.5% Bacto-yeast extract, 2% glucose, 0.09 M NaCl, 0.01 M MgCl.sub.2, and 100 .mu.g/ml ampicillin. Individual clones were isolated and grown overnight at 37.degree. C. in 2 ml of 2.times.YT medium containing 2% glucose with 50 .mu.g/ml ampicillin. One ml of the overnight cultures were centrifuged at .about.12,000 g for 5 min. The bacteria in the pellets were lysed and the DNAs were extracted with mini-prep method. The DNAs were digested with NotI and EcoRI restriction enzymes at 37.degree. C. overnight. The digested DNA mini-preps were subjected to electrophoresis on a 1% agarose gel, and those individual phage clones with .about.1300-bp inserts were selected for maxi-preparation of their DNAs using nucleobond DNA-binding columns (The Nest Group). The nucleotide sequences which encode the 8-mer, protease-susceptible peptide domains in the fusion proteins were determined.
In Vitro Degradation of Fibronectin by Recombinant mMCP-6-FLAG--Five .mu.g of purified mouse fibronectin (Alexis) was suspended in 1 mM EDTA and 25 mM sodium phosphate, pH 7.4, containing 0.01 U EK, 0.5 .mu.g recombinant pro-EK-mMCP-6-FLAG, 0.5 .mu.g recombinant mMCP-6-FLAG (activated with 0.01 U EK), or 0.5 .mu.g recombinant mMCP-7FLAG (activated with 0.01 U EK). After an incubated for various lengths of times, the resulting digests were subjected to SDS-PAGE. In one experiment, the N-terminal amino acid sequences; of the major fibronectin fragments in the mMCP-FLAG digest were determined.
mMCP-6-FLAG-Induced Emigration of Neutrophils Into the Peritoneal Cavity and mMCP-6-Induced Growth of Fibroblasts and their Adhesion to Fibronectin--This experiment is discussed below.
Results and Discussion
Generation of pro-mMCP-6 and pro-EK-mMCP-6-FLAG in Insect Cells, and EK Conversion of the Recombinant Pseudozymogen to Enzymatically Active Tryptase--Insect cells infected with the relevant construct secreted large amounts of pro-mMCP-6 and pro-EK-mMCP-6-FLAG into the conditioned media. Based on its deduced amino acid sequence, mMCP-6 has an overall net charge at pH 7.0 that is considerably more negative than any mouse mast cell chymase (Sali et al., J. Biol. Chem. 1993, 268:9023-9034). Nevertheless, because mMCP-6 does not dissociate easily from its serglycin proteoglycan, it is retained for >1 h in inflammatory sites (Ghildyal et al., J. Exp. Med. 1996, 184:1061-1073). Modeling studies suggested that this, unexpected feature of mMCP-6 is caused by an Arg/Lys rich domain that forms on the surface when the tryptase is properly folded. Like pro-mMCP-6, pro-EK-mMCP-6-FLAG bound to a heparin-Sepharose column that had been equilibrated in 100 mM NaCl/10 mM sodium phosphate, pH 5.5. Because both recombinant proteins dissociated from the heparin-Sepharose affinity column when the NaCi concentration of the buffer was raised to >300 mM, it was concluded that the secreted mMCP-6 pseudozymogen is properly folded. Pro-EK-mMCP-6-FLAG also could be readily purified using the immunoaffinity column.
As assessed by SDS-PAGE, the recombinant pseudozymogen decreased .about.2 kDa in size when incubated for 3 to 24 h with EK. Amino acid sequence analysis revealed that the resulting product possessed an N-terminal sequence of X-Y-Z which is identical to that of mature mMCP-6 deduced from its cDNA (Reynolds et al., J. Biol. Chem. 1991, 266:3847-3853). While recombinant and native mMCP-7 exhibit good catalytic activity in the absence of heparin (Ghildyal et al., J. Exp. Med. 1996, 184:1061-1073), it has been reported that human mast cell tryptases purified from the lung do not exhibit substantial enzymatic activity unless this glycosaminoglycan is present in the assay (Schwartz and Bradford, J. Biol. Chem. 1986, 261:7372-7379; Alter et al., Biochem. J. 1987, 248:821-827). The ability to purify pro-EK-mMCP-6-FLAG from the conditioned media by means of the immuno-affinity column allowed us to determine if the recombinant protease exhibits enzymatic activity in the absence of heparin. Recombinant mMCP-6-FLAG exhibited optimal enzymatic activity at .about.pH 7.4 and good enzymatic activity after a 3-h incubation with EK at 37.degree. C. at pH 5.2.
