Neutralizing activity of Usnic acid and β-cyclodextrins complex against SARS-CoV-2 spike pseudovirus

Abstract The rapid spread of SARS-CoV-2 and its infection severity require an urgent development of antiviral agents. In this respect, Usnic acid (UA), a natural dibenzofuran derivative, exerts antiviral activity against several viruses, though presenting very low solubility and high cytotoxicity. Here, UA was complexed with β-cyclodextrins (β-CDs), a pharmaceutical excipient used to improve drug solubility. The cytotoxic activity, tested on Vero E6 cells, revealed no effect for β-CDs alone whereas significant cytotoxicity for the UA/β-CDs complex was recorded at concentrations ≥ 0.05%. The neutralizing activity towards the fusion of SARS-CoV-2 Spike Pseudovirus showed no effects for β-CDs alone whereas the UA/β-CDs complex, when pre-incubated with the viral particles, efficiently inhibited the Pseudoviral fusion of about 90 and 82% at non-cytotoxic concentrations of 0.03 and 0.01%, respectively. In conclusion, although further evidences are needed to clarify the exact inhibition mechanism, UA/β-CDs complex could be useful in SARS-CoV-2 infection. Graphical Abstract


Introduction
SaRS-CoV-2 is a ssRna virus with a genome encoding structural, nonstructural, and accessory proteins (Harrison et al. 2020).the structural proteins include spike (S) glycoprotein which mediates viral entry through host specific receptor aCE2 (Wang et al. 2020). in addition to the efficacy of vaccines, there is an urgent requirement of drugs able to interfere, without side effects, with the viral entry into the cells of the nasal and oral cavity to avoid infection and transmission.usnic acid (ua) [2,6-diacetyl-7,9-d ihydroxy-8,9b-dimethyl-1,3(2H,9bH)-dibenzo-furandione], a dibenzofuran derivative biosynthesized by several species of lichens, such as Usnea florida (L.) F.H. Wigg., Usnea barbata (L.) F. H. Wigg. and Usnea longissima ach. (ingólfsdóttir 2002;Calcott et al. 2018), possesses different biological activities, including antibacterial, antifungal, antiparasitic, antiviral, immunostimulatory, antiinflammatory and antitumor (araujo et al. 2015;Wang et al. 2022).ua, characterized as a yellow pigmented solid substance, is naturally found in two enantiomeric forms, such as (−) levogyrous and (+) dextrogyrous, depending on the projection of the angular methyl group.ua gets its acidic feature from the phenolic hydroxyl groups: the enolic group 3-oH shows the strongest acidic character (pKa 4.4) (ingólfsdóttir 2002).ua isomers exhibit antiviral effects against Herpes simplex type 1 and polio type 1 viruses (perry et al. 1999) as well as the pandemic influenza virus a(H1n1) (Sokolov et al. 2012). in 100 female patients suffering from genital papillomavirus, the intravaginal treatment of ua and zinc sulfate improved the re-epithelization of lesions and the recurrence of infection (Scirpa et al. 1999).However, due to 'triketone' moiety cytotoxicity, the Zn-ua treatment exerted local irritant effects in 8% of the patients (Kristmundsdóttir et al. 2005;Jin et al. 2013;Luzina and Salakhutdinov 2018).Recent investigation on cytotoxicity mechanism showed that ua causes cell cycle dysregulation, dna damage, oxidative stress, and activation of nrf2 signaling pathway (Chen et al. 2017).another issue of ua is its low solubility in water, around 0.06 mg/ mL (nikolić et al. 2013).For this purpose, the use of a pharmacologically accessible form must be considered a priority to better address the balance of benefit/adverse effects. in this respect, β-cyclodextrins (β-Cds) could be a great candidate to improve the ua solubility (nikolić et al. 2013).β-Cds are a pharmaceutical excipient able to improve the solubility of various drugs through the formation of water-soluble drug-Cds complexes.toxicological studies have shown that orally administered β-Cds are non-toxic because of their low absorption into the circulation (arima et al. 2011).Here, we investigated the in vitro cytotoxic activity of β-Cds alone or complexed with ua on Vero E6 cells.Successively, the non-cytotoxic concentrations have been tested to investigate, for the first time, the neutralizing activity of the β-Cds alone and in combination with ua against a pseudovirus decorated with the S glycoprotein of SaRS-CoV-2.

