Characterisation of Nox4 Inhibitors from Edible Plants

Abstract

NADPH oxidases transport electrons from cytosolic NADPH through biological membranes to generate reactive oxygen species. NADPH oxidase 4, broadly expressed in humans, is an interesting pharmacological target, since its activity is deregulated in several diseases, including pulmonary fibrosis, diabetic nephropathy, and cardiac hypertrophy. Whereas several candidate NADPH oxidase 4 inhibitors were recently described, most of these compounds are either unspecific or toxic. Here we set out to identify new NADPH oxidase 4 inhibitors from edible plants, in an attempt to decrease the number of hits with toxic side effects. We screened a compound library prepared from edible plants for new bioactives with the ability to inhibit the activity of NADPH oxidase 4. Using both cell-based and cell-free assays, we identified several compounds with significant inhibitory activity towards NADPH oxidase 4. For selected compounds, the activity profile towards NADPH oxidase 2 and NADPH oxidase 5 was established, and controls were carried out to exclude general reactive oxygen species scavengers. A number of promising NADPH oxidase 4 inhibitors from edible plants was identified and characterised. Several new chemical entities are disclosed which act as NADPH oxidase 4 inhibitors, and the efficacies of our best hits, in particular several diarylheptanoids and lignans, are comparable to the best available pharmacological NADPH oxidase 4 inhibitors. These findings will provide valuable tools to study mechanisms of NADPH oxidase inhibition.

Key words

NADPH oxidase 4 - Nox4 - ROS - inhibitors - dehydrogenase domain - broken cell assay

• References

• 1 Perry BN, Govindarajan B, Bhandarkar SS, Knaus UG, Valo M, Sturk C, Carrillo CO, Sohn A, Cerimele F, Dumont D, Losken A, Williams J, Brown LF, Tan XL, Ioffe E, Yancopoulos GD, Arbiser JL. Pharmacologic blockade of angiopoietin-2 is efficacious against model hemangiomas in mice. J Invest Dermatol 2006; 126: 2316-2322
• 2 Lambeth JD. Nox enzymes and the biology of reactive oxygen. Nat Rev Immunol 2004; 4: 181-189
• 3 Lambeth JD, Krause KH, Clark RA. NOX enzymes as novel targets for drug development. Sem Immunopathol 2008; 30: 339-363
• 4 Geiszt M. NADPH oxidases: New kids on the block. Cardiovasc Res 2006; 71: 289-299
• 5 Martyn KD, Frederick LM, von Loehneysen K, Dinauer MC, Knaus UG. Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases. Cell Signal 2006; 18: 69-82
• 6 Luo Z, Chen Y, Chen S, Welch WJ, Andresen BT, Jose PA, Wilcox CS. Comparison of inhibitors of superoxide generation in vascular smooth muscle cells. Br J Pharmacol 2009; 157: 935-943
• 7 Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: Physiology and pathophysiology. Physiol Rev 2007; 87: 245-313
• 8 Royerpokora B, Kunkel LM, Monaco AP, Goff SC, Newburger PE, Baehner RL, Cole FS, Curnutte JT, Orkin SH. Cloning the gene for an inherited human disorder – chronic granulomatous-disease – on the basis of its chromosomal location. Nature 1986; 322: 32-38
• 9 Krause KH. Tissue distribution and putative physiological function of NOX family NADPH oxidases. Jpn J Infect Dis 2004; 57: S28-29
