Novel peptides which exhibit inhibitory activity toward serine proteases and methods for preparing and using same are disclosed. In one aspect, the present invention provides peptides comprising a generic inhibitory core having a functional site recognition sequence fused to the N-terminus. The functional site recognition sequence is adapted to provide enhanced selectivity and/or potency for a target protease.
This application is a continuation of Ser. No. 200,821, filed Jun. 1, 1988, abandoned, which is a continuation of Ser. No. 006,725, filed Feb. 6, 1987, abandoned which is a continuation-in-part application of co-pending application Ser. No. 840,810, filed Mar. 18, 1986 now abandoned.
BACKGROUND OF THE INVENTION
In its broadest aspect, the present invention, relates to enzyme inhibitors. More particularly, it relates to novel peptides which exhibit inhibitory activity toward serine proteases.
Protease inhibitor activities were first noted in human plasma by Fermi and Pernossi in 1894 Zgcar. Hyg. 18:83). Many investigations have been made to determine the various inhibitory activities present in plasma primarily by adding proteases of varying specificities and catalytic mechanisms to plasma. There are now recognized at least nine separate, well-characterized proteins in human plasma which share the ability to inhibit the activity of various proteases.
Several of the inhibitors have been grouped together, namely .alpha.-1-proteinase inhibitor, antithrombin III, antichymotrypsin, C1-inhibitor and .alpha.-2-antiplasmin. These are referred to as the .alpha.-1-proteinase inhibitor class. The protein .alpha.-2-macroglobulin inhibits members of all four catalytic classes: serine, cysteine, aspartic, and metalloproteases. However, the other types of protease inhibitors are class specific. The .alpha.-1-proteinase inhibitor group and inter-.alpha.-trypsin inhibitor inhibit only serine proteases, .alpha.-1-cysteine protease inhibitor inhibits only cysteine proteases, and .alpha.-1-anticollagenase inhibits only collagenolytic enzymes of the metalloenzyme class.
.alpha.-1-Proteinase inhibitor (antitrypsin, AT) is a glycoprotein of MW 51,000 with 394 amino acids and 3 oligosaccharide side chains and is present in human serum at 130 mg/100 ml or 23.6 .mu.M. It easily diffuses into tissue spaces and forms a 1:1 complex with a target protease, principally neutrophil elastase. The enzyme/inhibitor complex is then removed from circulation and catabolized by the liver and spleen. Human AT was originally named anti-trypsin because of its ability to inactivate pancreatic trypsin. Interest has focused on AT in both clinical and biochemical circles because many individuals with circulating levels of this inhibitor that are less than 15% of normal are susceptible to the development of lung disease (familial emphysema) at an early age (Eriksson (1965) Acta Med. Scan. 177 (Suppl. 432): 1-85). Therefore, it appears that this inhibitor represents an important part of the defense mechanism of the lung towards attack by proteases. Human AT is a single polypeptide chain with no internal disulfide bonds and only a single cysteine residue normally intermolecularly disulfide-linked to either cysteine or glutathione.
An important observation is that the reactive site of AT contains a methionine residue which is labile to oxidation. This oxidation to the corresponding sulfoxide which may be caused by cigarette smoke reduces the inhibitory activity of AT toward both pancreatic and neutrophil elastase. Inactive AT isolated from rheumatoid synovial fluid contains up to four methionine sulfoxide residues, two of which are at the P1 and P8 positions suggesting a connection to the tissue damage noted in this disease.
Human antithrombin III (AT III) is a serum glycoprotein (serum level=29 mg/100 mL or 4.7 .mu.M) that plays a major role in controlling serine proteases in the coagulation cascade scheme. Purified AT III is a single-chain molecule of MW 58,000 containing about 15% carbohydrate, and has six disulfide bonds. The major heparin binding site in AT III is in the N-terminus (PNAS (1984) 81, 289-293). The inactivation of proteases by AT III is enhanced 100 fold by the presence of heparin, an effect caused by the increase in binding to the protease.
Antichymotrypsin (ACT) is a plasma glycoprotein of MW 68,000 first isolated and characterized without knowledge of its function (Naturwissenschaften (1962) 49:133). It has since been shown to have inhibitory activity towards chymotrypsin, although its physiological role is thought to be the inhibition of leukocyte cathepsin G. This inhibition is brought about by formation of a 1:1 complex. This inhibitor is an acute phase protein, meaning that its concentration increases dramatically after traumatic events, e.g., surgery, burns, ulcerative colitis, and some cancers. The normal concentration of ACT in plasma is 25 mg/100 mL or 3.6 .mu.M.
