Even as the coronavirus disease 2019 (COVID-19) pandemic continues to take a heavy toll on the life and health of the global population – not to mention economic activity – effective therapies remain at a premium. Despite herculean drug development and repurposing efforts, few results have appeared to justify the investment.
A new study by researchers at The Hebrew University of Jerusalem in Israel reports the discovery of a promising group of E channel inhibitors with significant antiviral activity. This could be developed into new treatment agents for this often-deadly virus.
The research team has released its findings as a preprint on the bioRxiv* server.
The viral E protein
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), identified as the pathogenic agent in this pandemic, is an enveloped RNA virus with several membrane proteins. One of these is the E protein.
The E protein takes part in the viral lifecycle, being involved in its assembly, release and mechanism of disease in other coronaviruses. They appear to be key in viral infectivity. When the E protein is weakly expressed, the virion is also attenuated and may serve as a vaccine agent.
E proteins from SARS-CoV and SARS-CoV-2, among other similar proteins, are ion channels to transport cations into the cell.
These are classical targets for drug development, such as calcium channel blockers in hypertension, sodium channels in cystic fibrosis, and many others. With the influenza virus, aminoadamantine drugs suppress the activity of the viral M protein, which is an ion channel. However, these are no longer very useful due to the extensive development of resistance.
Screening by reciprocal assays
The current study used already approved drugs in libraries to screen around 3,000 drugs that inhibit E channels. The researchers looked at the hits for possible anti-SARS-CoV-2 activity.
As a first step, the E protein was expressed on bacteria in excess, causing increased membrane permeability and thus depressed growth. Inhibitors of the viral channel are thus easily identifiable by the restoration of bacterial growth.
They then screened the chemical library at concentrations of 50 μM, looking for hits. Each hit in this negative assay was then screened at different concentrations.
To rule out confounding factors that could also affect bacterial growth, they tested each shortlisted compound with a reciprocal inhibition test.
The second assay used bacteria that cannot take up potassium ions in normal cell media but thrive following the expression of a potassium channel even in low-potassium media. This positive assay yields results of channel blockade in the form of restricted growth.
Here again, hits were further tested in a dose-response assay.
Finally, a fluorescence-based test was used to detect alterations in the pH of the bacteria. If the bacteria are exposed to acidified media, and if they express a hydrogen ion channel, an obvious fluorescence is the result. Blockade of hydrogen ion channels will cause reduced fluorescence, therefore.
Of the eight hits from the two assays, six were active against the channel, as reflected by a reduction in fluorescence. The exceptions were 5-azacytidine and mebrofenin.
Cytotoxicity was measured for each compound with respect to cell metabolic activity markers.
Out of the large number of compounds tested, they found eight hits, to which another two were added from an earlier study on calcium channel blockers. These were tested against viruses in culture.
At a concentration of 10 μM, nine of the ten compounds inhibited viral growth, reducing the viral load by 98% in one case (5-azacytidine). Six reduced it by 40-50%, while the other two reduced it by up to a quarter. Memantine was the exception, probably due to its low affinity.
No toxicity against the host cells was observed, except for 5-azacytidine. The direct cytotoxicity of this drug may have contributed significantly to its very high antiviral activity rather than selective E channel inhibition.
What are the implications?
Repurposing is not only a reliable and valuable route towards drug discovery against specific targets, especially against viruses. It also uses approved drugs, reducing the time and chemical space required to bring out an effective agent.
Most repurposing studies have relied on in silico methods, while a few have used an experimental design. The approach used in this study was different in that the large library was screened for compounds with inhibitory activity against the E protein.
Not only are these channels highly suitable as drug targets, but their inhibitors may be rapidly and cheaply sought without expensive experimental workflows. The use of bacteria for screening could make it possible to identify many more inhibitors of the single protein target because of the inherent genetic selection in these microbes, conferring higher tolerance to toxicity.
The use of two reciprocal assays minimizes the chances of picking up the wrong hits.
The high screening concentrations used in this study allowed eight compounds to be identified – a high yield. Even at relatively lower affinities, such drugs can also have significant antiviral activity if modified further. Alternatively, they may have synergistic antiviral activity when combined with other selective inhibitors directed against other viral targets.
E channel inhibition may not be the only mechanism of antiviral activity operating among these compounds. Indeed, with 5-azacytidine, transcriptional networks may be widely modified by its known function as a DNA methyl donor.
The findings, therefore, present a hit list of approved drugs that may be used as a starting point for the synthesis of SARS-CoV-2 inhibitors. This approach could be used to identify other useful ion channel inhibitors as well, to help fight this and other viruses.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.