An Approach to Derive Functional Peptide Inhibitors of Transcription Factor Activity

Abstract

We report the development of a high-throughput, intracellular “transcription block survival” (TBS) screening platform to derive functional transcription factor antagonists. TBS is demonstrated using the oncogenic transcriptional regulator cJun, with the development of antagonists that bind cJun and prevent both dimerization and, more importantly, DNA binding remaining a primary challenge. In TBS, cognate TRE sites are introduced into the coding region of the essential gene, dihydrofolate reductase (DHFR). Introduction of cJun leads to TRE binding, preventing DHFR expression by directly blocking RNA polymerase gene transcription to abrogate cell proliferation. Peptide library screening identified a sequence that both binds cJun and antagonizes function by preventing DNA binding, as demonstrated by restored cell viability and subsequent in vitro hit validation. TBS is an entirely tag-free genotype-to-phenotype approach, selecting desirable attributes such as high solubility, target specificity, and low toxicity within a complex cellular environment. TBS facilitates rapid library screening to accelerate the identification of therapeutically valuable sequences.

In vitro single molecule and bulk phase studies reveal the AP-1 transcription factor cFos binds to DNA without its partner cJun

“…Each component was measured individually, and the sum of the spectra predicts the spectrum for the mixture in the absence of any interaction. The differences observed between the summed spectrum and the measured protein/DNA mixture spectrum provide information on binding ( 34 ). Upon addition of 10-fold excess of cFos or cJun to TRE DNA ( Fig.…”

Meeting Proceedings from ICBS 2022 – Uncovering Solutions for Diseases

“…The platform determined the 62-amino-acid peptide HingeW to be an antagonist, confirmed by in vitro binding studies. 68 Further truncation of peptides coupled with helix inducing can impart resistance to protease, increase target affinity, and increase cell permeability. Thus, shortened constructs of HingeW were designed, causing a concomitant decrease in cJun binding; however binding was recovered in part by the introduction of lactam bridges.…”

“…The TBS allows for screening of peptides encoded in a plasmid library to be screened dynamically for antagonism of cJun, which leads to activity of the murine DHFR and growth of E. coli colonies. The platform determined the 62-amino-acid peptide HingeW to be an antagonist, confirmed by in vitro binding studies . Further truncation of peptides coupled with helix inducing can impart resistance to protease, increase target affinity, and increase cell permeability.…”

Landscape of Global Gene Expression Reveals Distinctive Tissue Characteristics in Bactrian Camels (Camelus bactrianus)

“…With the rapid development of China's economy, camels primarily provide camel hair, meat, and milk, which is an important part of animal husbandry. Over the course of evolution, camels have developed several physiological adaptations to cold, heat, drought, and nutrient-poor conditions [7,8]. To adapt to the harsh living environment, they can store energy in their humps in the form of fat, enabling them to survive long periods without any food or water [9].…”