Selected GSNOR inhibitors were assessed
Selected GSNOR inhibitors were assessed for potential off-target activity with a panel of 54 transmembrane and soluble receptors, ion channels, and monoamine transporters. Off-target effects were estimated from the percent inhibition of receptor radio-ligand binding in the presence of 10μM of test compound. Typical binding assays were performed with a minimum of 6-control wells with/without vehicle for soluble compounds. Inhibition of 50% or greater was considered a positive response. Limited off-target activity was observed towards the δ2 opiate receptor for make money , , and similar to as reported earlier. Compound demonstrated potent GSNOR inhibition and a clean profile toward CYP inhibition, but exhibited 69% inhibition against the opioid peptide receptor (Mu). Compounds , , and were also screened for cytotoxicity towards the A549 epithelial lung cell line. The IC values for and were >250μM, and minimal cytotoxicity for (IC=160μM) was observed.
Compounds , , and were tested in mouse pharmacokinetic studies. Oral bioavailability of these compounds was 0.7%, 1.6%, and 0.49%, respectively, compared to 4.4% for reported earlier. The plasma clearance (CL) after intravenous (IV) administration was 54.1, 31.6, and 16.8ml/min/kg for , , and , respectively, compared to 37.7ml/min/kg for .
Compound was tested in a 5-day mouse toxicity study with intravenous QD dosing at 1, 10, or 50mg/kg. The results of this study suggest that treatment of male CD-1 mice with for 5days had no adverse effects. This study resulted in a no observable adverse effect level (NOAEL) of 50mg/kg for IV treatment. Compound was also tested in a 5-day mouse toxicity study with IV BID dosing at 5, 25, or 50mg/kg. To our surprise, the treatment of male CD-1 mice with for 5days resulted in significant adverse effects on numerous study endpoints. In particular, histological findings demonstrated toxicity to the liver, spleen, and thymus of treated animals. The NOAEL for could not be established from the study and was considered to be <10mg/kg/day.
The efficacy of GSNOR inhibitors was assessed in an animal model of asthma, a disease influenced by dysregulated GSNOR and altered function of NO, GSNO, and SNOs. In a mouse model of ovalbumin-induced asthma, compound attenuated methacholine-induced bronchoconstriction (airway hyper-responsiveness) and eosinophil infiltration into the lungs following a single IV dose administered 24h prior to the methacholine challenge. Efficacy was observed at doses ⩾0.01mg/kg compound . In a similar study, compound also attenuated methacholine-induced airway hyper-responsiveness and eosinophil infiltration into the lungs following a single IV dose of 1mg/kg administered 24h prior to the methacholine challenge.
In conclusion, based on the crystal structure of GSNOR inhibitors and crystal structure of ketoconazole with CYP 3A4, substitution of the imidazole at 2-position with small alkyl group and replacement of the phenyl ring with thiophene led to potent GSNOR inhibitors that demonstrated significantly reduced CYP inhibition. These findings validate the initial hypothesis of the imidazole as a key pharmacophore for the binding of GSNOR inhibitors to cytochrome enzymes causing CYP inhibition. However, despite the improved in vitro profile of GSNOR inhibitor , this compound exhibited a less attractive pharmacological profile than due to the surprise in vivo toxicity observed in the 5-day exploratory toxicity evaluation in mice. The studies described in this paper provided more insight into the understanding of GSNOR inhibition, the inhibitor–enzyme interaction, and structure–in vitro toxicity relationship of GSNOR inhibitors.
Signaling function of nitric oxide and its regulation Nitric oxide (·No) plays a crucial signaling role during plant growth and development and during stress response , , . ·No can affect enzyme/protein activity, translocation and protein function by posttranslational modifications. The predominant way of ·No action is S-nitrosylation – the reversible covalent attachment of ·No to cysteine thiols. Other ·No-dependent modifications are nitrosylation of metal centers of metalloproteins and the irreversible nitration of protein tyrosine residues , . As a free radical, ·No has a very short lifetime that restricts their effect to the local microenvironment. However, S-nitrosylated glutathione (S-nitrosoglutathione, GSNO) is a quite stable NO reservoir and ·No transport form, which can transfer its ·No moiety to proteins (trans-nitrosylation) , , , . GSNO level is regulated either by its production or degradation. The production depends on the availability of ·No and GSH and the presence of aerobic conditions, which promote their reaction . The primary conclusions regarding GSNO formation under aerobic conditions are that oxidation of ·No by oxygen is a prerequisite, and nitrosation occurs either through the formation of dinitrogen trioxide or though the addition of ·No to a glutathionyl radical formed during the reaction. GSNO can be degraded in non-enzymatic and enzymatic processes. Non-enzymatic processes include treatment with UV-light, high temperature or alkaline conditions. The enzymatic turnover mechanism is catalysed by GSNO reductase (GSNOR, At5g43940), which was originally identified in plants and other organisms as a glutathione-dependent formaldehyde dehydrogenase (GS-FDH; EC 188.8.131.52) , . Later it was demonstrated that the catalytic reaction involved the oxidation of a hydroxyl group of S-(hydroxymethyl)glutathione – a spontaneous adduct of glutathione and formaldehyde – to build S-formylglutathione leading to reclassification as S-(hydroxymethyl)glutathione dehydrogenase (EC 184.108.40.2064). In 2001 it was shown that this enzyme is involved in the S-nitrosothiol metabolism  and its GSNO degrading activity was described for Arabidopsis enzyme . GSNOR is a highly conserved enzyme in mammals, yeast and plants and is essential to protect cells under nitrosative stress , . GSNOR belongs to the class III alcohol dehydrogenase family (EC 220.127.116.11) . The crystal structure of GSNOR from mammals, yeast and plants revealed that the enzyme is a homodimer coordinating two zinc atoms per subunit , . Ammonia (NH3) and glutathione disulfide (GSSG) were main products of GSNOR according to the following scheme: