Author ORCID Identifier
https://orcid.org/my-orcid?orcid=0000-0002-0079-3501
Defense Date
2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Chemical Biology
First Advisor
Brian Fuglestad
Abstract
The regulation of cellular signaling across plasma membranes is a well-organized process through cross-talk of biomolecules such as proteins and membranes. Peripheral membrane proteins (PMPs) are a class of water soluble, membrane associated, proteins that play a critical role in transducing such signals. Due to the dynamic nature of their membrane interactions, relatively smooth surfaces, and general lack of deep ligand binding pockets, PMPs are commonly associated with an approximate 85% of the human proteome that is classified as ‘undruggable’ by current drug discovery methodologies. One such PMP, p47phox, contains a PX domain which is essential in activation of the NADPH Oxidase 2 (NOX2) pathway – where a mutation or knockout of the PX domain results in diminished enzymatic activity. After phosphorylation of the p47phox C-terminus, the PX domain translocates the entire trimeric cytosolic activating complex to NOX2, located within the plasma membrane, through its interactions with phosphoinositols and other phospholipids. While important for innate immune responses, NOX2 is susceptible to overactivation which results in an accumulation in cytotoxic levels of reactive oxygen species. This oxidative burst is implicated in various conditions such as cancers, cardiovascular diseases, and more. As an attractive therapeutic target, researchers have been striving for development of potent and selective NOX inhibitors which have thus far been unsuccessful due to a highly conserved catalytic domain across all seven NOX isoforms. This issue of selectivity highlights an emerging necessity for alternative approaches to NOX inhibition. Our research aims to overcome this complication through alternative means by targeting the pre-activation event induced by the PX domain of p47phox.
The small molecule inositol hexaphosphate (IP6) was studied against the PX domain of p47phox using an array of biochemical and biophysical assays to ascertain the tractability of small molecule inhibition of the protein domain. IP6 was found to inhibit the lipid binding and membrane anchoring events of p47phox-PX with high nM to low μM potency. Other tested inositol phosphates showed no apparent capacity to inhibit PX domain-membrane interactions. However, IP6 is found ubiquitously in mammalian tissues well above the inhibitory constants found within this study indicating a potential physiologically relevant regulatory mechanism of p47phox-PX translocation and NOX2 activation. Additionally, these results lead to understanding the capacity of using small molecules against the PX domain for selective NOX2 inhibition.
Motivated by these findings, we initiated a drug discovery campaign by employing a 2,000-fragment Nuclear Magnetic Resonance (NMR) Spectroscopy screen against p47phox-PX and discovered six high quality fragment hits ranging in affinity from approximately 500 μM to 3 mM, highlighting the tractability of the PX domain. Serving as starting points for fragment-based inhibitor design, these weakly binding small molecules can efficiently probe potential inhibitory surfaces of PMPs lacking deep ligand binding pockets, such as p47phox-PX, and will guide further elaborations into a drug-like molecule capable of breaking PX domain-lipid interactions and NOX2 deactivation. In conclusion, the activity of NOX2 is regulated through the complex’s scaffolding protein, p47phox. Small molecule inhibitor development which exploits this regulatory process can prove to be a robust therapeutic advantage for overcoming obstacles in NOX selective inhibition.
Rights
© Angela M. Develin
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
5-10-2024