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Targeting ROS production through inhibition of NADPH oxidases

Nature Chemical Biology volume 19, pages 1540–1550 (2023)Cite this article

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Abstract

NADPH oxidases (NOXs) are transmembrane enzymes that are devoted to the production of reactive oxygen species (ROS). In cancers, dysregulation of NOX enzymes affects ROS production, leading to redox unbalance and tumor progression. Consequently, NOXs are a drug target for cancer therapeutics, although current therapies have off-target effects: there is a need for isoenzyme-selective inhibitors. Here, we describe fully validated human NOX inhibitors, obtained from an in silico screen, targeting the active site of Cylindrospermum stagnale NOX5 (csNOX5). The hits are validated by in vitro and in cellulo enzymatic and binding assays, and their binding modes to the dehydrogenase domain of csNOX5 studied via high-resolution crystal structures. A high-throughput screen in a panel of cancer cells shows activity in selected cancer cell lines and synergistic effects with KRAS modulators. Our work lays the foundation for the development of inhibitor-based methods for controlling the tightly regulated and highly localized ROS sources.

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Fig. 1: The domain architecture and interaction partners of NOXs.
Fig. 2: The candidates from screen 1.
Fig. 3: Inhibitory activities of the selected screen 1 compounds on human NOX1, NOX2, NOX4 and NOX5.
Fig. 4: The crystal structures of the csNOX5 DH in complex.
Fig. 5: Hits derived from the in silico screen on csNOX5 DH where the C-terminal residues are in an open conformation (Fig. 4e).
Fig. 6: Structural insights on the binding mode of 3.

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Data availability

The data that support the findings of this study is available within the main text and its Supplementary Information file. The coordinates of the cocrystal structures of csNOX5 DH present in this study have been deposited in the PDB with accession codes 8CAK, 8CAL, 8CAO, 8CAP and 8CB0. Source data are provided with this paper. Data are also available from the corresponding author upon request.

References

  1. Moloney, J. N. & Cotter, T. G. ROS signalling in the biology of cancer. Semin. Cell Dev. Biol. 80, 50–64 (2018).

    Article  CAS  PubMed  Google Scholar 

  2. Bedard, K. & Krause, K.-H. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol. Rev. 87, 245–313 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Bromberg, Y. & Pick, E. Unsaturated fatty acids stimulate NADPH-dependent superoxide production by cell-free system derived from macrophages. Cell. Immunol. 88, 213–221 (1984).

    Article  CAS  PubMed  Google Scholar 

  4. Rossi, F. The O2−-forming NADPH oxidase of the phagocytes: nature, mechanisms of activation and function. Biochim. Biophys. Acta, Bioenerg. 853, 65–89 (1986).

    Article  CAS  Google Scholar 

  5. Sies, H. & Jones, D. P. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat. Rev. Mol. 21, 363–383 (2020).

    Article  CAS  Google Scholar 

  6. Lambeth, J. D. & Neish, A. S. Nox enzymes and new thinking on reactive oxygen: a double-edged sword revisited. Annu Rev. Pathol. 9, 119–145 (2014).

    Article  CAS  PubMed  Google Scholar 

  7. Al Ghouleh, I. et al. Binding of EBP50 to Nox organizing subunit p47phox is pivotal to cellular reactive species generation and altered vascular phenotype. Proc. Natl Acad. Sci. USA 113, E5308–E5317 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Liou, G.-Y. & Storz, P. Reactive oxygen species in cancer. Free Radic. Res. 44, 479–496 (2010).

    Article  CAS  PubMed  Google Scholar 

  9. Block, K. & Gorin, Y. Aiding and abetting roles of NOX oxidases in cellular transformation. Nat. Rev. Cancer 12, 627–637 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ogrunc, M. et al. Oncogene-induced reactive oxygen species fuel hyperproliferation and DNA damage response activation. Cell Death Differ. 21, 998–1012 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Adachi, Y. et al. Oncogenic Ras upregulates NADPH oxidase 1 gene expression through MEK-ERK-dependent phosphorylation of GATA-6. Oncogene 27, 4921–4932 (2008).

    Article  CAS  PubMed  Google Scholar 

  12. Suh, Y.-A. et al. Cell transformation by the superoxide-generating oxidase Mox1. Nature 401, 79–82 (1999).

    Article  CAS  PubMed  Google Scholar 

  13. Ju, H.-Q. et al. Mutant Kras- and p16-regulated NOX4 activation overcomes metabolic checkpoints in development of pancreatic ductal adenocarcinoma. Nat. Commun. 8, 14437 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Liang, S., Kisseleva, T. & Brenner, D. A. The role of NADPH oxidases (NOXs) in liver fibrosis and the activation of myofibroblasts. Front. Physiol. 7, 17 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Crosas-Molist, E. et al. The NADPH oxidase NOX4 represses epithelial to amoeboid transition and efficient tumour dissemination. Oncogene 36, 3002–3014 (2017).

