Tuesday, April 30, 2024
4/30/2024
PROJECT SUMMARY (ABSTRACT) In the past, protein function was viewed as dependent on a well-defined, folded three-dimensional structure; now, it is well established that disordered regions actively participate in the diverse functions mediated by proteins. Intrinsically disordered regions (IDRs) are abundant in the human proteome and these IDRs cultivate dynamic, transient interactions. The process of transcription is regulated by dynamic, IDR-containing proteins. Such proteins are overrepresented in multiple major disease pathways, making them ideal targets for drug discovery. Transcriptional coactivators are central proteins in transcriptional regulation; however, traditional drug discovery methods are less effective for targeting these dynamic domains, so there is a need for a directed approach to target these structures. IDRs evoke the conformational malleability of transcriptional protein domains in their essential protein-protein interactions (PPIs), thus conformational stabilization can be utilized as a modulation marker for dynamic domains. My research focuses on building a novel system to identify modulators of the dynamic transcriptional coactivator domains, KIX and HEAT and optimize modulators for use in therapeutics. Dysregulation of protein-protein interactions (PPIs) between these domains and their partnering transcription factors (TF) promotes several TF-dependent cancers. Representing the two major types of IDRs, the IDR within KIX acts as an allosteric site and the one in HEAT is an orthosteric site. My first aim is the identification of a variety of IDR modulators that functionally invoke conformational stabilization of these IDR-containing domains. There is a high correlation between conformational stability and thermostability; thus, for KIX primary screening, high-throughput differential scanning fluorimetry (HT-DSF) has been employed to observe IDR modulation by measuring change in protein thermostability upon ligand binding. With the preliminary KIX hits, follow-up fluorescence polarization (FP) screening will identify the hits that modulate the IDR in KIX such that its native PPIs are enhanced or inhibited. An expression and purification procedure will be developed to produce a stable HEAT construct. This domain will then be subjected to a HT-DSF and follow-up FP to define molecules that alter its IDR and native PPI binding. My second aim is the optimization of IDR modulators to be defined as therapeutic candidates. Collaborative structural assessment will then connect IDR modulation to modulation site, establishing structural models and defining ligand binding classes. Using the structural models, concerted high-throughput lead optimization will be performed for each ligand class to enhance their therapeutic potential. By implementing this system for the KIX and HEAT domains, it will be considered applicable for targeting an array of ‘undruggable’ dynamic domains and advancing the drug discovery efforts for this class of proteins.
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