Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 25 or more amino acid residues in defined sequence
Reexamination Certificate
1998-08-24
2001-08-14
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
25 or more amino acid residues in defined sequence
C514S012200, C435S004000
Reexamination Certificate
active
06274703
ABSTRACT:
FIELD AND OBJECTIVE OF THE INVENTION
This invention relates to methods for cell surface activation and inhibition. More particularly, the invention relates to methods of cell surface activation and inhibition that involve the interaction of an inhibitor of matrix metalloprotease known as TIMP-2, with the enzyme, gelatinase-A.
Matrix metalloproteases (MMPs) are ubiquitous in human disease and development. Most processes that involve a certain amount of tissue repair and damage are believed to be influenced by MMPs such as, for example, in degradation of type IV collagen that might occur in rheumatoid or osteoarthritis and remodeling of endothelial walls in restenosis. MMPs are also implicated in various aspects of cancer such as primary tumor formation, metastasis, and the vascularization of larger tumors (angiogenesis). It is also known that MMPs are involved in the conversion of inactive tumor necrosis factor (TNF) precursor into active TNF, which in turn is implicated in rheumatoid arthritis, Crohn's disease, multiple sclerosis, cachexia and sepsis.
Consequently, the screening for MMP inhibitors as potential drugs is of significant use in the medical and pharmaceutical fields.
MMPs are secreted by mammalian cells as zymogens and upon activation initiate tissue remodeling by proteolytic degradation of collagens and proteoglycans. Activation of the secreted proenzymes and interaction with their specific inhibitors, TIMP-1 and TIMP-2, determine the net enzymatic activity in the extracellular space.
TIMP-2 forms a specific complex with the proform of gelatinase-A (GelA) which is mediated by interaction with the C-terminal domain (GelA-CTD) of the enzyme. The amino acid sequence of the 72 kDa GelA is disclosed in Goldberg, U.S. Pat. No. 4,923,818, and its complex with TIMP-2 is disclosed in Goldberg published European Patent Application, EP 404,750. GelA is a multi-domain protein containing a catalytic domain, a domain with three type II fibronectin-like repeats, and a C-terminal domain.
Soluble GelA proenzyme is recruited to the cell surface where it is specifically activated by MT1-MMP, a membrane bound metalloprotease. The binding of GelA to cell surface and its subsequent activation is also mediated by GelA-CTD. Consequently, cell surface activation is inhibited in the presence of exogenously added excess of TIMP-2 or recombinant GelA-CTD.
It has not been known previously how the MT1-MMP that is inhibited by complex with TIMP-2 is able to cleave the GelA propeptide to initiate activation of the pro-enzyme. Resolution of this question is critical to an understanding of the mechanism by which GelA-CTD interacts with TIMP-2 and MT1-MMP on the cell surface.
BACKGROUND OF THE INVENTION
Literature references on the following background information and on conventional test method and laboratory procedures well known to the ordinary person skilled in the art, and other such state-of-the-art techniques as used herein, are indicated in parentheses, and appended at the end of the specification.
