WO2004067762A2 - Structure cristallisee hexamere a domaine nc1 de collagene de type iv - Google Patents

Structure cristallisee hexamere a domaine nc1 de collagene de type iv Download PDF

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WO2004067762A2
WO2004067762A2 PCT/US2004/002187 US2004002187W WO2004067762A2 WO 2004067762 A2 WO2004067762 A2 WO 2004067762A2 US 2004002187 W US2004002187 W US 2004002187W WO 2004067762 A2 WO2004067762 A2 WO 2004067762A2
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seq
group
sequence
hexamer
amino acids
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PCT/US2004/002187
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WO2004067762A3 (fr
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Muirathinam Sundaramoorthy
Billy Hudson
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University Of Kansas Medical Center
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the fields of crystallography, molecular biology, protein chemistry, angiogenesis, tumor growth and metastasis, and basement membrane assembly
  • the basement membrane (basal lamina) is a sheet-like extracellular matrix (ECM), which is a basic component of all tissues.
  • ECM extracellular matrix
  • the basal lamina provides for the compartmentalization of tissues, and acts as a filter for substances traveling between tissue compartments.
  • 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 ofthe associated cells.
  • Type IN collagen has been shown to be a major structural component of basement membranes, and consists of a family of six homologous chains, designated ⁇ l( ⁇ N) tlirough ⁇ 6(IN). Each chain is characterized by a non-collagenous ( ⁇ C1) domain at the carboxyl terminus; a long, helical collagenous domain in the middle region; and a 7S collagenous domain at the amino terminus.
  • ⁇ C1 non-collagenous domain at the carboxyl terminus
  • a long, helical collagenous domain in the middle region and a 7S collagenous domain at the amino terminus.
  • heterotrimer 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 NCI domains play the dominant role in this assembly, by determining the C-terminal dimeric association, leading to hexamer assembly.
  • the chain composition, and thus the properties of type IN collagen networks, are influenced by two factors.
  • the chain composition of networks is limited by chain availability: the six chains show a tissue-specific expression pattern, with the ⁇ l and o2 chains being ubiquitous, and the o3- ⁇ 6 chains having a more restricted tissue distribution.
  • the ⁇ C1 domain confers specificity to the chain-specific assembly of networks.
  • recognition sequences must exist within the ⁇ C1 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.
  • 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).
  • ⁇ eovascularization 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).
  • 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. i 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.
  • ECM extracellular matrix
  • the present invention provides a crystallized NCI domain hexamer of Type IV collagen, and methods for making the crystal, wherein the NCI domain hexamer is crystallized such that the three dimensional structure of the crystallized NCI domain hexamer can be determined to a resolution of at least 3 A or better.
  • 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 IN collagen ⁇ C1 domain hexamer produced by the methods of the invention, and identifying and synthesizing compounds that target regions of the NCI domain that have been identified by the analysis as being important for type IN 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.
  • the present invention provides novel polypeptides designed by the rational drug design methods ofthe present invention, based on an analysis ofthe type IV collagen ⁇ C1 hexamer structure disclosed herein. As a result of the information available from the crystal structure, it is possible to predict individual ⁇ C1 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 IN collagen hexamer.
  • Such therapeutic polypeptides can be used to inhibit or disrupt type IN 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.
  • Figure 1 Alignment of six human ⁇ Cl chains grouped as ⁇ l-like (1, 3, & 5) and ⁇ 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.
  • Figure 2. (a) ⁇ l chains and (b) a2 chains. Secondary structural elements are assigned based on the crystal structure. Both ⁇ l and ⁇ 2 structures contain ⁇ -strands ⁇ l- ⁇ lO and ⁇ l'- ⁇ lO' and a 3 ⁇ o helices gl and gl'. The differences in secondary structures are a 3 ⁇ o helix in ⁇ l and ⁇ -stand ⁇ p' in ⁇ 2 at the equivalent regions in the two sequences. The partner of ⁇ p' strand of ⁇ 2 chain is in one ofthe two ⁇ l chains. The corresponding region in ⁇ 2 and the other ⁇ l chains are extended structures. These regions marked by boxes. The secondary structures were from PROCHECK(61). Figure 3.
  • Figure 4. Illustration of ⁇ l monomer structure in the hexamer. Four ⁇ -sheet regions are identified as I, II, IF and II and three short 3 ⁇ o helices are also shown.
  • Figure 5. Topology diagram of NCI frimer 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 ⁇ lA chain. The ⁇ -sheets, I & II in the N-subdomain and I' & IF in the C- subdomain are identified. Each subdomain has 10 ⁇ -strands ( ⁇ l- ⁇ lO and ⁇ l'- ⁇ lO') and two short 3 ⁇ o (gl and g2') helices.
  • Figure 6. Generic interactions in the frimer. Six-strand ⁇ -sheets formed by interchain and intrachain 3D domain swapping interactions form the major force in the frimer organization. The sheets belonging to subdomains are shown in boxes to highlight such interactions. Central ⁇ 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 ⁇ lb- ⁇ 2 interface has more number of hydrogen bonds than the other interfaces.
  • Figure 7 Trimer-trimer interface. Comparison of essential hydrogen bonding interactions in the interface at "core” ( Figure 7A), "outer” (Figure 7B) and major-minor junction (Figure 7C) for ⁇ l- ⁇ l and ⁇ l- ⁇ 2 dimers (see text for details).
  • Type IN collagens are synthesized and assembled as heterotrimers inside the cells, which are then secreted extracellularly where hexamer assembly, and subsequent basement membrane (basal lamina) assembly, occurs.
  • the present work has elucidated the structure ofthe type IV collagen [(c l) 2 (o2)] ⁇ C1 hexamer.
  • Knowledge of this structure has utility in the design of compounds that can inhibit assembly of type IV collagen heterotrimers and hexamers, and thus are beneficial in the inhibition of angiogenesis, angiogenesis-mediated disorders, tumor growth, tumor metastasis, endothelial cell adhesion and/or proliferation, and basal lamina assembly.
  • Knowledge of the structure of the type IN collagen [(c l) 2 (o-2)] 2 ⁇ C1 hexamer structure provided by the present invention also has utility in the design of compounds that promote heterotrimer and hexamer assembly by providing tools and reagents for increasing the understanding of type IN collagen assembly, and thus also of basal lamina/basement membrane structure and function in general.
  • the invention provides a method for crystallizing a type IN collagen [(c ) ( ⁇ 2)] 2 ⁇ C1 hexamer to a resolution of less than about 3.0 A or better, preferably 2.2 A or better, and most preferably 2.0 A or better, wherein the type IV collagen [(c-l) 2 ( ⁇ 2)] NCI hexamer is present at a concentration of about 0.5 mg/ml to about 50 mg/ml, more preferably from about 1 mg/ml to about 15 mg/ml and most preferably about 10 mg/ml, and the crystallization takes place at 4°C to 32° C, more preferably from 10°C to 26°C, even more preferably at about 16° to 24°C, and even more preferably 20° C, to thereby obtain crystals of space group P2 ⁇ .
  • the crystallization in one embodiment, may occur using hanging drops and the vapor diffusion method over 10% (w/v) PEG 20K.
  • other crystallization methods may be used.
  • a temperature variation may be used to produce crystals, or crystallization in space may be used to improve resolution.
  • the crystallization in another embodiment, may occur over 20% PEG 3350.
  • other chemicals can be used in the place of PEG 20K or 3350.
  • organic chemicals e.g. isopropanol
  • inorganic chemicals e.g. (NH 4 ) 2 SO , NaH 2 PO 4
  • other molecular weight PEG may be used. Further details ofthe method are as described below.
  • the present invention provides a method for determining the three dimensional structure of the crystallized type IV collagen [(c l) 2 (o2)] 2 NCI hexamer, comprising the steps of crystallizing the type IV collagen [(c ) ( ⁇ 2)] 2 NCI hexamer as described above, and then analyzing the type IV collagen [(cd) 2 ( ⁇ 2)] 2 NCI hexamer to determine its three dimensional structure.
  • the analyzing is by x-ray diffraction.
  • Data sets generated from the diffraction analysis can be analyzed using any appropriate software, including but not limited to the DENZO and SCALEPACK programs of the HKL2000 suite (39), the SOLVE program (40), the RESOLVE (41) program, and/or the FFT program of CCP4 suite (42). Tracing of the polypeptides from the resulting analysis can be accomplished using any suitable software, including but not limited to the TOM FRODO graphics program (43).
  • the final structure analysis can be accomplished using any appropriate software, including but not limited to SETOR(45), GRASP(46), and SURFNET(47) graphics software packages, various utility programs in the CCP4 suite, and HBPLUS(48) and protein-protein interaction web server (http://www.biochem.ucl.ac.uk/bsm/ PP/server/).
  • Another aspect of the invention is to use the three-dimensional structure of the type IV collagen [( ⁇ l) 2 ( ⁇ 2)] hexamer to solve the three-dimensional structure of a different type IV collagen NCI domain hexamer crystal, or crystal of a mutant, homologue or co-complex of type IV collagen NCI domain hexamer.
  • a further aspect of this invention is to use the three-dimensional structure of type IN collagen [(c l) (o2)] 2 hexamer to design inhibitors of the assembly of heterotrimers and hexamers of type IN collagen, including the type IN collagen [(c ) 2 ( ⁇ 2)] 2 ⁇ C1 hexamer.
  • These inhibitors may be used as therapeutics to inhibit undesired angiogenesis, angiogenesis-mediated disorders, tumor growth, tumor metastasis, endothelial cell adhesion and/or proliferation, and basal lamina assembly.
  • This embodiment comprises:
  • target refers to compounds that will interact with this region, via covalent or non-covalent means.
  • the definitions of the various regions are discussed below.
  • the NCI domains drive the selection process for type IV collagen chain assembly, and thus analysis of NCI domain assembly correlates with type IV collagen assembly.
  • analysis of the [(o-l) 2 (o_2)] 2 NCI hexamer crystal structure provides insights into the structure of other hexamer types, as well as inhibiters of such assembly.
  • inhibiting assembly of heterotrimers and hexamers of type IV collagen means to inhibit initial assembly of such heterotrimers and/or hexamers, or to disrupt the assembly of already assembled heterotrimers and hexamers of type IN collagen ⁇ C1 domains.
  • the therapeutic compounds identified herein inhibit the initial assembly of such heterotrimers and/or hexamers of type IN collagen ⁇ C1 domains.
  • the inhibitors can comprise peptides, or antibodies directed against peptides derived from the critical regions that would be expected to interfere with type IN collagen heterotrimer and/or hexamer assembly.
  • small molecules that are identified based on their potential to inhibit such assembly.
  • Electronic screening of large, structurally diverse compound libraries, such as the Available Chemical Directory (ACD) can identify new structural classes of such modulators that would be expected to interact with the identified critical regions.
  • ACD Available Chemical Directory
  • knowledge of the type IN collagen [( ⁇ l) (o2)] 2 ⁇ C1 hexamer structure permits "de novo design" of compounds to inhibit assembly of any type IN collagen ⁇ C1 domain heterotrimers and/or hexamers.
  • Potential inhibitors can be examined in silico through the use of computer modeling, using a docking program such as GRAM, DOCK, or AUTODOCK [Dunbrack et al., 1997, supra]. These procedures can include computer fitting of candidate compounds to the type IV collagen [(C.1) 2 (G_2)] 2 ⁇ C1 hexamer to predict how the shape and chemical structure of the candidate compound will interfere with assembly of the type IV collagen heterotrimer and/or hexamer. Computer programs can also be used to estimate the attraction, repulsion, and steric hindrance of the candidate compound to the relevant binding site on the type IN collagen [(c ) 2 (Q_2)] 2 hexamer. Generally the tighter the fit (e.g., the lower the steric hindrance, and/or the greater the attractive force), the more potent the candidate compound will be, and the less likely that the candidate compound will induce significant side effects due to unwanted interactions with other proteins.
  • a docking program such as GRAM, DOCK, or AUTODOCK
  • Potential small molecule inhibitors can be obtained, for example, by screening random peptide libraries produced, for example, in recombinant bacteriophage (Scott and Smith, Science, 249:386-390 (1990); Cwirla et al., Proc. Natl. Acad. Sci., 87:6378-6382 (1990); Devlin et al., Science, 249:404-406 (1990)), or a combinatorial chemical library.
  • Candidate compounds selected in this manner can be systematically modified by computer modeling programs until one or more promising candidate compounds are identified.
  • candidate compounds are chemically synthesized, and their biological activity is assayed, as discussed below.
  • they can be complexed with the type IV collagen [(c ) 2 (o2)] NCI hexamer crystal for further X-ray diffraction analysis to map the interactions of the compound with the crystal structure.
