Alternative Approach to NADPH Oxidase Inhibition: Challenging Selectivity through Fragment Based Drug Design

Abstract

The regulation of cellular signaling across plasma membranes is a well‐organized process through cross‐talk of biomolecules such as proteins and lipids. Peripheral membrane proteins (PMPs) are a class of water soluble, membrane associated, proteins that play a critical role in transducing these signals. Due to the dynamic nature of their membrane interactions, relatively smooth surfaces, and lack of deep ligand binding pockets, PMPs are commonly associated with the ~85% of the human proteome that is classified as ‘undruggable’ by current drug discovery methodologies. One such PMP is p47phox that acts as an organizer/scaffold subunit essential for activation of transmembrane enzyme NADPH oxidase 2 (NOX2). The NOX2 complex is activated by translocation of the PX domain of p47phox (p47phox‐PX) through recognition of phospholipids (PIPs) localized in the plasma membrane. NOX2 is implicated in diseases such as cardiovascular, cancers, and neurodegenerative disorders, however, despite its importance, past efforts at inhibiting NOX2 activation have failed. The challenge lies in a lack of specificity among the NOX family of enzymes due to a highly conserved catalytic pocket. Inhibition of the complex prior to the activation event by targeting p47phox‐PX presents a novel alternative strategy for reviving NOX2 inhibition. Fragment‐based drug design (FBDD) was developed over the past twenty years and has already shown promise at targeting ‘undruggable’ proteins. Here, we initiate a FBDD study against p47phox‐PX. We discovered a small molecule binder, myo‐inositol‐1‐phosphate (I1P), that mimics the headgroup of p47phox‐PX’s natural PIP ligand. I1P has an ~800uM affinity towards the PX domain, as displayed by nuclear magnetic resonance spectroscopy (NMR) titrations, which will act as a scaffold for inhibitor design. Through a NMR based fragment screen, we identified 13 additional fragments that bind to p47phox‐PX. Initial characterization of fragment hits displayed a number of high uM to low mM affinities within the lipid headgroup recognition pocket, which is promising for fragment advancement towards inhibitors. Future endeavors to probe the interactions between fragments and the PX domain of p47phox will further optimize the affinity of the inhibitor to p47phox‐PX and increase selectivity against other PX domains. An array of biochemical, biophysical, and cellular assays will establish and optimize our inhibitor as a novel p47phox‐PX small molecule drug and deactivator of the NOX2 complex.