n6022 and Disease-Models--Animal

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

The nitric oxide (NO) metabolome and the NO metabolite-based neurovascular protective pathways are dysregulated after stroke. The major NO metabolite S-nitrosoglutahione (GSNO) is essential for S-nitrosylation-based signaling events and the inhibition of S-nitrosoglutahione (GSNO)-metabolizing enzyme GSNO reductase (GSNOR) provides protective effects following cardiac ischemia. However, the role of GSNOR and GSNOR inhibition-mediated increased GSNO/S-nitrosylation is not understood in neurovascular diseases such as stroke. Because age is the major risk factor of stroke and recovery in aged stroke patients is low and slow, we investigated the efficacy of GSNOR inhibition using a GSNOR selective inhibitor N6022 in a clinically relevant middle-aged cerebral ischemia and reperfusion (IR) mouse model of stroke. N6022 (5 mg/kg; iv) treatment of IR mice at 2 h after reperfusion followed by the treatment of the same dose daily for 3 days reduced the infarct volume and decreased the neurological score. Daily treatment of IR animals with N6022 for 2 weeks significantly improved neurological score, brain infarctions/atrophy, survival rate, motor (measured by cylinder test) and cognitive (evaluated by novel object recognition test) functions which paralleled the decreased activity of GSNOR, reduced levels of peroxynitrite and decreased neurological score. These results are the first evidence of a new pathway for the treatment of stroke via the inhibition of GSNOR. Based on the efficacy of N6022 in the stroke animal model and its use in human therapeutic studies without toxicity, we submit that GSNOR is a druggable target, and N6022 is a promising drug candidate for human stroke therapy.

Topics: Aging; Alcohol Dehydrogenase; Animals; Benzamides; Disease Models, Animal; Drug Delivery Systems; Male; Mice; Mice, Inbred C57BL; Pyrroles; Recovery of Function; Stroke

The nitric oxide (NO)-producing activity of endothelial nitric oxide synthase (eNOS) plays a significant role in maintaining endothelial function and protecting against the stroke injury. However, the activity of the eNOS enzyme and the metabolism of major NO metabolite S-nitrosoglutathione (GSNO) are dysregulated after stroke, causing endothelial dysfunction. We investigated whether an administration of exogenous of GSNO or enhancing the level of endogenous GSNO protects against neurovascular injury in wild-type (WT) and eNOS-null (endothelial dysfunction) mouse models of cerebral ischemia-reperfusion (IR).. Transient cerebral ischemic injury was induced by middle cerebral artery occlusion (MCAO) for 60 minutes in male adult WT and eNOS null mice. GSNO (0.1 mg/kg body weight, intravenously) or N6022 (GSNO reductase inhibitor, 5.0 mg/kg body weight, intravenously) was administered 30 minutes before MCAO in preinjury and at the reperfusion in postinjury studies. Brain infarctions, edema, and neurobehavioral functions were evaluated at 24 hours after the reperfusion.. eNOS-null mice had a higher degree (P< .05) of injury than WT. Pre- or postinjury treatment with either GSNO or N6022 significantly reduced infarct volume, improved neurological and sensorimotor function in both WT and eNOS-null mice.. Reduced brain infarctions and edema, and improved neurobehavioral functions by pre- or postinjury GSNO treatment of eNOS knock out mice indicate that GSNO can attenuate IR injury, likely by mimicking the eNOS-derived NO-dependent anti-ischemic and anti-inflammatory functions. Neurovascular protection by GSNO/N6022 in both pre- and postischemic injury groups support GSNO as a promising drug candidate for the prevention and treatment of stroke injury.

Topics: Alcohol Dehydrogenase; Animals; Behavior, Animal; Benzamides; Brain; Brain Edema; Disease Models, Animal; Enzyme Inhibitors; Infarction, Middle Cerebral Artery; Male; Mice, Inbred C57BL; Mice, Knockout; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Synthase Type III; Pyrroles; S-Nitrosoglutathione

The pyrrole based N6022 was recently identified as a potent, selective, reversible, and efficacious S-nitrosoglutathione reductase (GSNOR) inhibitor and is currently undergoing clinical development for the treatment of acute asthma. GSNOR is a member of the alcohol dehydrogenase family (ADH) and regulates the levels of S-nitrosothiols (SNOs) through catabolism of S-nitrosoglutathione (GSNO). Reduced levels of GSNO, as well as other nitrosothiols (SNOs), have been implicated in the pathogenesis of many diseases including those of the respiratory, cardiovascular, and gastrointestinal systems. Preservation of endogenous SNOs through GSNOR inhibition presents a novel therapeutic approach with broad applicability. We describe here the synthesis and structure-activity relationships (SAR) of novel pyrrole based analogues of N6022 focusing on removal of cytochrome P450 inhibition activities. We identified potent and novel GSNOR inhibitors having reduced CYP inhibition activities and demonstrated efficacy in a mouse ovalbumin (OVA) model of asthma.

Topics: Aldehyde Oxidoreductases; Animals; Asthma; Benzamides; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Design; Drug Evaluation, Preclinical; Enzyme Inhibitors; Humans; Imidazoles; Lung; Mice; Molecular Structure; Molecular Targeted Therapy; No-Observed-Adverse-Effect Level; Pyrroles; Receptors, Opioid, delta; S-Nitrosoglutathione; Structure-Activity Relationship