Crystallization of IGF-1
First Claim
1. A crystal formed by IGF-1 that diffracts x-ray radiation to produce a diffraction pattern representing the three-dimensional structure of the IGF-1.
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Abstract
Crystalline IGF-1 is provided along with a method for production thereof. Crystallizing IGF-1 comprises the steps of mixing an aqueous solution comprising IGF-1 with a reservoir solution comprising a precipitant to form a mixture; and crystallizing the mixture, optionally also recrystallizing and isolating the crystalline IGF-1. In addition, a method for identifying IGF-1 indirect agonists is provided using a detergent as a standard for the level of inhibition of binding of IGFBP-1 or IGFBP-3 to IGF-1 and/or using the coordinates of the binding pockets of IGF-1 to which a candidate indirect agonist binds for structure-based drug design.
53 Citations
50 Claims
- 1. A crystal formed by IGF-1 that diffracts x-ray radiation to produce a diffraction pattern representing the three-dimensional structure of the IGF-1.
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9. A method of crystallizing IGF-1 comprising the steps of:
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(a) mixing an aqueous solution comprising IGF-1 with a reservoir solution comprising a precipitant to form a mixed volume; and
(b) crystallizing the mixed volume. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30)
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28. A method of identifying indirect agonists of IGF-1 comprising the steps of:
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(a) comparing the ability of N,N-bis(3-D-gluconamidopropyl)-deoxycholamine to inhibit binding of IGFBP-1 or -3 to IGF-1 with the ability of a candidate indirect agonist of IGF-1 to so inhibit binding; and
(b) determining whether the candidate agonist inhibits such binding at least as well as N,N-bis(3-D-gluconamidopropyl)-deoxycholamine.
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- 31. A method of identifying indirect agonists of IGF-1 comprising co-crystallizing a candidate indirect agonist of IGF-1 with IGF-1 to form a co-crystalline structure and determining if the candidate agonist binds to one or both of two patches on IGF-1, wherein one patch has the amino acid residues Glu 3, Thr 4, Leu 5, Asp 12, Ala 13, Phe 16, Val 17, Cys 47, Ser 51, Cys 52, Asp 53, Leu 54, and Leu 57, and the second patch has the amino acid residues Val 11, Gln 15, Phe 23, Phe 25, Asn 26, Val 44, Phe 49, and Arg 55, and wherein binding occurs if there is at least one contact between each listed amino acid residue of a given patch and the candidate agonist that is less than or equal to 6 angstroms in the co-crystalline structure.
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35. A co-crystalline complex of IGF-1 and N,N-bis(3-D-gluconamidopropyl)-deoxycholamine.
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36. A method for determining a three-dimensional structure of IGF-1 comprising:
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(a) crystallizing the IGF-1;
(b) irradiating the crystalline IGF-1 to obtain a diffraction pattern characteristic of the crystalline IGF-1; and
(c) transforming the diffraction pattern into the three-dimensional structure of the IGF-1.
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37. A machine-readable data storage medium comprising a data storage material encoded with machine-readable data that, when read by an appropriate machine, displays a three-dimensional representation of a crystal of a molecule comprising IGF-1.
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38. An IGF-1 crystal with the structural coordinates shown in Appendix 1.
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39. A method of using a three-dimensional structure of IGF-1 derived from an IGF-1 crystal wherein the three-dimensional structure of IGF-1 includes an IGF-1 receptor-binding region, the method comprising identifying compounds having structures that interact with the receptor-binding region of the three-dimensional structure of IGF-1 and function as an IGF-1 agonist or antagonist.
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41. A method of identifying IGF-1 agonists or antagonists comprising the steps of:
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(a) crystallizing IGF-1 to form IGF-1 crystals, the IGF-1 crystals containing a group of amino acid residues defining an IGF-1 receptor-binding region;
(b) irradiating the IGF-1 crystals from step (a) to obtain a diffraction pattern of the IGF-1 crystals;
(c) determining a three-dimensional structure of IGF-1 from the diffraction pattern, the structure including an IGF-1 receptor-binding region; and
(d) identifying an IGF-1 agonist or antagonist having a three-dimensional structure that functionally duplicates essential IGF receptor-binding, solvent-accessible residues presenting the three-dimensional structure of the IGF-1 receptor-binding region, said IGF-1 agonist or antagonist having altered signal transduction capacity to IGF-1-responsive cells, as compared to IGF-1. - View Dependent Claims (42)
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43. A method of designing a compound that mimics the 3-dimensional surface structure of IGF-1 comprising the steps of:
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(a) determining the 3-dimensional structure of the IGF-1; and
(b) designing a compound that mimics the 3-dimensional surface structure of the IGF-1.
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44. A method for identifying a peptidomimetic that binds IGF-1 and blocks binding of an IGFBP or a receptor that binds to IGF-1 comprising the steps of:
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(a) searching a molecular structure database with the structural parameters or structural coordinates provided in Appendix 1; and
(b) selecting a molecule from the database that mimics the structural parameters or structural coordinates of the IGF-1. - View Dependent Claims (46, 48)
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45. A method for determining at least a portion of a three-dimensional structure of a molecular complex, said complex comprising IGF-1 and said method comprising the steps of:
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(a) determining the structural coordinates of a crystal of IGF-1;
(b) calculating phases from the structural coordinates;
(c) calculating an electron density map from the phases obtained in step (b); and
(d) determining the structure of at least a portion of the complex based on said electron density map.
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47. A method for evaluating the ability of a chemical entity to associate with IGF-1 or a complex thereof, the method comprising the steps of:
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(a) employing computational or experimental means to perform a fitting operation between the chemical entity and the IGF-1 or complex thereof, thereby obtaining data related to the association; and
(b) analyzing the data obtained in step (a) to determine the characteristics of the association between the chemical entity and the IGF-1 or complex thereof.
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49. A heavy-atom derivative of a crystallized form of IGF-1.
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50. A method of computationally or experimentally evaluating a chemical entity to obtain information about its association with a binding site of IGF-1 using a crystal of IGF-1 having the structural coordinates described in Appendix 1.
Specification