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  Crystallized structure of type iv collagen nc1 domain hexamerUSPTO Application #: 20070042965Title: Crystallized structure of type iv collagen nc1 domain hexamer Abstract: The present invention provides a crystallized NC1 domain hexamer of Type IV collagen, and methods for making the crystal, wherein the NC1 domain hexamer is crystallized such that the three dimensional structure of the crystallized NC1 domain hexamer can be determined to a resolution of at least 3 Å or better. The present invention also provides a method for designing compounds to inhibit angiogenesis, tumor growth, tumor metastasis, endothelial cell adhesion and/or proliferation, and/or basal lamina assembly, comprising analyzing the three dimensional structure of a crystallized Type IV collagen NC1 domain hexamer produced by the methods of the invention, and identifying and synthesizing compounds that target regions of the NC1 domain that have been identified by the analysis as being important for type IV collagen heterotrimer and hexamer assembly. The present invention also provides novel polypeptides designed by the rational drug design methods of the present invention, based on an analysis of the type IV collagen NC1 hexamer structure disclosed herein. (end of abstract) Agent: Mcdonnell Boehnen Hulbert & Berghoff LLP - Chicago, IL, US Inventors: Billy G. Hudson, Munirathinam Sundaramoorthy USPTO Applicaton #: 20070042965 - Class: 514016000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai, Cyclopeptides, 7 Or 8 Peptide Repeating Units In Known Peptide Chain The Patent Description & Claims data below is from USPTO Patent Application 20070042965. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE [0001] This application claims priority to U.S. Provisional Patent Application Ser. Nos. 60/308,523 filed Jul. 27, 2001; 60/351,289 filed Oct. 29, 2001; 60/366,854 filed Mar. 22, 2002; and 60/385,362 filed Jun. 3, 2002. FIELD OF THE INVENTION [0003] The present invention relates to the fields of crystallography, molecular biology, protein chemistry, angiogenesis, tumor growth and metastasis, and basement membrane assembly BACKGROUND OF THE INVENTION [0004] The basement membrane (basal lamina) is a sheet-like extracellular matrix (ECM), which is a basic component of all tissues. The basal lamina provides for the compartmentalization of tissues, and acts as a filter for substances traveling between tissue compartments. Typically the basal lamina is found closely associated with an epithelium or endothelium in all tissues of an animal, including blood vessels and capillaries. The basal lamina components are secreted by cells and then self assemble to form an intricate extra-cellular network. The formation of biologically active basal lamina is important to the development and differentiation of the associated cells. [0005] Type IV collagen has been shown to be a major structural component of basement membranes, and consists of a family of six homologous .alpha. chains, designated .alpha.1(IV) through .alpha.6(IV). Each .alpha. chain is characterized by a non-collagenous (NC1) domain at the carboxyl terminus; a long, helical collagenous domain in the middle region; and a 7S collagenous domain at the amino terminus. (Martin, et. al., 1988, Adv. Protein Chem. 39:1-50; Gunwar, et. al. 1991, J. Biol. Chem. 266:14088-14094). Three a chains assemble into triple helical molecules, the "heterotrimer." The heterotrimer, once formed in the endoplasmic lumen, is secreted into the extracellular space, where two such heterotrimers assemble into a hexamer via C-terminal interactions, and then into a supramolecular network through N-terminal associations. The NC1 domains play the dominant role in this assembly, by determining the C-terminal dimeric association, leading to hexamer assembly. [0006] The chain composition, and thus the properties of type IV collagen networks, are influenced by two factors. First, the chain composition of networks is limited by chain availability: the six a chains show a tissue-specific expression pattern, with the .alpha.1 and .alpha.2 chains being ubiquitous, and the .alpha.3-.alpha.6 chains having a more restricted tissue distribution. Second, the NC1 domain confers specificity to the chain-specific assembly of networks. Thus, as yet unidentified recognition sequences must