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Determining cell death
BETHESDA, Md.—Researchers at the National Center for Advancing Translational Sciences (NCATS), part of the National Institutes of Health, recently identified compounds that could potentially inhibit Zika virus replication and reduce its neurological damage.
According to Dr. Wei Zheng, an NCATS researcher, “The collaborators at Johns Hopkins University (JHU), Baltimore, and Florida State University (FSU), Tallahassee, found that Zika virus increases caspase-3 activity in human neural progenitor cells, resulting in cell death. Based on this information, NCATS researchers developed a high-throughput caspase-3 assay and identified lead compounds after a screening of approximately 6,000 compounds. This led to identification of lead molecules that inhibit Zika virus replication and reduce its ability to kill brain cells.”
Using NCATS’ drug repurposing screening robots, researchers identified compounds that were effective against Zika on the basis of antiviral activity and/or effect on Zika-related brain cell death. The compounds include emricasan, an investigational drug currently being evaluated in a clinical trial to reduce liver injury and fibrosis, and niclosamide, a U.S. Food and Drug Administration-approved drug for use in humans to treat worm infections. The researchers also identified nine cyclin-dependent kinase (CDK) inhibitors. CDK is usually involved in regulation of cellular processes as well as normal brain development, but the Zika virus can negatively affect this process.
Results were published in the August 29 issue of Nature Medicine. The NCATS screening effort builds on the initial research by JHU and FSU scientists, who discovered that the Zika virus infects brain cells early in development. Zika virus infection may be related to fetal microcephaly, an abnormally small head resulting from an underdeveloped and/or damaged brain, as well as being associated with neurological diseases such as Guillain-Barré syndrome in infected adults.
Zheng and his team led the drug repurposing screen to test three strains of Zika: Asian, African and Puerto Rican. The scientists first developed an assay using caspase 3, a protein that causes brain cell death when infected by the virus. The next step was screening 6,000 FDA-approved and investigational compounds, which resulted in the identification of more than 100 promising compounds. The team then evaluated the protective effect of these compounds in brain cells after Zika virus infection, and three lead compounds—emiracsan, niclosamide and a CDK inhibitor known as PHA-690509—were found to reduce neuronal cell death caused by Zika virus infection.
These compounds were effective either in inhibiting the replication of Zika or in preventing the virus from killing brain cells. For example, emricasan prevents cell death, and niclosamide and the nine CDK inhibitors stop the virus’ replication. The team also found that emricasan, when combined with one of the CDK inhibitors, prevented both cell death and virus replication. The team noted that the CDK inhibitors may be useful in treating non-pregnant patients who face an increased risk of Guillain-Barré syndrome and other conditions sparked by Zika infection, but cautioned that the use of emricasan and niclosamide during pregnancy for Zika infection will need to be evaluated in preclinical toxicology studies and clinical trials.
“We developed and tested two screening assays: the caspase-3 activity assay measuring Zika virus induced caspase-3 activity and the cell viability assay measuring Zika virus caused cell death,” Zheng explained. “Because the compound library screening requires robust and sensitive assays in a miniaturized assay format (such as a 1,536-well plate), only the caspase-3 activity assay worked for the high-throughput screening. The cell viability assay had a big variability and failed in the screening of large compound collections. The neural progenitor cells and astrocytes derived from human induced pluripotent stem cells are not easy to produce in large quantity for compound screening and cost is very high. The Zika virus solutions also had big variations from batch to batch. We were able to resolve and overcome these problems one by one.”
Zheng added, “Using the NCATS drug repurposing platform for emerging infectious diseases can help rapidly identify potential treatments for urgent needs such as the Zika virus. While identifying promising compounds is a first step, our goal at NCATS is to facilitate the translation of these findings for evaluation in the clinic. The release of all the compound screening data in this publication and in the public PubChem database opens the door to the research community to do just that.”
NCATS’ screening effort enabled the broader research team to quickly translate earlier discoveries toward work to develop treatments for Zika virus infection. Collaborators at both universities are performing mechanistic studies of these lead compounds and evaluation of their in-vivo efficacies in the mouse models of Zika virus infections and microcephaly. They are also working on the development of second generations of compound screening assays in order to facilitate additional drug screening for anti-Zika drug development.
JHU is working on a mouse model to study the neuroprotective effects of the compounds identified from the screen and studying the mechanism of action of the lead compounds. FSU is testing the efficacy of these compounds in a Zika virus mouse model and is also studying the mechanism of action of the lead compounds.
Zheng concluded, “NCATS’ role in the translational research is mainly catalytic. We have published the research results and deposited all the drug screening data into PubChem data base for open access by the entire research community. We hope that our collaborators, other Zika researchers and industrial partners will move the drugs to preclinical and clinical studies quickly.”