Discovery of S-Nitrosoglutathione Reductase Inhibitors: Potential Agents for the Treatment of Asthma and Other Inflammatory Diseases

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Pharmacokinetics and safety of cavosonstat (N91115) in healthy and cystic fibrosis adults homozygous for F508DEL-CFTR

Background

Cavosonstat (N91115), an orally bioavailable inhibitor of S-nitrosoglutathione reductase, promotes cystic fibrosis transmembrane conductance regulator (CFTR) maturation and plasma membrane stability, with a mechanism of action complementary to CFTR correctors and potentiators.

Nitric oxide negatively regulates abscisic acid signaling in guard cells by S-nitrosylation of OST1

The phytohormone abscisic acid (ABA) plays important roles in plant development and adaptation to environmental stress. ABA induces the production of nitric oxide (NO) in guard cells, but how NO regulates ABA signaling is not understood. Here, we show that NO negatively regulates ABA signaling in guard cells by inhibiting open stomata 1 (OST1)/sucrose nonfermenting 1 (SNF1)-related protein kinase 2.6 (SnRK2.6) through S-nitrosylation. We found that SnRK2.6 is S-nitrosylated at cysteine 137, a residue adjacent to the kinase catalytic site. Dysfunction in the S-nitrosoglutathione (GSNO) reductase (GSNOR) gene in the gsnor1-3 mutant causes NO overaccumulation in guard cells, constitutive S-nitrosylation of SnRK2.6, and impairment of ABA-induced stomatal closure. Introduction of the Cys137 to Ser mutated SnRK2.6 into the gsnor1-3/ost1-3 double-mutant partially suppressed the effect of gsnor1-3 on ABA-induced stomatal closure. A cysteine residue corresponding to Cys137 of SnRK2.6 is present in several yeast and human protein kinases and can be S-nitrosylated, suggesting that the S-nitrosylation may be an evolutionarily conserved mechanism for protein kinase regulation.Abscisic acid (ABA) plays critical roles in seed dormancy and germination, plant growth, and adaptation to environmental challenges (1, 2). Stresses, such as drought and high salt conditions, increase ABA concentration in plants as a result of ABA biosynthesis or ABA release from its inactive, conjugated forms (3). In the presence of ABA, the ABA receptors in the PYR1 (Pyrabactin Resistance 1)/PYL (PYR1-Like)/RCAR (Regulatory Component of ABA receptor) protein family bind to and inhibit the activity of clade A protein phosphatase 2Cs (PP2Cs), which are considered as coreceptors and negative regulators of ABA signaling (4–6). This process then results in the release of sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) from suppression by the PP2Cs. As central components of the ABA signaling pathway, the activated SnRK2s phosphorylate dozens of downstream effectors to regulate various physiological processes, including stomatal closure, root growth and development, seed dormancy, seed germination, and flowering (7).As the gateway for photosynthetic CO₂ uptake and transpirational water loss, stomata are critical for plant growth and physiology (8). ABA regulates stomatal movement and mutations in ABA biosynthesis genes (9), or in the PYL or SnRK2.6 (also known as OST1) genes cause open-stomata phenotypes (10). On the other hand, dysfunction of the PP2Cs or overexpression of RCAR1/PYL9 causes stomatal closure (5). Among the three SnRK2s, SnRK2.2, -2.3, and -2.6, which are most important for ABA signaling, SnRK2.6 is preferentially expressed in guard cells and plays a critical role in stomatal regulation, whereas SnRK2.2 and -2.3 are mainly expressed in seeds and young seedlings and are thus more important for seed germination and seedling growth (4, 11). SnRK2.6 phosphorylates the slow (S-type) anion channel associated 1 and inward potassium channel KAT1 (K⁺ channel in Arabidopsis thaliana 1) to cause stomatal closure (12, 13).ABA also triggers the generation of several second-messenger molecules, such as calcium, inositol phospholipids, and nitric oxide (NO) and reactive oxygen species (ROS) (14–17). These second messengers are involved in ABA regulation of stomatal closure and other physiological processes (15, 18–20). Among