Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

Area of Science:

• Molecular Biology
• Biochemistry
• Drug Discovery

Background:

• Heat shock protein 90 (Hsp90) is crucial for cellular processes, and its interactions with co-chaperones like FKBP51 and FKBP52 are key targets.
• The C-terminal MEEVD pentapeptide of Hsp90 mediates binding to the tetratricopeptide repeat (TPR) motif of co-chaperones.
• FKBP51 and FKBP52, despite structural similarities, have distinct roles in steroid hormone-dependent diseases, making selective targeting important.

Purpose of the Study:

• To develop and validate an assay for probing Hsp90-cochaperone interactions.
• To screen for small molecules that selectively inhibit Hsp90-FKBP51 or Hsp90-FKBP52 interactions.
• To identify potent and selective inhibitors of Hsp90-FKBP51 interactions for therapeutic applications.

Main Methods:

• Purification of glutathione S-transferase (GST)-tagged FKBP51 and FKBP52 proteins containing the TPR motif.
• Development of an amplified luminescent proximity homogenous assay (ALPHA) using GST-fused TPR proteins and an Hsp90 C-terminal peptide.
• Screening of small molecules using the ALPHA assay to identify inhibitors of Hsp90-FKBP51/52 interactions.

Main Results:

• Successfully purified GST-tagged FKBP51 and FKBP52.
• Established a homogeneous ALPHA assay to detect Hsp90-cochaperone interactions.
• Identified potent and selective small molecule inhibitors of Hsp90-FKBP51 interactions through screening.

Conclusions:

• The developed ALPHA assay is effective for probing Hsp90-cochaperone interactions.
• Selective inhibition of Hsp90-FKBP51 interactions is achievable.
• This approach holds significant therapeutic potential for diseases involving Hsp90-FKBP51/52 pathways.