Targeting ROS production through inhibition of NADPH oxidases.
Journal
Nature chemical biology
ISSN: 1552-4469
Titre abrégé: Nat Chem Biol
Pays: United States
ID NLM: 101231976
Informations de publication
Date de publication:
Dec 2023
Dec 2023
Historique:
received:
03
05
2023
accepted:
21
09
2023
medline:
27
11
2023
pubmed:
27
10
2023
entrez:
26
10
2023
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 37884805
doi: 10.1038/s41589-023-01457-5
pii: 10.1038/s41589-023-01457-5
doi:
Substances chimiques
NADPH Oxidases
EC 1.6.3.-
Reactive Oxygen Species
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1540-1550Subventions
Organisme : NIGMS NIH HHS
ID : R01 GM136859
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM136859
Pays : United States
Informations de copyright
© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.
Références
Moloney, J. N. & Cotter, T. G. ROS signalling in the biology of cancer. Semin. Cell Dev. Biol. 80, 50–64 (2018).
pubmed: 28587975
doi: 10.1016/j.semcdb.2017.05.023
Bedard, K. & Krause, K.-H. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol. Rev. 87, 245–313 (2007).
pubmed: 17237347
doi: 10.1152/physrev.00044.2005
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).
pubmed: 6090027
doi: 10.1016/0008-8749(84)90066-2
Rossi, F. The O
doi: 10.1016/0304-4173(86)90005-4
Sies, H. & Jones, D. P. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat. Rev. Mol. 21, 363–383 (2020).
doi: 10.1038/s41580-020-0230-3
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).
pubmed: 24050626
doi: 10.1146/annurev-pathol-012513-104651
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).
pubmed: 27540115
pmcid: 5018796
doi: 10.1073/pnas.1514161113
Liou, G.-Y. & Storz, P. Reactive oxygen species in cancer. Free Radic. Res. 44, 479–496 (2010).
pubmed: 20370557
doi: 10.3109/10715761003667554
Block, K. & Gorin, Y. Aiding and abetting roles of NOX oxidases in cellular transformation. Nat. Rev. Cancer 12, 627–637 (2012).
pubmed: 22918415
pmcid: 3711509
doi: 10.1038/nrc3339
Ogrunc, M. et al. Oncogene-induced reactive oxygen species fuel hyperproliferation and DNA damage response activation. Cell Death Differ. 21, 998–1012 (2014).
pubmed: 24583638
pmcid: 4013514
doi: 10.1038/cdd.2014.16
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).
pubmed: 18454176
doi: 10.1038/onc.2008.133
Suh, Y.-A. et al. Cell transformation by the superoxide-generating oxidase Mox1. Nature 401, 79–82 (1999).
pubmed: 10485709
doi: 10.1038/43459
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).
pubmed: 28232723
pmcid: 5333128
doi: 10.1038/ncomms14437
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).
pubmed: 26869935
pmcid: 4735448
doi: 10.3389/fphys.2016.00017
Crosas-Molist, E. et al. The NADPH oxidase NOX4 represses epithelial to amoeboid transition and efficient tumour dissemination. Oncogene 36, 3002–3014 (2017).
pubmed: 27941881
doi: 10.1038/onc.2016.454
Lin, X.-L. et al. Overexpression of NOX4 predicts poor prognosis and promotes tumor progression in human colorectal cancer. Oncotarget 8, 33586–33600 (2017).
pubmed: 28422720
pmcid: 5464892
doi: 10.18632/oncotarget.16829
Eser, S., Schnieke, A., Schneider, G. & Saur, D. Oncogenic KRAS signalling in pancreatic cancer. Br. J. Cancer 111, 817–822 (2014).
pubmed: 24755884
pmcid: 4150259
doi: 10.1038/bjc.2014.215
Durand, N. & Storz, P. Targeting reactive oxygen species in development and progression of pancreatic cancer. Expert Rev. Anticancer Ther. 17, 19–31 (2017).
pubmed: 27841037
doi: 10.1080/14737140.2017.1261017
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).
doi: 10.1038/s41392-020-0193-z
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).
pubmed: 12686516
doi: 10.1152/ajpcell.00525.2002
Aydin, E. et al. NOX2 inhibition reduces oxidative stress and prolongs survival in murine KRAS-induced myeloproliferative disease. Oncogene 38, 1534–1543 (2019).
pubmed: 30323311
doi: 10.1038/s41388-018-0528-1
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).
pubmed: 22550344
pmcid: 3418695
doi: 10.1182/blood-2011-11-391722
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).
pubmed: 33807114
pmcid: 8004739
doi: 10.3390/antiox10030498
Magnani, F. & Mattevi, A. Structure and mechanisms of ROS generation by NADPH oxidases. COSB 59, 91–97 (2019).