these second messengers, NO has been well documented to have important roles in ABA signal transduction. ABA induces NO generation in roots and guard cells (15, 21). Exogenous application of NO can trigger stomatal closure, whereas application of the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) inhibits stomatal closure (22), suggesting a positive role of exogenous NO in stomatal closure. Several studies have suggested that exogenous NO may affect stomatal responses to ABA by regulating an inward K⁺ channel (20), an anion channel (23), and the generation of nitrated cGMP (24), although the direct target of NO in ABA signaling remains unclear.In plants, NO is produced by the nitrite-dependent nitrate reductase pathway (15, 25) and a pathway dependent on the nitric oxide associated 1 (NOA1) protein (21), although NOA1 is not an NO synthase (26). NO regulates many physiological processes in plants, including responses to phytohormones, such as ABA, cytokinin, auxin, gibberellins, and salicylic acid, immunity against pathogens, senescence, and flowering (15, 27–31). The deficiency in NO generation in the nia1nia2noa1 triple mutant results in ABA-hypersensitive stomatal closure (21), suggesting a negative role of endogenous NO in ABA signaling. NO overaccumulates in Arabidopsis gsnor1(S-nitrosoglutathione reductase 1)/hot5 (sensitive to hot temperatures 5)/par2 (paraquat resistant 2) mutant plants that are impaired in the GSNO reductase gene (32–34). The gsnor1-3 mutant is hypersensitive to heat stress (33) and bacterial pathogen (28, 31), but is more resistant to oxidative stress (34). Characterization of gsnor1 mutant plants suggested that GSNOR regulates multiple developmental and metabolic programs in Arabidopsis (35). In cytokinin signaling, NO causes S-nitrosylation and inhibition of the histidine phosphotransfer protein AHP1 (Arabidopsis histidine phosphotransfer protein 1) (27). In salicylic acid signaling, the S-nitrosylation of the salicylic acid receptor NPR1 facilitates its oligomerization (31). In addition, NO regulates cell death in plant immunity by S-nitrosylation of the NADPH oxidase AtRBOHD (28).Here, we show that GSNO and Cys-NO (S-nitrosocysteine) can inhibit SnRK2.6 by S-nitrosylation. The S-nitrosylation of SnRK2.6 occurs at Cys137, which is adjacent to the catalytic loop of the kinase. A Cys137 to Ser mutation causes the kinase to be resistant to inhibition by GSNO in vitro, whereas a Cys137 to Trp mutation results in an inactive kinase in vitro and in vivo. Introduction of the Cys137 to Ser mutated form of SnRK2.6 into gsnor1-3/ost1-3 (open stomata 1–3, a null allele of snrk2.6) partially suppresses the defect in stomatal closure caused by overaccumulation of SNOs because of the gsnor1-3 mutation. Our results suggest that ABA induced S-nitrosylation of SnRK2.6 functions to negatively feedback regulate ABA signaling in plants. Moreover, the S-nitrosylation of cysteine137 is evolutionarily conserved in some AMPK/SNF1-related kinases and glycogen synthase kinase 3/SHAGGY-like kinases (SKs) in plants, yeast, and mammals, suggesting that S-nitrosylation–mediated inhibition may be a general regulatory mechanism for these eukaryotic protein kinases.     

Formaldehyde dehydrogenase: Beyond phase I metabolism

Formaldehyde dehydrogenase, formally Class III alcohol dehydrogenase (ADH3), has recently been discovered to partially regulate nitrosothiol homeostasis by catalyzing the reduction of the endogenous nitrosylating agent S-nitrosoglutathione (GSNO). Several studies have implicated this enzyme, and in particular GSNO reduction, as playing an important role in conditions such as asthma, cardiovascular disease, and immune function. While ADH3 has received considerable attention in the biomedical literature where it is often referred to as GSNO reductase (GSNOR), ADH3-mediated GSNO reduction has received comparatively less attention in the environmental toxicology community. Herein, evidences for a role of ADH3 in cell signaling through thiol homeostasis is highlighted, underscoring that the enzyme functions more broadly than to metabolize formaldehyde.