TREA

Serine protease inhibitors

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Information

  • Patent Grant
  • 4829052
  • Patent Number
    4,829,052
  • Date Filed
    Friday, May 8, 1987
    37 years ago
  • Date Issued
    Tuesday, May 9, 1989
    35 years ago
Information
Abstract
Synthetic polypeptides are disclosed which exhibit potent serine protease inhibition. Methods and compositions useful for treating conditions caused by unwanted serine protease activity are also disclosed.
Description
Claims
  • 1. A synthetic serine protease inhibitor having the following sequence: ##STR2## which exhibits inhibitory activity towards elastase in which X is selected from the group consisting of isoleucine, norvaline, valine, norleucine, methionine, leucine, alanine, glycine and phenylalanine.
  • 2. A serine protease inhibitor of claim 1 in which X is valine.
  • 3. A serine protease inhibitor of claim 1 in which X is iosleucine.
  • 4. A serine protease inhibitor of claim 1 exhibiting inhibitory activity toward elastase and cathepsin G in which X is phenylalanine.
  • 5. A serine protease inhibitor of claim 1 exhibiting inhibitory activity toward elastase and cathepsin G in which X is methionine.
  • 6. A serine protease inhibitor selected from the group consisting of
  • R V C P I I L M K C K K D S D C L A E C V C L E H G Y C G, H E E R V C P I I L M K C K K D S D C L A E C V C L E H G Y C G, R V C P I I L M E C K K D S D C L A E C V C L E H G Y C G, R V C P I I L M E C K K D S D C L A E C I C L E H G Y C G, H E E R V C P I I L M E C K K D S D C L A E C I C L E H G Y C G, M V C P I I L M K C K H D S D C L L D C V C L E D I G Y C G, M M C P I I L M K C K H D S D C L P G C V C L E H I E Y C G, R V C P V I L M K C K K D S D C L A E C V C L E H G Y C G, H E E R V C P V I L M K C K K D S D C L A E C V C L E H G Y C G, R V C P V I L M E C K K D S D C L A E C V C L E H G Y C G, R V C P V I L M E C K K D S D C L A E C I C L E H G Y C G, H E E R V C P V I L M E C K K D S D C L A E C I C L E H G Y C G, M V C P V I L M K C K H D S D C L L D C V C L E D I G Y C G, M M C P V I L M K C K H D S D C L P G C V C L E H I E Y C G, R V C P F I L M K C K K D S D C L A E C V C L E H G Y C G, H E E R V C P F I L M K C K K D S D C L A E C V C L E H G Y C G, R V C P F I L M E C K K D S D C L A E C V C L E H G Y C G, R V C P F I L M E C K K D S D C L A E C I C L E H G Y C G, H E E R V C P F I L M E C K K D S D C L A E C I C L E H G Y C G, M V C P F I L M K C K H D S D C L L D C V C L E D I G Y C G, M M C P F I L M K C K H D S D C L P G C V C L E H I E Y C G, R V C P M I L M K C K K D S D C L A E C V C L E H G Y C G, H E E R V C P M I L M K C K K D S D C L A E C V C L E H G Y C G, R V C P M I L M E C K K D S D C L A E C V C L E H G Y C G, R V C P M I L M E C K K D S D C L A E C I C L E H G Y C G, H E E R V C P M I L M E C K K D S D C L A E C I C L E H G Y C G, M V C P M I L M K C K H D S D C L L D C V C L E D I G Y C G, M M C P M I L M K C K H D S D C L P G C V C L E H I E Y