Discovery of Small Molecules to Dissect the Individual Roles of GACKIX-Activator Complexes

[摘要] In gene expression, the GACKIX motif interacts with multiple activators to regulate diverse physiological processes including cellular growth, hematopoiesis, synaptic plasticity, and lipid homeostasis; thus, dysregulation of these complexes is also linked to many diseases from leukemia to Alzheimer’s disease. Chemical tools are sought to understand the individual roles of activator-GACKIX complexes in transcription. However, the confounding problem has been finding small molecules that are selective in their interactions towards GACKIX because this small motif is found in multiple eukaryotic coactivators including CBP, its homolog p300 and the mediator subunit, MED15. In this dissertation, the site-directed strategy known as Tethering is applied to develop selective small molecule probes that target the KIX domain of one coactivator such as CBP versus p300.Tethering readily detects fragments with an intrinsic affinity for the target by amplifying the strength of these interactions via the formation of a covalent disulfide bond between a disulfide fragment and a cysteine-containing KIX target. Traditional liquid-chromatography mass spectrometry applied to Tethering screens ranked fragments by their ability to bind to the cysteine-containing KIX target. We developed a novel fluorescence polarization screen that streamlined the identification of small molecules inhibitors directed at the c-Myb and CREB- binding site of CBP KIX. More importantly, the FP Tethering screen has the potential to increase the applicability of Tethering to other important biological targets that would benefit from this approach.Tethering identified fragments that impacted the assembly or disassembly of activator binding partners with KIX. For example, KIX N627C tethered to 1-10 disrupted MLL binding and enhanced the binding affinity of KIX for pKID by 2-fold. By using mutants and tethered KIX complexes in transient state stopped-flow experiments, we showed that the stabilization of the ternary complex contributed to the positive cooperativity. In cells, 1-10 could be a useful probe to study the KIX interactions on CREB(pKID)-responsive genes because the 1-10-KIX N627C complex allosterically prolongs this interaction. The 1-10 disulfide was converted into a series of irreversible analogs that are compatible with the reducing environment in cells. This irreversible Tethering strategy demonstrated how fragments such as 1-10 selectivity have the potential to selectively target an engineered cysteine-containing KIX mutant of CBP in cellulo.In conclusion, I built upon the Tethering strategy to selectively target dynamic activator-GACKIX complexes with small molecules; these fragments along with other biochemical techniques demonstrated how KIX regulates its interactions with different partners.

[发布机构] University of Michigan

[效力级别] Tethering

[关键词] Transcriptional activator-coactivator interactions;Tethering;Covalent small molecule probes;GACKIX motif;Chemistry;Science;Chemistry