Evaluation of in vitro SARS‐CoV‐2 inactivation by a new quaternary ammonium compound: Bromiphen bromide

The pneumonia (COVID‐19) outbreak caused by the novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), which unpredictably exploded in late December of 2019 has stressed the importance of being able to control potential pathogens with the aim of limiting their spread. Although vaccines are well known as a powerful tool for ensuring public health and controlling the pandemic, disinfection and hygiene habits remain crucial to prevent infection from spreading and maintain the barrier, especially when the microorganism can persist and survive on textiles, surfaces, and medical devices. During the coronavirus disease pandemic, around half of the disinfectants authorized by the US Environmental Protection Agency contained quaternary ammonium compounds (QACs); their effectiveness had not been proven. Herein, the in vitro SARS‐CoV‐2 inactivation by p‐bromodomiphen bromide, namely bromiphen (BRO), a new, potent, and fast‐acting QAC is reported. This study demonstrates that BRO, with a dose as low as 0.02%, can completely inhibit SARS‐CoV‐2 replication in just 30 s. Its virucidal activity was 10‐ and 100‐fold more robust compared to other commercially available QACs, namely domiphen bromide and benzalkonium chloride. The critical micellar concentration and the molecular lipophilicity potential surface area support the relevance of the lipophilic nature of these molecules for their activity.

prevention strategies to limit the spread of SARS-CoV-2 and hopefully other unpredictable viral infections that could arise in the future.Indeed, although SARS-CoV-2 transmission mainly occurs through direct-route by aerosol respiratory droplets generated through coughing and sneezing by infected individuals, in a significant number of COVID-19 cases, epidemiological investigations could not find evidence of direct close contact with other confirmed patients; therefore, at least some of these cases may be caused by indirect transmission.The latter includes the contamination of hands or inanimate/inert surfaces (fomites) on which the virus can survive, followed by touching the mouth, nose, or eyes.Indeed, some viruses show the ability to transfer between and persist on different surfaces (from hours to days), including the human skin for that reason.Disinfection and hygiene habits remain crucial to avoid infection dissemination and to maintain the barrier.As a matter of fact, practical experience in different settings has widely demonstrated that the right choice of disinfectant agent, [1] stringent disinfection, and control measures may be highly effective in limiting person-toperson transmission.
As an enveloped virus SARS-CoV-2 is particularly sensitive to detergents and disinfectants, that is why World Health Organization recommends cleaning surfaces and hands with these classes of compounds.However, SARS-CoV-2 is a relatively novel virus and a biosafety level-3 (BSL-3) agent, hence experimental data assessing the virucidal effect of these compounds on this specific pathogen are still limited and need to be implemented.In fact, our current knowledge is mainly based on previous coronavirus studies reporting disinfectants, such as ethanol (>62% concentration), isopropanol, sodium hypochlorite, and quaternary ammonium compounds (QACs) combined with alcohol are able to contain coronavirus infections.In particular, QACs are organic compounds featuring a positively charged nitrogen atom, making them effective antimicrobial agents.
These compounds exhibit a wide range of structural diversity and can be tailored for specific applications.QACs are commonly used as disinfectants, preservatives, and surfactants due to their ability to disrupt cell membranes with well-known broad-spectrum antimicrobial activities, hence representing a repurposing opportunity as disinfectants against SARS-CoV-2.The hydrophilic head, a cationic ammonium group, is what gives them their distinctive properties.The ammonium group has four organic substituents, including alkyl or heterocyclic groups, which create the lipophilic tail.An anion, such as a halide or sulfate, balances the charge.Thanks to these specific features they eradicate surface bacteria and common viruses such as influenza by solvating and rupturing their lipid membranes or envelopes.Actually, QACs target microbial cell membranes through electrostatic interactions between the positively charged headgroup and negatively charged cytoplasmic membrane, then adsorption occurs, and insertion of their side chains into the intramembrane region takes place. [2,3][6] This success was confirmed, during the coronavirus pandemic disease when around half of the disinfectants authorized by the US Environmental Protection Agency contained QACs as active components against a wide range of microbes. [7,8]Indeed, their efficacy on SARS-CoV-2 inactivation has been recently demonstrated by some authors in different experimental conditions. [9,10]In particular, Ijaz et al. [9] demonstrated that 0.1% benzalkonium chloride (BAK) inactivates SARS-CoV-2 within 2 min in carrier test.Likewise, Xiling et al. [10] verified that the QAC disinfectants, di-Ndecyldimethylammonium bromide and di-N-decyldimethylammonium chloride, exhibit high efficiency with low dose effectiveness and short reaction times, claiming a more significant role of QACs in the global fight against COVID-19.
On the basis of these premises, finding a new virucidal compound and implementing experimental data concerning SARS-CoV-2 is persistently necessary.Notably, quite recently a new QAC, namely bromiphen bromide (BRO), has been characterized by means of liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and high-performance liquid chromatography analyses. [11,12]These studies revealed that BRO is stable under acidic and oxidizing conditions and also under thermal stress.BRO antimicrobial activity, mainly against Gram-positive strains, has been verified as well; but no one has so far proved that this new compound may display also antiviral activity and the minimum dose necessary to effectively inhibit SARS-CoV-2.Thus, in this study, we tested the virucidal effect of BRO on SARS-CoV-2 infection/ replication in comparison with two other commercially available QACs, domiphen bromide (DOM) and BAK (Figure 1).DOM was chosen because it is already used in commercially available pharmaceutical preparation and is very similar to BRO from a structural point of view.In fact, they differ for a bromide atom instead of a hydrogen one at the para position of the aromatic ring.analyses showed that all three QACs exert a cytotoxic effect at the highest concentrations used in the following experiments: 0.2%-0.002%(Figure 2a-c, MTT).Such cytotoxic effect abruptly disappeared at 0.0002% for all three tested compounds, while at 0.002% BRO and DOM showed a less pronounced cytotoxic effect compared to BAK.Considering their impact on cell viability, to effectively test their virucidal activity in an in vitro infection assay, all compounds were inactivated by incubation with a neutralizer solution.This was done before transferring the (virus + QAC) mix into the cell culture to neutralize any potential residual of their cytotoxic effect.Moreover, as shown in Figure 2d, the cytotoxicity of the neutralizer was also assessed by confirming that it did not affect cell viability from 1:10 dilution onwards.
Then, to evaluate the neutralizer capability of completely removing the cytotoxic effect, the highest BRO concentration (0.2%) was used according to the setting reported in Table 3 (see below in Section 4).
The results obtained are reported in Figure 3.In Test 1, the inhibition of viral replication was due to the cytotoxic effect exerted by BRO on VeroE6 cells, as shown by cytopathic effect (CPE) monitoring (Figure 3b).Notably, in Test 2, the neutralizer solution effectively inhibited BRO cytotoxicity when used in a 9:10 ratio (neutralizer:QAC) (Figure 3a).Moreover, neither the neutralizer (Test 3) nor the (disinfectant + neutralizer) mix (Test 4) had adverse effects on cell viability, indeed the virus was able to replicate.Neutralization analyses performed on DOM and BAK mirrored the results obtained with BRO (data not shown).Hence, the neutralizer was selected as a QAC inactivator in all the SARS-CoV-2 infection tests performed in the study.
Results show that BRO is able to inactivate SARS-CoV-2 just following 30 s of incubation, as shown in Figure 4a.In particular, a dose of 0.02% was sufficient to completely inhibit viral replication at T1 and to maintain such an effect over time.Conversely, the 0.002% concentration controlled viral replication at T1 and T2 but it was no longer efficacious at T3. Notably, the virucidal effect displayed by BRO showed a dose-response trend at T2 (Figure 4b).T1 and T3 were unfunctional timepoints to appreciate a dose-response trend as shown in Figure 4a.Indeed, at T1 there were no viral particles even in the untreated condition, whereas, at T3 the viral replication reached a plateau.
The same tests were performed by incubating SARS-CoV-2 with DOM (Figure 4c) or BAK (Figure 4d).Results mirrored those obtained by BRO treatment.However, BRO virucidal activity was 10-and 100-fold more efficient compared to DOM and BAK, respectively.Indeed, a concentration of 0.002% of DOM was unable to inhibit viral replication at T2 (Figure 4c).Moreover, BAK was even less efficacious compared to BRO and DOM as its virucidal activity was displayed and maintained over time only with a 0.2% concentration (Figure 4d).recently highlighted and virus inactivation below and above the determined CMC was confirmed. [13,14]r these reasons, it is worth knowing the self-assembling properties of the tested substances.Thus, the CMC of BRO was determined by measuring the electrical conductivity of aqueous dispersions at increasing concentrations.To understand completely the mechanism and how it influeces the virucidal activity highlighted and to support the determination of BRO CMC (never calculated before) the measurement was also performed on cetylpyridinium chloride (CPC), DOM, and BAK.
In general, the CMC of surfactants mainly depends on their hydrophobicity; this means that CMC values decrease rapidly, increasing the hydrophobic moiety of a molecule.
The obtained CMC values (Table 1) using conductometric measurements [23] were in good agreement with previously published results.The CMC value of BRO can therefore be considered reliable.
Conductivity measured for each dispersion at increasing percentages (Supporting Information: Table S1) and graphical representation according to Williams' method and Phillips' methods (Supporting Information: Figures S1-S4) are reported in Supporting Information.
From the obtained results, BRO shows the lowest CMC compared to DOM and BAK, highlighting a more pronounced hydrophobic nature of the new compound.Moreover, the CMC trend parallels the virucidal activity one.In fact, the lowest is the CMC the highest is the activity of the compound.It was also evidenced that BRO is active at a concentration (0.02%) lower than Dose-response trend of BRO at T2 (b).To avoid the QAC cytopathic effect, all QAC-virus mixtures were exposed to the neutralizer in a 9:10 ratio before inoculum on VeroE6 cells.BRO, DOM, and BAK virucidal effects occur at 0.002%, 0.02%, and 0.2% concentration, respectively.Results correspond to the absolute viral copy number of the SARS-CoV-2 N1 gene from cell supernatants that were quantified through a singlestep, real-time, RT-qPCR by referring to a standard curve from RT-qPCR C t values (IDT).Results are presented as mean ± SEM from at least n = 3 independent experiments, each performed in triplicate.BAK, benzalkonium chloride; BRO, bromiphen bromide; DOM, domiphen bromide; hpi, hours postinfection; QAC, quaternary ammonium compound; RT-qPCR, quantitative reverse transcription polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; UNT, untreated control.
T A B L E 1 Determined CMC according to conductometric analysis.
the CMC, suggesting that BRO activity in the tested solution is not due to organization in micelle aggregates.As already reported by Farcet et al. [14] for other detergents, the efficacy of BRO against virus can be detected below the CMC confirming that virus inactivation is not strictly related to micelle formation.Moreover, the difference in determined CMC for BRO and DOM that share structure similarities can be justified by diverse lipophilicity, which, in turn, can explain the better ability of BRO to disrupt biological membranes, thus resulting in a lower active concentration.Figure 5 reports the molecular lipophilicity potential (MLP) surface of DOM (Figure 5a) and BRO (Figure 5b).In MLP maps, color codes were used as follows: Furthermore, the analysis of the lipophilic character of both DOM and BRO through WLOGP, MLOGP, iLOGP, XLOGP3, and SILICOS-IT predictive models was performed via SwissADME software. [24]Table 2 reports estimations and consensus logP ow values for DOM and BRO.For BRO, the consensus logP ow value was calculated as 3.50.Since bromide substitution is known to increase lipophilicity, also considering the more classical Hammett constant, the result obtained is quite reasonable.At this point, SwissADME and VEGA ZZ [25] analysis results support each other.

