In silico and in vitro screening are promising in the identification of novel NADPH oxidases inhibitors

This thesis is concerned with the expression, purification, and membrane isolation of two members of the NADPH oxidase (NOXs) transmembrane enzyme family and the discovery of selective inhibitors. NOXs have a main role of producing Reactive Oxygen Species (ROS) in living organisms and so they are defined as the “professional ROS producers”. NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1 and DUOX2 are the seven human members of the family, all of which shares a NOX catalytic core. The latter comprises of a transmembrane (TM) domain and a cytosolic dehydrogenase (DH) domain which contains the active site. All NOXs utilize NADPH as substrate and electron donor. It binds at the DH domain and transfers electron to the FAD that is in the TM-DH interface. The reduced FAD (FADH2) transfers one electron at a time to the heme within the TM region (inner heme), then to the second heme in outer part of the TM region (outer heme) where molecular oxygen is finally reduced to superoxide (O2-) or hydrogen peroxide (H2O2). NOXs mainly differ in their mode of activation. NOX1-3 requires complex formation with the transmembrane subunit p22phox and cytosolic subunits for activation, while NOX4 in complex with p22phox is constitutively active. NOX5 and DUOX1/2 possess an EF-hand and depend on Ca2+ for activation, but the DUOXs require additional membrane components (DUOXA1/A2). NOXs play an important role in cell signaling and innate immunity at physiological ROS production. However, dysregulated NOXs are being implicated in different pathologies such as cancer, making NOXs important drug targets. Hence, it is necessary to understand how NOXs function and are regulated to develop potent and homolog-selective inhibitors. This thesis focuses on human NOXs (hNOXs): the expression of hNOX1, hNOX4 and the isolated DH of hNOX4, membrane isolation of hNOX1 and hNOX4, purification of hNOX1 and DH of hNOX4. The co-purification (pool-down) of NOX1-p22phox with the Trimera (a recombinant tripartite chimeric protein corresponding to the fusion of all cytosolic subunits) was optimized by reconstitution in nanodiscs for structural purpose. The hNOX1 and hNOX4 were over expressed in human cells and the isolated membranes were used to screen compound libraries. The DH of hNOX4 which was obtained following the “divide and conquer” approach (Magnani et al.) as the purification of full-length hNOX4 is still a challenge. The DH of hNOX4 was expressed in E. coli, purified, and used to screen inhibitor libraries. To trigger hNOX1 enzymatic activity, the tripartite chimera (Trimera) was used as the activator instead of adding the individual cytosolic proteins. Our inhibitor library comprises of two groups: the VAS and the M41 derived compounds. IC50 and Ki were only done with selected compounds on the DH of NOX4 based on the outcome of the primary screening. All the measured IC50 and Ki values gave results in the low µM range, confirming the good inhibitory activity of these compounds. This is a significant progress in the journey to homolog-selective NOX inhibitor discovery.

Lo screening in silico e in vitro è promettente nell'identificazione di nuovi inibitori della NADPH ossidasi. This thesis is concerned with the expression, purification, and membrane isolation of two members of the NADPH oxidase (NOXs) transmembrane enzyme family and the discovery of selective inhibitors. NOXs have a main role of producing Reactive Oxygen Species (ROS) in living organisms and so they are defined as the “professional ROS producers”. NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1 and DUOX2 are the seven human members of the family, all of which shares a NOX catalytic core. The latter comprises of a transmembrane (TM) domain and a cytosolic dehydrogenase (DH) domain which contains the active site. All NOXs utilize NADPH as substrate and electron donor. It binds at the DH domain and transfers electron to the FAD that is in the TM-DH interface. The reduced FAD (FADH2) transfers one electron at a time to the heme within the TM region (inner heme), then to the second heme in outer part of the TM region (outer heme) where molecular oxygen is finally reduced to superoxide (O2-) or hydrogen peroxide (H2O2). NOXs mainly differ in their mode of activation. NOX1-3 requires complex formation with the transmembrane subunit p22phox and cytosolic subunits for activation, while NOX4 in complex with p22phox is constitutively active. NOX5 and DUOX1/2 possess an EF-hand and depend on Ca2+ for activation, but the DUOXs require additional membrane components (DUOXA1/A2). NOXs play an important role in cell signaling and innate immunity at physiological ROS production. However, dysregulated NOXs are being implicated in different pathologies such as cancer, making NOXs important drug targets. Hence, it is necessary to understand how NOXs function and are regulated to develop potent and homolog-selective inhibitors. This thesis focuses on human NOXs (hNOXs): the expression of hNOX1, hNOX4 and the isolated DH of hNOX4, membrane isolation of hNOX1 and hNOX4, purification of hNOX1 and DH of hNOX4. The co-purification (pool-down) of NOX1-p22phox with the Trimera (a recombinant tripartite chimeric protein corresponding to the fusion of all cytosolic subunits) was optimized by reconstitution in nanodiscs for structural purpose. The hNOX1 and hNOX4 were over expressed in human cells and the isolated membranes were used to screen compound libraries. The DH of hNOX4 which was obtained following the “divide and conquer” approach (Magnani et al.) as the purification of full-length hNOX4 is still a challenge. The DH of hNOX4 was expressed in E. coli, purified, and used to screen inhibitor libraries. To trigger hNOX1 enzymatic activity, the tripartite chimera (Trimera) was used as the activator instead of adding the individual cytosolic proteins. Our inhibitor library comprises of two groups: the VAS and the M41 derived compounds. IC50 and Ki were only done with selected compounds on the DH of NOX4 based on the outcome of the primary screening. All the measured IC50 and Ki values gave results in the low µM range, confirming the good inhibitory activity of these compounds. This is a significant progress in the journey to homolog-selective NOX inhibitor discovery.