    Article  CAS  PubMed  Google Scholar 

  16. Lin, X.-L. et al. Overexpression of NOX4 predicts poor prognosis and promotes tumor progression in human colorectal cancer. Oncotarget 8, 33586–33600 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Eser, S., Schnieke, A., Schneider, G. & Saur, D. Oncogenic KRAS signalling in pancreatic cancer. Br. J. Cancer 111, 817–822 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Durand, N. & Storz, P. Targeting reactive oxygen species in development and progression of pancreatic cancer. Expert Rev. Anticancer Ther. 17, 19–31 (2017).

    Article  CAS  PubMed  Google Scholar 

  19. Chen, J. et al. Membranous NOX5-derived ROS oxidizes and activates local Src to promote malignancy of tumor cells. Curr. Signal Transduct. Ther. 5, 139 (2020).

    Article  CAS  Google Scholar 

  20. Brar, S. S. et al. NOX5 NAD(P)H oxidase regulates growth and apoptosis in DU 145 prostate cancer cells. Am. J. Physiol. Cell Physiol. 285, C353–C369 (2003).

    Article  CAS  PubMed  Google Scholar 

  21. Aydin, E. et al. NOX2 inhibition reduces oxidative stress and prolongs survival in murine KRAS-induced myeloproliferative disease. Oncogene 38, 1534–1543 (2019).

    Article  CAS  PubMed  Google Scholar 

  22. Aurelius, J. et al. Monocytic AML cells inactivate antileukemic lymphocytes: role of NADPH oxidase/gp91phox expression and the PARP-1/PAR pathway of apoptosis. Blood 119, 5832–5837 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Dakik, H. et al. Characterization of NADPH oxidase expression and activity in acute myeloid leukemia cell lines: a correlation with the differentiation status. Antioxidants 10, 498 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Magnani, F. & Mattevi, A. Structure and mechanisms of ROS generation by NADPH oxidases. COSB 59, 91–97 (2019).

    CAS  Google Scholar 

  25. Magnani, F. et al. Crystal structures and atomic model of NADPH oxidase. Proc. Natl Acad. Sci. USA 114, 6764–6769 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Massari, M., Nicoll, C. R., Marchese, S., Mattevi, A. & Mascotti, M. L. Evolutionary and structural analyses of the NADPH oxidase family in eukaryotes reveal an initial calcium dependency. Redox Biol. 56, 102436 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Liu, R. et al. Structure of human phagocyte NADPH oxidase in the resting state. eLife 11, e83743 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Noreng, S. et al. Structure of the core human NADPH oxidase NOX2. Nat. Commun. 13, 6079 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Augsburger, F. et al. Pharmacological characterization of the seven human NOX isoforms and their inhibitors. Redox Biol. 26, 101272 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Reis, J. et al. A closer look into NADPH oxidase inhibitors: validation and insight into their mechanism of action. Redox Biol. 32, 101466 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Rey, F. E., Cifuentes, M. E., Kiarash, A., Quinn, M. T. & Pagano, P. J. Novel competitive inhibitor of NAD(P)H oxidase assembly attenuates vascular O(2)(-) and systolic blood pressure in mice. Circ. Res. 89, 408–414 (2001).

    Article  CAS  PubMed  Google Scholar 

  32. Solbak, S. M. Ø. et al. Developing inhibitors of the p47phox-p22phox protein-protein interaction by fragment-based drug discovery. J. Med. Chem. 63, 1156–1177 (2020).

    Article  CAS  PubMed  Google Scholar 

  33. Gorgulla, C. et al. An open-source drug discovery platform enables ultra-large virtual screens. Nature 580, 663–668 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. REAL Database (Enamine, 2023); https://enamine.net/compound-collections/real-compounds/real-database

  35. Sterling, T. & Irwin, J. J. ZINC 15 – ligand discovery for everyone. J. Chem. Inf. Model. 55, 2324–2337 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. ZINC15 (Irwin and Shoichet Laboratories, 2023); https://zinc15.docking.org/

  37. Brandes, R. P., Weissmann, N. & Schröder, K. Nox family NADPH oxidases: molecular mechanisms of activation. Free Radic. Biol. Med. 76, 208–226 (2014).

