Identification of protein interaction antagonists using the repressed transactivator two-hybrid system

Area of Science:

• Molecular Biology
• Biochemistry
• Pharmacology

Background:

• The repressed transactivator (RTA) yeast two-hybrid system facilitates the genetic identification of interactions involving transcriptional activator proteins.
• Screening for inhibitors of protein-protein interactions is crucial for drug discovery and understanding biological pathways.

Purpose of the Study:

• To adapt the RTA yeast two-hybrid system for screening small molecule compound libraries to identify inhibitors of protein interactions.
• To validate the identified inhibitors in relevant biological contexts and assess their therapeutic potential.

Main Methods:

• Modification of the RTA yeast two-hybrid system to enable screening of small molecule libraries.
• Assay development using 3-aminotriazole (3-AT) to measure inhibition of protein-protein interactions.
• In vivo validation of identified compounds, including assessment of effects on calcineurin-dependent signaling and TGFbeta-R/SMAD pathways.

Main Results:

• The modified RTA system successfully screened a library of 23,000 small molecules, identifying inhibitors for four distinct protein interactions.
• Compounds inhibiting the FKBP12 and transforming growth factor beta receptor (TGFbeta-R) interaction were identified and validated.
• These compounds demonstrated inhibition of calcineurin-dependent signaling in T cells and affected TGFbeta-R/SMAD reporter gene expression.

Conclusions:

• The adapted RTA yeast two-hybrid system is effective for discovering small molecule inhibitors of protein interactions.
• The identified compounds show potential as novel therapeutic agents, particularly for modulating immune cell signaling pathways.
• This screening approach offers a valuable tool for identifying new drug candidates targeting protein-protein interactions.