New insights in the mechanism of the SARS-CoV-2 Mpro inhibition by benzisoselenazolones and diselenides

doi:10.21203/rs.3.rs-4082138/v1

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New insights in the mechanism of the SARS-CoV-2 M^pro inhibition by benzisoselenazolones and diselenides

https://doi.org/10.21203/rs.3.rs-4082138/v1

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published 21 Oct, 2024

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Although global vaccination campaigns relieved the SARS-CoV-2 pandemic in terms of morbidity and mortality, the capability of the virus to originate mutants may reduce vaccines efficiency, posing a serious risk to fall into the pandemic again. As a result, there is the need to develop small molecules able to tackle conserved viral targets, such as the main protease (M^pro). Here a series of benzisoselenazolones and diselenides were tested for their ability to inhibit M^pro, then, for the most potent compounds, the antiviral activity was measured in vitro, and the mechanism of action was investigated. Density functional theory and molecular docking procedures were also implemented to shed a light into the protein/compound interaction. Finally, a bioorganic model was set up to investigate the reaction between selenorganic compounds and biologically relevant thiols, to unravel possible metabolic pathways of such compounds. The overall results contribute to identify a series of novel Se-containing molecules active against SARS-CoV-2, and to clarify some important aspects in the mechanisms of action of such inhibitors targeting the SARS-CoV-2 M^pro.

Biological sciences/Chemical biology

Biological sciences/Drug discovery

Physical sciences/Chemistry

Benzisoselenazolones

diselenides

SARS-CoV-2 main protease inhibitors

glutathione

Ebselen

The Coronaviridae family was first globally recognized as a public threat during the emergence of the severe acute respiratory syndrome (SARS) in 2003¹ and, a decade later, its potential was confirmed by the emergence of the middle-east respiratory syndrome (MERS) ². Nonetheless, it was the unprecedented rapid emergence of SARS-CoV-2 and the resulting COVID-19 pandemic that triggered global research efforts to develop vaccines, therapeutics, and diagnostics to tackle this danger. While today effective vaccines are available, they do not prevent the infection and, in high-risk individuals, additional layers of protection are still desirable. Regrettably, despite the identification of several molecular targets of SARS-CoV-2^3–5, the number of compounds required to drive an effective drug discovery program remains relatively limited compared to the undergoing efforts, with the majority of research focused on drug repurposing^6,7.

Main protease (M^pro) and papain-like protease (PL^pro) are two viral enzymes essential for virus replication and evasion of the host immune responses. The absence of closely related cellular homologs makes these enzymes particularly attractive targets for the design of coronavirus-specific antiviral agents. Among the first compounds to be discovered to block their enzymatic activity, the organoselenium compound 2-phenyl-1,2-benzoisoselenazol-3-one (ebselen, compound 1a)^8,9 has been identified in a high-throughput screening study as a potent inhibitor of the M^pro, within a pool of existing small-molecules ¹⁰. Ebselen showed a potent inhibitory activity toward M^pro, with an IC₅₀ in the high nanomolar range (670 nM) and inhibitory activity toward the infectious virus with an EC₅₀ in the low micromolar range (4.67 µM)¹⁰. Subsequently, it was demonstrated that ebselen also inhibits the papain-like protease (PL^pro) from SARS-CoV-1 and SARS-CoV-2 (with an IC₅₀ = 2.26 µM for PL^pro of SARS-CoV-2)¹¹. The same authors of such pioneering study recently reported a series of ebselen analogues as potent inhibitors of nsp14 guanine N7‑methyltransferase¹².

Studies focusing on the identification of the mode of action of ebselen (1a) suggested that it acts as an irreversible inhibitor, covalently binding to the reactive cysteines in the PL^pro and M^pro active sites to form a stable selenyl sulfide. Tandem mass spectrometry and computational calculations proved that for SARS-CoV-2 the reactive cysteine of M^pro is Cys145 ^4,13, whereas molecular modeling suggested that Cys111 is the target of ebselen within the PL^pro active site ¹⁴. In addition, molecular dynamics simulation suggested that, besides a covalent inhibition, ebselen may interact and inhibit M^pro also non-covalently by binding in a pocket localized between the II and the III domains of the protein¹³.

The capability of ebselen to easily modify cysteines is well documented ^15–19, and this could represent a major issue for its actual clinical exploitability. On the other hand, several studies demonstrated an almost complete absence of toxic effects in vivo, and this has also been proved by the clinical trials in which 1a was evaluated^9,20. Furthermore, from a general standpoint, ebselen shows several additional pharmacological effects that have been pointed out to be favorable in the context of curing COVID-19 ²¹.

Beside ebselen, a few others selenium-containing compounds endowed with anti-SARS-CoV-2 activity have been reported in literature (Fig. 1).