C G, R V C P L I L M K C K K D S D C L A E C V C L E H G Y C G, H E E R V C P L I L M K C K K D S D C L A E C V C L E H G Y C G, R V C P L I L M E C K K D S D C L A E C V C L E H G Y C G, R V C P L I L M E C K K D S D C L A E C I C L E H G Y C G, H E E R V C P L I L M E C K K D S D C L A E C I C L E H G Y C G, M V C P L I L M K C K H D S D C L L D C V C L E D I G Y C G, M M C P L I L M K C K H D S D C L P G C V C L E H I E Y C G, R V C P A I L M K C K K D S D C L A E C V C L E H G Y C G, H E E R V C P A I L M K C K K D S D C L A E C V C L E H G Y C G, R V C P A I L M E C K K D S D C L A E C V C L E H G Y C G, R V C P A I L M E C K K D S D C L A E C I C L E H G Y C G, H E E R V C P A I L M E C K K D S D C L A E C I C L E H G Y C G, M V C P A I L M K C K H D S D C L L D C V C L E D I G Y C G, M M C P A I L M K C K H D S D C L P G C V C L E H I E Y C G, R V C P V I L Nle K C K K D S D C L A E C V C L E H G Y C G, R V C P V S L M K C K K D S D C L A E C V C L E H G Y C G, R V C P V I L L K C K K D S D C L A E C V C L E H G T C G, R V C G V I L M K C K K D S D C L A E C V C L E H G Y C G, R V C Hyp V I L M K C K K D S D C L A E C V C L E H G Y C G, R V C P V A L M K C K K D S D C L A E C V C L E H G Y C G, R V C P G I L M K C K K D S D C L A E C V C L E H G Y C G, H E E R V C P G I L M K C K K D S D C L A E C V C L E H G Y C G, R V C P G I L M E C K K D S D C L A E C V C L E H G Y C G, R V C P G I L M E C K K D S D C L A E C I C L E H G Y C G, H E E R V C P G I L M E C K K D S D C L A E C I C L E H G Y C G, M V C P G I L M K C K H D S D C L L D C V C L E D I G Y C G, and M M C P G I L M K C K H D S D C L P G C V C L E H I E Y C G.
  • 7. A pharmaceutical composition for inhibiting a target serine protease containing an effective amount of an inhibitor of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier therefor.
  • 8. The composition of claim 7 in which X is valine.
  • 9. The composition of claim 7 in which X is isoleucine.
  • 10. The composition of claim 7 in which X is phenylalanine.
  • 11. The composition of claim 7 in which X is methionine.
  • 12. A method for treating an individual having a physiological condition caused by unwanted serine protease activity which comprises administering to the individual a therapeutically effective amount of an inhibitor of claim 1.
  • 13. The method of claim 12 in which X is valine.
  • 14. The method claim 12 in which X is isoleucine.
  • 15. The method of claim 12 in which is phenylalanine.
  • 16. The method of claim 12 in which X is methionine.
  • 17. A pharmaceutical composition for inhibiting a target serine protease containing an effecting amount of an inhibitor of claim 6 or a pharmaceutically acceptable salt thereof and a pharmaceutically carrier therefor.
  • 18. A method for treating an individual having a physiological condition caused by unwanted serin protease activity which comprises administering to the individual a therapeutically effective amount of an ihibitor of claim 6.
Parent Case Info

