Discovery of New Neutrophil NADPH Oxidase Inhibitors

Neutrophils are well known to play a major role in acute inflammatory responses, and more recently, evidence has expanded the role of these cells in chronic inflammatory and autoimmune diseases. Superoxide anion release from infiltrating neutrophils, the so-called respiratory burst, is recognized to contribute to the pathogenesis of lung injury by damaging pulmonary interstitial tissue and causing an inflammatory response. NADPH oxidase plays a dominant role in superoxide anion production in human neutrophils. The NADPH oxidase is assembled by the cytosolic p47phox, p67phox, p40phox, and small GTP-binding protein Rac2 with flavocytochrome b558 complex on cell membranes. Dysregulation of NADPH oxidase plays an essential role in the development of the pathophysiology associated with neutrophil-dominated inflammation, including acute lung injury, acute respiratory distress syndrome, chronic obstructive pulmonary disease, and sepsis. Therefore, neutrophil NADPH oxidase is an attractive therapeutic target for inflammatory lung diseases. Currently, few drugs are used in clinical applications to restrain neutrophil function in inflammatory respiratory diseases. The purpose of this study is to research and development of new NAPDH oxidase inhibitors to treat the neutrophilic inflammatory diseases. In preliminary experiments, we have examined fifty-eight new synthetic compounds on the generation of superoxide anion and the release of elastase in formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF)-activated human neutrophils. Among these compounds, CYR5099 (4-(ω-aminoalkoxyimino)-1-alkyl-1H-benzo[f]indol-9(4H)-one derivative) showed specific inhibition of superoxide anion generation in fMLF-activated human neutrophils. We found that CYR5099 not only failed to scavenge superoxide anion in a cell-free assay, but also did not affect the cellular signal transduction pathways, including Src, Akt, ERK, JNK, p38 MAPK, and calcium. These results suggested that CYR5099 exerts its inhibitory effects on neutrophil respiratory burst by modulation of NADPH oxidase. Consistent with the hypothesis, CYR5099 inhibited the activity of NADPH oxidase in sodium dodecyl sulfate-reconstituted subcellular system. Certainly, the mechanisms of action of CYR5099 will be further investigated in this project. On the basis of these preliminary findings, we therefore propose a three-year project to conduct studies in the following aspects: (1) to examine the anti-inflammatory effects of CYR5099 in human neutrophils; (2) to investigate the mechanism of action of CYR5099 for inhibition of NADPH oxidase; (3) to identify the binding protein of CYR5099 in NADPH oxidase; (4) to establish the structure activities relationships of NADPH oxidase inhibitors and find lead compounds; (5) to confirm the anti-inflammatory effects of NADPH oxidase inhibitors in in vivo lung inflammatory models. Based on the importance of NADPH oxidase in a wide range of important inflammatory processes, we expect our research will provide new insight into the pharmacological profile of NADPH oxidase inhibitors in human neutrophils. In addition, these new inhibitors of neutrophil NAPDH oxidase can be used as lead compounds for further development of new anti-inflammatory drugs.

Project ID:PC10501-1764
External Project ID:MOST104-2320-B182-006-MY3