exist within the NC1 domain that direct the selection of chains to form triple helical protomers, and that direct triple helical protomers to form hexamers and, thus, collagen networks. While numerous type IV collagen hexamers are theoretically possible that differ in kind and a chain stochiometry, only three have been identified: [.alpha.1.sub.2.alpha.2].sub.2, [.alpha.3.alpha.4.alpha.5].sub.2, and [(.alpha.1.sub.2.alpha.2)(.alpha.5.sub.2.alpha.6)]. [0007] Angiogenesis, the process of formation of new blood vessels, plays an important role in physiological processes such as embryonic and postnatal development, as well as in wound repair. Formation of blood vessels can also be induced by pathological processes involving inflammation (e.g., diabetic retinopathy and arthritis) or neoplasia (e.g., cancer) (Folkman, 1985, Perspect, Biol. Med., 29, 10). Neovascularization is regulated by angiogenic growth factors secreted by tumor or normal cells as well as by the composition of the extracellular matrix and the activity of endothelial enzymes (Nicosia and Ottinetti, 1990, Lab. Invest., 63, 115). [0008] A common feature of all solid tumor growth is the requirement for a blood supply. Therefore, numerous laboratories have focused on developing anti-angiogenic compounds based on growth factors and their receptors. While this approach has led to some success, the number of growth factors known to play a role an angiogenesis is large. Therefore, the possibility exists that growth factor antagonists may have only limited use in treating cancer, since tumors and associated inflammatory cells likely produce a wide variety of factors that can induce angiogenesis. [0009] In this regard, a strategy that targets a common feature of angiogenesis, such as endothelial cell adhesion to the extracellular matrix (ECM), might be expected to have a profound physiological impact on tumor growth in humans. This notion is supported by the fact that antagonists of specific ECM cell adhesion receptors such as (.alpha.v.beta.3 and .alpha.v.beta.5 integrins can block angiogenesis. Furthermore, the .alpha.v.beta.3 integrin is expressed most prominently on cytokine-activated endothelial and smooth muscle cells, and has been shown to be required for angiogenesis. (Varner et al., Cell Adhesion and Communication 3:367-374 (1995); Brooks et al., Science 264:569-571 (1994)). Based on these findings, a potentially powerful new approach to anti-angiogenic therapy is to specifically target critical regulatory domains within distinct ECM components. [0010] Specific type IV collagen .alpha.(IV) NC1 domains have been demonstrated to be effective inhibitors of angiogenesis, tumor growth, tumor metastasis, cell binding to basement membranes, and assembly of Type IV collagen molecules (see, for example, U.S. Pat. Nos. 5,691,182; 5,856,184; 6,361,994; and 6,358,735). Despite the above, it would be of significant value to the art to identify further compounds capable of inhibiting these processes. [0011] It is therefore highly desirable to provide a method of deducing the crystal structure of type IV collagen NC1 domains, and of providing a method of using this structure to design compounds that inhibit assembly of the type IV collagen heterotrimer and/or the type IV collagen hexamer. SUMMARY OF THE INVENTION [0012] In one aspect, the present invention provides a crystallized NC1 domain hexamer of Type IV collagen, and methods for making the crystal, wherein the NC1 domain hexamer is crystallized such that the three dimensional structure of the crystallized NC1 domain hexamer can be determined to a resolution of at least 3 .ANG.or better. [0013] In another aspect, the present invention provides a method for designing compounds to inhibit angiogenesis, tumor growth, tumor metastasis, endothelial cell adhesion and/or proliferation, and/or basal lamina assembly, comprising analyzing the three dimensional structure of a crystallized Type IV collagen NC1 domain hexamer produced by the methods of the invention, and identifying and synthesizing compounds that target regions of the NC1 domain that have been identified by the analysis as being important for type IV collagen heterotrimer and hexamer assembly. Such compounds can be used to inhibit angiogenesis, tumor growth, tumor metastasis, endothelial cell adhesion and/or proliferation, and basal lamina assembly. [0014] In