The finding that the EK-activated tryptase readily cleaves tosyl-Gly-Pro-Lys-p-nitroanilide and tosyl-Gly-Pro-Arg-p-nitroanilide in the absence of heparin, indicates that the broad catalytic activity of this tryptase is not dependent on heparin-containing serglycin proteoglycans. However, the observation that mMCP-6-FLAG in the presence or absence of heparin, does not effectively cleave benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide, benzoyl-Pro-Phe-Arg-p-nitroanilide, or acetyl-Ile-Glu-Ala-Arg-p-nitroanilide indicates that mMCP-6 has a more restricted substrate specificity than trypsin. Models of the three-dimensional (3D) structures of mMCP-6 and mMCP-7 (Matsumoto et al., J. Biol. Chem. 1995, 270:19524-19531; Ghildyal et al., J. Exp. Med. 1996, 184:1061-1073) based on the crystallographic structure of bovine pancreatic trypsin suggests that seven loops form the substrate-binding cleft of each tryptase, as occurs for other serine proteases (Perona and Craik, Protein Sci. 1995; 4:2337-360). Relative to trypsin, 3 of the 7 loops in mMCP-7 have insertions that make its substrate-binding cleft deeper and more restricted than that of trypsin. Because similar insertions are found in the corresponding loops of mMCP-6, it is not surprisingly that this latter serine protease also has a restricted substrate specificity.
mMCP-6-Induced Emigration of Neutrophils Into the Peritoneal Cavity of BALB/c Mice--The mast cells that reside in the peritoneal cavity of BALB/c mice express mMCP-6 but not mMCP-7 (Stevens et al., Proc. Natl. Acad. Sci. USA 1994, 91:128-132). Because this observation suggests that mMCP-6, but not mMCP-7, cleaves specific proteins that reside in the peritoneal cavity, enzymatically active mMCP-6-FLAG was injected into the peritoneal cavity to assess whether or not the tryptase can induce an inflammatory reaction. Six to 36 h after mMCP-6-FLAG administration, a pronounced influx of neutrophils was observed in the peritoneal cavity. As typically seen in acute inflammatory responses (Robbins et al., "Inflammation and repair" in Pathologic Basis of Disease. 1994, 5th ed., W. B. Saunders Co., Philadelphia, Pa., pp 57-60), large numbers of eosinophils, lymphocytes, erythrocytes, basophils, and platelets, were not detected in the peritoneal exudate of the treated mice. However, unlike a typical inflammatory response where monocytes and eosinophils predominant at subsequent time points (Robbins et al., supra), kinetic experiments revealed that the mMCP-6-induced neutrophilia persisted for at least 3 days. Thus, the direct or indirect chemotaxis activity of mMCP-6 is relatively neutrophil specific. It also appears that tryptase treatment results in a relatively persistent recruitment of neutrophils into the peritoneal cavity. The observation that pro-mMCP-6-FLAG does not induce neutrophil emigration at the 36 h time point indicates that the induced inflammatory reaction is dependent on enzymatically active mMCP-6. Moreover, the observation that enzymatically active mMCP-7-FLAG 7-FLAG has very little, if any, neutrophil chemotaxis activity in this in vivo assay also documents the exquisite specificity of the tryptase effect.