Secreted metalloprotcases (MMPs) initiate tissue remodeling by degradation of extraccilular matrix (ECM) macromolecules (reviewed in 1-3). Normal physiological processes such as morphogenesis, tissue repair, and angiogenesis, are dependent upon spatial and temporal regulation of the activity of these enzymes, while malignant cells exploit these same proteases to promote invasion and metastasis (4-7). A clear understanding of the mechanisms governing regulation of MMP activity in extracellular space has remained an elusive goal. The MT1-MMP/GelA system (8-16) provides a first glimpse at a mechanism by which an activity of a soluble MMP, GelA (17), can be spatially regulated via its recruitment to the cell surface where the GelA proenzyme is converted into its active form. Transfection of Cos1 cells with MT1-MMP is sufficient to cause GelA binding to the cell surface and its activation (8,19). The cell surface activation of GelA involves a two step proteolytic processing of its propeptide. The first cleavage of the Asn
37
-Leu peptide bond is dependant on MT1-MMP (9), a membrane bound metalloprotease. This cleavage is also dependent on GelA having an intact C-terminal domain since a truncated form of the GCLA proenzyme lacking a C-terminal domain can not be activated by membrane bound MT1-MMP (13). Consequently the exogenously added recombinant GelA-CTD) is a competitive inhibitor of Asn
37
-Leu cleavage (9,10). Finally this reaction is inhibited in the presence of an excess of inhibitor, TIMP-2, while TIMP-1 has no effect. The consequent cleavage of propeptide is accomplished via an autoproteolytic, MT1-MMP independent mechanism (9,10,1 8,19) to generate a 62 kDa active GelA with an amino-terminal residue Tyr
81
. These data demonstrate that binding of GelA to the cell surface via its CTD is a prerequisite for enzyme activation. We have previously shown that two closely related proenzymes GelA and B form specific complexes with TIMP-2 and TIMP-1 respectively (20). These complexes are also formed via inhibitor interaction with the carboxyl-end domain of proenzyme (21,22). Thus TIMP-2 and cell surface binding activities of GelA-CTD appear to be interrelated. We have purified activated form of MT1-MMP using affinity chromatography approach (9) and demonstrated that it acts as cell surface TIMP-2 receptor with Kd=1.65×10
−9
M. The MT1-MMP-TIMP-2 complex in turn acts as a receptor for GelA-CTD (Kd=0.42×10
−9
M). The data we have presented support the hypothesis that the cell surface binding of GelA-CTD occurs via formation of a tri-molecular complex of activated MT1-MMP/TIMP-2/pro-GelA that promotes pro-GelA activation. This model, however, does not satisfactory resolve the GelA activation mechanism for the following reasons. The inhibitor TIMP-2 consists of two domains. The amino-terminal, inhibitory domain interacts with the active center of MMPs to form an inhibitory complex (23,24). The C-terminal domain binds to GelA-CTD. Thus the inhibitory complex of TIMP-2 with activated MT1-MMP can leave the C-terminal domain of the inhibitor exposed and available for interaction with GelA-CTD. In fact we have reported an analogous tri-molecular complex between GelB, TIMP-1 and activated interstitial collagens (22) where the collagens component of the complex was inhibited. Moreover the specific inhibition of soluble form of MT1-MMP by TIMP-2 has been recently demonstrated (25,26). Thus, the model of cell surface GelA activation that requires assembly of the MT1-MMP/TIMP-2/pro-GelA complex leaves unanswered the question of how the MT1-MMP inhibited by TIMP-2 is able to cleave the Asn
37
-Leu peptide bond to initiate activation of the pro-enzyme. An answer to this question demands a better understanding of the mechanism by which GelA-CTD interacts with TIMP-2 and MT1-MMP on the cell surface. We have recently reported the high resolution crystal structure of GelA-CTD (27). Here we report the results of extensive alanine scanning mutagenesis of solvent exposed GelA-CTD amino-acid residues and, using the coordinates of the GelA-CTD structure, define a TIMP-2 binding site on the surface of this domain. By comparison of the TIMP-2 binding site to the same regions in related MMP structures, we characterize structural features required for general TIMP binding and the specificity of TIMP-2- GelA-CTD interaction. We also report analysis of GelA activation inhibition activity of GelA-CTD mutants relative to that of wild type.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with the present invention, methods of cell surface activation and inhibition are provided which are useful for the screening of MMP inhibitors that are potentially useful for the treatment of diseases that involve tissue repair and damage and other diseases in which MMPs are implicated.
Critical to the methods of the invention is the discovery of a unique portion of the TIMP-2 binding site on the surface of the GelA-CTD domain, which has been determined herein to be the very strongly binding residue Asp
65
Low Christopher S. F.
Lukton David
Meyer Scott J.
Washington University
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