Mariana Castelôa
| Chasing NOX4: From VAS2870 to a Potent, Selective, Covalent Inhibitor |
| Mariana Castelôa 1, Marta Massari 2, Sara Marchese 2, Carla Lima 1, Joana Reis 2,3, Lorenzo Basile 2, Fernanda Borges 1,4, Sofia Benfeito 4, Andrea Mattevi 2, Daniel Chavarria 4 1 RISE-Health, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal 2 Department of Biology and Biotechnology Lazzaro Spallanzani, University of Pavia, Via Ferrata 9, 27100 Pavia, Italy 3 Department of Structural Biology, St. Jude Children’s Research Hospital, 262 Danny Thomas Pl, 38105 Memphis, TN, USA 4 RISE-Health, Department of Biomedicine, Pharmacology and Therapeutics Unit, Faculty of Medicine, University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto, Portugal |
| Abstract The NADPH oxidase (NOX) family comprises seven homologs (NOX1-5; DUOX1-2) that generate reactive oxygen species (ROS) and represent promising therapeutic targets in oxidative stress-related diseases. However, developing selective NOX inhibitors remains a major challenge due to the high structural conservation of the enzymes’ catalytic cores. Among known NOX inhibitors, VAS2870 stands out as a bona fide, non-assay-interfering compound that acts through covalent binding to a cysteine residue in the dehydrogenase domain. However, it shares common limitations with other NOX inhibitors, including lack of homolog selectivity, poor aqueous solubility, and cytotoxic effects [1,2]. In this work, a library of VAS2870 derivatives bearing distinct cysteine-reactive warheads was designed, synthesized, and screened against full-length NOX1, NOX2, NOX4, and NOX5. From this effort, a promising hit was identified. Subsequent hit-to-lead optimization, consisting of linker modification while maintaining the electrophilic warhead, was performed to improve potency and selectivity while confirming the utility of the chosen warhead. Ultimately, this optimization led to the discovery of a potent, non-interfering, selective NOX4 inhibitor with a favorable cytotoxicity profile. The results obtained will be presented in this communication. |
| References [1] Reis, J.; Gorgulla, C.; Massari, M.; Marchese, S.; Valente, S.; Noce, B.; Basile, L.; Törner, R.; Cox, H.; Viennet, T.; et al. Targeting ROS production through inhibition of NADPH oxidases. Nature Chemical Biology 2023, 19 (12), 1540-1550. [2] Reis, J.; Massari, M.; Marchese, S.; Ceccon, M.; Aalbers, F. S.; Corana, F.; Valente, S.; Mai, A.; Magnani, F.: Mattevi, A. A closer look into NADPH oxidase inhibitors: Validation and insight into their mechanism of action. Redox Biology, 2020, 32, 101466. Acknowledgments: This work was funded by FEDER funds through the Operational Program Competitiveness Factors COMPETE and national funds by the FCT-Foundation for Science and Technology under research grants for RISE-Health (UID/06397/2025, https://doi.org/10.54499/UID/06397/2025) and NOXIOUS (EXPL/BIA-BQM/0492/2021 – http://doi.org/10.54499/EXPL/BIA-BQM/0492/2021). This work was also funded by project IMPULSE and services of EU-OPENSCREEN (Horizon Europe: 101132028; DOI: 10.3030/101132028). M.C. (2022.13356.BD – https://doi.org/10.54499/2022.13356.BD), C.L. (UI/BD/154557/2023 – https://doi.org/10.54499/UI/BD/154557/2023) grants and D.C. (2024.07926.CEECIND) and S.B. (2023.06106.CEECIND/CP2833/CT0003 – https://doi.org/10.54499/2023.06106.CEECIND/CP2833/CT0003) contracts were also supported by FCT and FEDER/COMPETE funds. This research was also supported by the Associazione Italiana per la Ricerca sul Cancro (IG19808 to A.M., 26648 to M.M., and 28098 and 31561 to S.M.), the Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 800924 and MUR (Ministero Università Ricerca); grant no. PRIN 2020CW39SJ. This research was also funded by Regione Lombardia, regional law no. 9/2020, resolution no. 3776/2020. |