In particular, Yang, Zhang, O’Neill, and Hasnain tested a series of benzisoselenazolone analogs, previously reported as neuroprotective agents. They identified compounds 1b and 1c as being more active in inhibiting M^pro than 1a. At the same time, in an attempt to resolve the co-crystal structure with the protein, the authors reported that these compounds were able to transfer a hydrogenselenide unit to Cys145 through a S_NAr-like reaction taking place in the M^pro active site²². A related study was carried out by Kumar et al., in which it was also proved the selenylation of the very same cysteine also by other benzisoselenazolones ²³. Recently, Rana et al. reported a series of ebselen close analogs with anti-M^pro activity, with compounds 1d and 1e being the best in class. These derivatives showed an improved anti-viral activity not only in Vero cells but also in other cell lines chosen because they better mirrored the lung epithelium ²⁴. Zhang, Wang, et al. recently reported benzisoselenazolones bearing different aromatics at the N2 positions. In their study, authors identified compounds endowed with low nanomolar IC₅₀ and antiviral activity in the micromolar range ²⁵.

Besides benzisoselenazolones, selenides proved to be suitable substituents to improve the anti-SARS-CoV-2 activity of the natural product quercetin ²⁶. The resulting selenoquercetin analogs (2a and 2b) demonstrated a striking ability to inhibit M^pro, with compound 2b being active also in vitro at EC₅₀ in the low micromolar range and without showing any cytotoxicity ²⁷. Recently also other selenides were reported by some of us as capable of inhibiting SARS-CoV-2 in a cellular context ²⁸.

Taken together, these results clearly underline the value of selenorganic derivatives, in particular ebselen-like structures, as lead compounds to develop novel anti-SARS-CoV-2 agents.

In light of this, we herein report the anti-SARS-CoV-2 properties of ebselen-like derivatives (compounds 1f-s) and a series of closely related diselenides (compounds 3,4). Considering that in studies reporting an anti-SARS-CoV-2 activity the ebselen core is always decorated with aromatic and benzylic substituents, we sought to prepare benzisoselenazolones bearing aliphatic side chains in order to enlarge the structure-activity relationship (SAR) for this class of compounds. In addition, we provide mechanistic insight to prove that, for certain derivatives, a plausible metabolic link between benzisoselenazolones and the corresponding diselenides actually exists.

For the whole set of compounds herein reported, the anti-M^pro activity was initially assessed through an in vitro screening, and then mass spectrometry confirmed the covalent enzymatic inhibition. The most potent derivatives were then assayed in a cellular model of SARS-CoV-2 infection, by also investigating their mechanism of action. Not only benzisoselenazolones were found to be active, but also diselenides. A bioorganic, NMR-based model was purposely developed to get insight into the intracellular fate of the studied compounds.

Diphenyl diselenide (compound 8) and selenocystine (compound 6) are commercially available. Dibenzyl diselenide (compound 7) was prepared following the procedure reported in literature ²⁹. Ebselen-like compounds 1f-1l were prepared starting from 5 ^29,30, which was first coupled with amino acids protected as esters leading to compound 3f, h-l ³¹. The ester derivatives were then converted into the corresponding benzisoselenazolone 1f-l, following a procedure recently reported by some of us ³². Diselenides having an acid moiety were obtained starting from the corresponding esters through mild basic hydrolysis, leading to compound 4f, h-l (Scheme 1).

Diselenides 3n-q, s were prepared from the corresponding ebselen-like compounds (1n-q, s) ³³ via a sequential NaBH₄-mediated reduction of the Se-N bond and air oxidation of the so-formed selenolate anion.³⁴

As a first line of screening, the whole set of compounds was preliminarily assayed in vitro at the concentration of 40 µM, showing full inhibition of the enzymatic activity of M^pro (See Table S1). Then, the IC₅₀ was determined, and the results are collected in Tables 1–3.

The ebselen derivatives (1f-s) were generally more potent than the corresponding diselenides 3f-s and 4f-l. Derivatives 1k and 1i were the best in class among the compounds tested, with an IC₅₀ in the low nanomolar range. In all the cases, an anti-M^pro activity higher than that of ebselen and most of its derivatives reported in the literature was observed, highlighting that the replacement of the aromatic ring with an aliphatic one improves the ability to bind and inhibit the viral protease. On the contrary, terpenes-containing derivatives 1m-s displayed activity in the high nanomolar range.

Regarding the diselenides, compounds 5 and 7 were unable to exert any sort of inhibition, whereas selenocystine (compound 6) had an IC₅₀ in the high micromolar range. Surprisingly, also diphenyl diselenide 8 showed a low micromolar activity. The introduction of a carboxamide substituent in the ortho position with respect to the selenium atom preserved or even improved the activity, as in the case of compound 3f. From the comparison among compounds containing an ester functionality (3h-l) with the ones containing an acid moiety (4h-l), it clearly emerged that the latter were less active in the M^pro inhibition. Again, the terpene derivatives 3p-q and 3s were able to inhibit M^pro_, but they required a higher concentration compared to the other compounds, and compound 3n was completely inactive.

