A recent study published on the bioRxiv * prepress server assessed whether niclosamide could be used in the treatment of coronavirus disease 2019 (COVID-19).
Diagram of the effect of niclosamides on SARS-CoV-2 entry and spike protein-mediated syncytium formation. 1.) SARS-CoV-2 binds to and enters the ACE2 receptor of the host cell. Niclosamide has been shown to inhibit this in vitro entry step 2.) Viral replication generates many copies of the RNA genome. 3.) Infection results in increased expression of the viral spike protein (S) and the TMEM16F host cell in the plasma membrane. 4.) The S protein on the surface of an infected cell binds to the ACE2 receptor of an uninfected adjacent cell. 5.) Spike-dependent syncytial formation is mediated by calcium-dependent lipid scramblase TMEM16F to generate multinucleated infected cell bodies. Niclosamide, an inhibitor of TMEM16F, has been shown to block the formation of spike-dependent syncytia.
Fund
Coronavirus 2 (SARS-CoV-2) therapies for severe acute respiratory syndrome, which can stop or treat viral transmission, are still urgent.
Niclosamide, an oral anthelmintic agent authorized by the Food and Drug Administration (FDA), has broad biological functions, such as antibacterial, antiviral, and anticancer effects. Recent reports showed that niclosamide was a potent inhibitor of COVID-19 in vitro, arousing interest in its application in the prevention or treatment of SARS-CoV-2 infection.
However, there are several potential drawbacks to using niclosamide in COVID-19. These drawbacks included high cellular toxicity, significant polypharmacology, low bioavailability, and uncertain efficacy of niclosamide against emerging SARS-CoV-2 (VOC) concern variants.
About the study
In the current study, researchers at the University of Michigan used high-throughput imaging-based immunofluorescence experiments in two different cell models to determine the limitations and possibility of deploying niclosamide as a SARS-CoV antiviral. -2.
The team expanded the analyzes to 33 niclosamide structural analogs to establish a preliminary characterization of the link between structure and activity, potentially aiding the future generation of compounds that exhibit anti-SARS-CoV-2 potency and few outside impacts. the goal. In addition, they used high-content fluorescence images to estimate a selectivity index for niclosamide in two different cell types to examine the toxicity of niclosamide. The cell models used were the most physiologically relevant human lung adenocarcinoma cell line, H1437 and VeroE6.
In addition, in VeroE6 cells, the authors evaluated the efficacy of niclosamide against SARS-CoV-2 Alpha (B.1.1.7), wild-type (WA1), Beta (B.1.351), Gamma ( P.1) and Delta. (B.1.617.2) variants. Finally, they examined the impact of niclosamide therapy on COVID-19 by morphological characterization of Alpha-infected VeroE6 cells to determine the mechanism of action (MOA).
Results
The results of the study showed that niclosamide has a dramatically low selectivity rate on the VeroE6 and H1437 cell lines after 72 hours of exposure to the compound. This inference indicated that even if a compound exposure significantly appropriate for anti-SARS-CoV-2 efficacy occurs in the lungs, niclosamide would likely have a limited therapeutic window in the clinical setting.
Niclosamide is toxic at antiviral concentrations after long-term exposure. A.) Workflow for high-content anti-SARS-CoV-2 bioassay screening to determine infection inhibition and cytotoxicity. B.) 10-point dilution concentration-response curves for VeroE6 and H1437 cells with an initial concentration of 10 μM. VeroE6 cells were infected with the SARS-CoV-2 variant B.1.1.7 with a multiplicity of infections (MOI) of 0.1, while H1437 was infected with the SARS-CoV-2 WA1 variant with an MOI = 1 to achieve optimal infection 48 hours after infection. Data points represent the mean ± SEM for N = 10 replicates per condition. Curve adjustment was performed on GraphPad Prism 9.0 using a 4-parameter semi-log variable slope model. Representative overlay images for simulated treatment, vehicle, and 10 μM niclosamide (infected) (cyan = nuclei, magenta = SARS-CoV-2 N protein) are included.
