An FKBP12 binding assay based upon biotinylated FKBP12
C W Carreras1, H Fu, D V Santi
1Department of Pharmacological Sciences, Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, California 94545, USA. carreras@kosan.com
Analytical Biochemistry
|October 25, 2001
Summary
A new binding assay accurately measures FKBP12 ligand affinity. This method uses a fusion protein and competition with [3H]FK506, offering a rapid assessment for small molecule binding.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- FKBP12 is a crucial protein target for various therapeutic agents.
- Accurate measurement of FKBP12 ligand affinity is essential for drug discovery.
- Existing methods for assessing ligand binding can be time-consuming or complex.
Purpose of the Study:
- To develop and validate a novel, convenient, and rapid binding assay for FKBP12 ligands.
- To quantify the binding affinity (Kd values) of specific FKBP12 ligands using the developed assay.
Main Methods:
- Engineered a fusion protein by linking a biotinylation signal sequence to the human FKBP12 gene.
- Expressed the fusion protein in Escherichia coli with biotin ligase.
- Immobilized the fusion protein on avidin-coated plates for a competitive binding assay using [3H]FK506.
Main Results:
- The developed assay successfully measured the affinity of FKBP12 ligands.
- Obtained Kd values for FK520 and its analogs (32-hydroxyethyl indolyl FK520, 18-ene, 20-oxa FK520) consistent with prior research.
- Demonstrated the assay's ability to assess FKBP12 binding by small molecules.
Conclusions:
- A robust and efficient binding assay for FKBP12 ligands has been established.
- This assay facilitates the rapid screening and characterization of potential FKBP12-targeting compounds.
- The method provides reliable affinity data, supporting drug development efforts targeting FKBP12.
