Abstract 1117 Insights into p47phox Regulation and Inhibition: A Membrane Anchor of NADPH Oxidase 2

Regulation of signaling pathways plays a critical role in maintaining cellular homeostasis, and a lack thereof may result in aberrant activity of disease inducing processes. The NADPH Oxidase (NOX) family of transmembrane enzymes are susceptible to overactivation resulting in a cytotoxic burst of reactive oxygen species. One of seven isoforms, NOX2 is implicated in several disease states such as, but not limited to, cardiovascular disease, cancers, and neurodegenerative disorders. 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 conserved catalytic domain across all isoforms. Our research aims to overcome this complication through alternative means by targeting the pre-activation event induced by the peripheral membrane protein p47phox. The PX domain of p47phox has proven to be essential for NOX2 activation, where a mutation or knockout results in diminished enzymatic activity. While located in the cytosol in its resting state, the PX domain translocates p47phox and the cytosolic activating complex to NOX2 at the plasma membrane through its interactions with phosphoinositols and other phospholipids. We have found through several biochemical and biophysical assays that low micromolar quantities of soluble inositol phosphates (IPs) located in the cytosol are sufficient to inhibit membrane binding of p47phox-PX, which may serve to regulate NOX2 activation and have a physiological role. These results lead to understanding the capacity of using small molecules against the PX domain for selective NOX2 inhibition. We employed a 2,000-fragment NMR screen against p47phox-PX and discovered six high quality fragment hits ranging in affinity from 400uM to 4mM, highlighting the tractability of the PX domain. Serving as starting points for fragment-based inhibitor design, these weakly binding small molecules 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. Research reported in this abstract was supported by the VCU startup fund and the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM147221.