Beta Cyclodextrins Can Be Used As Antivirals Against SARS-CoV-2

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A new study by researchers from Spain led by scientist from the Cell Structure Lab, Centro Nacional de Biotecnologia, CNB – CSIC, Campus de Cantoblanco, Madrid -Spain has found that cheap natural cyclodextrins especially Beta Cyclodextrins can be used as antivirals against SARS-CoV-2.

The study findings were published on a preprint server and are currently being peer reviewed.
https://www.biorxiv.org/content/10.1101/2022.11.16.516726v1

Although pathogenic viruses pose a real and growing threat to public health, we have few medicines to prevent and treat viral infections. Here, a library of potential inhibitors of coronavirus infection was elaborated with a “based-on-knowledge” strategy. Examining the available information about what viruses use to complete their life cycle in cells and the description of the mechanism of action of drugs, we found compounds with potential to be used as antivirals to treat coronavirus infections.

The main advantage of this strategy compared to high-throughput analysis is that the list of candidates is limited, and that different protocols of infection and drug treatment can be tested, which increases the probabilities of identifying molecules with antiviral activity.

We have used a unique workflow involving biocomputational analysis and several biological assays to carefully select potential antivirals against different coronaviruses, including HCoV-229E and SARS-CoV-2. From our list of 116 compounds that target cell factors and pathways, 4 showed antiviral activity against both coronaviruses.

Results with β-CDs were particularly relevant as showed consistent efficacy in different cellular models including human pulmonary cells. Particularly promising is our finding suggesting that the mechanism of action of β-CDs is interfering with viral fusion.

Results from lipidomic analysis and transmission electron microscopy (TEM) showed that β- CDs may interfere with coronavirus infection by altering cholesterol and sphingomyelin content and disrupting the organization of membranes used by the virus.

This is supported by our TEM results that showed dose-dependent effects of β-CDs in all SARS-CoV-2 structures in infected cells. The integrity of DMVs was compromised and viral morphogenesis was impaired, with the production of abnormal viral particles that are sometimes trapped in DMVs and inside large vacuoles that could represent degradation compartments.

Our results confirm and broaden recent findings focused on HP-β-CDs (Bezerra et al., 2022), and with different methods further expand these results to other members of the cyclodextrin family and to SARS-CoV2 VOCs, including Omicron.

Given that cyclodextrins can be suited for oral, nasal or nebulized solutions, these results open different avenues to test diverse drug formulations that are known to be safe in humans (Stella and He, 2008; Tian et al., 2020). Further work should address the potential activity of these compounds in ameliorating SARS-CoV-2 infection in relevant animal models.

The well-known safety profiles of β-CDs render these molecules as ideal candidates to develop affordable prophylactic and therapeutic compounds against coronaviruses (Fatmi et al., 2021; Sorice et al., 2020). Such drugs, which are already approved for clinical use in nasal spray devices (Guard et al., 1989; Paolacci et al., 2021), may be easier to deploy in low income countries compared to vaccines, which often require cold storage and must be administered by trained personnel.

Given that β-CDs are widely used for compound encapsulation, they could be easily combined with other antivirals to potentiate activity and avoid viral resistance. Finally, the wide mechanism of action shown herein, which inhibits viral fusion with cellular membranes, could help to counteract other respiratory viruses, providing an arsenal to deploy in front of new variants of concern or future novel coronaviruses with pandemic potential.

Broad-spectrum antivirals such as β-CDs could be ultimately applied to counteract unknown emergent viruses yet to appear, but need rigorous assessment in preclinical models for further development.

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Cyclodextrins inhibit SARS-CoV-2 replication by interfering with viral fusion via cholesterol depletion

We had previously found that b-CD are in the catalytic pocket of the Mpro of SARS-CoV-2 by molecular modeling (Fig. 1C). This finding pointed to a possible explanation for the antiviral activity detected. All cyclodextrins, including α, b, g, HP- α, HP-b, HP-g and methyl-b-CDs, were screened by molecular modeling against the SARS-CoV-2 Mpro. Since is possible to inhibit the action of Mpro by targeting two allosteric sites -PDB codes 7AXM and 7AGA- (Günther et al., 2021)), we expanded the chemical space of search into the designed models.

Theory predicts that cyclodextrins can bind both active and allosteric Mpro sites, although significant dissimilarities appear depending on the size and nature of the macrocycle (Suppl. Table 3). To test whether this interaction occurred in vitro, the Mpro activity was measured in presence of increasing concentrations of methyl-b-CD (MbCD). While MbCD did not show activity, active GC376 control inhibited Mpro in a dose dependent manner (Fig. 8A).

We next explored alternative antiviral mechanisms of action of cyclodextrins. Given the well- known capacity of cyclodextrins to extract cholesterol from biological membranes (López et al., 2011), we performed a lipidomic analysis focusing on different lipids associated to cholesterol enriched domains in biological membranes. Calu-3 cells treated with increasing concentrations of methyl-b-CDs showed a reduction of free and ester cholesterol along with sphingomyelin, and did not affect phosphatidylcholine (Fig. 8B).

These results further confirm the capacity of cyclodextrins to alter the composition of cholesterol enriched domains actively involved in viral fusion processes. Taken together, these experiments along the pseudoviral fusion assays highlight the potential of cyclodextrins to inhibit SARS-CoV-2 replication by interfering with viral fusion via cholesterol depletion.

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