Screening of a Tryptase-Specific Phage Display Peptide Library with Recombinant mMCP-6-FLAG--The observation that recombinant mMCP-7-FLAG cleaves acetyl-Ile-Glu-Ala-Arg-p-nitroanilide much better than mMCP-6-FLAG in vitro and that mMCP-6-FLAG selectively induces neutrophil emigration in vivo indicates that the two mouse tryptases have different substrate specificities even though their overall amino acid sequences are quite similar. Thus, the tryptase-specific, phage peptide display library that helped us identify a physiologic substrate of mMCP-7 (Huang, et al., J Biol Chem. 1997, 272:31885-31893) was used to identify mMCP-6-preferred peptide substrates. When the library was subjected to 4 rounds of treatment with enzymatically-active mMCP-6-FLAG in the absence of heparin glycosaminoglycan, no specific peptide sequence in the hypervariable domain of the pIII fusion protein was obtained in the 30 arbitrarily selected clones (Table I). Nevertheless, the observation that only one of these mMCP-6-susceptible clones had the preferred mMCP-7-susceptible sequence in its pIII fusion protein (Huang, et al., J Biol Chem. 1997, 272:31885-31893) was further evidence that the two homologous tryptases degrade very different substrates. Another family of serine protease genes is present on chromosome 14 that encode cathepsin G (Heusel et al., Blood 1993, 81:614-1623), at least 5 granzymes (Burnet et al., Nature 1986, 322:268-271; Pham et al., Proc. Natl. Acad. Sci. USA 1996, 93:13090-13095), and at least 6 mast cell chymases (Gurish et al., J Biol Chem. 1993, 268:11372-11379; Hunt et al., J. Biol. Chem. 1995, 271:2851-2855). The observation that the two mouse tryptases are very similar in their overall primary sequences but very different in their preferred peptide substrates is further support that the chromosome 14 and chromosome 17 complexes of serine protease genes evolved so that mast cells and other hematopoietic effector cells that express varied members of the two families of serine proteases degrade different panels of proteins.
The two most prominent features of the peptides obtained by screening the library with mMCP-6-FLAG alone were the over and under representation of positively and negatively charged residues, respectively. One half of the selected clones had 3 or more Lys and/or Arg residues in the susceptible peptide, and 2 of the clones actually had 5 positively charged residues. These findings are consistent with the electrostatic properties of the mMCP-6 model which revealed that the substrate-binding pocket of mMCP-6 is more negatively charged than that in mMCP-7 (Ghildyal et al., J. Exp. Med. 1996, 184:1061-1073). The difference in the electrostatic potential of the pocket is due primarily to loop 3 which has a -3 net charge in mMCP-6 and a 0 net charge in mMCP-7.
When the phage peptide display library was subjected to 2 to 4 rounds of treatment with mMCP-6-FLAG in the presence of an equal amount of heparin glycosaminoglycan, a more limited number of sequences were obtained (Table II). Surprisingly, the 2 clones that were obtained repeatedly had dissimilar sequences of Thr-Pro-Leu-Leu-Lys-Ser-Trp-Leu (SEQ. ID NO. 64) and Arg-Asn-Arg-Gln-Lys-Thr-Asn-Asn (SEQ. ID NO. 65). The latter favored peptides and the other less favored peptides obtained in this selection process were similar in that each had a Pro residue, at least one Thr or Ser residue, and a net charge of only +1 or +2. The discovery that the favored peptide in this series had a Pro residue at its P4 site is of interest because Cromlish and coworkers (1987) found that a human mast cell tryptase purified from the pituitary will cleave three prohormones ex vivo that have Pro residues at their P4 sites and Lys/Arg residues at their P1 sites.
Despite these interesting findings, we speculated that the favored peptide from the phage display library which possesses a +3 charge probably is more physiologically relevant because its overall charge is similar to that generally obtained when the library was screened with mMCP-6 alone. Why only one +3 positively charged peptide was obtained and why this peptide was not present in the original 30 clones isolated when the library was screened with mMCP-6 alone remains to be determined experimentally. However, the electrostatic potential of the 3D model of mMCP-6 suggests that the putative heparin-binding domain on the surface of this tryptase resides closer to its active site than in all other mMCPs. Thus, it is likely that heparin sterically restricts the substrate-binding cleft of mMCP-6 by directly influencing one of the 7 loops that form the pocket. The discovery that the substrate specificity of a rat mast cell chymase is also altered by heparin (Le Trong et al., Proc. Natl. Acad. Sci. USA 1987, 84:364-367 ) now emphasizes the importance of serglycin proteoglycans in fine tuning the substrate specificities of certain members of the chromosome 14 and chromosome 17 families of serine proteases.
