NADPH oxidases (NOXs) are the only known human enzymes solely in charge of ROS production. In addition to their roles in the innate immunity and response to stressful conditions, NOXs are part of the redox signaling pathways that sustain cell proliferation, oncoprotein (e.g. RAS) driven cell transformation, and tumor microenvironment manipulation. NOXs are tightly controlled and understanding the molecular mechanisms underlying their isoform-specific regulation is an open issue with far-reaching implications for drug design.
In 2017, our group in Pavia has solved and published the first crystal structures of the dehydrogenase and transmembrane domains of a bacterial NOX5, 40% identical to human NOX5 [1-3]. Computer modelling was then used to view the overall structure of the NOX catalytic core. This landmark result revealed the structural basis for flavin reduction, “across-the-membrane” electron transfer, and ROS generation on the outer side of the membrane. In parallel to the structural studies, we have thoroughly investigated the putative NOX inhibitors that have been reported in the literature. This painstaking project led us to realize that virtually all known NOX inhibitors (31 of them were evaluated) suffer from off-targets effects, such as ROS scavenging and assay interference. This issue is so overwhelming that it was impossible to discern between the non-specific effects exerted by these compounds and their specific binding to NOXs (if any [4]). We have therefore employed the NOX5 dehydrogenase domain as our initial platform to carry on a drug design campaign. To this end, together with our collaborators at Harvard Medical School and Dana Farber Cancer Institute, we have conducted an ultra large-library computational screening using our NOX5 dehydrogenase domain PDB structure. We have evaluated the chosen library using a robust and high throughput workflow comprising primary and orthogonal assays as well as control assays to probe assay interfering compounds and ROS scavengers. Protein crystallography studies of the protein with the best hits have yielded for the first-time a crystal structure of this class of enzymes in complex with our studied inhibitors. Accordingly, our current efforts rely on the study and development of new and effective isoform-specific NOX inhibitors and the understanding of their effect on cancer model cells in which NOXs have a key role.
[1] Magnani, F., Nenci, S., Millana Fananas, E., Ceccon, M., Romero, E., Fraaije, M.W., Mattevi*, A. (2017) Crystal structures and atomic model of NADPH oxidase. Proc. Natl. Acad. Sci. USA 114, 6764-6769.
[2] Oosterheert, W., Reis, J., Gros, P., Mattevi, A. (2020) An Elegant Four-Helical Fold in NOX and STEAP Enzymes Facilitates Electron Transport across Biomembranes-Similar Vehicle, Different Destination. Acc. Chem. Res. 53, 1969-1980.
[3] Magnani, F., Mattevi*, A. (2019) Structure and mechanisms of ROS generation by NADPH oxidases. Curr. Opin. Struct. Biol.59, 91-97.
[4] Reis J, Massari M, Marchese S, Ceccon M, Aalbers FS, Corana F, Valente S, Mai A, Magnani F, Mattevi, A. (2020) A closer look into NADPH oxidase inhibitors: Validation and insight into their mechanism of action. Redox Biol. 32, 101466.
20/05/2022 Andrea Mattevi Department of Biology and Biotechnology University of Pavia, Italy