An Approach to Derive Functional Peptide Inhibitors of Transcription Factor Activity

Area of Science:

• Molecular Biology
• Drug Discovery
• Genetics

Background:

• Transcription factors are key regulators of gene expression, and their dysregulation is implicated in diseases like cancer.
• Developing small molecules that inhibit specific transcription factors, such as oncogenic cJun, is a significant therapeutic challenge.
• Existing methods for identifying transcription factor antagonists often face limitations in throughput and cellular context relevance.

Purpose of the Study:

• To develop and validate a high-throughput screening platform for identifying functional transcription factor antagonists.
• To demonstrate the utility of the platform by deriving antagonists for the oncogenic transcription factor cJun.
• To accelerate the discovery of novel therapeutic sequences targeting transcription factors.

Main Methods:

• Development of a "transcription block survival" (TBS) screening platform using the essential dihydrofolate reductase (DHFR) gene.
• Introduction of cJun-binding TRE sites into the DHFR coding region to create a cJun-dependent cell proliferation assay.
• Screening of a peptide library to identify sequences that bind cJun and inhibit its DNA binding and transcriptional activity.

Main Results:

• The TBS platform successfully identified a peptide antagonist that binds to cJun and prevents its DNA binding.
• The identified antagonist restored cell viability by alleviating cJun-mediated transcriptional repression of DHFR.
• In vitro validation confirmed the antagonist's ability to inhibit cJun function, demonstrating a tag-free genotype-to-phenotype approach.

Conclusions:

• The developed TBS platform is an effective, high-throughput method for discovering functional transcription factor antagonists.
• This approach facilitates the identification of sequences with desirable therapeutic attributes like specificity and low toxicity.
• TBS accelerates the discovery pipeline for novel therapeutics targeting transcription factors in a cellular environment.