    Article  CAS  PubMed  Google Scholar 

  38. Molina, D. M. et al. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341, 84–87 (2013).

    Article  CAS  Google Scholar 

  39. von Löhneysen, K., Noack, D., Hayes, P., Friedman, J. S. & Knaus, U. G. Constitutive NADPH oxidase 4 activity resides in the composition of the B-loop and the penultimate C terminus. J. Biol. Chem. 287, 8737–8745 (2012).

    Article  Google Scholar 

  40. Kean, K. M. et al. High-resolution studies of hydride transfer in the ferredoxin:NADP+ reductase superfamily. FEBS J. 284, 3302–3319 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Cox, J. A., Jeng, A. Y., Sharkey, N. A., Blumberg, P. M. & Tauber, A. I. Activation of the human neutrophil nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase by protein kinase C. J. Clin. Investig. 76, 1932–1938 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Yu, C. et al. High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines. Nat. Biotechnol. 34, 419–423 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Tsherniak, A. et al. Defining a cancer dependency map. Cell 170, 564–576.e516 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Germon, Z. P. et al. Blockade of ROS production inhibits oncogenic signaling in acute myeloid leukemia and amplifies response to precision therapies. Sci. Signal. 16, eabp9586 (2023).

    Article  CAS  PubMed  Google Scholar 

  45. García-Gómez, P. et al. NOX4 regulates TGFβ-induced proliferation and self-renewal in glioblastoma stem cells. Mol. Oncol. 16, 1891–1912 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Kaushik, D. et al. Nuclear NADPH oxidase-4 associated with disease progression in renal cell carcinoma. Transl. Res. 223, 1–14 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Takashiba, S. et al. Differentiation of monocytes to macrophages primes cells for lipopolysaccharide stimulation via accumulation of cytoplasmic nuclear factor kappaB. Infect. Immun. 67, 5573–5578 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Brüne, B. et al. Redox control of inflammation in macrophages. Antioxid. Redox Signal. 19, 595–637 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Jayavelu, A. K., Moloney, J. N., Böhmer, F.-D. & Cotter, T. G. NOX-driven ROS formation in cell transformation of FLT3-ITD-positive AML. Exp. Hematol. 44, 1113–1122 (2016).

    Article  CAS  PubMed  Google Scholar 

  50. Adane, B. et al. The hematopoietic oxidase NOX2 regulates self-renewal of leukemic stem cells. Cell Rep. 27, 238–254.e236 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Paolillo, R. et al. The NADPH oxidase NOX2 is a marker of adverse prognosis involved in chemoresistance of acute myeloid leukemias. Haematologica 107, 2562–2575 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Beretti, F. et al. The interplay between HGF/c-met axis and Nox4 in BRAF mutated melanoma. Int. J. Mol. Sci. 22, 761 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Song, N.-Y. et al. IKKα inactivation promotes Kras-initiated lung adenocarcinoma development through disrupting major redox regulatory pathways. Proc. Natl Acad. Sci. USA 115, E812–E821 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Shaw, A. T. et al. Selective killing of K-ras mutant cancer cells by small molecule inducers of oxidative stress. Proc. Natl Acad. Sci. USA 108, 8773–8778 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Lim, J. K. M. & Leprivier, G. The impact of oncogenic RAS on redox balance and implications for cancer development. Cell Death Dis. 10, 955 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Hillig, R. C. et al. Discovery of potent SOS1 inhibitors that block RAS activation via disruption of the RAS–SOS1 interaction. Proc. Natl Acad. Sci USA 116, 2551–2560 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Wang, X. et al. Identification of MRTX1133, a noncovalent, potent, and selective KRASG12D inhibitor. J. Med. Chem. 65, 3123–3133 (2022).

    Article  CAS  PubMed  Google Scholar 

  58. Lu, J. et al. NADPH oxidase 1 is highly expressed in human large and small bowel cancers. PLoS ONE 15, e0233208 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Schrödinger Release 2023-1: Maestro (Schrödinger, LLC, 2021).

  60. Morris, G. M. et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785–2791 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Alhossary, A., Handoko, S. D., Mu, Y. & Kwoh, C.-K. Fast, accurate, and reliable molecular docking with QuickVina 2. Bioinformatics 31, 2214–2216 (2015).

    Article  CAS  PubMed  Google Scholar 

  62. Koes, D. R., Baumgartner, M. P. & Camacho, C. J. Lessons learned in empirical scoring with smina from the CSAR 2011 benchmarking exercise. J. Chem. Inf. Model. 53, 1893–1904 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Quiroga, R. & Villarreal, M. A. Vinardo: a scoring function based on Autodock Vina improves scoring, docking, and virtual screening. PLoS ONE 11, e0155183 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  64. Sander, T. et al. DataWarrior: an open-source program for chemistry aware data visualization and analysis. J. Chem. Inf. Model. 55, 460–473 (2015).