  • the three-dimensional structure ofthe supplemental crystal can be determined by Molecular Replacement Analysis, which involves using a known three-dimensional structure as a search model to determine the structure of a closely related molecule or protein-ligand complex in a new crystal form. The measured X-ray diffraction properties of the new crystal are compared with the search model structure to compute the position and orientation of the protein in the new crystal.
  • any assay that can be used to test the effect ofthe candidate compounds on the in vitro or in vivo assembly of type IV collagen heterotrimers and/or hexamers can be used to verify the efficacy of the candidate compounds identified by the methods of the invention.
  • any assay that can be used to test the effect of the candidate compounds on angiogenesis, tumor growth, tumor metastasis, and endothelial cell adhesion and/or motility can be used to verify their inhibitory activity.
  • Such assays include, but are not limited to, the following.
  • the methods employed are as described in Boutaud et al., JBC 275 (39):30716-30724 (2000).
  • Native GBM hexamers are isolated by standard methods and dissociated by dilution ( ⁇ 50 ⁇ g/ml) into a solution of 50 mM formic acid buffered at pH 3.0 with Tris base. Under these conditions, complete dissociation to NCI monomers and dimers occurs, as can be verified by HPLC or FPLC gel filtration. The absence of salt from the buffer is optimal for complete hexamer dissociation.
  • Reassembly of the dissociated NCI domains is performed by changing the buffer to Tris-buffered saline (50mM Tris, pH 7.4, 150mM NaCl) by repeated dilution-concentration cycles. After incubating the NCI domains at a concentration of about 1 mg/ml for 24 hours at room temperature, in the presence or absence of the candidate compounds at a desired concentration(s), the reaction products are separated according to their molecular weights using gel filtration chromatography. Quantification ofthe relative amounts ofthe various species in the mixture is done by peak area analysis from the HPLC profiles.
  • Hexamer assembly from purified al- 6 NCI domains is carried out similarly.
  • the ratio of the NCI domains in the association mixture is preferably kept at 1:1.
  • the isolated NCI hexamers can subsequently be analyzed for composition by immunoprecipitation followed by Western blotting; for overall appearance (size and shape) by electron microscopy; and for molecular weight by sedimentation equilibrium ulfracentrifugation.
  • mice Experiments are performed with 1-3 month old Swiss Webster male mice. Following anesthesia, the thoracic aorta is excised under aseptic conditions and transferred to sterile MCDB 131 sterile growth medium (Clonetics, San Diego, CA) containing antibiotics. Fat is dissected away from the aorta and approximately six to eight 1 mm thoracic segments are obtained from each specimen. Segments are transferred to 48 well tissue culture plates. The wells of these plates are layered with 100 microliters of MatrigelTM (EHS basement membrane, Collaborative Biomedical Products, Bedford, MA) prior to fransfer ofthe aortic segments. The MatrigelTM is diluted 1 : 1 with MCDB 131 growth medium prior to use.
  • MatrigelTM EHS basement membrane, Collaborative Biomedical Products, Bedford, MA
  • the segments are centered in the wells and an additional 100 microliters of MatrigelTM is then placed over the specimens.
  • the aortic segments are therefore embedded in the basement membrane matrix.
  • Each well then receives 300 microliters of MCDB 131 growth medium.
  • the plates are placed in an incubator maintained at 37° C with 5% CO 2 . Specimens are observed daily over a 7 day period. Newly growing microvessels are counted using an inverted phase microscope at various times during the culture period.
  • the drug candidates are mixed with the MatrigelTM and with the MCDB 131 growth medium, and the growth of microvessels from the cultured tissue into the matrix is analyzed.
  • the drug candidates are injected intravenously into rats containing fibrin implants surgically placed subcutaneously, a modified version of the method described by Dvorak et al. ( Lab. Invest. 57(6):673-686 (1987)).
  • rats are given tail vein injections of either control, or various concentrations of the drug candidates.
  • the implants are then removed at appropriate times, and directly analyzed using an inverted microscope. The analysis involved counting the number of blood vessels per implant that grow into the fibrin in the control and experimental group.
  • Chick embryo CAM angiogenesis assay Angiogenesis is induced in the CAMs of 10 day old chick embryos with bFGF as described (Brooks et al, Cell 92:391-400 (1998)). Twenty four hours later, the embryos are systemically treated with various concentrations of the drug candidates, in a total volume of 100 ⁇ l of sterile phosphate buffered saline (PBS). Two days later, the embryos are sacrificed and the filter discs and CAM tissues removed. Angiogenesis is quantitated by counting the number of angiogenic blood vessel branch points in the confined area of the filter disc. The Angiogenic Index is defined as the number of branch points from experimental treatment minus control treatment.
  • Chick embryo tumor growth assay Briefly, single cell suspensions of distinct tumor types are applied to the CAM of
  • the tumors may include, for example, CS-1 Melanoma cells, HT1080 human fibrosarcoma cells, and Hep-3 human epidermoid carcinoma cells.
  • the embryos are injected systemically with varying concentrations of the drug candidates 24 hours later.
  • the embryos are allowed to incubate for a total of 7 days, at which time they are sacrificed.
  • the resulting tumors are resected and wet weights determined compared to control.
  • Immobilized NCI domains support human endothelial cell adhesion
  • endothelial cells In order for new blood vessels to form, endothelial cells must have the capacity to adhere and migrate through the ECM. Moreover, this endothelial cell-ECM interaction may facilitate signal transduction events required for new blood vessel formation.
  • Microtiter plates are coated with varying amounts ofthe drug candidates, followed by incubation with 1% bovine serum albumin (BSA) to block non-specific interactions.
  • BSA bovine serum albumin
  • Endothelial cells such as human ECV304 cells, are then allowed to attach to the immobilized polypeptides for varying time periods
  • Non-adherent cells are removed by washing and attached cells are quantified by measuring the optical density of crystal violet eluted from attached cells.
  • the ability ofthe drug candidates to support human endothelial cell migration can be tested in vivo.
  • drug candidates can be tested in the metastatic Lewis lung mouse tumor model using a standard protocol which is considered to be a good model of both metastasis and angiogenesis of lung tumors.
  • Guibaud et al., Anticancer Drugs 8:276-282 (1997); Anderson et al, Cancer Res. 56:715-718 (1996) See for example, Teicher et al., Anticancer Res. 18:2567-2573 (1998); Guibaud et al., Anticancer Drugs 8:276-282 (1997); Anderson et al, Cancer Res. 56:715-718 (1996)).
  • Drug candidates are administered intravenously once every 2 days for a desired number of doses starting one day after tumor inoculation. All animals are weighed twice a week throughout the study. Starting one day after the last treatment, 1 or more mice are periodically sacrificed from each control group to measure pulmonary tumor burden. The experiment is terminated when the lungs of control animals have sufficient tumor mass to provide meaningful evaluation. At that time, the lungs of all remaining animals are excised, weighed, and the number of tumor foci greater than 2 mm in diameter counted.
  • the present invention provides an inhibitor of type IV collagen assembly identified by any ofthe methods described above.
  • the present invention provides an inhibitor of one or more process selected from the group consisting of angiogenesis, tumor growth, tumor metastasis, endothelial cell adhesion, endothelial cell proliferation, and basal lamina assembly, identified by any ofthe methods described above.
  • the present invention provides novel polypeptides that 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.
  • polypeptide is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits are linked by peptide bonds.
  • the polypeptides described herein may be chemically synthesized or recombinantly expressed.
  • the polypeptides of the present invention are chemically synthesized.
  • Synthetic polypeptides prepared using the well known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids.
  • Amino acids used for peptide synthesis may be standard Boc (N ⁇ -amino protected N ⁇ -t-butyloxycarbonyl) amino acid resin with the standard deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154), or the base- labile N ⁇ -amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpino and Han (1972, J.
  • the polypeptides of the invention may comprise D-amino acids (which are resistant to L-amino acid- specific proteases in vivo), a combination of D- and L-amino acids, and various "designer" amino acids (e.g., 0-methyl amino acids, C ⁇ -methyl amino acids, and N ⁇ - methyl amino acids, etc.) to convey special properties.
  • D-amino acids which are resistant to L-amino acid-specific proteases in vivo
  • various "designer" amino acids e.g., 0-methyl amino acids, C ⁇ -methyl amino acids, and N ⁇ - methyl amino acids, etc.
  • Synthetic amino acids include ornithine for lysine, fluorophenylalanine for phenylalanine, and norleucine for leucine or isoleucine.
  • the polypeptides can have peptidomimetic bonds, such as ester bonds, to prepare peptides with novel properties.
  • a peptide may be generated that incorporates a reduced peptide bond, i.e., R ⁇ -CH 2 -NH-R 2 , where R and R are amino acid residues or sequences.
  • a reduced peptide bond may be infroduced as a dipeptide subunit.
  • Such a polypeptide would be resistant to protease activity, and would possess an extended half-live in vivo.
  • type IV collagens are synthesized and assembled as heterotrimers inside the cells, which are then secreted exfracellularly where hexamer assembly, and subsequent basement membrane assembly, occurs.
  • the polypeptides disclosed herein can work intra-cellularly to prevent heterotrimer assembly, which also necessarily inhibits hexamer assembly, and provide the desired therapeutic result.
  • the polypeptides disclosed herein can work exfracellularly, to inhibit hexamer assembly, and/or to disrupt assembled hexamers, providing the desired therapeutic result.
  • Such polypeptides can be selected based on their utility in inhibiting generic heterotrimer assembly (ie: not chain specific); specific heterotrimer assembly (ie: chain specific); generic hexamer assembly (ie: not chain specific); and/or specific hexamer assembly (ie: not chain specific).
  • generic heterotrimer assembly ie: not chain specific
  • specific heterotrimer assembly ie: chain specific
  • generic hexamer assembly ie: not chain specific
  • specific hexamer assembly ie: not chain specific
  • polypeptides consist of at least 8 contiguous amino acids of general formula I:
  • Rl is selected from the group consisting of L, M, A, V, norL, and I;
  • R2 is selected from the group consisting of F and Y;
  • R3 is selected from the group consisting of I, V, L, norL, A, and P;
  • R4 is selected from the group consisting of N, G, and H;
  • R5 is selected from the group consisting of N, D, Q, and E;
  • R6 is selected from the group consisting of N, Y, and H;
  • R7 is selected from the group consisting of F and Y.
  • This general formula I is derived from a consensus sequences of type IN collagen
  • Inter-CDSR inter-chain domain swapping region
  • This region is involved in interchain interactions within the heterotrimer, and a substantial portion of the sequence is also present at the hexamer interface, and thus is involved in hexamer assembly/stabilization.
  • peptides of general formula I are useful for inhibiting appropriate interchain interactions, and thus for disrupting optimal heterotrimer and hexamer assembly.
  • the polypeptides consists of at least 9, 10, 11, 12,
  • polypeptide consists of 14 amino acids of general formula I.
  • polypeptides consist at least 8 contiguous amino acids of general formula II, with the further limitation that R2 is F; R4 is N; R5 is selected from the group consisting of N and D; R6 is N; and R7 is F.
  • Polypeptides of this embodiment are derived from a consensus sequences of type IN collagen ⁇ C1 ⁇ l, c ⁇ , and ⁇ 5 domains at the Inter-CDSR.
  • polypeptides consist at least 8 contiguous amino acids of general formula I, with the further limitation that R2 is Y; R3 is selected from the group consisting of P and I; R5 is selected from the group consisting of D, Q, and E; R6 is selected from the group consisting of Y and H; and R7 is Y.
  • Polypeptides of this embodiment are derived from a consensus sequences of type IN collagen ⁇ C1 o-2, ⁇ 4, and a ⁇ domains at the Inter-CDSR.
  • the polypeptides according to formula 1 consist of at least 8 contiguous amino acids of a sequence selected from the group consisting of PFLFC ⁇ I ⁇ NC ⁇ FA ( ⁇ l) (SEQ ID ⁇ O:2); PFLFCNVNDVCNFA ( ⁇ 3) (SEQ ID NO:3); PFMFCNINNVCNFA ( ⁇ 5) (SEQ ID NO:4); PFLYCNPGDVCYYA (02) (SEQ ID NO:5); PFAYCNJ-HQVCHYA ( ⁇ 4) (SEQ ID NO:6); and PF ⁇ YCNINEVCHYA ( ⁇ 6) (SEQ ID NO:7).
  • These sequences represent the Inter-CDSR sequences from the individual type IV collagen ⁇ l- ⁇ 6 NCI domains.
  • the polypeptides consist of at least 9, 10, 11, 12, 13, or 14 amino acids of one of the recited sequences.
