The Development of a Small Molecule Transcriptional Activation Domain.

Abstract

The strict regulation of transcription is essential for the survival of an organism. Nearly every human disease is associated with mis-regulated transcription as either a cause or an effect. This realization has brought about significant research efforts to both better understand the mechanism of eukaryotic transcription and to create small molecules capable of modulating this complex process. Transcriptional activator proteins play a critical role in the control of gene expression. Minimally composed of a DNA binding domain and an activation domain, they are responsible for the levels and time-course of expression of a particular gene. The creation of activator artificial transcription factors (ATFs), non-natural replacements for activators, has provided mechanistic details about this process and has the potential as therapeutics. Chapter I details our current understanding of activators and progress that has been made in the creation of activator ATFs. Features of activators that contribute to their potency (the levels of transcription elicited) are poorly understood. Until recently, it was believed that the strength of an activator was directly correlated with its affinity for coactivator targets. Chapter II details our studies of activator ATFs that target the coactivator Med15 in order to reveal other functional contributors to activator potency. Specifically, these studies demonstrated the importance of binding site location and secondary interactions that prevent non-productive binding and proteolytic degradation to the potency of a transcriptional activator. Endogenous activators bind to an overlapping set of coactivator targets yet exhibit very little sequence homology. The permissive nature of coactivator binding sites led us to design a series of small molecules which presented functionality commonly observed in amphipathic activators to be used as an activation domain. Chapters III and IV detail the cell-free and cell-based assays which demonstrate the utility of an isoxazolidine scaffold for this purpose. Remarkably, they represent the first small molecule activation domain with activity in living cells. Early mechanistic work suggests that they function by a mechanism analogous to their natural protein counterparts. Future studies of these molecules include in-depth mechanistic investigations as well as structural optimizations to increase potency, experiments that are detailed in Chapter V.