The better activity of ebselen-derivatives in comparison with the diselenides could be ascribed to a higher electrophilicity of the former, which facilitates the reaction with reactive thiols and, as a consequence, the covalent inhibition of the protein. The greater electron deficiency of ebselen derivatives can be clearly deduced from the chemical nature of the two functional groups (a selenazolone and a diselenide) and is also confirmed spectroscopically by comparison of their ⁷⁷Se NMR chemical shifts, ranging from 804 to 935 ppm for benzisoselenazolones and from 439 to 450 ppm for the diselenides.

As a further evaluation of the electrophilicity of these compounds, the geometry of some selected ebselen-like and diselenides systems was optimized by DFT (b3lyp-d3/def2-tzvp level of theory), and the natural partial atomic charges were computed by using the Natural Population Analysis (NPA) as implemented in the NBO software suite. The results are shown in Table 4.

Table 4

NPA partial charge (in e) on the selenium atom (two values are shown for diselenide systems) on some ebselen-like and diselenides systems.
compound	q(Se)	compound	q(Se)
1a	0.607	3k	0.165/0.167
1f	0.613	3p	0.143/0.175
1i	0.619	5	0.173/0.186
1k	0.617	6	0.104/0.122
1p	0.608	8	0.151/0.153

The q(Se) value resulted to be inversely proportional to the IC₅₀ value; compounds having q(Se) > 0.610 e are the most active. This observation further supports the hypothesis of a nucleophilic attack of the sulfur of the cysteine to the selenium as a key step in the inhibition process in the tests in vitro. Noteworthy, some diselenides show an asymmetric charge distribution among the two selenium atoms. This is a consequence of a non-symmetrical involvement of the selenium atoms in weak interactions with the nearby chemical environment (Figure S1). For example, in 3p only one selenium is involved in two hydrogen bonds with the two amide groups, producing a slight polarization of the Se-Se bond.

Molecular docking was also used as an additional computational tool to investigate the binding of selected ligands in the active site of M^pro, and the steric constraints that may limit their access to the binding pocket. In particular, we focused on 1a (ebselen), 1i, and 1r, as representative examples of compounds with subtle structural difference leading to appreciable variations among their IC₅₀ values (see Table 1 and data reported in literature for 1a¹⁰). The results reported in Table 5 show that these compounds have a good affinity towards the binding site of M^pro, with docking scores of at least ‒6 kcal/mol, which is comparable with values previously found for other experimentally-validated binders of this protein.^26,35 The differences among the three compounds tested were negligible within the limit of the accuracy (about ± 0.3 kcal/mol) in the scoring function of the docking algorithm.³⁶ This finding suggests that the activity in the high nanomolar range observed for 1r, and in common with the other terpenes-containing derivatives, should not be ascribed to steric clashes within the binding site. We also observed that the affinities of these compounds did not change significantly by considering as flexible the side chains of protein residues His41/Cys145, indicating that rearrangements of the catalytic dyad of M^pro do not play an essential role in the binding.

In the docking poses, the minimum non-bonding distance found between the sulfur atom of Cys145 and the selenium was ~ 3.7 Å, as it could be expected considering their van der Waals radii (1.8 and 1.9 Å, respectively). However, we also verified (see Methods section) that each compound is able to form a covalent bond Se‒S (with a distance < 2.5 Å) and still fit within the active site of M^pro without requiring a local reorganization of the protein pocket. Therefore, although docking simulations cannot predict the fate of the molecular structure of these compounds, still they cannot exclude that it might be intact until the formation of the covalent bond Se‒S.

Table 5

Affinity (calculated considering the catalytic dyad His41/Cys145 in the binding pocket either as rigid or flexible) and minimum non-bonding distance between Se and S atoms observed in molecular docking for selected ebselen-like compounds.
compound	Affinity (kcal/mol) [rigid dyad]	Affinity (kcal/mol) [flexible dyad]	Minimum distance (Å) [Se···S]
1a	‒6.142	‒6.256	3.766
1i	‒5.938	‒6.290	3.606
1r	‒6.351	‒6.597	3.734