| CONCLUSION
Through this study we demonstrate that BRO can completely inhibit SARS-CoV-2 replication in just 30 s and with a dose as low as 0.02%, supporting the recommendation of BRO as a candidate commodity that could be used to kill SARS-CoV-2 and potentially other enveloped viruses.Importantly, BRO virucidal activity was 10-and 100-fold more robust compared to DOM and BAK, respectively.Moreover, the use of BRO would allow for a reduction in the dose of QAC disinfectants to be used, which would be extremely advantageous to minimize the load of QACs.Indeed, it cannot be overlooked that elevated QAC exposure has been supposed to favor the spread of antibiotic resistance and cause other environmental issues. [3,26,27]e mechanism of action of BRO is believed to occur as a result of perturbation of the virally modified, host-cell-derived, phospholipid bilayer glycoprotein envelope, and the associated spike glycoproteins that bind with the angiotensin-converting enzyme receptor required for infection of host cells likewise reported for other formulated microbicidal compounds and detergents. [28,29]As far as its behavior is concerned, BRO seems to be active even under its CMC thus maintaining activity also as unimer.CMC and MLP support that the virus inactivation is independent of the CMC but rather strictly related to the lipophilic nature of the molecules.It might be worth investigating further whether it is possible to define a limit on the lipophilic characteristic of a potentially active molecule.