  • the polypeptide consists of 14 amino acids of one ofthe recited sequences.
  • polypeptides of the present invention consist of at least 7 contiguous amino acids of general formula II:
  • Rl is selected from the group consisting of L, A, V, norL, and I;
  • R2 is selected from the group consisting of H, N, Q, and S;
  • R3 is selected from the group consisting of G, R, A, or is absent;
  • R4 is selected from the group consisting of R and Q; and
  • R5 is selected from the group consisting of N and H.
  • This general formula is derived from a consensus sequences of type IN collagen ⁇ C1 ⁇ l- ⁇ 6 domains at the intra-chain domain swapping region ("Intra-CDSR") that includes the 06 '-07' strands in the crystal structure, as further described below. This region is involved in monomer-monomer interactions within the heterotrimer, and a substantial portion of the sequence is also present at the hexamer interface, and thus is involved in hexamer assembly/stabilization. As such, peptides of this general formula are useful for inhibiting both heterotrimer and hexamer interactions of type IV collagen.
  • Intra-CDSR intra-chain domain swapping region
  • polypeptides consists of at least 8, 9, 10, 11,
  • polypeptide 12, or 13 amino acids of general formula II.
  • polypeptide consists of 13 amino acids of general formula II.
  • polypeptides consist at least 7 contiguous amino acids of general formula II, with the further limitation that R2 is H; R3 is R; R4 is G; and R5 is ⁇ .
  • Polypeptides of this embodiment are derived from a consensus sequence of the infra-CDSR sequences ofthe type IN collagen ⁇ l, ⁇ 3, and ⁇ 5 ⁇ C1 domains.
  • polypeptides consist at least 7 contiguous amino acids of general formula II, with the further limitation that R2 is selected from the group consisting of ⁇ , Q, and S; R3 is selected from the group consisting of G, R, and A; R4 is selected from the group consisting of R and Q; and R5 is H.
  • Polypeptides of this embodiment are derived from a consensus sequence of the infra-CDSR sequences of the type IN collagen o2, ⁇ 4, and ⁇ 6 ⁇ C1 domains.
  • polypeptides according to general formula II consist of at least 7 contiguous amino acids of a sequence selected from the group consisting of PFIECHGRGTC ⁇ ( ⁇ l and ⁇ 5) (SEQ ID ⁇ O:9); PFLECHGRGTCN ( ⁇ 3) (SEQ ID NO:10); PFIECNGGRGTCH (o2) (SEQ ID NO:ll); PFLECQGRQGTCH ( ⁇ 4) (SEQ ID NO:12); and PFIECSGARGTCH ( 6) (SEQ ID NO:13).
  • PFIECHGRGTC ⁇ ⁇ l and ⁇ 5
  • PFLECHGRGTCN ⁇ 3
  • PFIECNGGRGTCH o2
  • PFLECQGRQGTCH ⁇ 4
  • PFIECSGARGTCH SEQ ID NO:13
  • the polypeptides of this embodiment consist of at least 8, 9, 10, 11, 12, or 13 amino acids of one of the recited sequences, hi a most preferred embodiment, the polypeptides consist of 12 ( ⁇ l, ⁇ 3, ⁇ 5) or 13 ( ⁇ 2, ⁇ 4, 6) contiguous amino acids of any one the recited sequences.
  • the full length Intra-CDSR polypeptides e.g.: SEQ ID NO: 9, 10, 11, 12, or 13
  • polypeptides of the invention derived from the Intra-CDSR sequence of the ⁇ l-like NCI chains can thus be selected from the group consisting of at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids of a sequence selected from the group consisting of: ⁇ l : (E)(F)(R)(S)(A) PFIECHGRGTCN(Y)(Y)(A)(N)(A) (SEQ ID NO:14), c ⁇ : (E)(F)(R)(A)(S)PFLECHGRGTCN(Y)(Y)(S)(N)(S) (SEQ ID NO: 15); and c ⁇ : (E)(F)(R)(S)(A)PFIECHGRGTCN(Y)(Y)(A)(N)(S) (SEQ ID NO: 16); wherein the residues in parenthesis are the flanking sequences ofthe Intra-CDSR.
  • polypeptides of the invention derived from the Intra-CDSR sequence of the 02-like NCI chains can thus be selected from the group consisting of at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids of a sequence selected from the group consisting of: ⁇ 2: (D)(F)(R)(A)(T)PFIECNGGRGTCH(Y)(Y)(A)(N)(K) (SEQ ID NO: 17); ⁇ 4: (D)(F)(R)(A)(A)PFLECQGRQGTCH(F)(F)(A)(N)(K) (SEQ ID NO: 18); and ⁇ 6: (D)(F)(R)(A)(T)PFIECSGARGTCH(Y)(F)(A)(N)(K) (SEQ ID NO: 19); wherein the residues in parenthesis are the flanking sequences ofthe hitra-CDSR.
  • the Inter CDSR sequence while widely separated in the linear sequence of a given type IV collagen NCI domain from the hitra-CDSR sequence in the same ⁇ chain (separated by approximately 100 amino acids), is present in close spatial proximity (within approximately 2 amino acids) to the iter-CDSR sequence in the same ⁇ chain based on the derived crystal structure data.
  • the present invention provides chimeric polypeptides comprising:
  • the iter-CDSR and/or the Intra-CDSR portion of the chimeric polypeptides consists of 8, 9, 10, 11, 12, 13, or 14 amino acids of general formula I and 7, 8, 9, 10, 11, 12, 13 amino acids of general formula II, respectively.
  • the linker polypeptide consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.
  • the optimal length ofthe spacer depends, at least in part, on the length ofthe Inter-CDSR and hitra-CDSR, as well as the position of the sequences within the full length Inter-CDSR and Intra-CDSR used to create the chimera.
  • the spacer is preferably between 0-5 amino acids in length, more preferably between 1-4 amino acids in length, and most preferably 2-3 amino acids in length. Based on the teachings herein, it will be apparent to one of skill in the art to design further such chimeric polypeptides.
  • the Inter-CDSR polypeptide is selected from the group consisting of PFLFCNINNVCNFA (SEQ ID NO:2), PFLFCNVNDVCNFA (SEQ ID NO:3), PFMFCNINNVCNFA (SEQ ID NO:4), PFLYCNPGDVCYYA (SEQ ID NO:5), PFAYCNJ-HQVCHYA (SEQ ID NO:6), and PFIYCNTNEVCHYA (SEQ ID NO:7);
  • the Intra-CDSR polypeptide is selected from the group consisting of PFIECHGRGTCN (SEQ ID NO:9), PFLECHGRGTCN (SEQ ID NO:10), PFIECNGGRGTCH (SEQ ID NO:ll), PFLECQGRQGTCH (SEQ ID NO:12), and PFIECSGARGTCH (SEQ ID NO:13); and the linker polypeptide is 1, 2, 3, 4, or 5 amino acids; most preferably 2 amino acids.
  • polypeptides of the present invention consist of a sequence of an amino acids of general formula III: F(R1)T(R2) (SEQ ID NO:20) wherein Rl is selected from the group consisting of S and T; and R2 is selected from the group consisting of M and L.
  • This general formula III is derived from a consensus sequences of type FV collagen NCI ⁇ l- ⁇ 6 domains at the specificity region ("SR") between the 05-06 strands in the crystal structure, as further described below. This region is involved in specific recognition between monomers, by recognizing the specificity region partner ("SRP") in the monomer with which the SR of a given ⁇ chain interacts As such, peptides of general formula III are useful for inhibiting both heterotrimer and hexamer interactions of type IN collagen.
  • SRP specificity region partner
  • the SR polypeptides are selected from the group consisting of FSTM ( ⁇ l, ⁇ 2, ⁇ 5, and ⁇ 6) (SEQ ID ⁇ O:21), FTTM ( ⁇ 3) (SEQ ID NO:22) and FTSL ( ⁇ 4) (SEQ ID NO:23).
  • the SR polypeptides may optionally further include 0-5 amino acids at either or both the amino and carboxyl terminus that are derived from the same ⁇ chain, in order to provide appropriate secondary structural characteristics to the polypeptide for optimal inhibitory activity.
  • the polypeptides of the invention derived from the SR sequence ofthe NCI ⁇ chains can be selected from the group consisting of: ⁇ l X1-FSTM-Z1, wherein XI is 0, 1, 2, 3, 4, or 5 amino acids ofthe sequence SCLRK (SEQ ID NO: 24), and Zl is 0, 1, 2, 3, 4, or 5 amino acids of the sequence PFLFC (SEQ ID NO: 25) (the full sequence would thus be SCLRKFSTMPFLFC) (SEQ ID NO: 26); ⁇ 3: X3-FTTM-Z3, wherein X3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence SCLQR (SEQ ID NO: 27), and Z3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence PFLFC(SEQ ID NO: 25) (the full sequence would thus be SCLQRFTTMPFLFC) (SEQ ID NO:28); ⁇ 5: X5-FSTM-Z5, wherein X5 is 0, 1, 2, 3, 4, or 5 amino acids ofthe
  • SCLRR SEQ ID NO: 29
  • Z5 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence PFMFC (SEQ ID NO: 30) (the full sequence would thus be SCLRRFSTMPFMFC) (SEQ ID NO: 31);
  • polypeptides of the invention consist of an amino acid sequence of general formula IN:
  • R1MF(R2)K (SEQ ID ⁇ O:41) wherein Rl is selected from the group consisting of E, R, and D; and R2 is selected from the group consisting of K, R, and S.
  • This general formula IN is derived from a consensus sequences of type TV collagen ⁇ C1 ⁇ l, ⁇ 3, and ⁇ 5 domains at the specificity region partner ("SRP") located between the 08' and 09' strands, as discussed in more detail below.
  • SRP specificity region partner
  • This region is involved in specific recognition between monomers, by recognizing the specificity region ("SR") in the monomer with which the SRP of a given ⁇ chain interacts
  • peptides of general formula IV are useful for inhibiting both heterotrimer and hexamer interactions of type IV collagen.
  • the SRP polypeptides according to general formula IV are selected from the group consisting of EMFKK ( ⁇ l) (SEQ ID ⁇ O:42), RMFRK (c ⁇ ) (SEQ ID NO:43), and DMFSK ( ⁇ 5) (SEQ ID NO:44).
  • the SRP polypeptides are selected from the group consisting of SFQ (SRP of o2) (SEQ ID NO:45); LQF (SRP of ⁇ 4) (SEQ ID NO:46), and QQF (SRP of a.6) (SEQ ID NO:47).
  • SRP of o2 SEQ ID NO:45
  • LQF SRP of ⁇ 4
  • QQF SRP of a.6
  • the SRP polypeptides may optionally further include 0-5 amino acids at either or both the amino and carboxyl terminus that are derived from the same ⁇ chain, in order to provide appropriate secondary structural characteristics to the polypeptide for optimal inhibitory activity.
  • the SRP-containing polypeptides of this embodiment of the invention can thus be selected from the group consisting of: ⁇ l X1-EMFKK-Z1, wherein XI is 0, 1, 2, 3, 4, or 5 amino acids of the sequence TIERS (SEQ ID NO: 48), and wherein Zl is 0, 1, 2, 3, 4, or 5 amino acids of the sequence PTPST (SEQ ID NO: 49) (the full length sequence would thus be TI-ERSEMFKKPTPST) (SEQ ID NO: 50); ⁇ 3: X3-RMFRK-Z3, wherein X3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence SLNPE (SEQ ID NO: 51), and wherein Z3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence PIPST (SEQ ID NO: 52) (the full length sequence would thus be SLNPERMFRKPIPST) (SEQ ID NO:53); ⁇ 5: X5-DMFSK-Z5, wherein X5 is 0, 1, 2, 3, 4, or 5 amino acids of
  • TVKAD SEQ ID NO: 60
  • Z4 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence SSAPA (SEQ ID NO: 61) (the full length sequence would thus be TVKADLQFSSAPA) (SEQ ID NO: 62); and ⁇ 6: X6-QQF-Z6, wherein X6 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence TVEER (SEQ ID NO: 63), and wherein Z6 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence GELPV (SEQ ID NO: 64) (the full length sequence would thus be TVEERQQFGELPV) (SEQ ID NO: 65).
  • polypeptides ofthe invention consist of an amino acid sequence of general formula V:
  • R1AH(R2)QD SEQ ID NO:66 wherein Rl is selected from the group consisting of R and K; and R2 is selected from the group consisting of G and N.
  • This general formula V is derived from a consensus sequences of type IV collagen NCI domain 0-barrel-like core at the 04 strand, as discussed in more detail below. This region is involved in generic monomer-monomer interactions. As such, peptides of general formula V are useful for inhibiting both heterotrimer and hexamer interactions of type IN collagen.