Cytotoxic activity of β-cyclodextrins alone and Usnic acid and β-cyclodextrins complex on Vero E6 cells
all physico-chemical features of ua are reported in table S1.Briefly, ua, extracted from Usnea Longissima, appears as a yellow crystalline powder with purity corresponding to 98.7% and presenting a specific rotation of +502° (table S1).
Concerning the cytotoxicity assay, all the concentrations of the β-Cds alone (table S2) were non-cytotoxic after either 8 and 24h of incubation with Vero E6 (Figure S1a and S1b, respectively). of note, the cytotoxicity was considered significant for values of viability as lower than 70%, the threshold value to define a substance as cytotoxic (Cannella et al. 2019).
to preliminary explore the cytotoxic potential of the ua/β-Cds complex, different concentrations, ranging from 1 to 0.001% (table S2), were tested.
Results reported in table S3 indicated that: i) the ua/β-Cds complex was cytotoxic for Vero E6 cells at the concentrations of 1, 0.5, and 0.1% after 8 (cell viability < 55%) and 24h (cell viability < 42%) of incubation; ii) the concentration of 0.05% of the ua/β-Cds complex was at the threshold value of the cell viability (71%) at 8h, while at 24h of incubation the cell viability decreases (65%); iii) the concentration of 0.03% of the ua/β-Cds complex was non-cytotoxic, showing cell viability of 83 and 85% after 8 and 24h, respectively; iv) the concentrations of 0.01, 0.005 and 0.001% of the ua/β-Cds complex were non-cytotoxic for Vero E6 cells (cell viability > 90%) (table S3).
to better set the range of cytotoxic vs non-cytotoxic concentrations, also in term of reproducibility and significance, the solutions of the ua/β-Cds complex at 0.05, 0.03 and 0.01%, representing the closest concentrations to the cytotoxic threshold, were tested by further independent experiments (Figure S2).
these results strongly encourage the use of β-Cds as cargo for ua to decrease its cytotoxicity, especially when compared to other carriers.indeed, when ua was encapsulated with a copolymer of lactic and glycolic acid, the iC50 of encapsulated ua was 0.001% (Santos et al. 2005).Similarly, liposomes-loaded ua revealed a strong cytotoxic activity with a iC50 of about 0.002% (Lira et al. 2009).

Neutralizing activity of β-cyclodextrins alone and Usnic acid and β-cyclodextrins complex against SARS-CoV-2 Spike Pseudovirus
any natural substance or drug, capable of inhibiting viral entry through a competitive binding with cell receptors and/or with viral component, such as S glycoprotein (Rosa et al. 2023;Cutone et al. 2022), can exert an antiviral action and act as a protective barrier against infections.
the neutralizing assay was carried out using SaRS-CoV-2 S pseudovirus, on Vero E6 cells, at multiplicity of infection (moi) of 10, the optimal concentration to obtain an accurate luminescence test (Figure S3).β-Cds alone, at all the concentrations tested (from 0.5 to 0.0005%), did not show any inhibitory effect (data not shown).
the inhibition of the pseudovirus fusion by the ua/β-Cds complex at 0.03 and 0.01%, the highest concentrations non exerting cytotoxic activity, was tested according to the experimental scheme reported in supplementary material.
the results showed that the ua/β-Cds complex induced the highest neutralizing activity when pre-incubated with the pseudovirus at a concentration of 0.03% (Figure S4a).When the complex is added at the moment of infection, its activity still appears significant even if at lower extent respect to the pre-incubation of the complex with the viral particles (Figure S4a). the inhibition of pseudoviral fusion by the ua/β-Cds complex at the concentration of 0.03% significantly decreased when it was pre-incubated with the cells (Figure S4a).
the complex at the concentration of 0.01% showed the same inhibitory trend in all the experimental conditions, even if at lower extent (Figure S4b).
as reported by nikolić et al. (2013), ua alone presents a very poor solubility (around 0.06 mg/mL), whereas the complex with β-Cds increases such solubility to 0.3 mg/mL (molar ratio 1:1). in our experiments, we have prepared the complex with a w/w ratio of 1:1, presenting a similar solubility of 0.5 mg/mL (table S2). this increased solubility allows the ua to be applied at higher concentrations, without exerting significant cytotoxic activity (Figure S2), thus globally improving its neutralizing function against SaRS-CoV-2 S decorated pseudovirus (Figure S4).
our results suggest that the ua/β-Cds complex does not bind to the host receptors exploited by the virus to enter into the host cells (Hu et al. 2021;Cutone et al. 2022). in the light of our results and of two recent works (oh et al. 2022;Filimonov et al. 2022), the interaction occurring between ua and S glycoprotein could be considered as a possible mechanism of ua antiviral activity.In silico analysis showed that ua possesses high binding affinity (-6.02 kcal/mol) for the receptor binding domain (RBd) of S (Guthappa 2020).therefore, it is possible to hypothesize that the ua/β-Cds complex interacts with S, the sole structure present on the surface of pseudovirus able to mediate its fusion with host cell, however further studies are required to prove it.at our knowledge, this is the first study demonstrating the efficacy of ua in complex with the β-Cds in inhibiting SaRS-CoV-2 S-mediated cell fusion.Galla et al. (2023) have reported the efficacy of a new ua-based formulation (75 µg/mL ua + 150 µg/ mL β-Cd + 0.1% of tocopherol and tocotrienol + 1% hydroxypropyl methylcellulose) in hindering VSV-based SaRS-CoV-2 pseudoviral entry of about 22.5% compared to the infected untreated control, indicating that it is also able to create a barrier that can trap viruses (Galla et al. 2023).
overall, complexation of ua with β-Cds results in lowering cell cytotoxicity thus potentiating its antiviral activity against SaRS-CoV-2 and their variants, similar to another antiviral compound as remdesivir (oh et al. 2022).

Experimental
all details are provided in the supplementary material.

Conclusions
Even if further experiments are required to elucidate the molecular mechanism of action against SaRS-CoV-2 and demonstrate the actual safety, the ua/β-Cds complex could be useful in SaRS-CoV-2 infection.