Despite encouraging preliminary results, the authors demonstrated that niclosamide has a poor in vitro selectivity index for inhibiting SARS-CoV-2 as its antiviral activity coincides with its cytotoxicity. They also show that the efficacy of niclosamide against SARS-CoV-2 VOCs varies widely, ranging from 298 nM for the Beta variant to 1664 nM for the wild-type and was particularly effective against variants. with greater cell-to-cell spread, such as Alpha. According to an additional unicellular morphological characterization, niclosamide also hinders SARS-CoV-2 entry and cell-to-cell dissemination through syncytia.
The team discovered eight niclosamide analog compounds (compounds 34, 24, 22, 11, 10, 4, 3, 2) that were effective in both H1437 and VeroE6 cell models. Four of these (compounds 24, 11, 7, and 2) showed reduced cytotoxicity and improved potency than niclosamide in VeroE6. As a result, they believed there was potential to create niclosamide analogues with better qualities.
The potency of niclosamide depends on the SARS-CoV-2 variant. A.) Chronology of the trial for the 24-hour infection experiment. The test window was shortened to reduce niclosamide toxicity. B.) 10-point 10-point concentration-response curves for niclosamide against the different SARS-CoV-2 variants of concern (MOI = 0.1 for each variant) with a maximum concentration of 10 μM. The curves were equipped with GraphPad Prism 9.0 software using a 4-parameter semi-log variable slope model. The data for each variant were normalized to the average percentage of infected from their respective viral control. Data points represent the mean ± SEM for N = 3 replicates. C.) IC50 values for the potency of niclosamide against worrisome variants of SARS-CoV-2. Values were extracted from the curve fit using GraphPad 9.0 and include SEM error bars (WA1: 1664 ± 149 nM, B.1.1.7: 298 ± 23 nM, B.1.351: 440 ± 21 nM, B.1.617.2: 774 ± 774). 58 nM, P.1: 399 ± 34 nM). Significance was determined by Student’s T tests (* = P <0.05, ** = P <0.01).
In addition, current examination of the structure and activity revealed certain mechanistic aspects of niclosamide. Most importantly, after removal of the hydroxyl group from the protonophore, the two cellular models used in this study ceased to function. This inference indicated that nonspecific endolysosomal neutralization was the main MOA.
The potency of the analogues depended largely on how weakly acidic and lipophilic they were. Because there were carboxylic acid substituents, analogs with a predicted high acidity (pKa) were usually completely inactive. The team also found that other protonophores, such as oxyclozanide and carbonyl-p-trifluoromethoxyphenylhydrazone cyanide (FCCP), demonstrated anti-SARS-CoV-2 activity. This observation indicated that the ability of niclosamide to interfere with pH gradients was essential for its MOA.
Conclusions
Overall, the current study illustrated that niclosamide was not a good option for the treatment of COVID-19, given its limited bioavailability, nonspecific protonophore function, variant-dependent efficacy, weak selectivity index, and complex polypharmacology. However, the findings showed that the aniline and salicyl rings of niclosamide could be modified to alter selectivity and bioavailability while maintaining the action of the drug. Thus, niclosamide provides a useful chemical probe that could be exploited in a massive structure-activity relationship (SAR) effort to build better analogues in the future.
The researchers noted that longer niclosamide exposures to effective anti-SARS-CoV-2 concentrations are likely to result in noticeable side effects, which restricts practical use. Therefore, the safety of niclosamide in in vivo antiviral titers needs further research.
In addition, the development of de novo drugs for COVID-19 may benefit from additional research on the molecular variations behind SARS-CoV-2 variant-based responses to drugs such as niclosamide.
In addition, although current work reveals that niclosamide and similar mitochondrial decouplers have anti-SARS-CoV-2 potency, further research was needed to determine whether these MOAs are related to protonophore activity.
* Important news
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guided by clinical practice / health-related behavior, or treated as established information.
Magazine reference:
- In vitro evaluation and mitigation of the responsibilities of niclosamide as a treatment for COVID-19; Jesse W. Wotring, Sean M. McCarty, Khadija Shafiq, Charles J. Zhang, Theophilus Nguyen, Sophia R. Meyer, Reid Fursmidt, Carmen Mirabelli, Martin C. Clasby, Christiane E. Wobus, Matthew J. O’Meara, Jonathan Z. Sexton, bioRxiv preprint 2022, DOI: