Mutants of igf binding proteins and methods of production of antagonists thereof

A method for the determination of the atomic coordinates of a crystal suitable for X-ray diffraction obtained by

• (a) contacting IGF with a polypeptide consisting of the amino acids 39-91 of IGFBP-1, the amino acids 55-107 of IGFBP-2, the amino acids 47-99 of IGFBP-3, the amino acids 39-91 of IGFBP-4, the amino acids 40-92 of IGFBP-5, or the amino acids 40-92 of IGFBP-6 or a fragment thereof consisting at least of the 9th to 12th cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7th to 10th cysteine of IGFBP-6, to form a complex which exhibits restricted conformation mobility;
• and
• (b) obtaining a crystal from the complex so formed suitable for X-ray diffraction;
• (c) determining the atomic coordinates of said crystal.

A method for identifying a mutant of IGFBP (IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5 or IGFBP-6 or a mutant of a fragment thereof consisting at least of the 9th to 12th cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7th to 10th cysteine of IGFBP-6) having an enhanced binding affinity for IGF, comprising

• (a) constructing a three-dimensional structure of the complex of IGF and a polypeptide consisting of the amino acids 39-91 of IGFBP-1, the amino acids 55-107 of IGFBP-2, the amino acids 47-99 of IGFBP-3, the amino acids 39-91 of IGFBP4, the amino acids 40-92 of IGFBP-5, or the amino acids 40-92 of IGFBP-6 consisting at least of the 9th to 12th cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7th to 10th cysteine of IGFBP-6, based on the atomic coordinates of a crystal consisting of IGFI and said IGFBP or a fragment thereof;
• (b) employing said three-dimensional structure and modeling methods to identify said mutant of an IGFBP in which a residue within a distance of 5 Å to a hydrophobic amino acid residue of IGF is modified in that the hydrophobic interaction between IGF and said mutant of IGFBP is enhanced;
• (c) producing said mutant;
• (d) assaying said mutant to determine said enhanced binding affinity for IGF.

A method for identifying a mutant of IGFBP-5 with enhanced binding affinity for IGF-1, said method comprising

• (a) constructing a three-dimensional structure of the complex of IGF and IGFBP-5 defined by the atomic coordinates shown in FIGS. 5 and 6;
• (b) employing said three-dimensional structure and modeling methods to identify an amino acid residue in IGFBP-5 within a distance of 5 Å or shorter to an amino acid residue of IGFI, wherein said residue of IGFBP-5 can be modified hydrophobically in that the hydrophobic interaction between IGF and IGFBP-5 is enhanced;
• (c) producing said mutant;
• (d) assaying said mutant to determine said enhanced binding affinity for IGF.

A method for identifying a non-proteinaceous compound capable of binding to IGFBP, comprising

• (a) constructing a three-dimensional structure of a complex of insulin-like growth factor 1 or 11 and a polypeptide consisting of the amino acids 40-92 of insulin-like growth factor binding protein 5, amino acids 39-91 of IGFBP-1, amino acids 55-107 of IGFBP-2, amino acids 47-99 of IGFBP-3, amino acids 39-91 of IGFBP-4, amino acids 40-92 of IGFBP-5, amino acids 40-92 of IGFBP-6 or a fragment thereof consisting at least of the 9th to 12th cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7th to 10th cysteine of IGFBP-6, based on the atomic coordinates of a crystal consisting of IGF-I and said IGFBP;
• (b) employing said three-dimensional structure and modeling methods to identify a non-proteinaceous compound forming a complex with said IGFBP by hydrophobic binding with amino acids 49, 50, 70, 71 and 74 in the case of IGFBP-5 and in the case of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4 and IGFBP-6 with the corresponding amino acids according to Table 7;
• (c) producing said compound;
• (d) determining the binding between the compound and said IGFBP.