We then evaluated whether the anti-M^pro activity of the most potent compounds translate into the inhibition of the SARS-CoV-2 replication. The selected compounds were glycine derivatives 1f, 1g, 3f, and 4f, isoleucine derivatives 1i and 3i, valine derivatives 1k, 3k, and 4k, and benzisoselenazolones containing glutamic and aspartic esters 1j and 1k. Ebselen 1a was tested in parallel as a positive control. First, the cytotoxicity was evaluated at 100, 80, 60, 40, 20 µM concentration in Vero cells. No toxicity was observed for all compounds except for 3f that that was highly toxic at 100 µM (Figure S2). Next, confluent monolayers of Vero cells were infected with the virus in the presence or absence of the inhibitors. All compounds were tested at 100, 80, 60, 40, and 20 µM concentration (except for 3f, which was not tested at 100 µM). 2 days post-infection, the cytopathic effect was assessed, and toxicity for 1f, 1l, 3i was observed at 100 µM and for 1g at 100 and 80 µM, therefore these conditions were not collected for further analysis. Cytotoxicity for some compounds observed in infected cells as compared to XTT assay could be due to cellular stress caused by viral infection. All remaining samples were harvested and evaluated by means of quantitative PCR coupled with reverse transcription (RT-qPCR) (Figure S3). The EC₅₀ values were determined as summarized in Table 6, with the exception of compound 4f which was inactive. All compounds inhibited viral replication at low micromolar levels, with the ebselen-like compounds 1j and 1l endowed with the lowest, and mutually similar, EC₅₀ (7 and 8 µM, respectively). From a SAR standpoint, the double ester functionality seems to improve the antiviral activity of the compounds. The similarity of the EC₅₀ of the two compounds mirrors their activity toward the Mpro, against which they showed comparable potencies (see Tables 1 and 6). Compound 1i, which is a nanomolar M^pro inhibitor, inhibits the viral replication with remarkable activity. Unexpectedly, the most potent M^pro inhibitor, 1k, exerted an antiviral activity lower than expected on the basis of the biochemical assay, most probably for pharmacokinetic reasons. Among diselenides, the esters were way more potent than acids (see data for 3f vs 4f, and 3k vs 4k), regardless of their side chain.

Table 6

EC50 values of the tested compounds.a
compound	EC₅₀ (µM)
1a	12
1f	24
1g	15
1i	11
1j	8
1k	24
1l	7
3f	21.3
3i	11
3k	15
4f^b	> 100
4k	32
^aCompounds 1a, 1i, 1j, 1k, 3k and 4k were devoid of any toxicity up to 100 µM, compounds 1f, 1l, 3f and 3i up to 80 µM and compound 1g up to 60 µM; ^binactive in antiviral assay up to 100µM

Time of addition (TOA) experiments were also carried out to map the mode of action of the compounds. The most promising ebselen-like derivatives 1i and 1l, and diselenides 3i and 3k, together with 1a, were added at three different stages of virus infection at a concentration of 60 µM.

Assay I (PRE) was used to assess whether the compounds may render the cells resistant to the virus. As such, they were added before infection and cells were pre-incubated with compounds for 1 h at 37°C. Assay II (WITH) was to verify whether the compounds affect viral entry (i.e., early stages of replication). Thus, they were added with the virus, during the infection. Finally, the assay III (POST) in which compounds were added 2 h after infection was performed to assess their impact on viral replication and egress (i.e., late stages of replication).

As shown in Fig. 2, the compounds 1i, 1j, and 1l inhibited the SARS-CoV-2 virus at the early and late stages of the infection. We did not observe any effect of preincubation of cells or virions, suggesting that the inhibition occurs during the virus entry and replication. In the case of compounds 3i, 3k, and for ebselen (1a) used as a control, inhibition was observed at multiple stages, including in the pre-incubation assay, suggesting a different mechanism of action or an intracellular accumulation of the compounds. Conversely, the lack of activity of ebselen-like compounds 1i, 1j, and 1l could derive from their reaction/interaction with cellular thiols different from Cys145 of M^pro. Such a reaction would convert the compounds into their selenylsulfide analogs, whose activity against the viral protease remains to be proven.

The electrophilic reaction of selenium-containing compounds with nucleophilic thiols is a well-known process ³⁷, and this pro-oxidant property should explain why some of them are toxic. Among the thiols, glutathione (GSH) is the most abundant in living cells, especially under oxidative stress conditions ³⁸. For this reason, a direct interaction between every electrophilic selenium-containing compound and GSH may be considered as the first chemical event that involves them in the cellular environment. With the aim to improve our understanding of this topic, we set up an NMR-based model meant to study the reaction/interaction between selected compounds and GSH. In particular, beside an ¹H-NMR analysis, we selected the ⁷⁷Se NMR that represents an ease and reliable method to identify organoselenium derivatives in non-purified reactions mixtures directly performed in an NMR tube ³⁹. Unfortunately, limits associated with sensitivity and relaxation time hampered a quantitative interpretation of the obtained results.

The poor solubility of ebselen and its derivatives in buffered aqueous conditions forced us to select DMSO-d₆ as solvent for the reaction, even though it could not be considered totally inert based on its mild oxidant properties.

As reported by Back and coworkers, one molar equivalent of reduced glutathione rapidly converts 1 into 9 (Scheme 3) ⁴⁰. It is also known that the second equivalent of reductant (GSH) promotes the rapid and quantitative formation of the corresponding diselenide 3a. We demonstrated that this latter transformation can be activated by 5 mol % of starting from 9 and leading to the formation of diselenide 3a and oxidized glutathione in up to 50% conversion, accounting for a self-catalytic process according to the equilibrium depicted in Scheme 3 (Figure S4).

