Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Reexamination Certificate
2000-03-31
2003-03-18
Huff, Sheela (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
C530S350000, C435S183000
Reexamination Certificate
active
06534635
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to novel modified tissue inhibitors of metalloproteinase (hereinafter referred to as “TIMPs”).
The subject application claims priority based on the Japanese Patent Application No. 095142/1999. The contents of the Japanese application is hereby incorporated by reference.
Metastasis is a feature of malignant cancer and most life-threatening pathologies and therefore one of the important objects of cancer therapy is to arrest metastasis. In practice, metastasis is palliatively treated by surgery, radiation therapy or chemotherapy, but no therapy can definitively arrest it. However, the mechanism of metastasis has been gradually elucidated in recent years and the breakdown system of extracellular matrix (hereinafter referred to as “ECM”) is noted as a reflection of the metastatic potency of cancer.
More specifically, cancer cells begin to grow at the primary location and some of them discontinue adhering to surrounding cells so that they can escape from tumor tissues. However, tumor tissues are surrounded by dense ECM, and cancer cells cannot escape from there only via attack by physical motion without enzymatic breakdown. Metastatic cancer cells begin to move in the tissues by producing an enzyme that breaks down this barrier ECM. To further move to a distant location, cancer cells break vascular walls formed by robust ECM to enter the bloodstream. Then, they adhere to the inner membranes of the vascular walls at the second location, and enzymatically break down the vascular wall ECM again to escape from the vessel and infiltrate tissues by further breaking down surrounding ECM (“SAIBOU KOUGAKU” (Cell Technology), Vol. 17, No. 4, 1998, pp. 523-533).
In such a cascade of processes, breakdown of EMC seems to be most important for studying or diagnosing metastasis of cancer cells. Matrix metalloproteinases (hereinafter referred to as “MMPs”) (Docherty, A. J. P., O'Connell, J., Crabbe, T., Angal, S. and Murphy, G. (1992) Trends Biotechnol. 10, 200-207) are zinc-dependent endopeptidases that degrade components of ECM. MMPs play an essential role in tissue remodeling under physiological and pathological conditions such as morphogenesis, angiogenesis, tissue repair and tumor invasion (Docherty, A. J. P. et al., (1992) Trends Biotechnolol. 10, 200-207, supra.; Matrisian, L. M. (1992) Bioessays 14, 455-463; Stetler-Stevenson, W. G., Aznavoorian, S., and Liotta, L. A. (1993) Annu. Rev. Cell Biol. 9, 541-573). Most MMPs are secreted as zymogens and are activated by serine proteases or some activated MMPs.
At present, about twenty MMPs have been discovered, which have characteristic substrate specificities to degrade various collagens, glycoproteins, proteoglycans, etc. MMPs are grouped into several families by their substrate specificities and morphologies. For example, MMP-2 and -9 are also referred to as gelatinase A and gelatinase B, respectively, as members of the gelatinase family having a gelatin as a substrate. MMP-14 to -17 are the membrane-associated type, and belong to the MT-MMP family (membrane type-MMP). MMP-14 to -17 are referred to as MT1-MMP, MT2-MMP, MT3-MMP and MT4-MMP, respectively. Other families are the collagenase family (MMP-1, MMP-8, MMP-13 and MMP-18), stromelysin family (MMP-3 and MMP-10), etc.
The activities of activated MMPs are regulated by a family of specific inhibitors known as tissue inhibitors of metalloproteinases (hereinafter referred to as “TIMPs”). At present, four TIMPs have been identified, which efficiently inhibit MMPs except for MT-MMP. MT-MMP has selectivity in that it is efficiently inhibited by TIMP-2 and TIMP-3, but hardly inhibited by TIMP-1. TIMPs have a structure basically consisting of an N-terminal region and a C-terminal region. The MMP-inhibitory activity exists at the N-terminal region of TIMPs, and even recombinant TIMPs lacking the C-terminal region can inhibit MMPs.
Findings on the Mechanism of MMP-inhibitory Activity of TIMPs
Previous studies proposed hypotheses about the mechanism of MMP-inhibitory activity of TIMPs. For example, the following findings have been obtained about TIMP-2 and TIMP-1.