A computer search of a protein database with the sequence Arg-Asn-Arg-Gln-Lys-Thr (SEQ. ID NO. 1) present in the positively charged peptide revealed that a nearly identical sequence (i.e., Arg-Gly-Arg-Gln-Lys-Thr, SEQ. ID NO. 11) resides in the middle of each subunit of fibronectin and that this sequence is conserved from rats to humans. Fibronectin is an abundant protein in plasma and varied extracellular matrices and plays a central role in cellular adhesion. This adhesion protein is a dimer consisting of .about.220-kDa polypeptides that are disulfide bonded at the C terminus (Komblihtt et al., EMBO J. 1985, 4:1755-1759; Skorstengaard et al., Eur. J. Biochem. 1986, 161:441-453). Its primary structure can vary somewhat due to differential splicing of the transcript but each subunit consists of nearly 2400 residues. These subunits possess numerous conserved domains that enable fibronectin to interact simultaneously with different proteins on the cell's surface and in the extracellular matrix. For example, a domain near the N-terminus binds to varied native and denatured collagens, whereas the C-terminal half of the fibronectin contains adjacent domains that allow fibronectin to interact simultaneous with varied integrins and proteoglycans on the surface of the cell. In the case of fibroblasts, fibronectin forms focal adhesions with .beta..sub.1 integrins and syndecan proteoglycans thereby inducing synergistic signaling through distinct pathways (Woods and Couchman, Mol. Biol. Cell 1994, 5:183-192; Couchman and Woods, J. Cell. Biochem. 1996, 61:578-584). The in vitro adhesion of melanoma cells to fibronectin is also mediated by the cooperative action of .beta..sub.1 integrins and cell surface proteoglycans (Iida et al., J. Cell Biol. 1992, 118:431-444; Wahl et al., J. Leukocyte Biol. 1996, 59:789-796). The mMCP-6-susceptible sequence in fibronectin is at residues 1351 to 1356 between the collagen and integrin binding domains. Thus, the specific cleavage at this site should have a dramatic effect on the fibronectin-mediated adhesion of fibroblasts.
In Vitro Digestion of Fibronectin and Disruption of Fibronectin-Mediated Adhesion of Fibroblasts by mMCP-6-FLAG--Fibronectin was readily cleaved by the mMCP-6-FLAG/heparin complex in vitro but not by mMCP-7-FLAG either in the presence or absence of heparin. Fibronectin is susceptible to cleavage by a wide range of neutral proteases, including chymotrypsin (Ehrismann et al., J. Biol. Chem. 1982, 257:7381-7387), trypsin (Mosher and Proctor, Science 1980, 209:927-929), .alpha.-thrombin (Furie and Rifkin, J. Biol. Chem. 1980, 255:3134-3140), plasmin (Jilek and Hormann, Hoppe-Seyler's Z. Physiol. Chem. 1977, 358:133-136), plasminogen activator (Quigley et al., Proc. Natl. Acad. Sci. USA 1987, 84:2776-2780), cathepsin G (Vartio et al., J. Biol. Chem. 1981, 256:471-477), urokinase (Gold et al., Biochem. J. 1989, 262:529-534), elastase (McDonald and Kelley, J. Biol. Chem. 1980 255:8848-8858), and mast cell chymases (Vartio et al., J. Biol. Chem. 1981, 256:471-477). Because of its exquisite protease-susceptibility, fibronectin is routinely used to assess general neutral protease activities in samples. BALB/c mouse bone marrow-derived mast cells, developed in vitro using T cell-conditioned media, possess serine proteases in their granules that can readily degrade human fibronectin in vitro (DuBuske et al., J. Immunol. 