The efficacy of honokiol in inhibiting SARS-CoV-2 replication in cell culture

By Neha MathurAug 2 2022Reviewed by Danielle Ellis, B.Sc.

In a recent study posted to the bioRxiv* preprint server, researchers evaluated the efficacy of honokiol (HK) in inhibiting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication in cell cultures.

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Background

HK is a small lignan compound extracted from the barks and leaves of Magnolia trees. Studies have reported several biological effects of HK, including anti-inflammatory and anti-tumor properties. It has been shown to inhibit several viruses in cell cultures and modulate the host response to pathogens. HK has been linked to pathways involved in antiviral responses, including those using nuclear factor erythroid 2–related factor 2 (Nrf2), sirtuin 3 (Sirt3), and mammalian target of rapamycin (mTOR). It provided the rationale for assessing the effect of HK on SARS-CoV-2 infection in cell culture.

Moreover, amid continuously emerging SARS-CoV-2 variants, it is crucial to keep building the anti-SARS-CoV-2 arsenal to increase pandemic preparedness. In this endeavor, the researchers need direct-acting antivirals, compounds that modulate replication pathways of a broad range of viruses and repurposing existing compounds with favorable pharmacokinetics and safety profiles.

About the study

In the present study, researchers evaluated the inhibitory effects of HK in different cell models, varying its concentration, to observe how well it protected these cells against SARS-CoV-2 infection. First, they started with testing Vero E6 cells infected with SARS-CoV-2 using a cytopathic effect (CPE) reduction assay. Next, the team performed a viral load reduction (VLR) assay to confirm that the observed protection in the CPE reduction assays was due to HK only.

Vero E6 cells pretreated with HK for six hours were infected with SARS-CoV-2 at a multiplicity of infection (MOI)=1 for one hour. At 16 hours post-infection (hpi), they quantified viral titers in the supernatant using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR).

The researchers also performed a time-of-addition assay to identify HK inhibited which step of the viral replication cycle. They treated Vero E6 cells with 20 µM of HK at different time points. At zero hours, they infected cells with SARS-CoV-2 at an MOI of one and harvested supernatant at 10 hpi to quantify infectious virus titers in the supernatant. The team tested human lung cell line A549 expressing angiotensin-converting enzyme (ACE2)-transmembrane protease, serine 2 (TMPRSS2) in a VLR assay. They used cells pretreated with HK at increasing concentrations and infected with SARS-CoV-2 at an MOI of one.

Finally, the team treated Vero E6 cells with HK for six hours and then infected them with SARS-CoV-2 variants of concern (VOCs) Delta (B.1.617.2) and Omicron 237 (B.1.1.529) at an MOI of one. They determined the viral load at 16 hpi by RT-qPCR. Similarly, the researchers infected VeroE6 cells pretreated with HK for six hours at an MOI of one to evaluate its effectiveness against the Middle East Respiratory Syndrome coronavirus (MERS-CoV) and HCoV-229E.

Study findings

SARS-CoV-2 infected Vero E6 cells showed no signs of virus-mediated cytotoxicity for all HK concentrations tested. They showed increased viability, with a half maximal effective concentration (EC50) of ~7.8 µM. Also, the HK effect was cell line-independent as it inhibited SARS-CoV-2 replication in African green monkeys Vero E6 and human A549 cells expressing ACE2 and TMPRSS2.

The researchers observed a 99% reduction in viral RNA copies caused by HK in a dose-dependent manner. Furthermore, a plaque assay showed that 20 µM of HK caused an approximate three log reduction in infectious virus titer. The findings suggested that 20 µM HK specifically inhibited SARS-CoV-2 replication and protected cells from virus-induced CPE without causing measurable cytotoxicity.

The authors did not observe any difference in effectiveness between HK treatments initiated at any time between eight and one hour before infection. Time-of-addition analysis revealed maximal HK inhibition between two and eight hpi but lost its antiviral effect after eight hpi. It indicates that HK does not interfere with the early steps of the viral replication cycle but instead inhibits a post-entry replication step.

HK also inhibited MERS-CoV and HCOV-229E replication in cell culture. These viruses use dipeptidyl peptidase-4 (DPP4) and human aminopeptidase N as receptors, respectively, and not ACE2. Therefore, studies are currently studying the role of multiple host factors that may be responsible for the observed antiviral effects of HK. At 20 µM, HK inhibited both the Delta and Omicron VOCs, showing maximal antiviral effect against Delta, with a ~two-log reduction in copy number. Although its sensitivity to Omicron was the poorest, the researchers still observed a ~96% reduction in cells treated with 20 µM HK.

Conclusions

Overall, the oral administration of HK appeared to be well-tolerated and shall increase its acceptance as a therapeutic agent. Moreover, HK had good bioavailability, with HK getting adsorbed quickly and reaching its peak plasma concentration in 20 minutes and various tissues in barely five minutes. Later, it was eliminated slowly, with a half-life of ~290 minutes. During its intravenous administration, it showed a peak followed by elimination, ~56 minutes after a 10mg/kg dose. Previous human clinical trials showed that HK had no side effects on the majority of participants.

For SARS-CoV-2, HK could provide the additional benefit of reducing pathologic inflammation because of its anti-inflammatory effects in vivo. More importantly, it is safe, shows broad-spectrum antiviral effects against multiple CoVs, and reaches peak plasma levels within minutes of administration, all of which make it a viable compound for testing in animal studies and clinical trials as a SARS-CoV-2 drug candidate.

*Important notice: 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.