Magnani, F. et al. Crystal structures and atomic model of NADPH oxidase. Proc. Natl Acad. Sci. USA 114, 6764–6769 (2017).
pubmed: 28607049
pmcid: 5495252
doi: 10.1073/pnas.1702293114
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).
pubmed: 35998431
pmcid: 9421330
doi: 10.1016/j.redox.2022.102436
Liu, R. et al. Structure of human phagocyte NADPH oxidase in the resting state. eLife 11, e83743 (2022).
pubmed: 36413210
pmcid: 9711523
doi: 10.7554/eLife.83743
Noreng, S. et al. Structure of the core human NADPH oxidase NOX2. Nat. Commun. 13, 6079 (2022).
pubmed: 36241643
pmcid: 9568551
doi: 10.1038/s41467-022-33711-0
Augsburger, F. et al. Pharmacological characterization of the seven human NOX isoforms and their inhibitors. Redox Biol. 26, 101272 (2019).
pubmed: 31330481
pmcid: 6658998
doi: 10.1016/j.redox.2019.101272
Reis, J. et al. A closer look into NADPH oxidase inhibitors: validation and insight into their mechanism of action. Redox Biol. 32, 101466 (2020).
pubmed: 32105983
pmcid: 7042484
doi: 10.1016/j.redox.2020.101466
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).
pubmed: 11532901
doi: 10.1161/hh1701.096037
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).
pubmed: 31922756
doi: 10.1021/acs.jmedchem.9b01492
Gorgulla, C. et al. An open-source drug discovery platform enables ultra-large virtual screens. Nature 580, 663–668 (2020).
pubmed: 32152607
pmcid: 8352709
doi: 10.1038/s41586-020-2117-z
REAL Database (Enamine, 2023); https://enamine.net/compound-collections/real-compounds/real-database
Sterling, T. & Irwin, J. J. ZINC 15 – ligand discovery for everyone. J. Chem. Inf. Model. 55, 2324–2337 (2015).
pubmed: 26479676
pmcid: 4658288
doi: 10.1021/acs.jcim.5b00559
ZINC15 (Irwin and Shoichet Laboratories, 2023); https://zinc15.docking.org/
Brandes, R. P., Weissmann, N. & Schröder, K. Nox family NADPH oxidases: molecular mechanisms of activation. Free Radic. Biol. Med. 76, 208–226 (2014).
pubmed: 25157786
doi: 10.1016/j.freeradbiomed.2014.07.046
Molina, D. M. et al. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341, 84–87 (2013).
doi: 10.1126/science.1233606
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).
doi: 10.1074/jbc.M111.332494
Kean, K. M. et al. High-resolution studies of hydride transfer in the ferredoxin:NADP+ reductase superfamily. FEBS J. 284, 3302–3319 (2017).
pubmed: 28783258
pmcid: 5626627
doi: 10.1111/febs.14190
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).
pubmed: 2997297
pmcid: 424245
doi: 10.1172/JCI112190
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).
pubmed: 26928769
pmcid: 5508574
doi: 10.1038/nbt.3460
Tsherniak, A. et al. Defining a cancer dependency map. Cell 170, 564–576.e516 (2017).
pubmed: 28753430
pmcid: 5667678
doi: 10.1016/j.cell.2017.06.010
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).
pubmed: 36976863
doi: 10.1126/scisignal.abp9586
García-Gómez, P. et al. NOX4 regulates TGFβ-induced proliferation and self-renewal in glioblastoma stem cells. Mol. Oncol. 16, 1891–1912 (2022).
pubmed: 35203105
pmcid: 9067149
doi: 10.1002/1878-0261.13200
Kaushik, D. et al. Nuclear NADPH oxidase-4 associated with disease progression in renal cell carcinoma. Transl. Res. 223, 1–14 (2020).
pubmed: 32492552
pmcid: 8111697
doi: 10.1016/j.trsl.2020.05.009
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).
pubmed: 10531202
pmcid: 96928
doi: 10.1128/IAI.67.11.5573-5578.1999
Brüne, B. et al. Redox control of inflammation in macrophages. Antioxid. Redox Signal. 19, 595–637 (2013).
pubmed: 23311665
pmcid: 3718318
doi: 10.1089/ars.2012.4785
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).
pubmed: 27666490
doi: 10.1016/j.exphem.2016.08.008
Adane, B. et al. The hematopoietic oxidase NOX2 regulates self-renewal of leukemic stem cells. Cell Rep. 27, 238–254.e236 (2019).