another aspect, the present invention provides novel polypeptides designed by the rational drug design methods of the present invention, based on an analysis of the type IV collagen NC1 hexamer structure disclosed herein. As a result of the information available from the crystal structure, it is possible to predict individual NC1 domain sequences that are critical for assembly of the type IV collagen heterotrimer and/or hexamer. Thus, it is also possible to design therapeutic polypeptides that will interfere with those interactions, and to inhibit assembly of the type IV collagen heterotrimer and/or the type IV collagen hexamer. Such therapeutic polypeptides can be used to inhibit or disrupt type IV collagen assembly, and thus are useful to inhibit angiogenesis, angiogenesis-mediated disorders, tumor growth, tumor metastasis, endothelial cell adhesion and/or proliferation, and basal lamina assembly. BRIEF DESCRIPTION OF THE FIGURES [0015] FIG. 1. Alignment of six human .alpha.NC1 chains grouped as .alpha.1-like (1, 3, & 5) and .alpha.2-like (2, 4, & 6) families. The cysteine pairs intrachain disulfides are labeled with identical numbers at the bottom. Six segments that form the trimer-trimer interface are boxed and three major segments at the monomer-monomer are highlighted with larger font size. The most important segments forming generic and specific interactions are identified at the bottom with darkly shaded bars, respectively. [0016] FIG. 2. (a) .alpha.1 chains and (b) .alpha.2 chains. Secondary structural elements are assigned based on the crystal structure. Both .alpha.1 and .alpha.2 structures contain .beta.-strands .beta.1-.beta.10 and .beta.1'-.beta.10' and a 3.sub.10 helices g1 and g1'. The differences in secondary structures are a 3.sub.10 helix in .alpha.1 and .beta.-stand .beta.p' in .alpha.2 at the equivalent regions in the two sequences. The partner of .beta.p' strand of .alpha.2 chain is in one of the two .alpha.1 chains. The corresponding region in .alpha.2 and the other .alpha.1 chains are extended structures. These regions marked by boxes. The secondary structures were from PROCHECK (61). [0017] FIG. 3. Stereo diagram of deduced NC1 hexamer structure. The trimer-trimer interface ("Equatorial Plane"), collagen triple helical junction, and pseudo 3-fold axis or triple helix axis ("Polar Axis") are identified. The two trimers are related by a 2-fold NCS axis perpendicular to the polar axis and plane of the paper. This figure and FIGS. 5, 8, 9 and 10b were made using SETOR (45). [0018] FIG. 4. (a) Illustration of .alpha.1 monomer structure in the hexamer. Four .beta.-sheet regions are identified as I, II, II' and II and three short 3.sub.10 helices are also shown. [0019] FIG. 5. Topology diagram of NC1 trimer depicting interchain and intrachain 3D domain swapping interactions (generic assembly) and chain interfaces with different secondary structural elements (specific assembly). The secondary structural elements are labeled only for .alpha.1A chain. The .beta.-sheets, I & II in the N-subdomain and I' & II' in the C-subdomain are identified. Each subdomain has 10 .beta.strands (.beta.1-.beta.10 and .beta.1'-.beta.10') and two short 3.sub.10 (g1 and g2') helices. Additionally there are distinct secondary structures at the three interfaces--a parallel .beta.-sheet (.beta.p-.beta.p') at .alpha.1B-.alpha.2 interface and a 3.sub.10 helix (g1') and extended structure at .alpha.1A-.alpha.1B and .alpha.2-.alpha.1A interfaces. [0020] FIG. 6. a) Generic interactions in the trimer. Six-strand .beta.-sheets formed by interchain and intrachain 3D domain swapping interactions form the major force in the trimer organization. The sheets belonging to subdomains are shown in boxes to highlight such interactions. Central .beta. barrel-like core, shown inside the circle, also plays a role in packing and stabilizing this scaffold. (b) Unique secondary structures and prominent side chain interactions at the three interfaces are shown. The .alpha.1b-.alpha.2 interface has more number of hydrogen bonds than the other interfaces. Continue reading... Full patent description for Crystallized structure of type iv collagen nc1 domain hexamer Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Crystallized structure of type iv collagen nc1 domain hexamer patent application. 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