  • the polypeptides according to general formulaN are selected from the group consisting of RAHGQD ( ⁇ l, ⁇ 3, ⁇ 5) (SEQ ID NO: 67) and KAHNQD (02, ⁇ 4, ⁇ 6) (SEQ ID NO:68).
  • the 0-barrel polypeptides according to general formula V may optionally further include 0-5 amino acids at either or both the amino and carboxyl terminus that are derived from the same ⁇ chain, in order to provide appropriate secondary structural characteristics to the polypeptide for optimal inhibitory activity.
  • the 0-barrel-containing polypeptides of this embodiment of the invention can thus be selected from the group consisting of: ⁇ l X1-RAHGQD-Z1, wherein XI is 0, 1, 2, 3, 4, or 5 amino acids of the sequence VQGNE (SEQ ID NO: 69), and wherein Zl is 0, 1, 2, 3, 4, or 5 amino acids of the sequence LGTAG (SEQ ID NO: 70) (the full length sequence would thus be VQGNERAHGQDDLGTA) (SEQ ID NO: 71); ⁇ 3: X3-RAHGQD-Z3, wherein X3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence VQGNQ (SEQ ID NO: 72), and wherein Z3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence LGTLG (SEQ ID NO: 73) (the full length sequence would thus be VQGNQRAHGQDLGTLG) (SEQ ID NO:74); ⁇ 5: X5-RAHGQD-Z5, wherein X5 is 0,
  • R1G(R2)GQ (SEQ ID NO:85) wherein Rl is selected from the group consisting of E and Q;
  • R2 is selected from the group consisting of S, T, and G.
  • This general formula VI is derived from a consensus sequences of type IN collagen ⁇ C1 domain 0-barrel-like core at the 04' strand, as discussed in more detail below. This region is involved in generic monomer-monomer interactions. As such, peptides of general formula NI are useful for inhibiting both heterotrimer and hexamer interactions of type TV collagen.
  • polypeptides according to generalformulaVI are selected from the group consisting of EGSGQ ( ⁇ l, ⁇ 5) (SEQ ID ⁇ O:86), EGTGQ ( ⁇ 3) (SEQ ID NO:87), EGGGQ ( ⁇ 2, ⁇ 6) (SEQ ID NO:88) and QGGGQ ( ⁇ 4) (SEQ ID NO:89).
  • the 0-barrel polypeptides according to general formula VI may optionally further include 0-5 amino acids at either or both the amino and carboxyl terminus that are derived from the same ⁇ chain, in order to provide appropriate secondary structural characteristics to the polypeptide for optimal inhibitory activity.
  • the 0-barrel-containing polypeptides of this embodiment of the invention can thus be selected from the group consisting of: ⁇ l and ⁇ 5 X1-EGSGQ-Z1, wherein XI is 0, 1, 2, 3, 4, or 5 amino acids ofthe sequence TSAGA (SEQ ID NO: 90), and wherein Zl is 0, 1, 2, 3, 4, or 5 amino acids of the sequence ALASP (SEQ ID NO: 91) (the full length sequence would thus be TSAGAEGSGQALASP) (SEQ ID NO: 92); ⁇ 3: X3-EGTGQ-Z3, wherein X3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence TSAGS (SEQ ID NO: 93), and wherein Z3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence ALASP (SEQ ID NO: 91) (the full length sequence would thus be TSAGSEGTGQALASP) (SEQ ID NO:94); 02: X2-EGGGQ-Z2, wherein X2 is 0, 1, 2, 3, 4, or 5 amino acids
  • the polypeptides comprise sequences present at the hexamer interface, as determined from the deduced crystal structure.
  • Type IV collagens are synthesized and assembled as trimers inside the cells, which are then secreted exfracellularly where hexamer assembly, and subsequent basement membrane assembly, occurs.
  • Therapeutics such as those disclosed herein, can work intra-cellularly to prevent frimer assembly, thus inhibiting hexamer assembly, thus providing the desired therapeutic result.
  • therapeutics can work exfracellularly, which leaves frimer assembly uninhibited, but targets hexamer assembly.
  • polypeptides from regions at the hexamer interface can be used to inhibit hexamer formation or disrupt hexamer formation.
  • the polypeptides of the invention consist of an amino acid sequence of general formula VII: (R1)G(R2)(R3) (SEQ ID NO:103) wherein Rl is selected from the group consisting of Q and E; R2 is selected from the group consisting of N and Q; and R3 is selected from the group consisting of E, Q, and K.
  • This general formula VII is derived from a consensus sequences of type IV collagen NCI ⁇ l- ⁇ 6 domains at the hexamer interface at the end ofthe 03 strand up to the beginning of the 04 strand, as discussed in more detail below. This region is present at the hexamer interface, and is involved in hexamer assembly and stabilization.
  • peptides of general formula Nil are useful for inhibiting hexamer interactions of type IN collagen.
  • the polypeptides consist of general formula VII, with the further limitation that Rl is Q and R2 is ⁇ .
  • the formula is a consensus ofthe sequences present in the ⁇ l/ ⁇ 3/ ⁇ 5 ⁇ C1 domains for general formula VII.
  • the polypeptides according to general formula VII are selected from the group consisting of QGNE ( ⁇ l) (SEQ ID NO:104), QGNQ (c ⁇ ) (SEQ ID NO:105), and QGNK( ⁇ 5) (SEQ ID NO:106)
  • polypeptides according to general formula VII consist of EGQE (SEQ ID NO: 107), which is the sequence ofthe sequences present in the o-2/ ⁇ 4/ ⁇ 6 NCI domains in general formula VII.
  • the hexamer polypeptides selected from the group consisting of SEQ ID NOS: 104-107 may optionally further include 0-5 amino acids at either or both the amino and carboxyl terminus that are derived from the same ⁇ chain, in order to provide appropriate secondary structural characteristics to the polypeptide for optimal inhibitory activity.
  • Such polypeptides can thus be selected from the group consisting of: ⁇ l: X1-QGNE-Z1, wherein XI is 0, 1, 2, 3, 4, or 5 amino acids of the sequence SLLYV (SEQ ID NO: 108), and wherein Zl is 0, 1, 2, 3, 4, or 5 amino acids of the sequence RAHGQ (SEQ ID NO: 109) (the full length sequence would thus be SLLYVQGNERAHGQ) (SEQ ID NO: 110); ⁇ 3: X3-QGNQ-Z3, wherein X3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence SFLFV (SEQ ID NO: 111), and wherein Z3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence RAHGQ (SEQ ID NO: 109) (the full length sequence would thus be SFLFVQGNQRAHGQ) (SEQ ID NO: 112); ⁇ 5: X5-QGNK-Z5, wherein X5 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence SL
  • hexamer interface polypeptides according to general formula VII consists of 1 additional amino acid at both the amino and carboxy terminus ofthe ⁇ l- ⁇ 6 hexamer peptides, as follows: ⁇ l VQGNER (SEQ ID NO: 121) ⁇ 3 VQGNQR (SEQ ID NO: 122) ⁇ 5 VQGNKR (SEQ ID NO: 123) ⁇ 2 FEGQEK (SEQ ID NO: 124) ⁇ 4 LEGQEK (SEQ ID NO: 125) ⁇ 6 VEGQEK (SEQ ID NO: 126)
  • polypeptides ofthe invention consist of an amino acid sequence of general formula VIII: M(R1)M(R2)P (SEQ ID NO:127) wherein Rl is selected from the group consisting of S, N, or is absent; and
  • R2 is selected from the group consisting of A, Q, or is absent.
  • This general formula VIII is derived from a consensus sequences of type IN collagen ⁇ C1 ⁇ l- ⁇ 6 domains at the hexamer interface between the 08 and 09 strands, as discussed in more detail below. This region is present at the hexamer interface, and is involved in hexamer assembly and stabilization. As such, peptides of general formula NIII are useful for inhibiting hexamer interactions of type IV collagen.
  • polypeptides of general formula VIII are selected from the group consisting of MSMAP ( ⁇ l) (SEQ ID ⁇ O:128), MNMAP ( ⁇ 3) (SEQ ID NO:129), MSMQP ( ⁇ 5) (SEQ ID NO:130), and MMP ( ⁇ 2, ⁇ 4, and c ⁇ ) (SEQ ID NO: 131).
  • the hexamer polypeptides selected from the group consisting of SEQ ID NOS:128-131 may optionally further include 0-5 amino acids at either or both the amino and carboxyl terminus that are derived from the same ⁇ chain, in order to provide appropriate secondary structural characteristics to the polypeptide for optimal inhibitory activity.
  • Such polypeptides can thus be selected from the group consisting of: ⁇ l: X1-MSMAP-Z1, wherein XI is 0, 1, 2, 3, 4, or 5 amino acids of the sequence PEPMP (SEQ ID NO: 132), and wherein Zl is 0, 1, 2, 3, 4, or 5 amino acids of the sequence ITGEN (SEQ ID NO: 133) (the full length sequence would thus be PEPMPMSMAPITGEN) (SEQ ID NO: 134); c ⁇ : X3-MNMAP-Z3, wherein X3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence PALMP (SEQ ID NO: 135), and wherein Z3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence ITGRA (SEQ ID NO: 136) (the full length sequence would thus be PALMPMNMAPITGRA) (SEQ ID NO:137); ⁇ 5: X5-MSMQP-Z5, wherein X5 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence PEPMP (SEQ ID NO:132),
  • X2-MMP-Z2 wherein X2 is 0, 1, 2, 3, 4, or 5 amino acids ofthe sequence TAPLP (SEQ ID NO: 140), and wherein Z2 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence VAEDE (SEQ ID NO:141) (the full length sequence would thus be TAPLPMMPVAEDE) (SEQ ID NO: 142); ⁇ 4: X4-MMP-Z4, wherein X4 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence
  • AAPLP (SEQ ID NO:143), and wherein Z4 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence LSEEA (SEQ ID NO: 144) (the full length sequence would thus be AAPLPMMPLSEEA) (SEQ ID NO: 145); and ⁇ 6: X6-MMP-Z6, wherein X6 is 0, 1, 2, 3, 4, or 5 amino acids ofthe sequence TAPIP (SEQ ID NO:146), and wherein Z6 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence VSQTQ (SEQ ID NO:147) (the full length sequence would thus be TAPrPMMPVSQTQ) (SEQ ID NO: 148).
  • hexamer interface peptides according to general formula VIII consists of 3 additional amino acids at both the amino and carboxy terminus ofthe ⁇ l- ⁇ 6 hexamer peptides, as follows: ⁇ l PMPMSMAPITG (SEQ ID NO : 149); ⁇ 3: LMPMNMAPITG (SEQ ID NO:150); c ⁇ PMPMSMQPLKG (SEQ ID NO: 151); o2 PLPMMPVAE (SEQ ID NO: 152); ⁇ 4 PLPMMPLSE (SEQ ID NO: 153); and c ⁇ PIPMMPVSQ (SEQ ID NO: 154).
  • polypeptides ofthe invention consist of an amino acid sequence of general formula IX: AG(R1)(R2) (SEQ ID NO:155) wherein Rl is selected from the group consisting of A, S and D; and
  • R2 is selected from the group consisting of E and Q.
  • This general formula IX is derived from a consensus sequences of type JN collagen ⁇ C1 ⁇ l- ⁇ 6 domains between the 03' and 04' strands, as discussed in more detail below. This region is present at the hexamer interface, and is involved in hexamer assembly and stabilization. As such, peptides of general formula IX are useful for inhibiting hexamer interactions of type IN collagen.
  • polypeptides of general formula IX are selected from the group consisting of AGAE ( ⁇ l, ⁇ 5, and ⁇ 6) (SEQ ID ⁇ O:156), AGSE ( ⁇ 3) (SEQ ID NO:157), AGDE (o2) (SEQ ID NO:158), and AGDQ ( ⁇ 4) (SEQ ID NO:159).
  • the hexamer polypeptides selected from the group consisting of SEQ ID NOS: 156-159 may optionally further include 0-5 amino acids at either or both the amino and carboxyl terminus that are derived from the same ⁇ chain, in order to provide appropriate secondary structural characteristics to the polypeptide for optimal inhibitory activity.