It is known that in some instances the degradative action of serine proteases results in serious pathological conditions or disease states. For example, elastase is a protease which causes degradation and fragmentation of elastic fibers as a result of its protelytic activity on elastin the structural component of elastic fiber. Elastic tissue is rich in elastin and possesses a rubber-like property. Cartilaginous tissues present in the ear and epiglottis are considered elastic tissue. Tissue comprising the lungs, bronchi and skin also contain relatively large amounts of elastin and are considered elastic tissue. Elastase is required for turnover of damaged cells and the digestion of certain invading bacteria. However, excessive degradation of elastin has been associated with arthritis, atherosclerosis, certain skin diseases, pulmonary emphysema and adult respiratory-distress syndrome. Therefore, by inhibiting the activity of elastase it is possible to treat a wide variety of pathological conditions.
Proteases serve another important function in human physiology by mediating the activation of the complement system. The complement system consists of a complex group of proteins in body fluids which, working together with antibodies and other factors, play an important role as mediators of inflammation and defense against infections. The complement system is now understood to be composed of two distinct pathways, the "classical" pathway and the "alternative" pathway.
The classical pathway (CP) of complement activation is typically initiated by the union of antigen and antibody. Not all antigen-antibody reactions initiate the classical pathway. Immunoglobulins of the IgM class and IgG1, IgG2, or IgG3 subclass activate the classical pathway whereas IgG4, IgA, IgD and IgE do not. A conformational change presumably occurs after antigen binding to the Fab region of immunoglobulins that permits binding and activation of the first component of complement, C1. C1 is a macromolecular complex of three proteins (C1q, C1r and C1s), and requires calcium ions for both stability and reactivity. Binding of C1 to a suitably altered immunoglobulin leads first to a conformational change in the C1q subunit and later to the acquisition of enzymatic activity by the C1s subunit. Activated C1 (C1s), while bound to antibody, cleaves its natural substrates, C4 and C2, by limited proteolytic reactions. The activity of C1s is regulated by the endogenous serum protein, C1 esterase inhibitor (C1-inhibitor) which binds to the enzyme and thereby limits cleavage of C4 and C2. An inherited deficiency of C1-inhibitor results in uncontrolled cleavage of C4 and C2 and is manifested by recurrent attacks of angioedema (periodically recurring episodes of swelling of skin, mucous membranes, viscera and brain). C4 cleavage by C1s results in the formation of a small peptide (C4a) which is released in the fluid phase and a larger fragment, C4b, which can bind to the immune complex.
C2 is similarly cleaved by C1s into a small peptide (C2b) which is released into the fluid phase and a large fragment (C2a), which binds to C4b. The C4b2a complex thus formed possesses new proteolytic activity (C3 convertase) that is capable of cleaving the third component of complement, C3. Proteolytic cleavage of C3 by the C4b2a complex yields a small peptide, C3a, which is released into the fluid phase and a larger fragment (C3b), which possesses the ability to bind to immune complexes as well as to a variety of surfaces. Once bound, C3b forms a new C4b2a3b complex with surrounding C4b2a complexes, or C5 convertase, which is capable of cleaving native C5 to a small peptide C5a which is released to the fluid phase, and C5b which binds to the surface of the antigen. Bound C5b forms the basis for the stable macromolecular "membrane attack" complex with C6, C7, and C8. Binding of the final complement component C9 forms the attack sequence C5b6789 which inserts into the lipid bilayers of cell membranes and forms transmembrane channels that permit bidirectional flow of ions. This mechanism induces cellular injury and lysis.
The alternative pathway (AP) of complement activation is functionally a two-phase system in which six proteins participate. This pathway bypasses the early-acting components, C1, C4 and C2 and leads directly to proteolytic cleavage of C3 and ultimately to the assembly of the terminal attack complex, C5b-C9. The first phase is initiation in which particle-bound C3b fulfills a recognition function. The second phase is one of amplification by means of a positive feedback loop involving bound C3b, Factor B, Factor D, and unbound C3.