Diselenide 3a, synthesized according to literature ¹¹, was analyzed by means of ⁷⁷Se NMR in DMSO-d6 and a peak at 442 ppm was observed. By the addition of a one molar equivalent of GSH, the signal of selenylsulfide 9 at 547 ppm appeared. Not all the diselenide was consumed in the reaction, indicating that the reaction with GSH is not as fast as that involving compound 1 (Figure S5). Diselenide 3k was subjected to the same investigations, showing a behavior similar to that of the derivative 3a (Figure S6).

Two things emerge clearly from the proposed mechanism: 1) under reducing conditions ebselen 1a cannot be considered as the final chemical entity responsible for the interaction with M^pro; 2) both 1a and the corresponding diselenide 3a in the presence of a reducing thiol establish an equilibrium between 9, 10 and 3a, that it was demonstrated to be prone to further thiol exchange processes. When this mixture was reacted with N-acetylcysteine (NAC), ⁷⁷Se NMR showed the formation of another compound having a chemical shift compatible with that of the selenylsulfide 11 (Figure S7). This indicate that the above-described equilibria can be further modulated by thiol exchange processes.

These data suggest that a particular attention should be paid in the interpretation of the data produced by the enzymatic inhibition test. In fact, when it is performed under non-reducing conditions, it may not be able to correctly interpret the molecular mechanism and consider the molecular species actually involved in a real biological environment, losing its predictability.

For this reasons, freshly prepared compounds 1a and 3a were assayed in parallel against M^pro both in the presence and absence of DTT (as a reducing thiol). The results are summarized in Table 7.

As also reported by Wang⁴¹, the anti M^pro activity of 1a drops dramatically in the presence of DTT, and this is also true for diselenide 3a, that, in the presence of the reducing agent, displays an IC₅₀ in the high micromolar range. As expected, in the absence of a reducing agent, ebselen is more potent that its diselenide, mirroring the higher electrophilicity of the selenium atom. In the presence of DTT, both 1a and 3a produced the same effect in terms of IC₅₀ (considering that 3a is a dimer), in accordance with the mechanism proposed in Scheme 3.

Clearly, this mechanism require that that Se-Se bond should be polarized. As an example, glutathione is not able to reduce diphenyl diselenide 8 even when used in a large excess. In our cases, the amide functionality adjacent to the selenium atom can establish a non-bonded interaction able to modulate the electrophilicity and the redox properties of the selenium atom. This underlines that probably not all the diselenides (and not all the ebselen-derivatives) share the same reactivity and mechanism in the enzyme inhibition. Beside the covalent inhibition, a non-covalent mechanism of action can reasonably be speculated for some of them. This is the case of compound 8, which is still able to inhibit M^pro, but thought a non-covalent mechanism as recently proposed by Orian and Rocha.⁴²

HRMS analysis performed under reducing conditions (for the presence of DTT, see SI) evidenced that both selenazolones and diselenides bind covalently the M^pro, but not in the same manner and not selectively. The mass of apo M^pro is ~ 33.8 kDa, in line with previous observations.¹⁰ In experiments with ebselen 1a, and the analogous 1i, 1l, and 1k, a mass corresponding to M^pro in covalent complexes with one, two, or three intact molecules was observed. In contrast, 3a and 3i, afforded peaks corresponding to M^pro in covalent complexes with one, two, or three halves of the intact compounds. In the case of 1j, the increment in M^pro mass did not correspond to either the intact molecule or half of it. Finally, 3k did not produced relevant covalent modification of M^pro.

To rule out that the inhibition could arise from compound 9, we tested by molecular docking simulations whether it could sterically bind within the active site of M^pro, but the results suggest that the GSH moiety would remain solvent-exposed, and sterically hamper the anchoring of the compound with the selenium in a position favorable to form a covalent bond with Cys145. This finding is coherent to the observations recently reported by Teixeira da Rocha ⁴³.

Finally, as a further investigation of the mechanism showed by those compounds that were proved to inhibit viral replication when administered before virus addition, an entry inhibition assay was set up. Compounds 1a, 3i, and 3k were tested for their ability to inhibit the spike (S) protein-mediated internalization of pseudoviruses, using the VSV-G-glycoprotein-decorated pseudoviruses as control. Such a system allows one for the identification of a direct interference with the entry process and the assessment of the selectivity of this process. As shown in Fig. 3, compound 1a showed the ability to block the entry of both pseudoviruses when tested at 60 µM, indicating that it interferes with the viral entry process in an unspecific manner. In contrast, compounds 3i and 3k did not inhibit the entry of the pseudoviruses.