  • polypeptides can thus be selected from the group consisting of: ⁇ l: X1-AGAE-Z1, wherein XI is 0, 1, 2, 3, 4, or 5 amino acids of the sequence VMHTS (SEQ ID NO: 160), and wherein Zl is 0, 1, 2, 3, 4, or 5 amino acids of the sequence GSGQA (SEQ ID NO: 161) (the full length sequence would thus be VMHTSAGAEGSGQA) (SEQ ID NO: 162); ⁇ 3: X3-AGSE-Z3, wherein X3 is 0, 1, 2, 3, 4, or 5 amino acids ofthe sequence
  • IMFTS (SEQ ID NO: 163), and wherein Z3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence GTGQA (SEQ ID NO: 164) (the full length sequence would thus be J-MFTSAGSEGTGQA) (SEQ ID N0:165); ⁇ 5: X5-AGAE-Z5, wherein X5 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence MMHTS (SEQ ID NO: 166), and wherein Z5 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence GSGQA (SEQ ID NO: 161) (the full length sequence would thus be MMHTSAGAEGSGQA) (SEQ ID NO: 167); ⁇ 2: X2-AGDE-Z2, wherein X2 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence LMHTA (SEQ ID NO: 168), and wherein Z2 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence GGGQS (SEQ ID NO: 169) (the full length sequence would thus be LMHTAAGDEGGGQ
  • polypeptides of the invention consist of at least 5 amino acids ofthe sequence of general formula X:
  • Rl is selected from the group consisting of H, N, Q, and S;
  • R2 is selected from the group consisting of G, R, A, or is absent;
  • R3 is selected from the group consisting of R and Q R4 is selected from the group consisting of N and H;
  • R5 is selected from the group consisting of F and Y;
  • R6 is selected from the group consisting of F and Y.
  • polypeptide consists of at least 6, 7, 8, 9,
  • polypeptide consists of 12 amino acids of general formula X.
  • This general formula X extensively overlaps with the Intra-CDSR, discussed above, and is present within the 06'-
  • polypeptides are as described above for general formula X, with the exception that R2 is selected from the group consisting of G, R, A; and R4 is H. Polypeptides of this embodiment are derived from the consensus sequence ofthe 02/4/6 of general formula X.
  • polypeptides of general formula X are selected from the group consisting of ECHGRGTC ⁇ YY ( ⁇ l/3/5) (SEQ ID ⁇ O:176), ECNGGRGTCHYY ( ⁇ 2) (SEQ ID NO:177), ECQGRQGTCHFF ( ⁇ 4) (SEQ ID NO:178), and ECSGARGTCHYF (c ⁇ ) (SEQ ID NO:179).
  • polypeptides of the invention consist of an amino acid sequence of general formula XI:
  • R1(R2)T(R3)K SEQ ID NO:180 wherein Rl is selected from the group consisting of P, S, and A;
  • R2 is selected from the group consisting of S, E, and D;
  • R3 is selected from the group consisting of L and V.
  • This general formula XI is present overlapping with the 09' strand in the crystal structure, as discussed in more detail below. This region is present at the hexamer interface, and is involved in hexamer assembly and stabilization. As such, peptides of general formula XI are useful for inhibiting hexamer interactions of type IN collagen.
  • R3 is L (as in ⁇ 2/4/6/l/5).
  • R2 is selected from D and E (0/2/4/5/6).
  • the polypeptide according to general formula XI is selected from the group consisting of PSTLK ( ⁇ l) (SEQ ID ⁇ O:181), PSTVK ( ⁇ 3) (SEQ ID NO:182), SETLK ( ⁇ 5 and ⁇ 6) (SEQ ID NO:183), ADTLK (o2) (SEQ ID NO:184), and PDTLK ( ⁇ 4) (SEQ ID NO:185).
  • the hexamer polypeptides selected from the group consisting of SEQ ID NOS:181-185 may optionally further include 0-5 amino acids at either or both the amino and carboxyl terminus that are derived from the same ⁇ chain, in order to provide appropriate secondary structural characteristics to the polypeptide for optimal inhibitory activity.
  • Such polypeptides can thus be selected from the group consisting of: ⁇ l: X1-PSTLK-Z1, wherein XI is 0, 1, 2, 3, 4, or 5 amino acids of the sequence FKKPT (SEQ ID NO: 186), and wherein Zl is 0, 1, 2, 3, 4, or 5 amino acids of the sequence AGELR (SEQ ID NO: 187) (the full length sequence would thus be FKKPTPSTLKAGELR) (SEQ ID NO: 188); ⁇ 3: X3-PSTVK-Z3, wherein X3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence FRKPI (SEQ ID NO: 189), and wherein Z3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence AGELE (SEQ ID NO: 190) (the full length sequence would thus be FRKPrPSTNKAGELE) (SEQ ID ⁇ O:191); ⁇ 5: X5-SETLK-Z5, wherein X5 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence FSKPQ
  • polypeptides of the invention consist of an amino acid sequence of general formula XII: A(R1)RND (SEQ ID NO:204) wherein Rl is selected from the group consisting of S, Q, and R.
  • Rl is selected from the group consisting of S, Q, and R.
  • This general formula XII is present in the highly conserved loop connecting the ⁇ 7 and ⁇ 8 strands in the crystal structure. This region is present at the hexamer interface, and is involved in hexamer assembly and stabilization. As such, peptides of general formula XII are useful for inhibiting hexamer interactions of type IV collagen.
  • polypeptide according to general formula XII is selected from the group consisting of ASRND ( ⁇ l, c ⁇ , c ⁇ , cQ) (SEQ ID NO:205), AQRND ( ⁇ 4) (SEQ ID NO:206), and ARRND ( ⁇ 6) (SEQ ID NO:207).
  • the hexamer polypeptides selected from the group consisting of SEQ ID NOS:205, 206, and 207 may optionally further include 0-5 amino acids at either or both the amino and carboxyl terminus that are derived from the same ⁇ chain, in order to provide appropriate secondary structural characteristics to the polypeptide for optimal inhibitory activity.
  • Such polypeptides can thus be selected from the group consisting of: ⁇ l and ⁇ 5: X1-ASRND-Z1, wherein XI is 0, 1, 2, 3, 4, or 5 amino acids ofthe sequence NVCNF (SEQ ID NO: 208), and wherein Zl is 0, 1, 2, 3, 4, or 5 amino acids of the sequence YSYWL (SEQ ID NO: 209) (the full length sequence would thus be NVCNFASRNDYSYWL) (SEQ ID NO: 210); ⁇ 3: X3-ASRND-Z3, wherein X3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence DVCNF (SEQ ID NO: 211), and wherein Z3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence YSYWL (SEQ ID NO: 209) (the full length sequence would thus be DVCNFASRNDYSYWL) (SEQ ID NO:212); ⁇ 2: X2-ASRND-Z2, wherein X2 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence
  • polypeptides of the invention consist of an amino acid sequence of general formula XIII: (R1)(R2)(R3)N(R4) (SEQ ID NO:221) wherein Rl is selected from the group consisting of Y and F; R2 is selected from the group consisting of Y and F; R3 is selected from the group consisting of A and S; and R4 is selected from the group consisting of A, S, and K.
  • This general formula XIII is present in the highly conserved loop connecting the ⁇ 7' and ⁇ 8' strands in the crystal structure. This region is present at the hexamer interface, and is involved in hexamer assembly and stabilization. As such, peptides of general formula XIII are useful for inhibiting hexamer interactions of type IV collagen. hi further preferred embodiments, the polypeptide according to general formula
  • XIII is selected from the group consisting of YYANA ( ⁇ l) (SEQ ID NO:222) YYSNS ( ⁇ 3) (SEQ ID NO:223) YYANS ( ⁇ 5) (SEQ ID NO:224) YYANK (02) (SEQ ID NO:225) FFANK ( ⁇ 4) (SEQ ID NO:226) and YFANK( ⁇ 6) (SEQ ID NO:227).
  • the hexamer polypeptides selected from the group consisting of SEQ ID NOS:222-227 may optionally further include 0-5 amino acids at either or both the amino and carboxyl terminus that are derived from the same ⁇ chain, in order to provide appropriate secondary structural characteristics to the polypeptide for optimal inhibitory activity.
  • Such polypeptides can thus be selected from the group consisting of: ⁇ l: X1-YYANA-Z1, wherein XI is 0, 1, 2, 3, 4, or 5 amino acids of the sequence RGTCN (SEQ ID NO: 228), and wherein Zl is 0, 1, 2, 3, 4, or 5 amino acids of the sequence YSFWL (SEQ ID NO: 229) (the full length sequence would thus be RGTCNYYANAYSFWL) (SEQ ID NO: 230); ⁇ 3: X3-YYSNS-Z3, wherein X3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence RGTCN (SEQ ID NO: 228), and wherein Z3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence YSFWL (SEQ ID NO: 229) (the full length sequence would thus be RGTCNYYSNSYSFWL) (SEQ ID NO:231); ⁇ 5: X1-YYANS-Z2, wherein XI is 0, 1, 2, 3, 4, or 5 amino acids of the sequence RGTCN
  • the present invention provides novel polypeptides derived from the hypervariable region of the type IV collagen ⁇ chain NCI domain sequences located between the 08 'and the 09' sfrands, which are identified from the crystal structure as being present at the monomer-monomer interface, and which include the SRP and are involved in providing appropriate secondary structure for optimal interactions between the SR and the SRP.
  • the polypeptides consist of at least 7 amino acids of a sequence selected from the group consisting of lERSEMFKKPT ( ⁇ l) (SEQ ID NO:238), LNPERMFRKPI ( ⁇ 3) (SEQ ID NO:239), VDVSDMFSKPQ ( ⁇ 5) (SEQ ID NO:240), 1PEQSFQGSPS (02) (SEQ ID NO:241), VKADLQFSSAPA ( ⁇ 4) (SEQ ID NO:242), and VEERQQFGELPV ( 6) (SEQ ID NO:243).
  • the polypeptides consist of at least 8, 9, 10, 11, or 12 amino acids of a sequence selected from the group consisting of SEQ ID NO:235-240.
  • SEQ ID NOS:238-243 may optionally further include 0-5 amino acids at either or both the amino and carboxyl terminus that are derived from the same ⁇ chain, in order to provide appropriate secondary structural characteristics to the polypeptide for optimal inhibitory activity.
  • Such polypeptides can thus be selected from the group consisting of: ⁇ l: X1-IERSEMFKKPT-Z1, wherein XI is 0, 1, 2, 3, 4, or 5 amino acids of the sequence FWLAT (SEQ ID NO: 244), and wherein Zl is 0, 1, 2, 3, 4, or 5 amino acids of the sequence PSTLK (SEQ ID NO: 181) (the full length sequence would thus be FWLATIERSEMFKKPTPSTLK) (SEQ ID NO: 245); 03: X3-LNPERMFRKPI-Z3, wherein X3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence FWLAS (SEQ ID NO: 246), and wherein Z3 is 0, 1, 2, 3, 4, or 5 amino acids of the sequence PSTVK (SEQ
  • the present invention provides other polypeptides that include multiple regions identified as being important for inhibiting monomer-monomer interactions (and thus heterotrimer assembly), and/or trimer-trimer interactions (and thus hexamer assembly).
  • Polypeptides according to this aspect of the invention include the following:
  • SR plus the hiter-CDSR ⁇ l FSTMPFLFCNTNNVCNFA (SEQ ID NO: 253) ⁇ 3 FTTMPFLFCNVNDVCNFA (SEQ ID NO: 254) ⁇ 5 FSTMPFMFCNINNVCNFA (SEQ ID NO: 255) ⁇ 2 FSTMPFLYCNPGDVCYYA (SEQ ID NO: 256) ⁇ 4 FSTLPFAYCNIHQVCHYA (SEQ ID NO: 257) 6: FSTMPF ⁇ YCN ⁇ NEVCHYA (SEQ ID NO: 258)
  • Inter-CDSR plus contiguous hexamer interface region ⁇ l PFLFCN ⁇ NNVCNFASRND (SEQ ID NO: 259) ⁇ 3 PFLFCNNNDNCNFASRND (SEQ ID NO: 260) ⁇ 5 PFMFCNTNNVCNFASRND (SEQ ID NO: 261) o2 PFLYCNPGDVCYYASRND (SEQ ID NO: 262) ⁇ 4 PFAYCNIHQVCHYAQRND (SEQ ID NO: 263) 6 PFIYCNINEVCHYARRND (SEQ ID NO: 264)
  • SR plus the hiter-CDSR plus contiguous hexamer interface region ⁇ l FSTMPFLFCNTNNNCNFASRND (SEQ ID NO: 265) ⁇ 3 FTTMPFLFCNVNDVCNFASRND (SEQ ID NO: 266) ⁇ 5 FSTMPFMFCNINNNCNFASRND (SEQ ID NO: 267) 02 FSTMPFLYCNPGDVCYYASRND (SEQ ID NO: 268) ⁇ 4 FSTLPFAYCN ⁇ HQVCHYAQRND (SEQ ID NO: 269) ⁇ 6 FSTMPFIYCN ⁇ NEVCHYARRND (SEQ ID NO: 270)
  • Intra-CDSR plus contiguous hexamer interface region ⁇ l and ⁇ 5 : PFIECHGRGTCNYY (SEQ ID NO:271)
  • the present invention provides methods for inhibiting angiogenesis, angiogenesis-mediated disorders, tumor growth, tumor metastasis, endothelial cell adhesion and/or proliferation, and basal lamina assembly, comprising administering to a subject in need thereof an amount effective to inhibit angiogenesis, angiogenesis-mediated disorders, tumor growth, tumor metastasis, endothelial cell adhesion and/or proliferation, and basal lamina assembly of one or more polypeptides of the invention, antibodies against such polypeptides, or pharmaceutical compositions thereof.
