The Development of Assays for Detecting Proximal Phosphorylation and Covalent Modification of Proteins

Bioassays are important tools in translational research for several disciplines including medicinal chemistry, molecular recognition and chemical biology. Achieving success in bioassays represents important milestones in many pharmaceutical pipelines and disease diagnostics, and as such, must be designed and fine-tuned to produce meaningful, robust, and reproducible results. This thesis focuses on the design and development of unique bioassays for two important medicinal chemistry fields of research. The first is the selective detection of proximally phosphorylated biomolecules, including peptides, proteins, nucleotides, and phospholipids. Proximal phosphorylation affects many cellular functions including protein localization, degradation, signalling cascades, gene expression, and the cell cycle. Importantly, the dysregulation of proximal phosphorylation motifs has been implicated in aberrant protein states, including the overactivation of kinases in oncology and protein aggregation in neurodegenerative diseases. Similarly, phosphate-induced anionic charge within phospholipid membranes is an important biomarker for mammalian cell apoptosis and can be used to selectively detect bacterial infections. To achieve this goal, a previously discovered fluorescent excimer chemosensor was characterized and next generation chemosensor libraries were designed and synthesized. Chemosensors were used for proximal phosphate detection in numerous biologically-relevant applications with a variety of assay types including fluorimetry, microscopy, polyacrylamide gels and flow cytometry. The second objective in assay development was to assess a modular electrophilic warhead platform for novel covalent inhibitors. Through nucleophilic aromatic substitution methodology, selectively derivatized polyfluorinated benzene rings were synthesized to construct an electrophilic warhead library that could be electronically and sterically tuned for specific protein interactions. To assess the inherent reactivity of the electrophilic warhead, a fluorescent chemosensor-based fluoride-release assay was developed which revealed a diverse range of intrinsic cysteine reactivities. As a proof-of-concept, the electrophilic warhead platform was applied to a clinical covalent tubulin inhibitor for improved potency and pharmacokinetic properties. The modified tubulin inhibitors were assessed in a variety of bioassays that demonstrated both the success and potential of the warhead platform. Across both objectives, the development of bioassays has produced technologies and insights that can overall benefit chemical biology, molecular recognition, and medicinal chemistry research.

Department dc:contributor.department

Chemistry

Year dc:date.issued

2021

Author dc:creator

• Cabral, Aaron David

Advisor dc:contributor.advisor

• Gunning, Patrick T.

• Assay Development
• Chemosensor
• Covalent
• Drug Discovery
• Electrophilic Warhead
• Proximal Phosphorylation

Handle dc:identifier.uri

http://hdl.handle.net/1807/130819

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/130819