• 10 Chen K, Craige SE, Keaney Jr. JF. Downstream targets and intracellular compartmentalization in Nox signaling. Antiox Redox Signal 2009; 11: 2467-2480
• 11 Geiszt M, Kopp JB, Varnai P, Leto TL. Identification of Renox, an NAD(P)H oxidase in kidney. Proc Natl Acad Sci USA 2000; 97: 8010-8014
• 12 Ago T, Kitazono T, Ooboshi H, Iyama T, Han YH, Takada J, Wakisaka M, Ibayashi S, Utsumi H, Iida M. Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase. Circulation 2004; 109: 227-233
• 13 Yang S, Madyastha P, Bingel S, Ries W, Key L. A new superoxide-generating oxidase in murine osteoclasts. J Biol Chem 2001; 276: 5452-5458
• 14 Ellmark SH, Dusting GJ, Fui MN, Guzzo-Pernell N, Drummond GR. The contribution of Nox4 to NADPH oxidase activity in mouse vascular smooth muscle. Cardiovasc Res 2005; 65: 495-504
• 15 Cucoranu I, Clempus R, Dikalova A, Phelan PJ, Ariyan S, Dikalov S, Sorescu D. NAD(P)H oxidase 4 mediates transforming growth factor-beta1-induced differentiation of cardiac fibroblasts into myofibroblasts. Circ Res 2005; 97: 900-907
• 16 Gorin Y, Ricono JM, Kim NH, Bhandari B, Choudhury GG, Abboud HE. Nox4 mediates angiotensin II-induced activation of Akt/protein kinase B in mesangial cells. Am J Physiol Renal Physiol 2003; 285: F219-229
• 17 Mahadev K, Motoshima H, Wu X, Ruddy JM, Arnold RS, Cheng G, Lambeth JD, Goldstein BJ. The NAD(P)H oxidase homolog Nox4 modulates insulin-stimulated generation of H2O2 and plays an integral role in insulin signal transduction. Mol Cell Biol 2004; 24: 1844-1854
• 18 Li J, Stouffs M, Serrander L, Banfi B, Bettiol E, Charnay Y, Steger K, Krause KH, Jaconi ME. The NADPH oxidase NOX4 drives cardiac differentiation: Role in regulating cardiac transcription factors and MAP kinase activation. Mol Biol Cell 2006; 17: 3978-3988
• 19 Uchizono Y, Takeya R, Iwase M, Sasaki N, Oku M, Imoto H, Iida M, Sumimoto H. Expression of isoforms of NADPH oxidase components in rat pancreatic islets. Life Sci 2006; 80: 133-139
• 20 Serrander L, Cartier L, Bedard K, Banfi B, Lardy B, Plastre O, Sienkiewicz A, Forro L, Schlegel W, Krause KH. NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation. Biochem J 2007; 406: 105-114
• 21 Sampson N, Koziel R, Zenzmaier C, Bubendorf L, Plas E, Jansen-Durr P, Berger P. ROS signaling by NOX4 drives fibroblast-to-myofibroblast differentiation in the diseased prostatic stroma. Mol Endocrinol 2011; 25: 503-515
• 22 Schroder K, Zhang M, Benkhoff S, Mieth A, Pliquett R, Kosowski J, Kruse C, Luedike P, Michaelis UR, Weissmann N, Dimmeler S, Shah AM, Brandes RP. Nox4 is a protective reactive oxygen species generating vascular NADPH oxidase. Circ Res 2012; 110: 1217-1225
• 23 Li Y, Mouche S, Sajic T, Veyrat-Durebex C, Supale R, Pierroz D, Ferrari S, Negro F, Hasler U, Feraille E, Moll S, Meda P, Deffert C, Montet X, Krause KH, Szanto I. Deficiency in the NADPH oxidase 4 predisposes towards diet-induced obesity. Int J Obes (Lond) 2012; 36: 1503-1513