1984, 133:1535-1541) into 8 or more fragments. Because this population of mast cells expresses mMCP-2 (Ghildyal et al., J. Biol. Chem. 1992, 267:8473-8477), mMCP-5 (McNeil et al., Proc. Natl. Acad. Sci. USA 1991, 89:11174-11178), mMCP-6 (Reynolds et al., J. Biol. Chem. 1991, 266:3847-3853 ), and mMCP-7 (McNeil et al., supra), it has not been ascertained which, if any, of these granule mMCPs degrade fibronectin in vitro. There are nearly 200 positively charged (Arg+Lys) residues in each subunit of fibronectin. Thus, it is not much of a surprise that this adhesion protein is susceptible to digestion by recombinant mMCP-6-FLAG. The novel finding is the specificity of the enzymatic attack when mMCP-6-FLAG is bound to heparin. Only 2 fragments are obtained after a 60-min incubation of fibronectin with mMCP-6-FLAG. N-terminal amino acid analysis of the amino acid sequence of the generated fragments is used to confirm that the preferred cleavage site in fibronectin is Arg-Gly-Arg-Gln-Lys-Thr (SEQ. ID NO. 11).
Swiss albino mouse skin-derived 3T3 fibroblasts exhibit homotypic, contact inhibition in vitro. However, these cells will become less adhesive and divide in vitro when they are trypsin treated. To determine if mMCP-6-FLAG could specifically alter the growth and/or adhesion of these cells, the fibroblasts were allowed to attach to replicate fibronectin-coated culture dishes and then were incubated for 15 min at 37.degree. C. with buffer alone or buffer containing either pro-EK-mMCP-6-FLAG, mMCP-6-FLAG, mMCP-7-FLAG, or trypsin. The fibroblasts which were exposed to buffer alone, pro-EK-mMCP-6-FLAG, or mMCP-7-FLAG continued to adhere to the fibronectin-coated culture dishes. Many of the cells in these cultures also exhibited the classical stellate shape of a fibroblast bound to its matrix via focal adhesion sites. In contrast, both trypsin and mMCP-6-FLAG rapidly induced the cultured fibroblasts to round up. Moreover, very few fibroblasts remained attached to the culture dish after a 40 min incubation with either protease. SDS-PAGE/immunoblot analysis of the supernatants from the result cultures confirmed that fibronectin was degraded in the mMCP-6-FLAG-treated cultures but not in the pro-EK-mMCP-6-FLAG or mMCP-7-FLAG treated cultures.
Although Forsberg-Nilsson and coworkers (Scand. J. Immunol. 1996, 44:267-272) recently reported that a mast cell tryptase purified from human lung is not mitogenic for cultured human foreskin fibroblasts, Ruoss and coworkers (J. Clin. Invest. 1991, 88:493-499) reported that a tryptase purified from dog mastocytoma tissue is mitogenic for cultured Chinese hamster lung fibroblasts. Hartman and coworkers (Am. J. Physiol. 1992, 262:L528-L534 ) reported that a tryptase purified from human lung is mitogenic for cultured rat, hamster, and human fibroblasts but not for rat smooth muscle cells, and Cairns and Walls (J. Immunol. 1996, 156:275-283) reported that tryptases purified from human lung is mitogenic for the H292 human epithelial cell line. Mast cells express two or more tryptases in all species that have been examined. Moreover, strain-dependent expression of tryptase expression has been noted in mast cells of the mouse (Ghildyal et al., J. Immunol. 1994, 153:2624-2630; Hunt et al., J. Biol. Chem. 1996, 271:2851-2855) and rat (Lutzelschwab et al., J. Exp. Med. 1996, 185:13-29). The discovery that mMCP-7-FLAG treated mouse fibroblasts do not lose their contact inhibition, continue to adhere to fibronectin, and do not increase their rate of proliferation, suggests that the apparently conflicting data in the above human studies probably is the result of functionally different tryptases in the analyzed preparations.
