Exploiting the allosteric regulation of Bruton's Tyrosine Kinase for small molecule screens
Date
2023-05
Authors
Kueffer, Lauren
Major Professor
Advisor
Andreotti, Amy H
Chen, Stone
Nelson, Scott
Underbakke, Eric
Sacco, Randy
Committee Member
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Abstract
Bruton’s Tyrosine Kinase (BTK) is a non-receptor tyrosine kinase and is a member of the TEC family of kinases. BTK plays an integral role in many immunological cellular signaling pathways and has become a target for therapeutic development. BTK is composed of, from N- to C- terminus, the regulatory Pleckstrin Homology – Tec Homology (PHTH), Src-homology 3 (SH3), Src-homology 2 (SH2) domains followed by the catalytic Kinase domain. All domains N- terminal to the Kinase domain play a role in regulating the activity of BTK. The nearest full- length structure of BTK is that of the SH3-SH2-Kinase fragment in the autoinhibitory conformation whereby the SH3 and SH2 domains dock onto the backside of the Kinase domain, maintaining autoinhibition. The PHTH domain plays two roles: autoinhibition by binding to the Kinase domain and localizing BTK to the membrane by dimerization at PIP3 lipids. Existing drugs targeting BTK all bind to the active site of Kinase domain and patients taking these drugs may acquire resistance mutations rendering drug treatment ineffective. Therefore, the allosteric regulatory mechanisms in BTK should be exploited for therapeutic development.
This dissertation explores harnessing the allosteric regulatory interactions of BTK for the development of a small molecule screen and the characterization of small molecules allosterically targeting BTK. Since the allosteric regulatory mechanisms are being explored in this dissertation, the first chapter provides a historical perspective of allostery, kinases, and targeting kinases in instances of disease. The second chapter is a modified manuscript published in the journal Frontiers in Cell and Developmental Biology that details the current understanding of existing active site inhibitors, mechanisms of drug resistance, and the allosteric regulatory mechanisms of BTK. The final two chapters provide the ways in which we have capitalized on
the allosteric regulatory mechanisms of BTK for small molecule screening. The third chapter is a preliminary manuscript to be submitted for publication and details the screening approach taken against the SH3-SH2-Kinase fragment of BTK and the characterization of the hits received from the screen. The fourth chapter outlines the progress for developing a cellular based screen targeting the regulatory mechanisms of the BTK PHTH domain. Overall, this dissertation provides the strategies we have taken for targeting BTK allosterically with small molecules. This work highlights the importance of the allosteric regulation of BTK and why targeting BTK allosterically is promising in a clinical setting.
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dissertation