Characterization of Serine/Threonine Kinase MRCKα and Screening of Novel MRCKα Inhibitors

Candidate: Loughlin Kelly
Supervisor: Dr. Michael Olson

Abstract:

Cancer metastasis, responsible for roughly 90% of cancer deaths, involves complex mechanisms significantly regulated by the actomyosin cytoskeleton. Central regulators include serine/threonine kinases such as Myotonic Dystrophy-related Cdc42-binding kinases (MRCKs). This thesis investigates MRCKα, aiming to characterize its structure and biological roles, identify novel substrates, and evaluate potential inhibitors. Attempts to crystallize MRCKα and MRCKβ kinase domains complexed with inhibitor BDP9066 were unsuccessful. To identify MRCKα substrates, two approaches were employed: analogue-sensitive kinase assay (ASKA) and kinase inactivation assay using FSBA in whole-cell lysates. Although multiple MRCKα mutants were generated, suitable analogue-sensitive mutants were not identified. The FSBA assay effectively inhibited endogenous kinases but faced challenges with protein precipitation, limiting its utility. Fluorescence polarization assays identified three novel MRCK inhibitors (TJS-73, TJS-75, TJS-78). Despite lower potency compared to BDP9066, these candidates showed significant MRCKα inhibition, offering promising scaffolds for optimization. Overall, this thesis underscores the complexity of kinase characterization and the therapeutic potential of MRCK inhibitors, highlighting the need for further development.