From the screening of some benzisoselenazolones and diselenide analogues, a series of new M^pro inhibitors has been identified. Computational investigations proved that the inhibition is likely covalent with a direct proportionality between the IC₅₀ and the electrophilicity of selenium among the tested compounds. Most of the M^pro inhibitors are endowed with antiviral activity measured in a cell context without major cytotoxicity, thus leading to positive selectivity index values, even if the non-specific activity of selenium compounds is well-described and reported in several cases^41,44–46. Time of addition studies proved that, although the antiviral potency (expressed in terms of EC₅₀ values) is similar among the compounds, differences are still present. Ebselen, as an example, is able to inhibit viral replication when added either pre-, with- and post–infection, whereas benzisoselenazolones 1i, 1j and 1l are devoid of any activity when the virus is added to the cells. This indicates that minor structural variations in these compounds have an impact on their specific antiviral properties, but not in their overall ability to cause an M^pro inhibition.

We have also demonstrated that the antiviral activity cannot be traced back in a simplistic manner to the direct interaction of benzisoselenazolones with residue Cys145 of M^pro. Its marked electrophilicity leads it to react rapidly and quantitatively with glutathione, most likely leading to a mixture in which the glutathionated adduct is in a dynamic equilibrium with the corresponding selenol and diselenide. This equilibrium can be obtained also starting from the diselenide, and it is prone to further modulation thought thiol exchange processes that could be envisioned as those responsible for the non-selective selenenylation of different free cysteine of the enzyme. Furthermore, it was demonstrated that ebselen also acts by inhibiting the spike (S) protein-mediated internalization of pseudoviruses, depicting a multifaced non-selective mechanism of action that involves both benzisoselenazolone derivatives and the corresponding diselenides. A more generalist approach is therefore necessary in the interpretation of the mechanism of action of these derivatives, and any reductionist simplification may miss important details of their functioning.

DFT studies

All the geometry optimizations were performed with the Orca code⁴⁷, version 4.1.0 at the B3LYP/def2-TZVP level and def2/J auxiliary basis. Dispersion effects were taken into account using the Grimme D3-parametrized empirical dispersion correction, with the Becke–Johnson (BJ) damping function^48,49. Frequency calculations were carried out at the same level of theory, to ensure that the stationary structures had no imaginary frequencies. The solvent effects were modelled using the Conductor-Like Polarizable Continuum Model (CPCM), with water as solvent.

NBO atomic charges has been computed using the NBO6 suite of software⁵⁰.

Molecular docking

Molecular docking was performed by using AutoDock Vina, version 1.2.3 ³⁶, following a protocol already used to test the binding of other ligands to the main protease of SARS-CoV-2 ^35,51. The structure of M^pro was taken from the crystal conformation of the ligand-free enzyme (Protein Data Bank entry: 6Y2E ⁵²), and considered either fully rigid or partly flexible in the sole side chains of the catalytic dyad His41/Cys145. Compounds 1a, 1i, and 1r, simulated as representative examples of ebselen-like ligands, were built by using the molecular editor Avogadro, version 1.2.0 ⁵³, and full flexibility was allowed for rotations around their dihedral angles. The binding of Se-GSH to the active site was also probed in the same way. In all cases, a blind search was carried out within a volume encompassing the whole protein, and with an exhaustiveness 16 times larger than the default value ⁵⁴.

Additional simulations were run to test whether compounds 1a, 1i, and 1r could be covalently bond with the catalytic Cys145 without requiring a structural reorganization of the active site of M^pro. In this case, M^pro was built from the complex with an ebselen-derivative inhibitor (Protein Data Bank entry: 7W9G ⁵⁴), in which the sole coordinates of the selenium atoms bound to S-Cys145 are reported. Since AutoDock Vina is not able to simulate a covalent docking, each of our compounds was modeled as an adduct already bound to the (modified) residue Cys145, and with the same C^α‒S‒Se geometry present in the crystallographic structure. Afterwards, the self-docking of the fully-flexible side chain of such Cys-modified residue and the rest of the rigid protein structure was simulated. The existence of bound conformations of the compounds that fit into the protein active site was verified by visual inspection, as well as by binding affinities (< ‒15 kcal/mol) incompatible with extensive steric clashes (which would lead instead to large positive values).

Chemistry

Compound 6 and 8 were purchased from Sigma Aldrich and used without further purifications. Compounds 1a, 1f-1l were prepared as we previously reported in Nascimento et al. ³², compound 3a was prepared as reported in Weglarz-Tomczak et al. ¹¹, compounds 3f, 3h-3l, 4f, 4h-4l and 5 were prepared as reported in Sancineto et al. ³¹. Compound 7 was prepared as reported in Krasowska et al. ²⁹. Diselenides 3n-q, s were prepared from the corresponding ebselen-like compounds (1n-q, s) ³³ via a sequential NaBH₄-mediated reduction of the Se-N bond and air oxidation of the so-formed selenolate anion ³⁴. Spectral data are superimposable to those reported in literature.

NMR experiments

NMR experiments were carried out at 25°C on a Bruker Avance NEO 600 MHz spectrometer equipped with Cryoprobe Prodigy and operating at 600 MHz for ¹H, and 114.45 MHz for ⁷⁷Se experiments. ¹H, and ⁷⁷Se chemical shifts (δ) are reported in parts per million (ppm) and they are relative to TMS 0.0 ppm and the residual solvent peak of DMSO-d6 at δ 2.35 ppm. ⁷⁷Se experiments were referenced to PhSe₂ (⁷⁷Se δ = 463 ppm in CDCl₃).