The mechanism by which the dog and human mast cell tryptases induce proliferation of fibroblasts and epithelial cells in vitro was not deduced in the Ruoss et al. (J. Clin. Invest. 1991, 88:493-499), Hartmann et al. (Am. J. Physiol. 1992, 262:L528-L534), and Cairns and Walls (J. Immunol. 1996, 156:275-283) studies but it appears that they do not stimulate cellular division via the thrombin receptor. While it is now well established that fibronectin plays a central role in cell adhesion, it has become increasingly apparent that certain proteolytically-derived fragments of fibronectin possess potent bioactivities in some in vitro systems. For example, the C-terminal 140- to 120-kDa fragment of fibronectin that presumably contains both its integrin- and syndecan-binding domains induces expression of certain metalloproteases and their inhibitors in fibroblasts and other cell types (Werb et al., J. Cell Biol. 1989, 109:877-889; Huhtala et al., J. Cell Biol. 1995, 129:867-879 ; Kapila et al., Matrix Biol. 1996, 15:251-261). Relevant to our study, it has been shown that comparable fragments of fibronectin are chemotactic for fibroblasts (Seppa et al., Cell Biol. Int. Reports 1981, 5:813-819) and neutrophils (Odekon et al., Immunol. 1991, 74:114-120). The discovery that neutrophils are selectively recruited into the peritoneal cavity of BALB/c mice when recombinant mMCP-6-FLAG, but not recombinant mMCP-7-FLAG, is injected into this site, now suggests that the neutrophil emigration in this in vivo assay is mediated, in part, by a generated large-sized fragment of fibronectin that lacks its collagen binding domain. Thus, our discovery that the tryptase mMCP-6 (but not the tryptase mMCP-7) specifically cuts fibronectin between its collagen- and integrin-binding domains has important implications for mast cell-mediated control of fibrosis and inflammation.
Although mMCP-6 and mMCP-7 have different substrate specificities, both tryptases alter integrin-mediated signaling pathways. mMCP-7 does this by attacking fibrinogen which is the ligand for the .alpha..sub.M .beta..sub.2, .alpha..sub.X .beta..sub.2, .alpha..sub.IIb, .beta..sub.3, and .alpha..sub.V .beta..sub.3, family of integrins (Springer Nature 1990, 346:425-434; Wahl et al., J. Leukocyte Biol. 1996, 59:789-796), whereas mMCP-6 does this by attacking fibronectin which is the ligand for the .alpha..sub.2 .beta..sub.1, .alpha..sub.3 .beta..sub.1, .alpha..sub.4 .beta..sub.1, .alpha..sub.5 .beta..sub.1, .alpha..sub.V .beta..sub.1, .alpha..sub.IIb .beta..sub.3, .alpha..sub.V .beta..sub.3, and .alpha..sub.4 .beta..sub.7 family of integrins (Springer, supra; Wahl et al., supra) Although their roles in asthma have not been deduced, linkage analysis (De Sanctis et al., Nature Genetics 1995, 11:150-154) has implicated the region of chromosome 17 where the mMCP-6 and mMCP-7 genes reside as one of three candidate loci for the inheritance of intrinsic airway hyperresponsiveness. In addition, low molecular weight inhibitors of tryptic enzymes block antigen-induced airway constriction and tissue inflammatory response in Ascaris suum-sensitized sheep (Clark et al., Am. J. Respir. Crit. Care Med. 1995, 152:2076-2083). Our data suggest that mast cell tryptases play central roles in mast cell-mediated inflammation by controlling different integrin-dependent signaling pathways.
TABLE III presented below includes references to the GenBank Accession numbers of selected sequences presented in the Sequence Listing, followed by the claims and the abstract.
