With the rapid spread of the virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) throughout the world, causing the current COVID-19 pandemic, there is an urgent need for new antivirals and vaccines to combat its spread. The current study published on the preprint server bioRxiv* in June 2020 shows some of the results of the extensive repurposing of already FDA-approved drugs.
Novel Coronavirus SARS-CoV-2 Colorized scanning electron micrograph of a cell (green) infected with SARS-COV-2 virus particles (purple), isolated from a patient sample. Image captured at the NIAID Integrated Research Facility (IRF) in Fort Detrick, Maryland. Credit: NIAID
Approved antiviral drugs target viral proteins that are essential for successful viral infection – these are called direct-acting antivirals. On the other hand, host-targeted antivirals are those which block physiological pathways in the host cell that are taken over by the virus for successful replication, assembly, and other activities required for established viral infection.
The Study: Screening for Antiviral Activity in COVID-19
Earlier, remdesivir, which was developed against the Ebola RNA-dependent RNA polymerase, was repurposed for use in SARS-CoV-2. Other drugs that are being trialed for this new use after being approved for other indications include chloroquine and hydroxychloroquine (HCQ), based on in vitro studies. It must be stressed that the latter drug has not lived up to its hype in clinical trials.
The current study developed a screening tool to identify effective anti-SARS-CoV-2 antivirals from the existing library of over 3,000 molecules and drugs. While testing, the researchers found that the human hepatocyte cell line Huh7.5 was readily infected with SARS-CoV-2. Screening in this human cell line, they validated 23 drugs that were active in dose-response experiments and showed a favorable selective index versus toxicity. These candidates targeted a wide variety of cellular activities, but few were active in Vero cells. However, one class, the chloroquines, and their derivatives were active in both cell types. They found that remdesivir showed antiviral activity in Calu-3 culture, which is derived from lung epithelium, but not HCQ, or several quinolines.
In lung epithelial cells, they found that viral entry and drug response was quite different in these cells, being independent of pH conditions and dependent on the presence of the protease TMPRSS2. On the other hand, in the earlier cultures, a low pH was necessary, while viral entry required acid-dependent endosomal protease activity.
They found seven antivirals in lung cells, of which 7 have already been tested, and 3 are approved. These antivirals show essential host target proteins and can be switched into clinical use rapidly once their efficacy is proved since they have already passed safety and dosage tests.
Camostat, which inhibits TMPRSS2 activity, is very active in these cells as well, but not in the two other cell types already mentioned. This may mean that different types of cells bind the virus leading to viral entry by different modes.
Direct-acting antivirals can act against viral infection in many cell types, which is an advantage observed with remdesivir. However, host-targeted antivirals inhibit significant steps in the viral lifecycle and are conserved so that their expression is likely to remain unchanged across a variety of cell types. Despite this theoretical framework, the researchers observed that there was a significant disparity in the antiviral activity shown by most drugs across the different cell types.
Viral Infection Occurs with All 3 Cell Types
The researchers observed that the use of Vero cells allows direct-acting antivirals to be screened because these cells allow viral entry and infection. Huh7.5 cells, on the other hand, also readily allow infection with SARS-CoV-2, perhaps because they lack innate immune signaling. The IC50 at which the drugs remdesivir and HCQ inhibit replication in these two cell types is over 50-fold apart.
The increased antiviral activity of these drugs is echoed by nanchangmycin. All these suggest greater sensitivity to some antivirals and may help to differentiate those that are selectively active against human host targets.
Antiviral Efficacy Varies with Cell Culture
Using Calu-3 cells, they found that remdesivir had antiviral activity but not HCQ or other chloroquine derivatives. On the other hand, these are antiviral in Vero and HuH7.5 cell lines. This suggests, say the researchers, “there are major differences in the requirement for endosomal acidification during infection of SARS-CoV-2 in lung epithelial cells.”
Endosomal acidification is essential because the endosomal protease cathepsin cleaves the viral S protein only at low pH. The cathepsin inhibitor Z-FA-FMK thus successfully inhibited viral activity in both Vero and Huh7.5 cell types because these cells need acid pH for viral entry.
The researchers resorted to testing in Calu-3 cells to find the model that was most relevant for human infection. They used dose-response testing to identify the most significant antiviral activity and found nine drug candidates. The presence of several kinase inhibitors in the list of active drugs suggests that these cell signal facilitators are required for viral infection.
Cyclosporin: Mechanism of Action
Cyclosporin is one of the drugs on the list, an FDA-approved immunosuppressant drug, readily available and with a low IC50, and selective in its action on both Calu-3 and Huh7.5 cells. It acts by binding to cyclophilin A, which blocks the activation of the phosphatase calcineurin.
Calcineurin is a phosphate-cleaving enzyme crucial for the nuclear factor in activated T cells to be translocated inside the nucleus. Molecules that inhibit calcineurin activity in T cells are used for their immunosuppressant activity.
Cyclosporin is known to have antiviral activity against multiple viruses, including other coronaviruses (CoVs), but this depends on cyclophilin rather than calcineurin pathways. The researchers found antiviral activity in four of the drug analogs, namely, isocyclosporin A, cyclosporin A, B, and C.
These were all active, but the IC50 increased from the first to the fourth. A derivative of cyclosporin, PSC 833, has a similar antiviral activity to cyclosporin C but does not suppress immunity. Similarly, cyclophilin A inhibitor TMN355 has 27-fold activity compared to cyclosporin A.
NIM811 is a second derivative of cyclosporin that is powerfully antiviral without causing immunosuppression. This indicates a second line of evidence that cyclophilin inhibition is the mechanism of the antiviral activity of cyclosporin rather than calcineurin inhibition. The antiviral activity of cyclosporin and its derivatives are preserved across cell lines, which indicates that they all act through the same mechanisms. Significantly, none of them inhibit viral activity in Vero cells.
Finally, the calcineurin inhibitor FK506, which binds immunophilin FKBP and not cyclophilin A, to produce a block of the phosphatase enzyme, is not antiviral but does have a powerful immunosuppressant effect. Neither do NFAT inhibitors (NFAT being another primary target of calcineurin) have an antiviral effect.
In short, the inhibition of the enzymatic activity of cyclophilin A is not essential in the antiviral activity of cyclosporine, but rather, it works by blocking cyclophilin-dependent viral infection via some other mechanism. Thus, the researchers conclude, “Cyclosporins are potent antivirals against SARS-CoV-2 in lung epithelial cells, and that this activity is independent of Calcineurin and NFAT.”
The researchers also discuss other drugs like salinomycin, ebastine, other protease inhibitors (including Z-FA-FMK), natural antivirals, metabolic inhibitors, kinase inhibitors, mTOR inhibitors, and some cannabinoid receptor inhibitors, which show varying degrees of antiviral effect against SARS-CoV-2.
The study also highlights the need to garner much more data about how the culture conditions affect the antiviral effects observed with the use of different drugs, and to validate these observations in lung epithelial cells resembling the actual viral target. “Altogether,” the researchers sum up, “these studies highlight the roles of cellular genes in viral infection, cell-type differences, and our discovery of nine broadly active antivirals suggest new avenues for therapeutic interventions.”
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.