Design and Synthesis of Non-Peptidic Transcription Factors.-

Abstract

Transcription is important for the determination of cellular phenotype through the regulation of gene expression and its mis-regulation can lead to abnormal cell function. Transcriptional activators are essential for high fidelity transcription, responsible for seeking out particular genes and up-regulating them to precise levels in a signalresponsive fashion. Molecules that can reconstitute the function of transcriptional activators, artificial transcription activators, are highly desirable commodities as mechanistic tools and transcription-based therapeutics. Transcriptional activators control the specificity and extent of gene upregulation through two domains: the DNA binding domain (DBD) confers specific binding to DNA and the transcriptional activation domain (TAD) dictates the level of gene expression. Many questions surrounding how natural transcriptional activation domains function has hindered the development of TAD replacements despite their likely advantages in terms of stability, delivery, and and/or immunogenic properties. To address the need for the development and characterization of small molecule xv TADs we have employed a combination of organic chemistry and biological evaluations to produce a class of isoxazolidines that functionally mimic natural TADs. We identified the first small molecule, an amphipathic isoxazolidine, that reconstitutes transcription in living cell culture. Additionally, the amphipathic isoxazoldine alone can competitively inhibit the DNA localized-isoxazolidine, indicating that it is the isoxazolidine moiety that makes contacts with the transcriptional machinery that are important for activation. Many different peptide sequences can function as activators and we hypothesized this feature would translate to other suitably functionalized small molecules. Indeed, other isoxazolidine and non-isoxazolidine TADs activated transcription in cell culture. With an array of small molecule TADs, we designed activator artificial transcription factors and tested them for activation in cell culture.