Small Molecule Modulators of Transcription.

Abstract

Transcriptional activators are key components in the regulation of gene expression, being critical for precise, high fidelity transcription. Misregulation of transcription is a hallmark of many diseases, including diabetes, leukemias, and other cancers. Transcription-targeted therapeutics have been a long sought after goal in the treatment of these diseases, however progress has been slow as the molecular recognition events that occur during transcriptional activation are not well understood. Transcriptional activators consist minimally of a DNA-binding domain (DBD) responsible for gene specificity, and a transcriptional activation domain (TAD) that recruits the transcriptional machinery through direct binding interactions.

The Mapp lab has developed a small molecule isoxazolidine, that when tethered to a DBD, reconstitutes activator function in cells, however the mechanism remained an open question. To address this, an isoxazolidine TAD (iTAD) was prepared containing benzophenone, a photoactive group, for using in photocrosslinking experiments. The Creb binding protein (CBP) was identified as on of several target of iTAD 1 through in vitro crosslinking experiments. Furthermore, the binding site of iTAD 1 was found to overlap that of the natural activators MLL, Jun, Tat, and Tax within the KIX domain of CBP. The relevance of CBP in iTAD 1-mediated activation was further explored in a cellular 2-hybrid system, where it was shown that the activity of the small molecule correlated with the availability of CBP, and more specifically, the KIX domain. These data, coupled with the multiprotein binding profile and in vitro binding studies suggest that iTAD 1 functions in a manner similar to endogenous activators. The overlapping binding site of iTAD 1 with natural activators was further explored using a luciferase-based assay to test the ability of iTAD 1 and related isoxazolidines to inhibit the binding of the endogenous activators MLL and Jun. iTAD 1 was found to inhibit the activity of the minimal TADs of both MLL and Jun. Furthermore, it was shown to decrease the expression of cyclin D1, a gene tightly regulated by Jun. Several analogs were synthesized to achieve further potency or specificity; however, no compound was more potent than iTAD 1, though several trends in toxicity were observed.