  • Angiogenesis-mediated disorders refers to diseases and conditions with accompanying undesired angiogenesis, including but not limited to solid and blood-borne tumors, diabetic retinopathy, rheumatoid arthritis, retinal neovascularization, choroidal neovascularization, macular degeneration, corneal neovascularization, retinopathy of prematurity, corneal graft rejection, neo vascular glaucoma, retrolental fibroplasia, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections
  • polypeptides, or antibodies against such polypeptides may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc.
  • conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc.
  • polypeptides, or antibodies against such polypeptides are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration.
  • the polypeptides, or antibodies against such polypeptides may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration.
  • polypeptides, or antibodies against such polypeptides of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.
  • saline water
  • polyethylene glycol propylene glycol
  • carboxymethyl cellulose colloidal solutions ethanol
  • corn oil corn oil
  • peanut oil cottonseed oil
  • sesame oil sesame oil
  • tragacanth gum and/or various buffers.
  • Other adjuvants and modes of administration are well known in the pharmaceutical art.
  • the carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.
  • the amount or dosage range of the polypeptides, antibodies against such polypeptides, or pharmaceutical compositions employed is one that effectively inhibits angiogenesis, angiogenesis-mediated disorders, tumor growth, tumor metastasis, and/or endothelial cell-extracellular matrix interactions.
  • An inhibiting amount ofthe polypeptides that can be employed ranges generally between about 0.01 ⁇ g/kg body weight and about 10 mg/kg body weight, preferably ranging between about 0.05 ⁇ g/kg and about 5 mg/kg body weight.
  • polypeptides, antibodies against such polypeptides, or pharmaceutical compositions thereof may be administered by any suitable route, including orally, parentally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.
  • parenteral as used herein includes, subcutaneous, intravenous, intra-arterial, intramuscular, intrasternal, infratendinous, intraspinal, intracranial, infrathoracic, infusion techniques or intraperitoneally.
  • the polypeptides are administered intravenously or subcutaneously.
  • polypeptides, antibodies against such polypeptides, or pharmaceutical compositions thereof may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions).
  • the polypeptides and antibodies against such polypeptides ofthe invention may be applied in a variety of solutions. Suitable solutions for use in accordance with the invention are sterile, dissolve sufficient amounts of the polypeptides, and are not harmful for the proposed application.
  • one or more of the disclosed polypeptides, antibodies against such polypeptides, or pharmaceutical compositions thereof are used so as to target more than one region of type IV collagen for inhibition of assembly.
  • peptides that target different hexamer regions can be used in combination to increase their inhibitory effect.
  • combining a peptide targeting monomer- monomer interactions with a peptide that targets hexamer assembly can provide an additive inhibitory effect.
  • Other combinations are well within the knowledge of one of skill in the art, based on the teachings herein.
  • NCI hexamer was isolated from bovine eye lenses purchased from Pel-Freeze Biologicals (Rogers, AR) (37). Briefly, LBM was prepared by sonication ofthe lenses in the presence of 1 M NaCl and protease inhibitors (38). To cleave the NCI domain from the full-length type IV collagen, the LBM preparation was digested with bacterial coUagenase at 37° C. The NCI hexamer was purified by using DE-52 and S-300 column chromatography.
  • Multiwavelength anomalous diffraction (MAD) data sets were collected at peak, inflection and two remote wavelengths using a single crystal soaked in 0.5 M KBr for 1 min and flash-frozen in cold N stream (Table 1).
  • the heavy atom soak screens were carried out at beamlines 1-5 and 9-2 of Stanford Syncrhortron Radiation Laboratory (SSRL) and beamline X8C of National Synchrotron Light Source (NSLS) at Brooldiaven National Laboratory.
  • the Br-MAD data sets used in this study were collected at SSRL and processed using DENZO and SCALEPACK programs of HKL2000 suite (39).
  • the Br " sites were located using SOLVE program (40) and 37 highest peaks (> 6 ⁇ ) were used for phasing the reflections at 2.2 A resolution.
  • the resulting phases were improved by solvent flattening using RESOLVE (41) and the electron density map was calculated using FFT program of CCP4 suite (42).
  • Polypeptides of two ⁇ l chains and one ⁇ 2 chain (chains A-C) were traced using the TOM FRODO graphics program (43).
  • the complete asymmetric unit was generated using non- crystallographic symmetry ("NCS") relations obtained from Br " sites — first the second frimer (chains D-F) was generated to complete one hexamer and then the second hexamer (chains G-L) was generated from the first hexamer.
  • NCS non- crystallographic symmetry
  • the 2.0 A data set collected at 0.8856 A ( ⁇ 4 ) was used for model refinement using CNS program (44) and 5% of the data were set aside for monitoring Rfr ee -
  • the bovine LBM NCI hexamer composed of ⁇ l and ⁇ 2 chains, crystallizes in monoclinic space group R2 ⁇ (A-form) with four hexamers per asymmetric unit. This is different from the crystal forms reported for mouse EHS tumor NCI (49) and human placenta NCI hexamers (50), which crystallized with two hexamers and one hexamer in the asymmetric unit respectively.
  • the intensity statistics of the preliminary diffraction data suggested the presence of pseudo-translation symmetry along the c axis in LBM NCI crystals. An extensive search for heavy atom derivatives using soaking experiments was not successful.
  • the map was fitted with human NCI ⁇ l and ⁇ 2 sequences (Fig. 2) since neither of the bovine sequences is available.
  • Four each of the ⁇ l and ⁇ 2 sequences of other mammalian species are known, which share more than 95% sequence identity among them. More than 95% of the residues of the human sequences fit experimental electron density map. Differences between the human sequences and the map were found for residues Ilel5Thr, Ser22Pro, Prol29Gln in ⁇ l chain and Asp96Glu, Glu97Asp, and Glyl76Ala in ⁇ 2 chain.
  • the sequences are numbered so that the residue after the last Gly-Xaa-Yaa repeat of the collagenous region is counted as the first residue in both ⁇ chains.
  • the 12 chains in two hexamers have been assigned chain IDs A-L in the order of ⁇ l, ⁇ l and ⁇ 2 in each frimer.
  • the map shows disorder for 5-6 residues at N- and two residues at C-termini of all the chains.
  • the final model includes two hexamers, 36 Br " ions, 48 glycerol molecules and 1139 water molecules.
  • the final R-factor and R free of the refinement are 0.168 and 0.197 respectively. More than 90% of the residues are within the most favorable regions in Ramachandran map and Arg76 and Serl48 of the first ⁇ l chain, Serl48 of the second ⁇ l chain and Arg75, Glu95 and Alal45 of ⁇ 2 chain in each frimer lie in the disallowed region. Only a handful of residues are in multiple conformations.
  • the two hexamers in the asymmetric unit are similar with no apparent differences due to crystal contacts.
  • the hexamer comprising chains A-F is used to describe the model
  • the overall structure ofthe hexamer is illustrated in Fig. 3.
  • the two trimers in the hexamer are related by a 2-fold NCS axis at the interface ("equatorial plane") and the monomers within a trimer are related by a pseudo 3-fold symmetry coinciding with the triple helix axis ("polar axis").
  • the two ⁇ l chains in the frimer are identical and the ⁇ 2 chain has a similar overall structure.
  • the C ⁇ atoms of 214 matching residues in one of the ⁇ l chains and the ⁇ 2 chain superimpose with an RMS deviation of 0.9 A.
  • Each chain can be divided into two homologous subdomains, N- and C-subdomains.
  • the two subdomains fold in a similar topology and C ⁇ atoms of 96 matching residues of two subdomains of ⁇ l chain superimpose with an RMS deviation of 1.0 A.
  • the 12 invariant cysteine residues form six disulfides, three in each subdomain, at conserved positions (Fig. 2 and 5).
  • the major difference between the two subdomains occurs at the regions encompassing Pro86- Pro95 in the N-subdomain and Ilel96-Thr209 in the C-subdomain, which are least conserved in the six human sequences (Fig. 1).
  • Each subdomain has two ⁇ -sheets — a three-strand anti-parallel sheet (I & Y) close to the triple helical junction and a six-strand anti-parallel sheet (II & IF) close to the hexamer interface, which consists of the regions of interactions between the two trimers that make up the hexamer (Fig. 4and 5).
  • the ⁇ - sheet I is formed by the three non-contiguous strands ( ⁇ l, ⁇ lO and ⁇ 2) of the sequence belonging to the first half of the polypeptide.
  • ⁇ -sheet II only four strands ( ⁇ 4, ⁇ 3, ⁇ 8, and ⁇ 9) belong to the first half of the sequence and the remaining two sfrands ( ⁇ 6' and ⁇ 7') form a part of the second half of the sequence.
  • a ⁇ -hairpin structure from the second half of the sequence (the "infra-chain domain swapping region", or "Intra-CDSR") swaps into the N-subdomain to form a six-strand ⁇ -sheet.
  • the region in the C-subdomain corresponding to the six-strand ⁇ -sheet in the N-subdomain lacks two strands to fo ⁇ n a similar ⁇ -sheet in the isolated monomer structure.
  • ⁇ 6- ⁇ 7 hairpin in the N- terminal half which corresponds to the ⁇ 6'- ⁇ 7' hairpin in the C-terminal involved in the domain swapping interaction, extends out in the monomer structure.
  • Trimer Organization Two chains of the ⁇ l NCI domain and one chain of the ⁇ 2 NCI domain form the trimer structure with a pseudo 3 -fold molecular symmetry. Since each chain is made up of topologically similar subdomains, there is even a pseudo 6-fold symmetry.
  • the topology diagram ofthe trimer is shown in Fig. 5.
  • the trimer structure is approximately cone-shaped with a base diameter of about 65 A and a hollow core of about 12-14.0 A inner diameter. This is about the same of as the diameter ofthe collagen triple helix, with N-te ⁇ nini of all three chains coming together at the vertex of the cone where the triple helical collagenous domain links with the NCI domain.
  • the trimer is tightly packed tlirough several interchain hydrophobic and hydrogen bonding interactions (Table 2). Residues of five segments in the N-subdomain of one chain make contact with those of seven segments in the C-subdomain of the second chain, and constitute the "monomer-monomer interface", which consists of the regions of monomer-monomer interaction within the trimer. The most important interactions are confined to one N- subdomain segment and two C-subdomain segments (Fig. 1). There are two levels of monomer-monomer interactions, one essential for the "generic trimer” assembly and the other dictating the NCl chain specificity of the monomer-monomer interactions within the trimer.
  • ⁇ ASA interface solvent accessible area
  • M main chain
  • S side chain
  • the hexamer contains two such trimers; the monomer-monomer interfaces in the second trimer are ⁇ lD- ⁇ 2F; ⁇ lE- ⁇ 2F;and ⁇ lD- ⁇ lE.
  • Each of these six-strand ⁇ -sheets is formed by four strands ( ⁇ 4/4 ⁇ ⁇ 3/3', ⁇ 8/8', ⁇ 9/9') in one half of the sequence and the remaining two strands ( ⁇ 6'/6, ⁇ 777) are contributed by the other half of the same chain ( ⁇ 6/ ⁇ l; the Inter-CDSR) or adjacent chain ( ⁇ 6'/ ⁇ l/; the "Infra-chain domain swapping region", or "Intra-CDSR").
  • the amino acid sequences of all the strands with the exception of ⁇ 9, are highly conserved in ⁇ chains within and across the species.
  • the six topologically similar ⁇ -sheets formed in cyclical fashion give the pseudo 6-fold symmetry appearance for the trimer (Fig. 6a).
  • the outermost strand ( ⁇ 9//39') lies on the surface parallel to the equatorial plane of the hexamer interface forming a part of the outer ring and the innermost strand ( ⁇ 4/ ⁇ 4') runs nearly parallel to the polar axis or pseudo 3 -fold axis in the core.
  • the angle between these two strands within each sheet is about 75° giving it a right-handed twist.