For the experiments reported in Figure S4, a solution of compound 9 (21 mg, 0.036 mmol), prepared as reported in Sands et al. ⁴⁰, in 1 ml of DMSO-d6 was prepared, then 0.0018 mmol of GSH (from a 0.4 M stock solution in D₂O) were added. Sequential ¹H and ⁷⁷Se NMR spectra were recorded.

For the experiment reported in Figure S5, a solution of compound 3a (10 mg, 0.018 mmol) in 1 ml of DMSO-d6 was prepared, then a stoichiometric amount of GSH (0.018 mmol from the above-mentioned stock solution) was added. Sequential ¹H and ⁷⁷Se NMR spectra were recorded.

For the experiment reported in Figure S6, a solution of 3k (12 mg, 0.018 mmol) in 1 ml of DMSO-d6 was prepared, then a stoichiometric amount of GSH (0.018 mmol from the above-mentioned stock solution) was added. Sequential ¹H and ⁷⁷Se NMR spectra were recorded.

For the experiment of Figure S7, a solution of 1a (5.1 mg, 0.018 mmol) in 1 ml of DMSO-d6 was prepared, then a stoichiometric amount of GSH (0.018 mmol from the above-mentioned stock solution) was added. Sequential ¹H and ⁷⁷Se NMR spectra were recorded. To the same sample, a stoichiometric amount of N-acetyl cysteine (NAC, from a 0.4 M stock solution in D₂O) was then added. Sequential ¹H and ⁷⁷Se NMR spectra were recorded to reveal the formation of a 1:1 mixture of two selenylsulfides 9 and 11. To the same sample, 0.018 mmol of GSH was later added, sequential ¹H and ⁷⁷Se NMR spectra were recorded, and a higher amount of 9 was observed as revealed by the heights of the ⁷⁷Se resonance peak. Finally, to the same sample, 0.036 mmol of NAC was added, and a higher amount of 11 was observed as revealed by the heights of the ⁷⁷Se resonance peak.

Cells and viruses

Vero cells (Cercopithecus aethiops; kidney epithelial; ATCC CCL-81) and HeLa cells overexpressing ACE2 (HeLa^ACE2) cells were maintained in Dulbecco-modified Eagle's medium (DMEM, high glucose, ThermoFisher Scientific, Poland) supplemented with 5% heat-inactivated fetal bovine serum (FBS, ThermoFisher Scientific, Poland). The medium was supplemented with penicillin (100 U/mL, ThermoFisher Scientific, Poland) and streptomycin (100 µg/ml, ThermoFisher Scientific, Poland). Cells were cultured at 37°C in an atmosphere containing 5% CO₂ and humidity. Every two weeks, cells were tested for mycoplasma contamination.

Reference SARS-CoV-2 strain 026V-03883 was kindly granted by Christian Drosten, Charité—Universitätsmedizin, Berlin, Germany, by the European Virus Archive—Global (EVAg); https://www.european-virus-archive.com/, accessed on 15 April 2021).

SARS-CoV-2 stock was generated by infecting monolayers of Vero cells. The cells were incubated at 37°C under 5% CO₂. The virus-containing medium was collected at day 2 post-infection (p.i.), aliquoted, and stored at -80℃. Control samples from mock-infected cells were prepared in the same manner.

Virus yields were assessed by titration on fully confluent cells in 96-well plates according to the method of Reed and Muench.⁵⁵ Plates were incubated at 37℃, and the cytopathic effect (CPE) was scored by observation under an inverted microscope.

Time of addition assay

To discover the mechanism of action of the tested compounds, the inhibitors were added at three different stages of virus infection: PRE – compounds were added before infection, and cells were pre-incubated with compounds for 1 h at 37°C; WITH – compounds were added to the virus, during the infection; POST – compounds were added after infection (2 h post-infection). In details, the Vero cells were seeded in a culture medium on a 96-well plate 1 day before infection. Fully confluent cells were inoculated with 1600 TCID₅₀/ml of the SARS-CoV-2 virus, either in the presence or absence of the inhibitors. Mock control and medium control were also included. Cells were then incubated for 2 h at 37°C and 5% CO₂. Afterward, the cells were washed twice with PBS. Only in the POST-treatment version, each compound was applied into the cells monolayer, whereas in other conditions (PRE and WITH) the medium was applied into the cells monolayer. Cell culture supernatants were collected after 24 h for viral RNA isolation. The SARS-CoV-2 experiment was performed in triplicate biological and technical replications.

