am-36 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

Determination of extent of infarction in animal models of cerebral ischemia is most commonly achieved by either classical histology (thionin staining) and light microscopy or staining with 2,3, 5-triphenyltetrazolium chloride (TTC). These techniques have limitations and we now describe a novel technique and its validation for assessment of the neuroprotective activity of AM-36, a novel arylalkypiperazine compound with combined antioxidant and sodium channel blocking activity. AM-36 (1.8 mg/kg i.p.) or vehicle, was administered 30 min, 24 and 48 h after endothelin-1-induced middle cerebral artery occlusion in conscious rats. Rats were killed at 72 h, brains removed and frozen in liquid nitrogen prior to coronal sectioning. Using a simple apparatus relying on basic principles of light propagation and a computerised image analysis system, ischemic damage in unstained slide-mounted sections was clearly visualised and measured. AM-36 significantly reduced the area of infarct in both cortex and striatum. The method was verified by thionin staining, and light microscopy. Linear regression analysis showed a highly significant correlation between methods at 72 h for infarct area in the cortex and striatum. Highly significant correlations between methods were found at 3 and 24 h after ischemia. Our method quickly and clearly delineates areas of damage in a manner superior to conventional staining methods.

Topics: Animals; Brain Ischemia; Cerebral Cortex; Disease Models, Animal; Image Processing, Computer-Assisted; Male; Microscopy, Video; Neostriatum; Nerve Degeneration; Neuroprotective Agents; Piperazines; Rats; Rats, Wistar; Stroke; Time Factors