Methods of identifying inhibitors of FGF23 binding to the binary FGFR-klotho complex for the treatment of hypophosphatemia

• US 8,889,426 B2
• Filed: 05/18/2012
• Issued: 11/18/2014
• Est. Priority Date: 10/30/2009
• Status: Active Grant

• Current Assignee: New York University
• Original Assignee: New York University
• Inventors: Mohammadi, Moosa, Goetz, Regina, Eliseenkova, Anna V.
• Primary Examiner(s): Saoud, Christine J
• Application Number:
  US13/475,094
• Publication Number:
  US 20120288886A1
• Time in Patent Office: 914 Days
• Field of Search: None
• US Class Current: 436/501
• CPC Class Codes: A61K 31/59 Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
• CPC Class Codes: A61K 38/17 from animals; from humans enzyme inhibitors A61K38/005
• CPC Class Codes: A61K 38/1709 from mammals
• CPC Class Codes: A61K 38/1825 Fibroblast growth factor [FGF]
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: G01N 21/84 Systems specially adapted for particular applications
• CPC Class Codes: G01N 30/00 Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation G01N3/00, G01N5/00, G01N7/00, G01N9/00, G01N11/00, G01N13/00, G01N15/00, G01N17/00, G01N19/00, G01N21/00, G01N22/00, G01N23/00, G01N24/00, G01N25/00, G01N27/00, G01N29/00 take precedence
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92

Abstract

The present invention is directed to a method of treating hypophosphatemia in a subject. The present invention is also directed to a method of screening for compounds suitable for treatment of hypophosphatemia associated with elevated or normal FGF23. This method involves providing FGF23, FGFR-Klotho complex, and one or more candidate compounds. The FGF23, the FGFR-Klotho complex, and the candidate compounds are combined under conditions effective for the FGF23 and the binary FGFR-Klotho complex to form a ternary complex if present by themselves. This method also involves identifying the candidate compounds, which prevent formation of the complex as being potentially suitable in treating hypophosphatemic conditions associated with elevated or normal FGF23. A method of screening the specificity of compounds which prevent formation of the FGF23-Klotho-FGFR complex is also disclosed.

Claims

1. A method of screening for compounds suitable for treatment of a hypophosphatemic condition, said method comprising:

providing a receptor binding fragment of FGF23 consisting of the amino acid sequence of SEQ ID NO;

12 or amino acid residues 1 to 21 of SEQ ID NO;

11;

providing binary FGFR-Klotho complex;

providing one or more candidate compounds;

combining the receptor binding fragment of FGF23, the binary FGFR-Klotho complex, and the candidate compounds under conditions effective for the receptor binding fragment of FGF23 and the binary FGFR-Klotho complex to form a ternary complex if present by themselves; and

identifying the candidate compounds which prevent formation of the ternary complex and bind the binary FGFR-Klotho complex as being suitable in treating hypophosphatemia associated with elevated or normal FGF23.

2. The method according to claim 1, wherein the Klotho has the amino acid sequence of SEQ ID NO:

7.

3. The method according to claim 1, wherein a plurality of candidate compounds are tested.

4. The method according to claim 1, wherein the FGF receptor has the amino acid sequence of SEQ ID NO:

9.

5. The method according to claim 1, wherein the method is carried out using surface plasmon resonance spectroscopy.

6. The method according to claim 1, wherein the method is carried out using size-exclusion chromatography.

7. The method according to claim 1, wherein the method is carried out using a pull-down assay.

8. The method according to claim 1, wherein the method is carried out using co-immunoprecipitation.

9. The method according to claim 1, wherein the method is carried out in a cellular assay.

10. The method according to claim 1, wherein the receptor binding fragment of FGF23 consists of the amino acid sequence of SEQ ID NO:

12.

11. The method according to claim 1, wherein the receptor binding fragment of FGF23 consists of amino acid residues 1 to 21 of SEQ ID NO:

11.

12. The method according to claim 1, wherein the one or more candidate compounds comprise a polypeptide.

13. The method according to claim 1, wherein the one or more candidate compounds comprise an FGF23 fragment.