Structure-driven drug discovery of NADPH Oxidase 2 inhibitors and the design of a chimeric protein as a tool for the specific activation of NADPH Oxidase 1

NADPH oxidases are the only family of enzymes entirely committed to the production of ROS (Reactive Oxygen Species) as their principal product. ROS are known to serve fundamental roles in several biological processes, such as in cell signaling and host defense mechanisms. On the other end, ROS are very unstable molecules that tend to interact with the biological macromolecules altering their structure and function. For this reason, NOXs are required for normal cellular processes, but they can also contribute to the development of several diseases. NOXs dysregulation, and hence an excessive ROS production, have been indeed widely associated with chronic inflammation, cardiovascular diseases, neurological disorders, and cancer. Given the key roles they assume in the main cellular physiological or pathological processes, the human NADPH oxidases emerge as appealing and valuable drug targets. With the ambition of successfully targeting these enzymes, on one hand a structure-driven drug discovery workflow, focused on the search for isoenzyme specific NOX inhibitors was undertaken. On the other hand, a strategy to deeply investigate their still unknown mechanism of activation from a biochemical and structural point of view was outlined. Here, the results of the quest for a potential inhibitor specifically targeting the human NOX2, the first identified and most studied isoform, are presented. Starting from a structure-driven in silico screening platform, a panel of NOX2 inhibitors was identified, evaluated in vitro on the endogenous NOX2 from human native cells by means of cell-free assays, and later optimized through Structure-Activity Relationship (SAR). This led to the identification of few lead compounds that were extremely promising and that were selected for following evaluations on cancer cell lines. The second part of this work of thesis is instead focused on the isoform NOX1, physiologically activated only upon stimulus by the interaction with three cytosolic partners. In this regard we have designed and developed for the first time a chimeric single- chain protein as a tool for specifically triggering the human NOX1 activation in vitro. The construct of this chimeric protein was designed starting with computational analysis by taking advantage of the chimeric activator specific for NOX2 that was already available and used in the laboratory. The great advantage of this tool is that having a single protein rather than multiple ones can reduce the variability during enzymatical assays and, in addition, minimize the time and effort needed for the expression and purification of different proteins, allowing faster and more reliable experiments. The data provided here are still very preliminary, but all together they set the basis to implement the use of this tool that will allow new insight into the biochemical and structural characterization of NOX1.