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Development and synthesis of molecules to study endogenous proteins : new inhibitors of NADPH Oxidase isoform 2 and efforts towards the development of a traceless affinity molecule

University of British Columbia

The study of proteins is essential to further our understanding of their functions in both healthy and diseased organisms. While several techniques are available for the study of isolated proteins, far fewer are available to study them in their native environments. The ability to understand how proteins interact with other biological molecules (other proteins, hormones, neurotransmitters, etc.) can allow us to develop specific drugs for specific interactions, potentially lessening side effects. In this thesis, two approaches to studying the function of living cells have been investigated. The first approach involves the design and synthesis of enzyme inhibitors. The second focuses on the design of a universal protein tagging method. Chapter two of this thesis focuses on the synthesis of novel NADPH oxidase (NOX) inhibitors based on Vasopharm’s VAS2870 (which contains a triazolopyrimidine backbone). The reactive oxygen species (ROS) generated by NOX have been implicated in the development of several neurodegenerative diseases (such as Alzheimer’s and Parkinson’s). The first inhibitor described contains a p-azidomethyl substituent for future tagging studies. Initial ROS inhibition experiments show that this modification does not interfere with the inhibitor’s activity. The second inhibitor replaces a reactive sulfur-carbon bond of the original inhibitor with a carbon-carbon bond. This compound did not show any inhibitory effects, indicating that the sulfur-carbon bond is necessary for the activity of VAS2870 and the p-azidomethyl derivative. This finding may call into question the conclusions of previous neurobiological studies. Chapter three describes efforts toward the development of a traceless affinity molecule. This molecule is composed of a ligand, cleavable linker, and chemical tag. When the linker is cleaved, the ligand is free of the tag, and can dissociate. Cleavage of the linker, an α-silyl-β-lactone, can be achieved via a Peterson olefination with a lysine residue outside of the ligand binding site. The result is a tag-protein complex, which enables the visualization of the protein in real time. In addition, the location of the tag is such that it should not interfere with the native functions of the protein. This study establishes the pioneering work necessary to design viable cleavable linkers for future protein labelling

Text

2019-07-08

Attribution-NoDerivatives 4.0 International

http://hdl.handle.net/2429/70936

Chemistry

University of British Columbia

UBCO

http://creativecommons.org/licenses/by-nd/4.0/