Claims
We claim:
1. A peptide selected from the group consisting of: Arg-Asn-Arg-Gin-Lys-Thr (SEQ.ID NO.1), Arg-Asn-Arg (SEQ.ID NO.2), Arg-Asn-Arg-Gln (SEQ.ID NO.3), Arg-Asn-Arg-Gln-Lys (SEQ.ID NO.4), Asn-Arg-Gln-Lys-Thr(SEQ.ID NO.5), Arg-Gln-Lys-Thr (SEQ.ID NO.6), Gln-Lys-Thr (SEQ.ID NO.7), Arg-Gln-Lys (SEQ.ID NO.8), Asn-Arg-Gln (SEQ.ID NO.9), and Arg-Gln-Lys (SEQ.ID NO.10).
2. The peptide of claim 1, wherein said peptide is selected from the group consisting of: Arg-Asn-Arg (SEQ.ID NO.2), Arg-Asn-Arg-Gln (SEQ.ID NO.3), Arg-Asn-Arg-Gln-Lys (SEQ.ID NO.4), Asn-Arg-Gln-Lys-Thr (SEQ.ID NO.5), Arg-Gln-Lys-Thr (SEQ.ID NO.6), Gln-Lys-Thr (SEQ.ID NO.7), Arg-Gln-Lys (SEQ.ID NO.8), Asn-Arg-Gln (SEQ.ID NO.9), and Arg-Gln-Lys (SEQ.ID NO.10).
3. The peptide of claim 1, wherein said peptide contains 1, 2, 3, 4, or 6 conservative amino acid substitutions, provided that the number of conservative amino acid substitutions cannot exceed the number of amino acids in the peptide.
4. The peptide of claim 1, wherein the amino acids are covalently coupled by non-hydrolyzable peptide bonds.
5. A tryptase-6 complex inhibitor that is a functionally equivalent peptide of SEQ.ID NO. 1, said functionally equivalent peptide having the formula: wherein: P is a peptide selected from the peptides of claim 1, 2, 3, or 4; X is an N-terminal peptide containing from zero to five amino acids of N-terminal to amino acids 1351-1356 of SEQ.ID NO. 12; Y is a C-terminal peptide containing from zero to five amino acids of C-terminal to amino acids 1351-1356 of SEQ.ID NO. 12; wherein said functionally equivalent peptide competitively inhibits cleavage of a peptide having SEQ.ID NO. 1 by the tryptase-6 complex.
6. The tryptase-6 complex inhibitor of claim 5, wherein the tryptase-6 complex is a human tryptase-6 complex.
7. A method for selecting a tryptase-6 complex inhibitor comprising: determining whether a tryptase-6 complex cleaves a peptide that is or that contains the amino acid sequence of SEQ.ID NO. 1 in the presence of a putative tryptase-6 complex inhibitor.
8. The method of claim 6, wherein the tryptase-6 complex is a human tryptase-6 complex.
9. The method of claim 6, wherein the tryptase-6 complex is an mMCP-6 complex.
10. The method of claim 6, wherein the tryptase-6 complex protease inhibitor is contained in a phage display library.
11. A peptide having the sequence: Arg-Asn-Arg-Gln-Lys-Thr (SEQ.ID NO. 1) and functionally equivalent fragments thereof that contain from 3 to 6 amino acids and wherein said functionally equivalent fragments competitively inhibit cleavage of a peptide having SEQ.ID NO. 1 by a tryptase-6 complex.
12. The peptide of claim 10, wherein said peptide is SEQ.ID NO. 1.
13. The peptide of claim 10, wherein said peptide is SEQ.ID NO. 2.
14. The peptide of claim 10, wherein said peptide is SEQ.ID NO. 3.
15. The peptide of claim 10, wherein said peptide is SEQ.ID NO. 4.
16. The peptide of claim 10, wherein said peptide is SEQ.ID NO. 5.
17. The peptide of claim 10, wherein said peptide is SEQ.ID NO. 6.
18. The peptide of claim 10, wherein said peptide is SEQ.ID NO. 7.
19. The peptide of claim 10, wherein said peptide is SEQ.ID NO. 8.
20. The peptide of claim 10, wherein said peptide is SEQ.ID NO. 9.
21. The peptide of claim 10, wherein said peptide is SEQ.ID.NO. 10.
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