Pseudoviruses experiments

To verify the activity of the tested compound on PRE infection step, the assay with pseudoviruses was conducted. Briefly, HeLa^ACE2 cells were seeded in 96-well plates, cultured for 24 h at 37°C with 5% CO₂, and pre-incubated with tested compounds (60 µM) for 30 min at 37°C and then transduced with pseudoviruses harboring VSV-G or S-SARS-CoV-2 proteins or lacking the fusion protein (ΔEnv) in the presence of polybrene (4 mg/ml, Sigma-Aldrich, Poland). After 4 h of incubation at 37°C, unbound virions were removed by three washes with PBS, and cells were further cultured for 72 h at 37°C with 5% CO2. Cells were lysed in Bright-Glo luciferase assay buffer (Promega, Poland) and transferred onto white 96-well plates. Luminescence levels were measured on SpectraMax iD5 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA).

Isolation of Nucleic Acids, Reverse Transcription, and Quantitative PCR

A viral DNA/RNA kit (A&A Biotechnology, Gdansk, Poland) was used for nucleic acid isolation from cell culture supernatants. RNA was isolated according to the manufacturer’s instructions. Viral RNA was quantified using quantitative PCR coupled with reverse transcription (RT-qPCR) (GoTaq Probe 1-Step RT-qPCR System, Promega, Poland) using a CFX96 Touch real-time PCR detection system (Bio-Rad, Munich, Germany). The reaction was carried out in the presence of the probes and primers (Fwd: CAC ATT GGC ACC CGC AAT C; Rev: GAG GAA CGA GAA GAG GCT TG; probe: 6FAM-ACT TCC TCA AGG AAC AAC ATT GCC A-BHQ-1). The heating scheme was as follows: 15 min at 45°C and 2 min at 95 ℃, followed by 40 cycles of 15 s at 95 ℃ and 1 min at either 58 ℃ or 60 ℃. In order to assess the copy number of the N gene, standards were prepared. The PCR product was amplified and cloned into pTZ57R/T plasmids using an InsTAclone PCR cloning kit (Thermo Scientific). The resulting plasmid was linearized, and its concentration was assessed using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA); the number of copies was deducted based on the Avogadro constant. Eight 10-fold serial dilutions were used as a qPCR template to develop a standard curve.

Statistical Analyses

The results are expressed as mean ± standard error of the mean (SEM). The statistical significance of the data presented in the manuscript was assessed with the non-parametric Kruskal–Wallis test, and P values below 0.05 were considered significant unless stated otherwise. Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA). For the determination of the half-maximal inhibitory concentration (IC₅₀), a dose–response curve fit was performed by using a nonlinear regression model.

Author Contributions: Conceptualization, JK, JS, YL, KP, CS; methodology, AD, JK, JS, YL, CS; formal analysis, LS, FM, AD, CSc, VC, JK, GC, BR, AK-P, JL; investigation, LS, FM, AD, AJP, MOF, CSc, VC, JK, YZ, GC, VN, BR, AK-P, YL; data curation, LS, JK, GC, YL, KP; resources, JK, JS,YL, KP, CS; software, GC, BR; writing original draft preparation, LS, JK, GC, CS; writing review and editing, LS, AD, JK, GC, BR, AK-P, HY, YL, KP, CS; visualization, LS; supervision, JK, CS, YK, KP, CS; funding acquisition, JS, YL, KP, CS. All authors have read and agreed to the published version of the manuscript.

Acknowledgments: This work was undertaken under the umbrella of the Selenium Sulfur Redox & Catalysis Network (SeSRedCat).

Funding: (CS) This research was supported by Department of Pharmaceutical Sciences with the project DELPHI "Dipartimenti di Eccellenza 2018-2022" and by University of Perugia with project “Ricerca di Base”.

(KP) This work was supported by the DURABLE project, co-funded by the European Union, under the EU4Health Programme (EU4H) (https://health.ec.europa.eu/funding/eu4health-programme-2021-2027-vision-healthiereuropean-union_en#work-programmes), and ERAnet ICRAD—project Musecov: Multiscale Eco-evolution of Coronaviruses: from surveillance toward emergence prediction (https://www.era-learn.eu/network-information/networks/icrad/1st-icrad-call-2019/multiscale-eco-evolution-of-coronaviruses-from-surveillance-toward-emergence-prediction)

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Patient and Public Involvement: Patients or human data were not involved in this study

Data Availability Statement: Data are provided within the manuscript and supplementary information files. Further details are available under reasonable request to the corresponding authors: Ying Lei, Krzysztof Pyrc and Claudio Santi

Conflicts of Interest: The authors declare no conflict of interest.

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Tables 1 to 3 and 7 are available in the Supplementary Files section.

Schemes 1 to 3 are available in the Supplementary Files section.

No competing interests reported.

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New insights in the mechanism of the SARS-CoV-2 M^pro inhibition by benzisoselenazolones and diselenides

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DFT studies

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Cells and viruses

Time of addition assay

Pseudoviruses experiments

Isolation of Nucleic Acids, Reverse Transcription, and Quantitative PCR

Statistical Analyses

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References

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