Diagram of niclosamides effect on SARS-CoV-2 entry and spike protein-mediated syncytia formation. 1.) SARS-CoV-2 binds to the ACE2 receptor of the host cell and enters. Niclosamide has been shown to inhibit this entry step in vitro 2.) Viral replication generates many copies of the RNA genome. 3.) Infection results in an increased expression of viral spike (S) protein and host cell TMEM16F at the plasma membrane. 4.) The S protein at the surface on an infected cell binds to the ACE2 receptor of an adjacent uninfected cell. 5.) Spike-dependent syncytia formation is mediated by the calcium-dependent lipid scramblase TMEM16F to generate multinucleated infected cell bodies. Niclosamide, an inhibitor of TMEM16F, has been shown to block spike-dependent syncytia formation.

Researchers analyze the potential of niclosamide as a SARS-CoV-2 treatment

A recent study posted to the bioRxiv* preprint server has assessed whether niclosamide can be used in the treatment of coronavirus disease 2019 (COVID-19).

Diagram of the effect of niclosamides on SARS-CoV-2 and entry spike protein-mediated syncytia formation. 1.) SARS-CoV-2 binds to the host cell’s ACE2 receptor and enters. Niclosamide has been shown to inhibit this entry step in vitro. 2.) Viral replication generates many copies of the RNA genome. 3.) Infection results in increased expression of viral spike (S) protein and host cell TMEM16F at the plasma membrane. 4.) The S protein on the surface of an infected cell binds to the ACE2 receptor of an adjacent uninfected cell. 5.) Spike-dependent syncytia formation is mediated by the calcium-dependent lipid scramblase TMEM16F to generate multinucleated infected cell bodies. Niclosamide, an inhibitor of TMEM16F, has been shown to block peak-dependent syncytia formation.

Background

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) therapies, which can halt or treat viral transmission, are still urgently needed.

Niclosamide, the Food and Drug Administration (FDA) authorized oral anthelmintic, has broad biological functions, including antibacterial, antiviral and anticancer effects. Recent reports showed that niclosamide was a potent inhibitor of COVID-19 in vitrowhich sparked interest in its application in the prevention or treatment of SARS-CoV-2 infections.

Nevertheless, there are several potential drawbacks to using niclosamide in COVID-19. These drawbacks include high cellular toxicity, significant polypharmacology, low bioavailability, and uncertain efficacy of niclosamide against emerging SARS-CoV-2 variants of concern (VOCs).

About the study

In the present study, the University of Michigan researchers used high-content imaging-based immunofluorescence experiments in two different cell models to determine the limitations and possibility of using niclosamide as an antiviral agent against SARS-CoV-2.

The team extended the analyzes to 33 structural analogs of niclosamide to establish a preliminary characterization of the structure-activity relationship, potentially enabling future generation compounds with anti-SARS-CoV-2 potency and little off-target effects can help. Furthermore, they used high-content fluorescence imaging to estimate a selectivity index for niclosamide in two different cell types to investigate the toxicity of niclosamide. The cell models used were the more physiologically relevant human lung adenocarcinoma cell line, H1437 and VeroE6.

In addition, the authors assessed the effectiveness of niclosamide against the SARS-CoV-2 Alpha (B.1.1.7), wild-type (WA1), Beta (B.1.351), Gamma (P.1) and Delta (B) in VeroE6 cells. .1.617.2) variants. Finally, they examined the impact of niclosamide therapy on COVID-19 using the morphological characterization of VeroE6 cells infected with Alpha to determine the mechanism of action (MOA).

Results

The study results showed that niclosamide has a dramatically low selectivity index in VeroE6 and H1437 cell lines after 72 hours of exposure to the compound. This inference indicated that even if significantly adequate exposure to the compound 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 prolonged exposure.  A.) Workflow for high-level anti-SARS-CoV-2 bioassay screening to determine infection inhibition and cytotoxicity.  B.) 10-point, 2-fold dilution concentration-response curves for VeroE6 and H1437 cells with a starting concentration of 10 M. VeroE6 cells were infected with SARS-CoV-2 B.1.1.7 variant with a multiplicity of infection (MOI) of 0.1, while H1437 were infected with SARS-CoV-2 WA1 variant with a MOI = 1 to achieve optimal infection at 48 h post-infection.  Data points represent mean ± SEM for

Niclosamide is toxic at antiviral concentrations after prolonged exposure. A.) Workflow for high-level anti-SARS-CoV-2 bioassay screening to determine infection inhibition and cytotoxicity. B.) 10-point, 2-fold dilution concentration-response curves for VeroE6 and H1437 cells with a starting concentration of 10 M. VeroE6 cells were infected with SARS-CoV-2 B.1.1.7 variant with a multiplicity of infection (MOI) of 0.1, while H1437 were infected with SARS-CoV-2 WA1 variant with a MOI = 1 to achieve optimal infection at 48 h post-infection. Data points represent mean ± SEM for N = 10 replicates per condition. Curve fitting was performed in GraphPad Prism 9.0 using a semi-log 4-parameter variable slope model. Representative overlay images for mock, vehicle, and 10 M Niclosamide treatment (infected) are included (cyan = nuclei, magenta = SARS-CoV-2 N protein).