    Article  CAS  PubMed  Google Scholar 

  65. Durocher, Y., Perret, S. & Kamen, A. High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res. 30, E9 (2002).

    Article  PubMed  PubMed Central  Google Scholar 

  66. Zhang, J. et al. Transient expression and purification of chimeric heavy chain antibodies. Protein Expr. Purif. 65, 77–82 (2009).

    Article  CAS  PubMed  Google Scholar 

  67. Zhao, B., Summers, F. A. & Mason, R. P. Photooxidation of Amplex Red to resorufin: implications of exposing the Amplex Red assay to light. Free Radic. Biol. Med. 53, 1080–1087 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Kabsch, W. XDS. Acta Crystallogr. D. Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D. Biol. Crystallogr. 67, 235–242 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D. Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  71. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D. Biol. Crystallogr. 67, 355–367 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D. Struct. Biol. 75, 861–877 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  74. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    Article  CAS  PubMed  Google Scholar 

  75. Wiegand, T. et al. Sedimentation yields long-term stable protein samples as shown by solid-state NMR. Front. Mol. Biosci. 7, 17 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Corsello, S. M. et al. Discovering the anticancer potential of non-oncology drugs by systematic viability profiling. Nat. Cancer 1, 235–248 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Song, M.-g et al. NRF2 signaling negatively regulates phorbol-12-myristate-13-acetate (PMA)-induced differentiation of human monocytic U937 cells into pro-inflammatory macrophages. PLoS ONE 10, e0134235 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  78. Ianevski, A., He, L., Aittokallio, T. & Tang, J. SynergyFinder: a web application for analyzing drug combination dose–response matrix data. Bioinformatics 33, 2413–2415 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

Research on NOX proteins was supported by the Associazione Italiana per la Ricerca sul Cancro (IG19808 to. A.M., Fellowships grant nos. 26648 to M.M. and 28098 to S.M.), the European Union’s 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 funded also by Regione Lombardia, regional law no. 9/2020, resolution no. 3776/2020. H.A. acknowledges support from the National Institutes of Health (grant no. R01 GM136859) and Claudia Adams Barr Program for Innovative Cancer Research. We thank R. Nagarajan for the useful discussions and the ‘Centro Grandi Strumenti’ of the University of Pavia for technical support.

Author information

Author notes

  1. These authors contributed equally: Joana Reis, Christoph Gorgulla, Marta Massari, Sara Marchese, Sergio Valente.

Authors and Affiliations

  1. Department of Biology and Biotechnology Lazzaro Spallanzani, University of Pavia, Pavia, Italy

    Joana Reis, Marta Massari, Sara Marchese, Lorenzo Basile & Andrea Mattevi

  2. Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    Joana Reis, Christoph Gorgulla, Ricarda Törner, Huel Cox III, Thibault Viennet, Moon Hee Yang & Haribabu Arthanari

  3. Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA

    Joana Reis, Christoph Gorgulla, Ricarda Törner, Huel Cox III, Thibault Viennet, Moon Hee Yang & Haribabu Arthanari

  4. Department of Physics, Faculty of Arts and Sciences, Harvard University, Cambridge, MA, USA

    Christoph Gorgulla

  5. Department of Drug Chemistry and Technologies, Sapienza University of Rome, Rome, Italy

    Sergio Valente, Beatrice Noce & Antonello Mai

  6. Broad Institute of Harvard and MIT, Cambridge, USA

    Melissa M. Ronan, Matthew G. Rees & Jennifer A. Roth

  7. Department of Precision Medicine, University of Campania Luigi Vanvitelli, Naples, Italy

    Lucia Capasso, Angela Nebbioso & Lucia Altucci

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  1. Joana Reis
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Contributions

J.R., S.M., M.M. and L.B prepared csNOX5 dehydrogenase, NOX4 dehydrogenase, NOX1, NOX2, NOX4 and NOX5. J.R., S.M., M.M. and L.B. performed protein crystallography and biochemical and cellular-based experiments. C.G. performed virtual screening studies. S.V. and B. N. performed and supervised compound chemical synthesis. R.T. and T.V. performed and supported NMR experiments. H.C. performed AML cell experiments. M.H.Y. performed and analyzed experiments on KRAS cell lines. M.M.R., M.G.R. and J.A.R. performed and analyzed all the PRISM cancer line experiments. L.C., A.N. and L.A. performed and supervised cancer cell experiments. J.R., M.M., S.M., S.V., A.M., H.A. and A.M. conceived the experiments and wrote the manuscript. All the authors read and approved the manuscript.

