Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 15 to 23 amino acid residues in defined sequence
Patent
1995-05-18
1999-10-26
Elliott, George C.
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
15 to 23 amino acid residues in defined sequence
530300, C07K 700, C07K 708, C07K 1400
Patent
active
059731109
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to methods for inhibiting the bio-activity of enzymes. More specifically, the invention comprises molecules having cytotoxic T-lymphocyte antigen properties, and methods of inhibiting the proteolytic activity of cysteine protease using such molecules.
DESCRIPTION OF THE RELATED ART
Cytotoxic T-Lymphocyte Antigen-2 (abbreviated CTLA-2) is a molecule expressed by activated T-cells and mast cells. Complementary DNA (cDNA) for two distinct but homologous forms of CTLA-2 are known, namely CTLA-2.alpha. and CTLA-2.beta. (Denizot et al., 1989, Eur. J. Immunol., 19:631-635).
The present specification describes the identification of the gene encoding for peptides with CTLA-2 activity, and the expression, purification and characterization of such peptides having CTLA-2 activity. The identified gene for CTLA-2.beta. codes for a protein consisting of 138 amino acids (15,900 g/mole) including a putative leader sequence. Removal of the hydrophobic N-terminus results in a protein of 110 amino acids (12,800 g/mole). There are a total of five cysteine residues in the molecule, two of which are in the putative leader sequence. Therefore, the mature protein should contain three cysteine residues indicating the formation of disulfide linked dimers.
The CTLA-2.alpha. gene codes for a protein which is 98% homologous (90% identical) to CTLA-2.beta. at the protein level. CTLA-2.alpha. has a total of three cysteines in the full-length protein but only one in the mature form. Therefore, CTLA-2.alpha. also has the potential of forming a disulfide linked dimer.
The presently disclosed invention includes methods for inhibiting certain cysteine proteases with molecules having CTLA-2.alpha. and CTLA-2.beta. activity. Members of the cysteine protease family all have cysteine in their active sites, and the family includes the enzyme papain found in the papaya plant, a developmentally regulated proteinase in the Dictyostelium slime mold, Chinese goosberry actinidin, and the mammalian lysosomal Cathepsins B, H, and L (Portnoy et al., 1986, J. Biol. Chem., 261:14697).
Cathepsins are proteolytic enzymes found in most mammalian cells and their functions include cellular autolysis and tissue degredation (Bohley et al., 1992, Experimentia, 48:151; Funabiki et al., 1990, Int. J. Biochem. 22:1303). Cathepsin L is a major lysosomal protease and is responsible for bulk turnover of intracellular protein. The cDNA sequence for CTLA-2.beta. is known to be 40% homologous to the pro-region of mouse Cathepsin L (Denizot et al., id.), but there is no homology to the active protease sequence.
The present disclosure for the first time provides experimental evidence indicating that a protein homologous to the pro-region of a cysteine protease acts as an inhibitor of that cysteine protease. Specifically, the results hereinbelow show, inter alia, that purified CTLA-2.beta. inhibits the proteolytic activity of the cysteine proteases papain, Cathepsin H and Cathepsin L.
There is a need in the art for inhibitors of cysteine proteases as these enzymes have been implicated in the formation of new foci of metastatic carcinoma (Aoyama et al., 1990, Biochem. 87:8296; Keren et al., 1988, Cancer Res., 48:1416; Sloane et al., 1984, Cancer Metastases Rev. 3:249; Stearns et al., 1990, Arch. Biochem. Biophys., 283:447). Inhibition of cysteine proteases is reported to be a good candidate for cancer therapy (Nakajima et al., 1991, Cancer Biology, 2:115).
In addition, there is further data implicating the role of cysteine proteases in the pathology of a number of diseases including: rheumatoid arthritis (Trabandt et al., 1990, Matrix, 10:349); glomerulonephritis (Baricos et al., 1991, Arch. Biochem. Biophys., 288:468); emphysema (Manson et al., 1986, Biochem. J., 233:925); osteoporosis (Delaisse et al., 1991, Biochem. J. 279:167); and Alzheimer's disease (Cole et al., 1989, Neurochemical Res., 14:933). Cysteine protease inhibitors should prove useful in the treatment of such disease states.
There have been a number of dif
REFERENCES:
patent: 5189144 (1993-02-01), Asada et al.
S. Gal and M. Gottesman "Isolation and sequence of a cDNA for human pro-(cathepsin L)" Biochem. J. 253(1): 303-306 (1988).
B.R. Tron et al., "Sequence and expression of the cDNA for MEP (major excreted protein), a transformation-regulated secreted cathepsin" Biochem. J. 246(3): 731-735 (1987).
L.J. Joseph et al., "Complete nucleotide and deduced amino acid sequences of human and murine preprocathepsin L" J. Clin. Invest. 81(5): 1621-1629 (1988).
F. Denizot et al., "Novel structures CTLA-2alpha and CTLA-2beta expressed in mouse activated T cells and mast cells and homologus to cysteine proteinase proregions" Eur. J. Immunology 19: 631-635 (1989).
Brownell Elise
Delaria Katherine
Muller Daniel
Wallace Linda
Bayer Corporation
Elliott George C.
McGarry Sean
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