This is a continuation-in-part application of co-pending application Ser. No. 873,014 filed June 11, 1986, now abandoned. In its broadest aspect, the present invention relates to enzyme inhibitors. More particularly, it relates to novel polypeptid-es which exhibit inhibitory activity toward serine proteases. The mechanistic class of proteolytic enzymes known as serine proteases is widely distributed in nature having been identified in animals, microbes and insects. The assignment of members to this class was originally made on the basis of their enzymatic mechanisms. Subsequently, members of this class have demonstrated a great deal of sequence and structural homology. Serine proteases are typified by a catalytic triad consisting of aspartic acid, histidine and serine at the active site. Serine proteases can be easily identified since the active site serine can be irreversibly and covalently modified by diisopropylfluorophosphate. Serine protease inhibitors have been found in microbes and in the tissues and fluids of plants, animals, insects and other organisms. The naturally occurring serine protease inhibitors are usually, but not always, polypeptides and proteins which have been classified into families primarily on the basis of the disulfide bonding pattern and the sequence homology of the reactive site. The reactive site is defined as the portion of the primary sequence that directly interacts with the protease. Studies have indicated that most inhibitors inhibit by a common mechanism in which the inhibitor is actually a poor substrate which is tightly bound and only very rarely experiences the catalytic hydrolysis of a particular bond within the reactive site. However, if the pH is drastically changed from neutrality hydrolysis of the reactive site peptide bond occurs. In addition to their normal physiological function, serine proteases have been implicated in a number of pathological conditions in humans including pulmonary emphysema, various clotting disorders and inflammatory processes. One illustration of the importance of the catalytic activity of serine proteases is provided by the role of human neutrophil elastase and one of its natural inhibitors, .alpha.-1-proteinase inhibitor (.alpha.-P1), in the pathogenesis of emphysema. In the lungs of healthy individuals there is a balance between the levels of elastase and its inhibitors. The elastase serves in the repair and turnover of connective tissues (elastin) and the .alpha.-1-proteinase inhibitor is involved in the regulation and clearance of elastase. The term elastin refers to a heterogeneous, highly cross-linked and highly insoluble polypeptide, that is the main component of elastic connective tissue in the body. Disruption of the elastase/.alpha.-1-proteinase inhibitor balance leads to increased elastin degradation and, hence, to elastic tissue destruction. New elastin can be synthesized but the proper network of elastic fibers which is laid down during lung development is not achieved. A prolonged imbalance leads to an irreversible dilation of pulmonary airways and damage to the respiratory tissues of the lung, a condition known as pulmonary emphysema. This imbalance may occur in a number of ways. An example is the case of familial emphysema first identified in Scandinavia. Individuals found to have a homozygous genetic deficiency for active forms of serum .alpha.-1-proteinase inhibitor have been shown to be more likely to develop emphysematous symptoms, especially when exposed to other risk factors such as cigarette smoking. As another example, oxidants from the condensate of cigarette smoke have been shown to drastically reduce the elastase binding affinity of .alpha.-1-proteinase inhibitor by oxidizing a methionine residue within the reactive site. Furthermore, the serum of smokers has been shown to have dramatically higher levels of oxidized .alpha.-1-proteinase inhibitor when compared to non-smokers. It has been concluded that the balance has been disrupted by the inactivation of the inhibitor. A final example involves both elevated levels of elastase and simultaneously lower levels of functional .alpha.-1-proteinase inhibitor. The inflammatory response to foreign particulate matter from air pollution or cigarette smoke leads to elevated levels of polymorphonuclear leukocytes in the lungs. These cells disrupt the protease/protease inhibitor balance by secretion of proteolytic enzymes, e.g., elastase. They also secrete oxidants including myeloperoxidase which appear to oxidatively inactive .alpha.-1-proteinase inhibitor. Thus, imbalances created through the removal of a control mechanism or through the prolonged induction of elevated levels of elastase eventually leads to an undesirable pathological situation, damage to connective tissue and diminished pulmonary function. .alpha.-1-Proteinase inhibitor (antitrypsin, AT) is a single-chain glycoprotein of MW 51,000 with 394 amino acids with no disulfide bridges and 3 oligosaccharide side chains that is present in human serum at 130 mg/100 ml or 23.6 [2 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 rapidly removed from circulation and catabolized by the liver and spleen. Human AT was originally named antitrypsin because of its ability to inactivate pancreatic trypsin. There are many practical problems in the use of naturally-occuring mammalian serine protease inhibitors, or related materials, as therapeutic agents. The relatively large naturally occuring mammalian serine protease inhibitors pose a problem since large polypeptides can be more difficult to produce and administer to the patient than smaller polypeptides. These naturally occuring inhibitors lack stabilizing disulfide bridges and are heat labile. Also, the oligosaccharide side chains have been shown to have an effect on the life-lime of the inhibitors in vivo. Thus, the production of these inhibitors with the proper oligosaccharide component is another serious difficulty in their use as therapeutic agents. The description involving elastase given above furnishes but one example of a situation where the control of serine protease activity is useful and desirable. The scope of this invention includes, but is by no means limited to, inhibition of human elastase. It is therefore the goal of the present invention, in its broadest aspect, to provide novel serine protease inhibitors. Accordingly, it should be recognized that this invention is applicable to the control of catalytic activity of serine proteases in any appropriate situation including, but not necessarily limited to, medicine, biology, agriculture, and microbial fermentation. These and other objects and advantages of the present invention will be recognized by those skilled in the art from the following description and representative examples.

Non-Patent Literature Citations (4)
Entry
Wieczorek et al., Biochemical and Biophysical Research Communications, vol. 126, No. 2, pp. 646-652 (1985).
Kupryszewski et al., Experientia, vol. 41, pp. 1422-1423 (1985).
Rudinger, Peptide Hormones, Parsons (Ed.), U Park Press, Baltimore, pp. 1-7 (1976).
Joubert, F. J., "Trypsin Isoinhibitors from Momordica Repens Seed" 1984 Phytochemistry, vol. 23, No. 7, pp. 1401-1405.
Continuation in Parts (1)
Number Date Country
Parent 873014 Jun 1986