• 24 Laleu B, Gaggini F, Orchard M, Fioraso-Cartier L, Cagnon L, Houngninou-Molango S, Gradia A, Duboux G, Merlot C, Heitz F, Szyndralewiez C, Page P. First in class, potent, and orally bioavailable NADPH oxidase isoform 4 (Nox4) inhibitors for the treatment of idiopathic pulmonary fibrosis. J Med Chem 2010; 53: 7715-7730
• 25 Jaquet V, Scapozza L, Clark RA, Krause K-H, Lambeth JD. Small-molecule NOX inhibitors: ROS-generating NADPH oxidases as therapeutic targets. Antiox Redox Signal 2009; 11: 2535-2552
• 26 Ugusman A, Zakaria Z, Hui CK, Nordin NA. Piper sarmentosum inhibits ICAM-1 and Nox4 gene expression in oxidative stress-induced human umbilical vein endothelial cells. BMC Complement Alternat Med 2011; 11: 31
• 27 Jaquet V, Marcoux J, Forest E, Leidal KG, McCormick S, Westermaier Y, Perozzo R, Plastre O, Fioraso-Cartier L, Diebold B, Scapozza L, Nauseef WM, Fieschi F, Krause KH, Bedard K. NADPH oxidase (NOX) isoforms are inhibited by celastrol with a dual mode of action. Br J Pharmacol 2011; 164: 507-520
• 28 Jiang JX, Chen X, Serizawa N, Szyndralewiez C, Page P, Schroder K, Brandes RP, Devaraj S, Torok NJ. Liver fibrosis and hepatocyte apoptosis are attenuated by GKT137831, a novel NOX4/NOX1 inhibitor in vivo . Free Radic Biol Med 2012; 53: 289-296
• 29 Doussiere J, Gaillard J, Vignais PV. The heme component of the neutrophil NADPH oxidase complex is a target for aryliodonium compounds. Biochemistry 1999; 38: 3694-3703
• 30 OʼDonnell BV, Tew DG, Jones OT, England PJ. Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase. Biochem J 1993; 290: 41-49
• 31 Kim JA, Neupane GP, Lee ES, Jeong BS, Park BC, Thapa P. NADPH oxidase inhibitors: a patent review. Expert Opin Ther Pat 2011; 21: 1147-1158
• 32 Borbely G, Szabadkai I, Horvath Z, Marko P, Varga Z, Breza N, Baska F, Vantus T, Huszar M, Geiszt M, Hunyady L, Buday L, Orfi L, Keri G. Small-molecule inhibitors of NADPH oxidase 4. J Med Chem 2010; 53: 6758-6762
• 33 Wind S, Beuerlein K, Eucker T, Muller H, Scheurer P, Armitage ME, Ho H, Schmidt H, Wingler K. Comparative pharmacology of chemically distinct NADPH oxidase inhibitors. Br J Pharmacol 2010; 161: 885-898
• 34 Gaggini F, Laleu B, Orchard M, Fioraso-Cartier L, Cagnon L, Houngninou-Molango S, Gradia A, Duboux G, Merlot C, Heitz F, Szyndralewiez C, Page P. Design, synthesis and biological activity of original pyrazolo-pyrido-diazepine, -pyrazine and -oxazine dione derivatives as novel dual Nox4/Nox1 inhibitors. Bioorg Med Chem 2011; 19: 6989-6999
• 35 Borbely G, Huszar M, Varga A, Futosi K, Mocsai A, Orfi L, Idei M, Mandl J, Keri G, Vantus T. Optimization of important early ADME(T) parameters of NADPH oxidase-4 inhibitor molecules. Med Chem 2012; 8: 174-181
• 36 Lener B, Koziel R, Pircher H, Hutter E, Greussing R, Herndler-Brandstetter D, Hermann M, Unterluggauer H, Jansen-Durr P. The NADPH oxidase Nox4 restricts the replicative lifespan of human endothelial cells. Biochem J 2009; 423: 363-374
• 37 Stockl P, Hutter E, Zwerschke W, Jansen-Durr P. Sustained inhibition of oxidative phosphorylation impairs cell proliferation and induces premature senescence in human fibroblasts. Exp Gerontol 2006; 41: 674-682