Corresponding authors

Correspondence to Antonello Mai, Haribabu Arthanari or Andrea Mattevi.

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Competing interests

C.G. and H.A. are cofounders of Quantum Therapeutics Inc., a company that uses computational methods for drug development and the work presented here has no overlap. The remaining authors declare no competing interests.

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Nature Chemical Biology thanks Fabien Cailliez and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Pipeline for human NOX inhibitor discovery.

A graphic representation of the experimental workflow and selection criteria employed for the discovery of human NOX inhibitors.

Extended Data Fig. 2 The crystal structures of the csNOX5 DH in complex with 3 (a), 4 (b), 6 (c), and 2 (d).

The panels show the close-up views of the bound inhibitors and their neighboring residues. The inhibitor 2Fc-Fc maps are countered at 1.2−1.4 σ levels and were calculated prior to the inclusion of the inhibitors in the crystallographic refinement.

Extended Data Fig. 3 Cell-based assays to monitor the efficacy of NOX inhibitors.

(a-b) Dose-response curves are graphed on a logarithmic scale with percentage of inhibition as a function of compound concentration. The conducted assay followed ROS production and determined the EC50 of 3 on NOX2 (left, pink) and 1 on NOX5 (right, orange). Data are given as the mean ± s.d, n = 2 independent experiments for NOX2, n = 3 independent experiments for NOX5. DMSO and inhibitors experiments were performed in parallel. (c-d) Western blot protein quantification after exposure to increasing temperatures without and with the inhibitor, and (e-f) CETSA analysis - data were obtained i) in the presence of 3 (red) and DMSO (blue) on NOX2; ii) in the presence of 1 (red) and DMSO (blue) on NOX5. Overall, the thermal shifts for 3 on NOX2 and 1 on NOX5 are 2.1 °C and 4.3 °C, respectively. 1 also induces a stabilizing effect on NOX4 with a thermal shift of 1.3 °C whilst 3 causes a thermal shift of 1.2 °C on NOX4. Data are given as the mean ± s.d, n = 2 independent experiments for NOX2, n = 4 independent experiments for NOX5. DMSO and inhibitors experiments were performed in parallel. The solid lines represent the best fit of the data to the saturation binding curve model within the GraphPad Prism software.

Source data

Extended Data Fig. 4 Results from PRISM experiments and leukaemia cell line studies on 3 and 15.

(a) Average area under the curves (AUCs) of blood, lymphocyte, central nervous system, and kidney cancer cell lines, divided by sub-lineages: acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia (HRCL) in the blood lineage; non-Hodgkin lymphoma (NNHL), Hodgkin lymphoma (HDGL), lymphoma unspecified (LYMU), adult T-cell lymphoma (ATL) in the lymphocyte lineage; meningioma (MNNG), glioma (GLIM), medulloblastoma (MDLL), primitive neuroectodermal tumor (PNET) in the central nervous system lineage; malignant rhabdoid tumor (MLRT), renal cell carcinoma (RNCC), unspecified carcinoma (NA) in the kidney lineage. The PRISM assay comprised two immortalized non-cancerous cell lines (MMNK1 and HA1E). Both 1 and 3 did not show any activity against them (AUC > 0.98); mean ± s.d., n = 3 biologically independent experiments. Boxplots show medians, 25th and 75th percentiles, with bars showing the standard error of the mean. (b) Dose-response to 3 by the PRISM-identified cell lines (mean ± s.d., n = 3 independent experiments). (c) Cell viability studies (ATP quantification) on the U937 cells incubated for 72 hours with 3 (left) and 15 (right) with and without phorbol 12-myristate13-acetate (PMA; mean ± s.d., n = 3 independent experiments; 95% confidence interval for variable dose-response fit). For comparison, the lack of activity of 1 and 3 against a non-cancerous cell line is shown in Supplementary Fig. 56.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–3, Figs. 1–106, note on the chemical syntheses and uncropped western blots/gels.

Reporting Summary

Supplementary Data

Raw data for Supplementary Figs. 1–36, 40–44, 47–51, 53 and 55–57.

Source data

Source Data Fig. 2

Raw data for Fig. 2b

Source Data Fig. 3

Raw data for Fig. 3a

Source Data Fig. 5

Raw data for Fig. 5b,c

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Raw data for Extended Data Fig. 3a,b,e,f

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Raw data for Extended Data Fig. 4

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Reis, J., Gorgulla, C., Massari, M. et al. Targeting ROS production through inhibition of NADPH oxidases. Nat Chem Biol 19, 1540–1550 (2023). https://doi.org/10.1038/s41589-023-01457-5

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