Despite encouraging preliminary results, the authors showed that niclosamide has a poor in vitro selectivity index to inhibit SARS-CoV-2, as its antiviral activity coincides with its cytotoxicity. They further show that the effectiveness of niclosamide against the SARS-CoV-2 VOCs varies widely, ranging from 298 nM for the beta variant to 1664 nM for the wild-type and that it was particularly effective against variants with a greater cell-to-cell-to-cell ratio. cell proliferation, such as Alpha. According to additional single-cell morphological characterization, niclosamide also hinders SARS-CoV-2 entry and cell-to-cell diffusion via syncytia.

The team discovered eight niclosamide analog compounds (compounds 34, 24, 22, 11, 10, 4, 3, 2) that were effective in both the 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 a potential to make niclosamide analogs with better properties.

The potency of niclosamide is dependent on the SARS-CoV-2 variant.  A.) Analysis timeline for 24-hour infection experiment.  The test window was shortened to reduce the toxicity of niclosamide.  B.) 10-point 2-fold concentration-response curves for niclosamide against the different SARS-CoV-2 variants of concern (MOI = 0.1 for each variant) with a top concentration of 10 M. Curves were fitted with GraphPad Prism 9.0 software using a semi-log 4-parameter variable slope model.  Data for each variant were normalized to the mean percentage of infected from the respective viral control.  Data points represent mean ± SEM for

The potency of niclosamide is dependent on the SARS-CoV-2 variant. A.) Analysis timeline for 24-hour infection experiment. The test window was shortened to reduce the toxicity of niclosamide. B.) 10-point 2-fold concentration-response curves for niclosamide against the different SARS-CoV-2 variants of concern (MOI = 0.1 for each variant) with a top concentration of 10 M. Curves were fitted with GraphPad Prism 9.0 software using a semi-log 4-parameter variable slope model. Data for each variant were normalized to the mean percentage of infected from the respective viral control. Data points represent mean ± SEM for N = 3 replicates. C.) IC50 values ​​for the potency of niclosamide against SARS-CoV-2 variants of concern. Values ​​were extracted from curve fitting with GraphPad 9.0 and included 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 ± 58 nM, P.1: 399 ± 34 nM). Significance was determined using Student’s T-tests (* = P<0.05, ** = P<0.01).

In addition, the current structure-activity study revealed certain mechanistic aspects of niclosamide. Most importantly, after the removal of the hydroxyl group from protonophore, both cell models used in this study ceased to function. This inference indicated that nonspecific endolysosomal neutralization was the major MOA.

The potency of analogs was highly dependent on how weakly acidic and lipophilic they were. Because carboxylic acid substituents were present, analogs with a high predicted acidity (pKa) were usually completely inactive. The team also found that other protonophores, such as oxyclozanide and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), showed 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 present study illustrated that niclosamide was not a good choice for COVID-19 treatment, given its limited bioavailability, non-specific protonophore function, variant-dependent efficacy, weak selectivity index, and complex polypharmacology. Nevertheless, the findings showed that the aniline and salicyl rings of niclosamide can be modified to alter selectivity and bioavailability while preserving drug action. Thus, niclosamide provides a useful chemical probe that can be used in a massive structure-activity relationship (SAR) effort to build better analogs in the future.

The researchers pointed out that longer exposure to niclosamide at effective anti-SARS-CoV-2 concentrations is likely to lead to noticeable side effects, limiting its practical use. Hence, the safety of niclosamide at antiviral titers in vivo need further investigation.

In addition, de novo drug development for COVID-19 may benefit from additional research into the molecular variations behind SARS-CoV-2 variant-based responses to drugs such as niclosamide.

In addition, although the current work shows that niclosamide and similar mitochondrial uncouplers have anti-SARS-CoV-2 potential, additional research was needed to determine whether these MOA are linked to protonophore activity.

*Important announcement

bioRxiv publishes preliminary scientific reports that have not been peer-reviewed and therefore should not be considered conclusive, that should guide clinical practice/health-related behavior or be treated as established information.

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