Among the MMP family, gelatinase A (MMP-2) and gelatinase B (MMP-9) are critical in the invasion of tumor cells across basement membranes because of their strong activity against type IV collagen, a major component of basement membranes (Liotta, L. A. (1986) Cancer Res. 46, 1-7; Collier. I. E., Wilhelm, S. M., Elsen, A. Z., Marmer, B. L., Grant G. A., Seltzer, J. L., Kronberger, A., He, C., Bauer E. A., and Goldberg, G. I. (1988) J. Biol. Chem. 263, 6579-6587; Wilhelm, S. M., Collier, I. E., Marmer, B. L., Eisen, A. Z., Grant G. A., and Goldberg, G. I. (1989) J. Biol. Chem. 264, 17213-17221). Unlike other zymogens of MMPs, progelatinase A is not activated by serine proteases or soluble MMPs and had been reported to be activated by a MMP-like activity on the surface of cancer and fibroblastic cells (Overall, C. M., and Sodek, J. (1990) J. Biol. Chem. 265, 21141-21151; Brown, P. D., Levy, A. T., Margulies, I. M., Liotta, L. A., and Stetler-Stevenson, W. G. (1990) Cancer Res. 50, 6184-6191; Ward, R V., Atkinson, S. J., Slocombe, P. M., Docherty, A. J., Reynolds, J. J., and Murphy, G. (1991) Biochim. Biophys. Acta. 1079, 242-246; Azzam, H. S. and Thompson, E. W. (1992) Cancer Res 52, 4540-4544).
Sato et al. (Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. (1994) Nature 370, 61-65) identified a novel membrane-type MMP, named MT-MMP as an activator of progelatinase A on the cell surface. The cell-mediated activation of progelatinase A includes two steps of processing; MT-MMP-catalyzed cleavage of progelatinase A at a peptide bond between Asn-37 and Leu-38 firstly converts the zymogen into an intermediate form, and then autocatalytic cleavage of an Asn-80-Tyr-81 bond converts the intermediate form into a mature one (Strongin, A. Y., Marmer, B. L., Grant, G. A., and Goldberg, G. I. (1993) J. Biol. Chem. 268, 14033-14039). Several studies suggest that both steps are greatly accelerated by binding of (pro)gelatinase A onto the cell surface, and therefore, the receptor of (pro)gelatinase A on the cell surface is important for the activation. Carboxy-terminal hemopexin-like domain of gelatinase A is reported to be essential for the interaction with the cell-surface receptor (Strongin, A. Y., Marmer, B. L., Grant, G. A., and Goldberg, G. I. (1993) J. Biol. Chem. 268, 14033-14039; Strongin, A. Y., Collier, I., Bannikov, G., Marmer, B. L., Grant, G. A., and Goldberg, G. I. (1995) J. Biol. Chem. 270, 5331-5338).
Recent studies (Brooks, P. C., Silletti, S., von Schalscha, T. L., Friedlander, M., and Cheresh, D. A. (1996) Cell 92, 391-400; Kinoshita, T., Sato, H., Takino, T., Itoh, M., Akizawa, T., and Seiki, M. (1996) Cancer Res. 56, 2535-2538; Pei, D. Q., and Weiss, S. J. (1996) J. Biol. Chem. 271, 9135-9140; Will, H., Atkinson, S. J., Butler, G. S., Smith, B., and Murphy, G. (1996) J. Biol. Chem. 271, 17119-17213; Lichte, A., Kolkenbrock, H., and Tschesche, H. (1996) FEBS Lett. 397, 277-282) suggest that transmembrane domainless variants of MT-MMP convert progelatinase A to the intermediate form but hardly to the mature one. It is also reported that cell-mediated processing of mutant progelatinase A of which active site residue is replaced by site-directed mutagenesis, does not produce the mature form of the mutant (Atkinson, S. J., Crabbe, T., Cowell, S., Ward, R. V., Butler, M. J., Sato, H., Seiki, M., Reynolds, J. J., and Murphy, G. (1995) J. Biol. Chem. 270, 30479-30485; Sato, H., Takino, T., Kinoshita, T., Imal, K., Okada, Y., Stetler-Stevenson, W. G., and Seiki, M. (1996) FEBB Lett. 385, 238-240).
These studies suggest the importance of cell-associated activity of gelatinase A for the conversion of intermediate form of gelatinase A to its mature form.
On the other hand, the crystal structures of complexes of TIMPs and MMPs have also been studied.
The crystal structure of the complexformed between TIMP-1 and stromelysin suggests that the free &agr;-am
Higashi Shouichi
Miyazaki Kaoru
Davis Natalie
Huff Sheela
Nixon & Vanderhye P.C.
Oriental Yeast Co. Ltd.
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