The alternative pathway can be activated by the introduction of a wide variety of substances into serum. These include lipopolysaccharides (e.g., bacterial endotoxins), complex polysaccharides (e.g. inulin, zymosan), and immune complexes containing immunoglobulins of the IgA or IgD classes that cannot activate the classical pathway. Surface constituents of some intact cells (e.g. rabbit erythrocytes, certain bacteria and fungi) activate the alternative complement pathway in human serum. This property of foreign cells provides a mechanism for their recognition in the complete absence of antibody. The alternative pathway may therefore be thought of as a phylogenetically older first line of defense against invading microorganisms. The actual mechanism that activates the alternative complement patyway is controversial because there is no counterpart to the recognition unit C1q of the classical pathway. The current view is that native C3 is undergoing limited proteolytic reactions at all times, i.e. normal catabolism. The C3b fragments formed transiently must be near enough to a suitable surface to attach before the metastable binding site on the C3b molecule decays. The regulatory proteins of the alternative pathway are Factor H and Factor I. Factor H controls the alternative pathway by directly binding to C3b or to the C3bBb complex. When bound to C3b it blocks the formation of the C3bBb complex and when bound to previously existing C3bBb complex it dissociates Bb from the complex. Factor I functions as an endopeptidase cleaving C3b which is complexed with Factor H. C3b which escapes Factor H and binds to a suitable surface can interact with Factor B to form a stable, catalytically inactive bimolecular complex C3bB. This complex, if it escapes from inactivation by Factor H and I, is the precursor of both the C3 and C5 convertases of the alternative pathway. Factor B, when complexed with C3b, becomes susceptible to cleavage by Factor D.
Factor D is not consumed and can activate many C3bBb complexes. The activated C3bBb complex produced is the alternative pathway C3 convertase and is able to cleave free C3 to produce more C3b, which in turn can combine with more Factor B. This positive feedback loop is the central theme of the alternative pathway. Because of this mechanism, deposition of very few molecules of C3b on a biological particle can lead to the subsequent placement of many more molecules of C3bBb on the surface. This results in the opsonization (engulfment) of the particle facilitating its clearance by phagocytic cells and the generation of C3a which functions in the inflammatory process. C3bBb which has been activated by Factor D can be protected from inactivation activity of Factor H by addition of properdin (Factor P) which stabilizes the alternative pathway C3 convertase about eight-fold at 37.degree. C. As the amplification phase continues, the C3bBbP complex binds one additional molecule of C3b, forming C3bBbPC3b, which can cleave C5, producing C5a and C5b. Generation of C5b and its binding to the surface of the particle results in the self assembly of the membrane attack complex C5b-9.
As with nearly all complex physiological pathways, there are situations in which activation of complement is triggered to the detriment of the host. This type of activation often results in grave pathological conditions. Exemplary of these conditions are autoimmune hemolytic anemia, rheumatoid arthritis, allergy complement activation, systemic lupus erythematosus, ankylosing spondylitis and myasthenia gravis. The presence of conditions such as those described above, provokes a much recognized and as yet unmet need for synthetic serine protease inhibitors for use as therapeutic agents.
It should be understood that the pathways and conditions noted above are only exemplary and the present invention is not limited to these states. Rather, the serine protease inhibitors of this invention have broad application in the inhibition of serine protease activity.
Accordingly, it is therefore the overall object of the present invention to provide novel peptides which exhibit inhibitory activity toward serine proteases.
It is an object of the present invention to provide serine protease inhibitors exhibiting relatively high activity at relatively low concentrations.
It is another object of the present invention to provide serine protease inhibitors exhibiting selectivity for certain key proteases involved in complement activation.
It is yet another object of the present invention to provide serine protease inhibitors exhibiting selectivity for certain key proteases involved in blood clotting and clot degradation.
These and other objects and advantages of the present invention will be recognized by those skilled in the art from the following description and illustrative examples.
D-enzyme compositions are described comprising an amino acid residue sequence that defines an polypeptide able to catalyze an enzymatic reaction. The D-enzyme has an amino acid residue sequence consisting essentially of D-amino acids.
A method is disclosed for blocking the SEC receptor and also inhibiting the neurotoxic effects of amyloid-.beta. protein by treating SEC receptor-bearing cells with a synthetic inhibitor peptide, e.g. peptide PB145.
The invention relates to the use of complement inhibitors, especially of C1 inactivator or of factors I or H, for the preparation of a pharmaceutical and for the prophylaxis and therapy of chronic inflammatory intestinal disorders, inflammatory skin disorders and purpura.
Detection or determination of a protease in a sample by incubating the sample with a substrate of the protease and observing proteolytic cleavage of said substrate. The substrate is a modified proenzyme containing a recognition site, e.g., an activation site, cleavable by said protease. Proteolytic cleavage of the modified proenzyme is detected by observing the resulting activity using a suitable substrate of the activated proenzyme. The protease may be e.g. an aspartic protease or a metalloprotease, and the modified proenzyme e.g. pro-urokinase having a mutant activation site which is cleavable by the protease to be determined.
The invention provides methods of treating and preventing vascular diseases, by inhibiting cysteine proteases active at sites of vascular injury, particularly cathepsins K and S, by administering cystatin C or TGF-.beta..sub.1. Cystatin C is severely reduced in both atherosclerotic and aneurysmal aortic lesions. Also provided is a method of diagnosing a patient at risk of, or with, vascular injury by detecting low levels of circulating cystatin C.