| Experimental design
The primary endpoint of the study was to assess the virucidal effect of the BRO compound on SARS-CoV-2 and to establish its minimal effective dose.The secondary endpoints were: (i) to test the CPE of these disinfectants, (ii) to compare the BRO effect to two other QACs already commercially available, namely DOM and BAK, and (iii) to identify BRO critical micellar concentrations.
To assess all these aims a specific viral titer was exposed to scalar disinfectant concentrations and its virucidal effect was tested in an in vitro infection assay as later described.

| Cell lines, virus, and reagents
VeroE6 (CRL-1586™, African green monkey kidney epithelial cells) cells were purchased from the American Type Culture Collection  concentration was assessed by TCID 50 endpoint dilution assay as previously described. [30]The TCID 50 for viral titers exposed to the disinfectants in each test was 2.5 × 10 5 TCID 50 /mL.
All the experiments with the SARS-CoV-2 virus were performed in the BSL-3 facility; before sample analysis outside the BSL-3 area, the virus was disabled according to institutional safety guidelines.
The following reagents were used in the cell culture assays: BRO was synthesized by our laboratories according to the procedures already published [11,12,31] ; DOM and BAK were purchased from Merck KGaA and used as commercially distributed.

| MTT assay
The cytotoxic effect of BRO, DOM, and BAK was evaluated by means of MTT assay: VeroE6 cells were seeded in 96-well plates (2 × 10 4 per well) for 24 h and treated with different BRO, DOM, and BAK concentrations: 0.02%-0.002%-0.0002%-0.00002%-0.000002%-0.0000002%.The concentrations were decided by considering the active doses of BRO, DOM, and BAK as previously reported. [12]After 72 h, cell viability was assessed by the MTT method.
Briefly, 30 μL of MTT (final concentration, 0.5 mg/mL) was added to each well under sterile conditions, and the 96-well plates were incubated for 4 h at 37°C.Supernatants were removed, and dimethyl sulfoxide (100 μL/well) was added.The plates were then agitated on a plate shaker for 5 min.The absorbance of each well was measured at 490 nm with a Bio-Rad automated EIA analyser (Bio-Rad Laboratories).The viability of untreated cells (control) was considered 100%, while the other conditions were expressed as percentages of control.

| Neutralizer efficacy assessment
Since all the highest concentrations of the tested compounds proved to exert a cytotoxic effect on VeroE6 cell lines in an MTT assay, a composite that was able to stop the disinfectant's action (neutralizer) was employed.Neutralizing agents need to have an appropriate disabling effect on the chemical disinfectant and must not display detrimental or adverse effects on the virus and cell line used in the in vitro infection assay.To verify all these conditions, we conceived six series of tests for each compound to depict the neutralization efficacy as shown in Table 3.
We performed this by incubating the highest QAC concentrations (0.2%) used in the subsequent experiments, with/without neutralizer (9:10 QAC concentration) in a test tube for 5 min and adding 2.5 × 10 5 TCID 50 /mL of virus suspension and mixing well.The mixture was then used in an in vitro infection assay and viral replication was assessed by the culture-PCR (C-RT-PCR) method, [32] as detailed in the following.In parallel, the CPE induced by SARS-CoV-2, QACs, and/or neutralizers was assessed.The neutralizer composition has been previously described in Wood et al.'s [33] study.T A B L E 3 Tests performed to assess the neutralizing agent efficacy.

