Crystallization of IGF-1

• US 7,596,455 B2
• Filed: 06/20/2006
• Issued: 09/29/2009
• Est. Priority Date: 02/09/2001
• Status: Expired due to Fees

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Ultsch, Mark, Schaffer, Michelle, Vajdos, Felix
• Primary Examiner(s): Noakes; Suzanne M.
• Assistant Examiner(s): Lee; Jae W
• Application Number:
  US11/471,896
• Publication Number:
  US 20060293507A1
• Time in Patent Office: 1,197 Days
• Field of Search: None
• US Class Current: 702/27
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/65 Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
• CPC Class Codes: C07K 2299/00 Coordinates from 3D structures of peptides, e.g. proteins or enzymes

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.

Claims

1. A method of designing a compound that has a 3-dimensional structure that mimics the 3-dimensional surface structure of human IGF-1 as set forth in SEQ ID NO:

1 comprising the steps of;

(a) determining the 3-dimensional structure of the human IGF-1 (SEQ ID NO;

     1) by X-ray diffraction of an orthorhombic crystal of said human IGF-1 that has approximately the following cell constants a=31.831 Å

, b=71.055 Å

, c=65.995 Å

, and a space group of C222₁, and α

=β

=γ

=90°

; and

(b) designing a compound that has a 3-dimensional structure that mimics the 3-dimensional surface structure of the human IGF-1.

2. A method for identifying a compound that binds human IGF-1 as set forth in SEQ ID NO:

1 and blocks binding of an IGF-binding protein (IGFBP) or a receptor that binds to said human IGF-1 comprising the steps of;

(a) determining the structural coordinates of a crystal of the human IGF-1 as set forth in SEQ ID NO;

1 by X-ray diffraction of an orthorhombic crystal of said human IGF-1 that has approximately the following cell constants a=31.831 Å

, b=71.055 Å

, c=65.995 Å

, and a space group of C222₁, and α

=β

=γ

=90°

;

(b) searching a molecular structure database with the structural coordinates determined in step (a); and

(c) selecting a compound from the molecular structure database that has a 3-dimensional structure that associates, in a fitting operation, with the 3-dimensional structure of human IGF-1 as generated by the structural coordinates of the human IGF-1, wherein the compound so identified binds to human IGF-1 and blocks binding of an IGFBP or a receptor that binds to human IGF-1 is identified.

3. A method for determining at least a portion of a 3-dimensional structure of a molecular complex, said complex comprising human IGF-1 as set forth in SEQ ID NO:

1 and said method comprising the steps of;

(a) determining the phases and structural coordinates from a crystal of the human IGF-1 as set forth in SEQ ID NO;

1 by X-ray diffraction of an orthorhombic crystal of said human IGF-1 that has approximately the following cell constants a=31.831 Å

, b=71.055 Å

, c=65.995 Å

, and a space group of C222₁, and α

=β

=γ

=90°

, wherein the structural coordinates are set forth in Appendix I;

(b) combining the phases as determined in step (a) with data collected from a different human IGF-1 molecular complex than that set froth in Appendix I to calculate a new electron density map containing said human IGF-1 complex; and

(c) determining the 3-dimensional structure/structural coordinates of at least a portion of the complex based on said electron density map.

4. A method for evaluating the ability of a chemical entity to associate with human IGF-1 as set forth in SEQ ID NO:

1, the method comprising the steps of;

(a) performing a fitting operation between a three-dimensional model defined by the structural coordinates of a chemical entity and a three-dimensional structure of defined by the structural coordinates of the human IGF-1 as set forth in SEQ ID NO;

1, wherein said 3-dimensional structure of the human IGF-1 is determined by X-ray diffraction of an orthorhombic crystal of said human IGF-1 that has approximately the following cell constants a=31.831 Å

, b=71.055 Å

, c=65.995 Å

, and a space group of C222₁ , and α

=β

=γ

=90°

, thereby obtaining data related to the association;

(b) analyzing the data obtained in step (a) to determine the characteristics of the association between the chemical entity and the human IGF-1 and

(c) determining the efficiency with which said chemical entity associates with IGF-1 by computational and experimental evaluation.

5. A method of designing a compound that mimics the 3-dimensional surface structure defined by the structural coordinates determined from crystalline human IGF-1 as set forth in SEQ ID NO:

1 comprising;

(a) designing a compound that mimics the 3-dimensional surface structure of an orthorhombic crystal of human IGF-1 as set forth in SEQ ID NO;

1 by defining a solvent-accessible surface of the IGF-1 and identifying compounds having structural coordinates that position appropriate functional groups in 3-dimensional structural arrangements similar to said solvent accessible surface, wherein the structural coordinates of the 3-dimensional surface structure of an orthorhombic crystal of human IGF-1 as set forth in SEQ ID NO;

1 are determined from the crystalline human IGF-1 by X-ray diffraction analysis, and have approximately the following cell constants;

a=31.831 Å

, b=71.055 Å

, c=65.995 Å

, and a space group of C222₁, and α

=β

=γ

=90°

;

and

(b) determining the efficiency with which said compound associates with N, N-bis-(3-D-gluconamidopropyl)-deoxycholamine by computational and experimental evaluation.

6. The method of claim 5, wherein step (a) or step (b) comprises computational evaluation of the interaction of said compound with N, N-bis-(3-D-gluconam idopropyl)-deoxycholamine.

7. The method of claim 5, wherein step (b) comprises experimental evaluation of the interaction of said compound with N, N-bis-(3-D-gluconamidopropyl)-deoxycholamine.

8. A method for identifying a compound that binds human IGF-1 as set forth in SEQ ID NO:

1 and blocks binding of an IGF-binding protein (IGFBP) or a receptor that binds to said human IGF-1 comprising the steps of;

(a) searching a molecular structure database for a compound that has a 3-dimensional structure that associates, in a fitting operation, with the 3-dimensional structure of the human IGF-1 having the structural coordinates determined by X-ray diffraction of an orthorhombic crystal of human IGF-1 as set forth in SEQ ID NO;

1 having approximately the following X-ray diffraction cell constants;

a=31.831 Å

, b=71.055 Å

, c=65.995 Å

, and a space group of C222₁, and α

=β

=γ

=90°

;

(b) selecting a compound from the molecular structure database that has a 3-dimensional structure that associates, in a fitting operation, with the 3-dimensional structure of said human IGF-1 as set forth in step (a); and

(c) determining the efficiency with which said compound binds the human IGF-1 as set forth in SEQ ID NO;

1, or blocks binding of the IGF-binding protein (IGFBP) or the receptor that binds to said human IGF-1 by computational and experimental evaluation.