  • the ⁇ 4/ ⁇ 4' strands from all the six ⁇ -sheets form a parallel ⁇ barrel-like core of about 14 A diameter even though there are no backbone hydrogen bonds between them (Fig. 6a).
  • ⁇ 4/4' strands have a mixture of hydrophobic and hydrophilic residues, with the former pointing to the core and the latter pointing towards the adjacent strand.
  • the ⁇ 4 strands contain long chain hydrophilic amino acids so that they form more direct hydrogen bonds with the backbone atoms of the ⁇ 4' strand of the neighboring chain indicating stronger interchain interactions.
  • the interactions between ⁇ 4' and ⁇ 4 within a chain are mainly mediated tlirough solvent molecules.
  • the six-strand ⁇ -sheets are essential structural components in the organization ofthe generic trimer structure through 3D domain swapping interactions and the compact ⁇ barrel-like core structure. However, they may play only a limited role in the chain specific assembly of the trimer. Therefore, compounds that target the Intra- CDSR, the hiter-CDSR, and the B4l ⁇ 4' based ⁇ barrel-like core, such as peptides derived from these regions, can be used to inhibit generic monomer-monomer interactions, and thus to inhibit trimer assembly.
  • these additional main chain hydrogen bond interactions between the two chains are found only at the ⁇ lB- ⁇ 2 interface (i.e.: which includes the interaction of the SR of ⁇ l and the SRP of ⁇ 2), but not in ⁇ 2- ⁇ lA (i.e.: which includes the interaction of the SRP of ⁇ l and the SR of 02) or ⁇ lA- ⁇ lB (i.e.: which includes the interaction of the SR of ⁇ l and the SRP of ⁇ l) interfaces, due to the presence of the 3 ⁇ o helical structure in ⁇ l chains rather than the extended structure present in ⁇ 2 chain.
  • a preferred inhibitor of specific trimer assembly would target the SR, which is identical in ⁇ l and ⁇ 2, and thus such an inhibitor would be expected to interfere with interactions at each interface within the monomer-monomer interface, and thus to inhibit trimer assembly. Also preferred would be an inhibitor that targets the 02 SRP, which is required for the additional H-bonding interactions seen at the ⁇ lB- ⁇ 2 interface.
  • Lys56( ⁇ lB) The side chain of Lys56( ⁇ lB) is sandwiched between the backbone of the loop preceding the parallel ⁇ -sheet in ⁇ 2 chain and the contiguous bonds of backbone and side chain of Glnl20( ⁇ 2). In this tightly locked position, Lys56( ⁇ lB) assumes a linear conformation to form two strong hydrogen bonds with the carbonyl of Ilel94( ⁇ 2) and the carboxyl of Aspl21( ⁇ 2), and two more weak interactions with the carbonyls of Glnl20( ⁇ 2) and Glul96( ⁇ 2).
  • the ⁇ l-like (ie: ⁇ l/3/5 family) region corresponding to the parallel ⁇ -sheet of ⁇ 2 chain is the 3 ⁇ o helix, which spans a longer sequence.
  • Lys56( ⁇ lA) is not quite parallel to the backbone bonds, which provides more room for this lysine to adopt a different rotamer conformation to form only weak hydrogen bond with the carbonyl oxygen of Ilel96( ⁇ lB).
  • This may also be influenced by the presence of hydrophobic Thrl24 in ⁇ l chains in place of hydrophilic Aspl21 in ⁇ 2.
  • Arg55( ⁇ 2) is docked in similar position as Lys56 of ⁇ l chains in other two interfaces with one sfrong hydrogen bond interaction with carbonyl of Ilel96( ⁇ lA).
  • Arg55/Ala54 and Gly98/Glu95 make differences in hydrogen bonding patterns at the interfaces.
  • the Arg55( ⁇ 2)/Lys56(ol) is an important residue for optimal ⁇ l- OZmonomer-monomer interactions, and compounds targeting this region, such as peptides including LRKF (SEQ ID NO:294) ( ⁇ l) or LARF (SEQ ID NO:295) ( ⁇ 2), can be used to inhibit the assembly of specific monomer-monomer interactions. Since this region precedes the SR, this region can be combined with the SR to form a longer peptide that will interfere with multiple aspects of specific monomer-monomer interactions, and thus be even more effective at inhibiting trimer assembly.
  • the regions Ilel94-Glul96 ( ⁇ 2), Ilel96 ( ⁇ l) and Glnl20-Aspl21( ⁇ 2) also are involved in optimal ⁇ l- ⁇ 2monomer-monomer interactions, and compounds targeting these region, such as peptides including IPE (SEQ ID NO:294) (02 184-196), IER (SEQ ID NO:295) ( ⁇ l 196-198) or QD (SEQ ID NO:296) (o2 120-121), can be used to inhibit the assembly of specific monomer-monomer interactions, and thus to inhibit trimer assembly.
  • IPE SEQ ID NO:294
  • IER SEQ ID NO:295
  • QD SEQ ID NO:296
  • the ⁇ lB- ⁇ 2 interface (i.e.: which includes the interaction ofthe SR of ⁇ l and the SRP of ⁇ 2) has the maximum number of contact residues, the highest proportion of hydrophilic atoms, and contains more hydrogen bonds than the other monomer-monomer interfaces (Table 2).
  • the buried surface area is largest for ⁇ lA- ⁇ lB interface (i.e.: which includes the interaction of the SR of ⁇ l and the SRP of ⁇ l). From these observations, it is evident that the ⁇ lB- ⁇ 2 interface is formed predominantly through hydrogen bonding interactions and the ⁇ lA- ⁇ lB interface is stabilized by more hydrophobic forces.
  • packing considerations may also play an important role in determining chain stochiometry in the trimer.
  • the ⁇ l and ⁇ 2 NCI chains fold in a similar tertiary structure with a low RMS deviation, the relative orientation of the two subdomains in each NCI chain is different near the triple helical junction.
  • the region encompassing Thrl3-Tyr30 of the N- subdomain in the ⁇ 2 chain is farther from its equivalent region Aspl21-Tyrl38 ofthe C- subdomain in the ⁇ 2 chain compared to the relative orientations of similar regions in the ⁇ l structure.
  • peptides designed to interfere with monomer-monomer interactions are preferably delivered into the cell, where such monomer-monomer assembly occurs.
  • the peptides can be used to disrupt assembled trimers that have been secreted by the cell.
  • Hexamer Assembly The type IV collagen trimer, once formed in the endoplasmic lumen, is secreted into the extracellular space where it assembles into the hexamer, and then into a supramolecular network through N- and C-terminal associations.
  • the NCI domains play the dominant role in this assembly, by determining the C-terminal dimeric association, leading to hexamer assembly.
  • the foot-ball shaped hexamer is made up of two identical trimers, each containing two ⁇ l chains and one ⁇ 2 chain as described in the previous section.
  • Each protomer ie: the complete type IV collagen trimer, including NCI domains
  • Each protomer formed by the tightly intertwined trimer is considered as a single entity so that the hexamer can be analyzed relative to other homodimeric protein complexes (43).
  • Gap Index 1.24 2.2 Percentage of polar and non-polar atoms are 45.5 and 54.5 respectively.
  • the two ⁇ C1 trimers are related by a 2-fold ⁇ CS axis in lying the equatorial plane and perpendicular to the pseduo 3-fold axis of symmetry within an individual trimer (Fig. 4).
  • This symmetry constraint may be partly influenced by a few differences in the interface residues of ⁇ l like and ⁇ 2 like sequences in addition to more efficient packing.
  • the hexamer interface is formed by the nearly flat surfaces of the two trimers, with an RMS deviation of 1.9 A for all the hexamer interface atoms from the mean plane (Fig. 9a). This is significantly lower than the average planarity value of 3.5 A for 32 homodimers discussed in a recent review (43).
  • the hexamer interface formed by six segments each ofthe three monomers, with a total of 109 residues per trimer, is nearly circular, with the major and minor axial lengths of the mean plane measuring approximately 69 and 61 A respectively.
  • This flat circular hexamer interface covers about 4400 A 2 of solvent accessible area per trimer, which correlates with the observation of larger molecules having larger interfaces (54).
  • Such a large interface facilitates strong interaction between the trimers, involving both hydrophobic and hydrophilic residues.
  • the polar (45.5%) and non-polar atoms (54.5%) in the hexamer interface are nearly in equal proportions, underscoring the importance of both types of interactions in hexamer stabilization. The discussion thus far focused on the overall nature of the hexamer interface.
  • each monomer of one trimer makes contact with two monomers of the other trimer, designated as the "major” and “minor” contacts based on the extent of the contact area and number of hydrogen bonds.
  • the two monomers making major contact is referred to as "dimer” in a similar sense as the term used in the denaturation experiments of hexamers (55).
  • the 2-fold NCS between the two trimers results in only one "homodimer” formed by two ⁇ l chains ( Figure 7A), with the remaining two “heterodimers” formed by ⁇ l and ⁇ 2 chains ( Figure 7A-B).
  • the two 6-strand ⁇ -sheets, II and IF, formed by the 3D domain swapping interactions play as crucial role in the formation of hexamer assembly as in the case of trimer organization.
  • the hexamer interface is populated with ⁇ - turns connecting ⁇ 3-/34 and ⁇ 3'- ⁇ 4' in the core. These turns along with the remaining sfrands of the ⁇ -sheets 11/11' position a large number of conserved residues for extensive hydrogen bonding interactions at the hexamer interface.
  • the core ⁇ -turns (two per monomer contributed by the two equivalent subdomains) in the two trimers pack in staggered configuration such that each turn in one trimer contacts with two turns in the other trimer.
  • the turns in the N-subdomains are of type I'/III' containing hydrophilic amino acids in the second (Asn39/Gln38) and third positions (Glu40/39).
  • the C- subdomain turns are of type II in ⁇ l chains and type IF in ⁇ 2 chains with small hydrophobic amino acids, Alal49/146-Glyl50/147-Alal51/As ⁇ l48, with Alal49 ⁇ l or Aspl48 of ⁇ 2 introducing a ⁇ -bulge.
  • the hydrophilic side chains of turns in the N- subdomain participate in hydrogen bonds and hydrophobic residues of turns in C- subdomain pack through hydrophobic interaction as well as stacking interaction of peptide planes (Fig. 7A).
  • the conserved Glu40(39) penetrates between the N- and C-subdomains of a monomer chain in the other trimer to form a hydrogen bond with the side chain of the conserved Gln37(36).
  • the Glu40 residues in the ⁇ l- ⁇ l dimer form a strong hydrogen bond with each other that is missing in ⁇ l- ⁇ 2 dimers.
  • the packing of the turns and side chains appear to be tight at the core interface in CPK models indicating strong van der Waals interactions in additions to the obvious hydrogen bonding interactions.
  • compounds that target the core regions of major contact at the hexamer interface can be used to inhibit hexamer assembly.
  • peptides derived from these regions can be used to inhibit hexamer assembly at the core region of major contact.
  • peptides including the /33-/34 connecting region or the /33'-/34' connecting region can be used to inhibit hexamer assembly at the core region of major contact.
  • peptides derived from these regions can be used to inhibit hexamer assembly.
  • peptides including the sequence ASRND (SEQ ID NO:201) ( ⁇ l) or YYANA (SEQ ID NO:218) ( ⁇ l), or the corresponding sequences in the other alpha chains can be used to inhibit hexamer assembly at the outer region of major contact.
  • Major-minor junction is the area of the hexamer interface where two chains from one trimer contact two chains ofthe other trimer. There are two types of junctions, one involving three ⁇ l and one ⁇ 2 chains, and the other involving two each of ⁇ l and ⁇ 2 chains. The hydrogen bonding pattern in the two junctions is highly conserved ( Figure 7C). Both ⁇ l- ⁇ l and ⁇ 2- ⁇ 2 form a Asnl87(185)- Tyrl89(188) (NYY) (SEQ ID NO:297) hydrogen bond pairs in the interface.
  • Asnl87(185) forms a pair of hydrogen bonds with Arg76(75) of another chain (within the outer region of major contact discussed above) from the opposite trimer.
  • the multiple hydrogen bonds formed by Asnl87(185) involving residues from two different chains is probably one of the major factors stabilizing the trimer-trimer interface. Therefore, compounds that target major-minor junction at the hexamer interface, such as peptides derived from these regions, can be used to inhibit hexamer assembly.
  • peptides including the sequence NYY (SEQ ID NO:288) ( ⁇ l) (such as ECHGRGTCNYY (SEQ ID NO: 172)), or corresponding sequences in the other ⁇ chains, all of which is present at the hexamer interface (and which includes a large portion of the Intra-CDSR), or ASRND (SEQ ID NO:201) ( ⁇ l (which includes the ARG76(75) residue), or corresponding sequences in the other ⁇ chains, can be used to inhibit hexamer assembly at the major-minor junction.