| In vitro SARS-CoV-2 infection assay and virucidal effect evaluation
BAK was selected as a benchmark because, in addition to belonging to the same chemical class as BRO, it is present in several medical devices already in the market.F I G U R E 1 Structures of Domiphen bromide (DOM), Bromiphen bromide (BRO), and Benzalkonium chloride (BAK).

2. 1 . 3 |
Determination of critical micellar concentration and lipophilic analysis BRO, as well as other QACs, is an amphiphilic molecule, commonly known as a surfactant, composed of a hydrophobic and a hydrophilic portion.Based on these chemical-physical characteristics, QACs' activity has often been associated with micelle formation.Micelles are supramolecular aggregates; in a physiological environment, hydrophilic heads are exposed outside, in touch with the aqueous environment, while the hydrophobic tails are packed together to form the core.The physical state of surfactants as unimers or micelles has a significant impact on their behavior.The discussion about the role of micelle formation and surfactant ability to disrupt biological membranes is still open, even if the nondependence of the activity with respect to the critical micelle concentration (CMC) value was
hydrophilic surfaces are defined by blue regions; violet/purple regions show the most lipophilic surfaces.Last, intermediate lipophilic surfaces are colored yellow.The violet/purple zones that represent the lipophilic surfaces are more intense in BRO (Figure 5b) than in DOM (Figure 5a).On the other hand, in the MLP diagram referred to DOM the yellow intermediate lipophilic area originating from the unsubstituted aromatic region is more dominant.Therefore, as shown by MLP maps, BRO is expected to be more lipophilic than DOM.

F I G U R E 5
MLP diagrams of (a) DOM and (b) BRO.BRO, bromiphen bromide; DOM, domiphen bromide; MLP, molecular lipophilicity potential.T A B L E 2 Estimations of physicochemical properties and LogP ow values for DOM and BRO.

The 2 . 5 ×
10 5  TCID 50 /mL viral suspension was mixed with each of the three QACs at different concentrations and allowed to react for 30 s.Then, 0.1 mL of the reaction solution was added to a test tube containing 0.9 mL of the neutralizer solution (1:10) and mixed for 5 min, before undergoing two 10-fold series dilutions with DMEM (ECB7501L; Euroclone) as reported in Figure6.The diluted samples containing 25 TCID 50 /mL and 0.0002% QAC were seeded onto a 24well cell culture plate (1.5 × 10 5 VeroE6 cells/well) with cells growing into monolayers and three wells for each concentration.After 1 h at 37°C and 5% CO 2 cells were rinsed two times with warm phosphate buffer saline, replenished with DMEM with 10% FBS medium, with 100 U/mL penicillin and 100 μg/mL streptomycin and observed daily for cytopathic effect (CPE).Viral replication was assessed by an integrated C-RT-PCR method at 18 (T1), 48 (T2), and 72 (T3) hpi in cell culture supernatants, as well as by analyzing SARS-CoV-2induced CPE.RNA was extracted from VeroE6 cell culture supernatant by the Maxwell RSC Instrument with Maxwell RSC Viral Total Nucleic Acid Purification Kit (Promega).Real-time PCR was performed on a CFX96 (Bio-Rad) using the 2019-nCoV CDC qPCR Probe Assay emergency kit (IDT), which targets two regions (N1 and N2) of the nucleocapsid gene of SARS-CoV-2 (N2 data not shown).Reactions were performed according to the following thermal profile: initial denaturation (95°C, 10 min) followed by 45 cycles of 15 s at 95°C (denaturation) and 1 min at 60°C (annealing-extension).Viral copy quantification was assessed by creating a standard curve from the quantified 2019-nCoV_N positive Plasmid Control (IDT).