Small Molecule Transcriptional Activation Domains.

Abstract

Transcriptional activators are essential for high fidelity transcription, responsible for seeking out particular genes and up-regulating them to precise levels in a signal responsive fashion. Indeed the altered transcription patterns observed in disease states can often be attributed to malfunctioning and/or misregulated transcriptional activators. Thus, molecules that can reconstitute the function of transcriptional activators, artificial transcription activators, are highly desirable commodities as mechanistic tools and transcription-targeted therapeutics. Transcriptional activators control the specificity and extent of gene upregulation through two domains: the DNA binding domain (DBD) is responsible for the former and the transcriptional activation domain (TAD) dictates the level of gene expression. It has proven quite challenging to identify TAD replacements with functional properties comparable to the natural system despite their likely advantages in terms of stability, delivery, and and/or immunogenic properties. This is likely due to the many open questions surrounding how natural transcriptional activation domains function.

To address the need for the development and characterization of small molecule TADs we have employed a combination of organic chemistry, NMR spectroscopy, and biological evaluations to a class of isoxazolidines that functionally mimic natural TADs. We prepared five stereochemically pure isoxazolidine isomers, each of which contained identical functional groups (benzyl, isobutyl and hydroxyl) arranged in different positions around the isoxazolidine ring. All of these amphipathic isoxazolidines functioned as TADs in a cell-free assay, revealing that analogous to endogenous TADs, a particular positioning of functional groups is not required for transcription function. Similar functional trends were observed in a cellular assay. We further demonstrated that the small molecule TADs interact with at least a subset of the same coactivator proteins as do natural TADs. In particular, interaction with the KIX domain of Creb Binding Protein is correlated with transcription function, although binding interactions with Tra1, Med15 and Med23 are also observed. These molecules are thus anticipated to be an excellent starting point for the design of more potent small molecule regulators of transcription.