  • peptides containing the sequence ASRND can interfere with hexamer assembly by interfering with interactions at both the outer region of major contact and the major-minor junction.
  • peptides that target the Intra-CDSR and extend to contain the 2 additional Y residues from the sequence "NYY” can be used to inhibit trimer assembly, as well as hexamer assembly.
  • residues that are located at the hexamer interface, and that are believed to be important for hexamer assembly include (1) MSMAP (SEQ ID NO: 129) (residues 91-95 ⁇ l)/MMP (SEQ ID NO: 132) (02), and corresponding sequences in the other o chains; (2) PSTLK (SEQ ID NO:177) (residues 208-212 in ⁇ l; /39'strand; ADTLK in 02 (SEQ ID NO: 180)), and corresponding sequences in the other ⁇ chains; (3) FCNINNNCNFA (SEQ ID NO:289) ( ⁇ l AND ⁇ 5-co-extensive with the hiter-CDSR), and corresponding sequences in the other ⁇ chains:
  • peptides containing these sequences, or portions thereof, can be used to inhibit hexamer assembly.
  • Disulfide cross-linking is a recurring theme in collagen assembly and is believed to play an important role in the stabilization of the trimeric structure (11). Fibrillar procollagens are believed to form interchain disulfide bonds catalyzed by protein disulfide isomerase in either the C-telopeptide or C-propeptide (56, Kiovu, 1987 #343). Interchain disulfides have been proposed to form both in the collagenous and NCI domains of type IV collagen. Whereas the interchain disulfides in the collagenous domains are formed within a protomer to stabilize the collagen triple helix, those in the NCI domains are believed to occur between the protomers to stabilize the network at the C-terminus.
  • Disulfide exchange between NCI domains of similar ⁇ chains from two different protomers was proposed as one of the major stabilizing forces in the hexamer assembly (57).
  • the human placenta derived NCI hexamer dissociated as dimers and monomers.
  • the dimers were shown to be crosslinked predominantly by disulfide bridges.
  • Langeveld et al (55) comparing the NCI hexmers isolated from several BMs revealed rather complex results.
  • the disulfides in the NCI monomer are arranged in three tiers with Cys20-Cysl l l and Cysl30-Cys225 are close to the triple helical junction, Cys65-Cys71 and Cysl76-Cysl82 are close to the interface and Cys53- Cysl08 and Cysl64-Cys222 lies in between.
  • the disulfide pairs Cys20-Cyslll and Cys53-Cysl08 in the monomers of ⁇ lA- ⁇ lD dimer are about 70 A and 50 A apart respectively.
  • disulfide exchange if any, exists only for the Cys65- Cys71 and Cysl76-Cysl82 pairs.
  • non-collagenous domains There is very little crystallo graphic data available on non-collagenous domains.
  • the only available structures of non-collagenous domains are those of endostatins (58,59), which are homologous fragments of single chains from types XNIII and XN collagens.
  • the present work provides the first unambiguous structural basis for the chain stochiometry of the type IV collagen ⁇ l. ⁇ 2 network, as well as the structural basis for chain specific assembly of type IV collagen.
  • the ⁇ C1 monomer folds into a novel tertiary structure and the close ended-trimer of ( ⁇ l) 2 . ⁇ 2 is organized through unique 3D domain swapping interactions. These features must be conserved in all type IV collagen networks, from all species, due to overall sequence similarity and very high sequence identity of the regions participating in domain swapping.
  • the chain specificity is determined by the differences in the primary sequences of the hypervariable regions of the ⁇ C1 domains of the constituent chains, which manifest as different secondary structures at the monomer-monomer interfaces.
  • the hexamer structure is stabilized by the extensive hydrophobic and hydrophilic interactions at the trimer-trimer interface without a need for disulfide cross-linldng.
  • the crystal structure of LBM ⁇ C1 hexamer and the denaturation studies of ⁇ C1 hexamers from several BMs suggest an alternative conformation must exist in hexamers that are cross-linked by interchain disulfides. Some hitherto unknown enzymatic process might be responsible for folding the same amino acid sequences into different conformations in different tissues.
  • polypeptide synthesis of the polypeptides described below was carried out according to standard procedures. Polypeptide samples were aliquoted and stored frozen at -20°C. They were only thawed once prior to individual experiments to prepare the working dilution of each substance. In order to analyze the activity of the compounds in inhibiting angiogenesis, a Mafrigel-based endothelial cell alignment assay was used. Briefly, suspended human umbilical vein endothelial cells (HUVEC, purchased from Promocell, Germany) were seeded on top of polymerized Matrigel (BD Biocoat Matrigel Matrix 24-well plates; BD Biosciences, Cat. No.
  • Angiogenesis We next tested the effects of systemic administration of soluble peptides in the chick embryo CAM angiogenesis assay. Angiogenesis was induced in the CAMs of 10 day old chick embryos with bFGF as described (Brooks et al, Cell 92:391- 400 (1998)). Twenty four hours later the embryos were systemically treated with 30 micrograms per embryo (with the exception of experiment 9, in which the dose was 20 micrograms per embryo) of peptide in a total volume of 100 ⁇ l of sterile phosphate buffered saline (PBS). Two days later the embryos were sacrificed and the filter discs and CAM tissues removed.
  • PBS sterile phosphate buffered saline
  • Angiogenesis was quantitated by counting the number of angigogenic blood vessel branch points in the confined area of the filter disc.
  • the Angiogenic hidex is defined as the number of branch points from experimental treatment minus control treatment (The data is normalized by subtracting out number of branch points in non-bFGF-treated CAM (NT).) "Mean” is the number of branch points; “SD” is the Standard deviation; and “N” is Number of data points.
  • Tumor growth We then analyzed tumor growth in the CAM assay. Using Peptides 4, 10, and 11, we showed that peptide 4 strongly inhibited tumor growth in this model, peptide 11 inhibited tumor growth to a lesser extent relative to untreated controls, and peptide 10 showed minimal effect on tumor growth relative to untreated controls

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Abstract

La présente invention concerne un hexamère à domaine NC1 cristallisé de collagène de type IV, ainsi que des procédés de fabrication de ce cristal. Selon l'invention, l'hexamère à domaine NC1 est cristallisé de manière que sa structure tridimensionnelle puisse être déterminée avec une résolution d'au moins 3 Å. L'invention concerne également un procédé d'élaboration de composés destinés à inhiber l'angiogenèse, la croissance tumorale, les métastases tumoraux, l'adhésion et/ou la prolifération de cellules endothéliales, et/ou des ensembles de lame ventrale. Ledit procédé consiste à analyser la structure tridimensionnelle d'un hexamère à domaine NC1 cristallisé de collagène de type IV obtenu au moyen du procédé selon l'invention, et à identifier et synthétiser des composés ciblant des régions du domaine NC1 dont l'importance pour l'ensemble hétérotrimère et hexamère de collagène de type IV a été identifiée par l'analyse. L'invention concerne par ailleurs des polypeptides élaborés au moyen des procédés selon l'invention d'élaboration rationnelle de substances thérapeutiques, sur la base de l'analyse de la structure hexamère à domaine NC1 de collagène de type IV.
PCT/US2004/002187 2003-01-27 2004-01-27 Structure cristallisee hexamere a domaine nc1 de collagene de type iv WO2004067762A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7122517B2 (en) 2001-07-27 2006-10-17 Kansas University Medical Center Crystallized structure of type IV collagen NC1 domain hexamer
EP2649095A2 (fr) * 2010-12-10 2013-10-16 Aleksander S. Popel Peptides mimétiques dérivés de collagène de type iv et leur utilisation pour traiter des maladies dépendantes de l'angiogenèse et de la lymphangiogenèse

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5593900A (en) * 1989-07-07 1997-01-14 Tryggvason; Karl Immunological methods for the detection of the human type IV collagen α5 chain
US5856184A (en) * 1994-06-30 1999-01-05 Yale University Isolated domains of type IV collagen to modify cell and tissue interactions
WO2001051523A2 (fr) * 2000-01-07 2001-07-19 Beth Israel Deaconess Medical Center Proteines et fragments de proteines anti-angiogeniques et leurs procede d'utilisation
US6277558B1 (en) * 1990-11-30 2001-08-21 Kansas University Medical Center α-3 chain type IV collagen polynucleotides
US6361994B1 (en) * 1995-06-30 2002-03-26 University Of Kansas Medical Center Method for inhibiting angiogenesis and tumors with the isolated NC1 α1 chain monomer of type IV collagen
WO2003012122A2 (fr) * 2001-07-27 2003-02-13 University Of Kansas Medical Center Structure cristallisee de l'hexamere du domaine nc1 du collagene de type iv

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5593900A (en) * 1989-07-07 1997-01-14 Tryggvason; Karl Immunological methods for the detection of the human type IV collagen α5 chain
US6277558B1 (en) * 1990-11-30 2001-08-21 Kansas University Medical Center α-3 chain type IV collagen polynucleotides
US5856184A (en) * 1994-06-30 1999-01-05 Yale University Isolated domains of type IV collagen to modify cell and tissue interactions
US6361994B1 (en) * 1995-06-30 2002-03-26 University Of Kansas Medical Center Method for inhibiting angiogenesis and tumors with the isolated NC1 α1 chain monomer of type IV collagen
WO2001051523A2 (fr) * 2000-01-07 2001-07-19 Beth Israel Deaconess Medical Center Proteines et fragments de proteines anti-angiogeniques et leurs procede d'utilisation
WO2003012122A2 (fr) * 2001-07-27 2003-02-13 University Of Kansas Medical Center Structure cristallisee de l'hexamere du domaine nc1 du collagene de type iv

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
COLORADO P C ET AL: "ANTI-ANGIOGENIC CUES FROM VASCULAR BASEMENT MEMBRANE COLLAGEN" CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, BALTIMORE, MD, US, vol. 60, no. 9, 1 May 2000 (2000-05-01), pages 2520-2526, XP001019102 ISSN: 0008-5472 *
MAESHIMA YOHEI ET AL: "Distinct antitumor properties of a type IV collagen domain derived from basement membrane" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 275, no. 28, 14 July 2000 (2000-07-14), pages 21340-21348, XP002204077 ISSN: 0021-9258 *
MAESHIMA YOHEI ET AL: "Extracellular matrix-derived peptide binds to alphavbeta3 integrin and inhibits angiogenesis" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 276, no. 34, 24 August 2001 (2001-08-24), pages 31959-31968, XP002296029 ISSN: 0021-9258 *
PETITCLERC E ET AL: "NEW FUNCTION FOR NON- COLLAGENOUS DOMAINS OF HUMAN COLLAGEN" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 275, no. 11, 17 March 2000 (2000-03-17), pages 8051-8061, XP002144125 ISSN: 0021-9258 *
SUNDARAMOORTHY MUNIRATHINAM ET AL: "Crystal structure of NC1 domains. Structural basis for type IV collagen assembly in basement membranes" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 277, no. 34, 23 August 2002 (2002-08-23), pages 31142-31153, XP002296030 ISSN: 0021-9258 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7122517B2 (en) 2001-07-27 2006-10-17 Kansas University Medical Center Crystallized structure of type IV collagen NC1 domain hexamer
EP2649095A2 (fr) * 2010-12-10 2013-10-16 Aleksander S. Popel Peptides mimétiques dérivés de collagène de type iv et leur utilisation pour traiter des maladies dépendantes de l'angiogenèse et de la lymphangiogenèse
EP2649095A4 (fr) * 2010-12-10 2014-05-07 Aleksander S Popel Peptides mimétiques dérivés de collagène de type iv et leur utilisation pour traiter des maladies dépendantes de l'angiogenèse et de la lymphangiogenèse
US9056923B2 (en) 2010-12-10 2015-06-16 The Johns Hopkins University Mimetic peptides derived from collagen type IV and their use for treating angiogenesis- and lymphagiogenesis-dependent diseases
US10106597B2 (en) 2010-12-10 2018-10-23 The Johns Hopkins University Mimetic peptides derived from collagen type IV and their use for treating angiogenesis- and lymphangiogenesis-dependent diseases
US10774131B2 (en) 2010-12-10 2020-09-15 The Johns Hopkins University Mimetic peptides derived from collagen type IV and their use for treating angiogenesis- and lymphangiogenesis-dependent diseases
US11155603B2 (en) 2010-12-10 2021-10-26 The Johns Hopkins University Mimetic peptides derived from collagen type IV and their use for treating angiogenesis- and lymphangiogenesis- dependent diseases

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