pubmed: 30943405
pmcid: 6931909
doi: 10.1016/j.celrep.2019.03.009
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).
pubmed: 35172562
pmcid: 9614539
doi: 10.3324/haematol.2021.279889
Beretti, F. et al. The interplay between HGF/c-met axis and Nox4 in BRAF mutated melanoma. Int. J. Mol. Sci. 22, 761 (2021).
pubmed: 33451139
pmcid: 7828605
doi: 10.3390/ijms22020761
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).
pubmed: 29311298
pmcid: 5789942
doi: 10.1073/pnas.1717520115
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).
pubmed: 21555567
pmcid: 3102385
doi: 10.1073/pnas.1105941108
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).
pubmed: 31852884
pmcid: 6920345
doi: 10.1038/s41419-019-2192-y
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).
pubmed: 30683722
pmcid: 6377443
doi: 10.1073/pnas.1812963116
Wang, X. et al. Identification of MRTX1133, a noncovalent, potent, and selective KRASG12D inhibitor. J. Med. Chem. 65, 3123–3133 (2022).
pubmed: 34889605
doi: 10.1021/acs.jmedchem.1c01688
Lu, J. et al. NADPH oxidase 1 is highly expressed in human large and small bowel cancers. PLoS ONE 15, e0233208 (2020).
pubmed: 32428030
pmcid: 7237001
doi: 10.1371/journal.pone.0233208
Schrödinger Release 2023-1: Maestro (Schrödinger, LLC, 2021).
Morris, G. M. et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785–2791 (2009).
pubmed: 19399780
pmcid: 2760638
doi: 10.1002/jcc.21256
Alhossary, A., Handoko, S. D., Mu, Y. & Kwoh, C.-K. Fast, accurate, and reliable molecular docking with QuickVina 2. Bioinformatics 31, 2214–2216 (2015).
pubmed: 25717194
doi: 10.1093/bioinformatics/btv082
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).
pubmed: 23379370
pmcid: 3726561
doi: 10.1021/ci300604z
Quiroga, R. & Villarreal, M. A. Vinardo: a scoring function based on Autodock Vina improves scoring, docking, and virtual screening. PLoS ONE 11, e0155183 (2016).
pubmed: 27171006
pmcid: 4865195
doi: 10.1371/journal.pone.0155183
Sander, T. et al. DataWarrior: an open-source program for chemistry aware data visualization and analysis. J. Chem. Inf. Model. 55, 460–473 (2015).
pubmed: 25558886
doi: 10.1021/ci500588j
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).
pubmed: 11788735
pmcid: 99848
doi: 10.1093/nar/30.2.e9
Zhang, J. et al. Transient expression and purification of chimeric heavy chain antibodies. Protein Expr. Purif. 65, 77–82 (2009).
pubmed: 19007889
doi: 10.1016/j.pep.2008.10.011
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).
pubmed: 22765927
pmcid: 3501008
doi: 10.1016/j.freeradbiomed.2012.06.034
Kabsch, W. XDS. Acta Crystallogr. D. Biol. Crystallogr. 66, 125–132 (2010).
pubmed: 20124692
pmcid: 2815665
doi: 10.1107/S0907444909047337
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D. Biol. Crystallogr. 67, 235–242 (2011).
pubmed: 21460441
pmcid: 3069738
doi: 10.1107/S0907444910045749
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D. Biol. Crystallogr. 60, 2126–2132 (2004).
pubmed: 15572765
doi: 10.1107/S0907444904019158
Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D. Biol. Crystallogr. 67, 355–367 (2011).
pubmed: 21460454
pmcid: 3069751
doi: 10.1107/S0907444911001314
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).
pubmed: 31588918
pmcid: 6778852
doi: 10.1107/S2059798319011471
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
pubmed: 15264254
doi: 10.1002/jcc.20084
Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).
pubmed: 8520220
doi: 10.1007/BF00197809
Wiegand, T. et al. Sedimentation yields long-term stable protein samples as shown by solid-state NMR. Front. Mol. Biosci. 7, 17 (2020).
pubmed: 32154263
pmcid: 7047159
doi: 10.3389/fmolb.2020.00017
Corsello, S. M. et al. Discovering the anticancer potential of non-oncology drugs by systematic viability profiling. Nat. Cancer 1, 235–248 (2020).
pubmed: 32613204
pmcid: 7328899
doi: 10.1038/s43018-019-0018-6
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).
pubmed: 26222138
pmcid: 4519053
doi: 10.1371/journal.pone.0134235
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).
pubmed: 28379339
pmcid: 5554616
doi: 10.1093/bioinformatics/btx162