Kinetic and Cellular Characterization of Novel Inhibitors of S-Nitrosoglutathione Reductase

Abstract

S-Nitrosoglutathione reductase (GSNOR) is an alcohol dehydrogenase involved in the regulation of S-nitrosothiols (SNOs) in vivo. Knock-out studies in mice have shown that GSNOR regulates the smooth muscle tone in airways and the function of ␤-adrenergic receptors in lungs and heart. GSNOR has emerged as a target for the development of therapeutic approaches for treating lung and cardiovascular diseases. We report three compounds that exclude GSNOR substrate, S-nitrosoglutathione (GSNO) from its binding site in GSNOR and cause an accumulation of SNOs inside the cells. The new inhibitors selectively inhibit GSNOR among the alcohol dehydrogenases. Using the inhibitors, we demonstrate that GSNOR limits nitric oxide-mediated suppression of NF-B and activation of soluble guanylyl cyclase. Our findings reveal GSNOR inhibitors to be novel tools for regulating nitric oxide bioactivity and assessing the role of SNOs in vivo.

S-Nitrosoglutathione Reductase in Human Lung Cancer

“…Inhibitors of GSNO reductase are in development (as are nitrogen oxide donors) for the treatment of asthma and cystic fibrosis (15,(26)(27)(28)(29). Our data suggest that decreasing the activity of GSNO reductase may change airway biology to favor the development of lung cancer, particularly in the context of exposure to smoke.…”

“…Consistent with a previous report (3), CSNO increased the activity of Ras ( Figure 6G). In the H1650 cancer cell line, this increase tended to be augmented by the GSNO reductase inhibitor C3 (26), although this change did not reach significance ( Figure 6G). The weak effect of C3 may reflect the decreased expression and activity of GSNO reductase in cancer cells at baseline (Figures 1, 2, and 4).…”

“…S-nitrosothiols were assayed as described elsewhere (18). The activity of Ras was assayed by ELISA (catalogue number 17-497; Millipore, Jaffrey, NH) by Raf-1RBD binding, using Fluostar Omega (BMG, Offenburg, Germany) in the presence of GSH (2 mM) and NADH (300 mM), with or without either preincubation with S-nitrosocysteine (CSNO, 10 mM) or the GSNO reductase inhibitor, 5-chloro-3{2-[4-ethoxyphenyl)(ethyl)amino]-2-oxoethyl}-1H-indole-2-carboxylic acid (C2, 100 mM; gift of P. Sanghani [26]). …”

Pharmacological In Vivo Inhibition of S-Nitrosoglutathione Reductase Attenuates Bleomycin-Induced Inflammation and Fibrosis

“…Together, these considerations have suggested that SPL-334 may provide a multifactorial approach to the treatment of ILD as an inducer of antioxidant enzymes to limit oxidant damage (Chen and Kunsch, 2004), as an anti-inflammatory agent (Sanghani et al, 2009;Ferrini et al, 2013), and as a bronchodilator (Ferrini et al, 2013). To assess this possibility of a protective and curative effect of SPL-334 on ILD, we have tested preclinically the preventive and therapeutic effects of SPL-334 in vivo in the bleomycin (BLM) lung injury mouse model.…”

“…GSNO is present in high levels in lung lining fluid and has been shown to exert bronchodilatory activity with a 100-fold higher potency than theophylline (Gaston et al, 1993(Gaston et al, , 1994. By inhibiting GSNOR, SPL-334 inhibits the catabolism of GSNO, which increases the cellular pool of GSNO and the level of nitroso groups on critical proteins, leading to smooth muscle relaxation, decreased inflammation via inhibition of the nuclear factor-kB (NF-kB) pathway, activation of antioxidant pathways via activation of Nrf-2, and increased mucin clearance (Nozik-Grayck et al, 2002Que et al, 2005;Sanghani et al, 2009;Wu et al, 2010;Kelleher et al, 2011;Foster et al, 2012;Ferrini et al, 2013).…”