• 38 Caldwell SE, McCall CE, Hendricks CL, Leone PA, Bass DA, McPhail LC. Coregulation of NADPH oxidase activation and phosphorylation of a 48-kD protein(s) by a cytosolic factor defective in autosomal recessive chronic granulomatous disease. J Clin Invest 1988; 81: 1485-1496
• 39 Grant JA, Gallardo M, Pickup B. A fast method of molecular shape comparison: A simple application of a Gaussian description of molecular shape. J Computat Chem 1996; 17: 1653-1666
• 40 Mills JE, Dean PM. Three-dimensional hydrogen-bond geometry and probability information from a crystal survey. J Comput Aided Mol Des 1996; 10: 607-622
• 41 Stielow C, Catar RA, Muller G, Wingler K, Scheurer P, Schmidt HH, Morawietz H. Novel Nox inhibitor of oxLDL-induced reactive oxygen species formation in human endothelial cells. Biochem Biophys Res Commun 2006; 344: 200-205
• 42 ten Freyhaus H, Huntgeburth M, Wingler K, Schnitker J, Baumer AT, Vantler M, Bekhite MM, Wartenberg M, Sauer H, Rosenkranz S. Novel Nox inhibitor VAS2870 attenuates PDGF-dependent smooth muscle cell chemotaxis, but not proliferation. Cardiovasc Res 2006; 71: 331-341
• 43 Stefanska J, Pawliczak R. Apocynin: molecular aptitudes. Mediators Inflamm 2008;
• 44 Liou KT, Shen YC, Chen CF, Tsao CM, Tsai SK. The anti-inflammatory effect of honokiol on neutrophils: mechanisms in the inhibition of reactive oxygen species production. Eur J Pharmacol 2003; 475: 19-27
• 45 Banfi B, Tirone F, Durussel I, Knisz J, Moskwa P, Molnar GZ, Krause KH, Cox JA. Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5). J Biol Chem 2004; 279: 18583-18591
• 46 Foti MC, Amorati R. Non-phenolic radical-trapping antioxidants. J Pharm Pharmacol 2009; 61: 1435-1448
• 47 Fato R, Bergamini C, Bortolus M, Maniero AL, Leoni S, Ohnishi T, Lenaz G. Differential effects of mitochondrial complex I inhibitors on production of reactive oxygen species. Biochim Biophys Acta 2009; 1787: 384-392
• 48 Helmcke I, Heumuller S, Tikkanen R, Schroder K, Brandes RP. Identification of structural elements in Nox1 and Nox4 controlling localization and activity. Antiox Redox Signal 2009; 11: 1279-1287
• 49 Lyle AN, Deshpande NN, Taniyama Y, Seidel-Rogol B, Pounkova L, Du P, Papaharalambus C, Lassegue B, Griendling KK. Poldip2, a novel regulator of Nox4 and cytoskeletal integrity in vascular smooth muscle cells. Circ Res 2009; 105: 249-259
• 50 Zhang L, Nguyen MV, Lardy B, Jesaitis AJ, Grichine A, Rousset F, Talbot M, Paclet MH, Qian G, Morel F. New insight into the Nox4 subcellular localization in HEK293 cells: first monoclonal antibodies against Nox4. Biochimie 2011; 93: 457-468
• 51 Nisimoto Y, Jackson HM, Ogawa H, Kawahara T, Lambeth JD. Constitutive NADPH-dependent electron transferase activity of the Nox4 dehydrogenase domain. Biochemistry 2010; 49: 2433-2442
• 52 Kleinschnitz C, Grund H, Wingler K, Armitage ME, Jones E, Mittal M, Barit D, Schwarz T, Geis C, Kraft P, Barthel K, Schuhmann MK, Herrmann AM, Meuth SG, Stoll G, Meurer S, Schrewe A, Becker L, Gailus-Durner V, Fuchs H, Klopstock T, de Angelis MH, Jandeleit-Dahm K, Shah AM, Weissmann N, Schmidt HH. Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration. PLoS Biol 2010; 8: e1000479
• 53 Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev 2001; 46: 3-26