New Results

Ebselen derivatives are very potent dual inhibitors of SARS-CoV-2 proteases - PLpro and Mpro in in vitro studies

Mikolaj Zmudzinski, Wioletta Rut, Kamila Olech, Jarosław Granda, Mirosław Giurg, Małgorzata Burda-Grabowska, Linlin Zhang, Xinyuanyuan Sun, Zongyang Lv, Digant Nayak, Malgorzata Kesik-Brodacka, Shaun K. Olsen, Rolf Hilgenfeld, Marcin Drag
doi: https://doi.org/10.1101/2020.08.30.273979

Abstract

Proteases encoded by SARS-CoV-2 constitute a promising target for new therapies against COVID-19. SARS-CoV-2 main protease (Mpro, 3CLpro) and papain-like protease (PLpro) are responsible for viral polyprotein cleavage - a process crucial for viral survival and replication. Recently it was shown that 2-phenylbenzisoselenazol-3(2H)-one (ebselen), an organoselenium anti-inflammatory small-molecule drug, is a potent, covalent inhibitor of both the proteases and its potency was evaluated in enzymatic and anti-viral assays. In this study, we screened a collection of 23 ebselen derivatives for SARS-CoV-2 PLpro and Mpro inhibitors. Our studies revealed that ebselen derivatives are potent inhibitors of both the proteases. We identified three PLpro and four Mpro inhibitors superior to ebselen. Our work shows that ebselen constitutes a promising platform for development of new antiviral agents targeting both SARS-CoV-2 PLpro and Mpro.

1. Introduction

In the winter of 2019, an outbreak of pneumonia with flu-like symptoms emerged in Wuhan, China.1,2 Shortly thereafter, the disease-causing pathogen was isolated and analyzed, leading to identification of the novel, highly contagious human beta-coronavirus SARS-CoV-2 (formerly known as 2019-nCoV).3 By the end of August 2020, with over 24.5 million people diagnosed with Coronavirus Disease 2019 (COVID-19), the death toll exceeded 830,000 patients worldwide.4 With neither vaccines nor drugs targeting the virus available, various strategies have been employed to accelerate finding an effective therapy to fight the pathogen.5 One of these strategies is drug repurposing - establishing therapeutic properties for already approved substances for new medical applications. This strategy can be supported by computational analysis, which can lower the costs, speed up the process in comparison with de-novo development of new therapeutics and serve as a first stage in screening vast libraries of active compound.6–10 Drug repositioning has been already successfully used in fighting COVID-19.11 A bright example here is remdesivir, an antiviral agent targeting viral RNA-dependent RNA polymerase (RdRp) that was designated to treat Ebola but has shown efficacy shortening recovery time and reducing mortality as well as serious adverse effects in COVID-19 patients.12 Nonetheless, current treatment options are critically limited and finding new therapeutics for COVID-19 patients constitutes a leading challenge for the scientific community.

To address the problem, medicinal chemists identified druggable targets among viral non-structural proteins (nsps), two of them being proteases. The SARS-CoV-2 main protease (Mpro, 3CLpro, nsp5) and the papain-like protease (PLpro, nsp3 papain-like protease domain) enable viral replication in host cells by processing the viral polyprotein and generating 16 nsps, crucial for virus replication. SARS-CoV-2 Mpro generates 13 viral nsps, making it a key player in the process of virus replication and maturation.13–15 Mpro is a dimeric cysteine protease with a structure highly conserved among human coronaviruses. Unusual preference for a glutamine residue at the P1 position of the substrate cleavage site sets Mpro apart from known human proteases. This feature can be beneficial for design and synthesis of effective, broad-spectrum antiviral agents with minimum side effects.7,13,16–19 SARS-CoV-2 PLpro is a viral cysteine protease proposed as an excellent target for COVID-19 treatment due to its pathophysiological roles. PLpro processes viral polyprotein and generates proteins nsp1-3. Moreover, the protease also alters the host immune response by deubiquitinating and deISGylating proteins within infected cells.20–23 Thus, PLpro inhibition would not only block the replication of the virus, but would also limit the dysregulation of cellular signaling mediated by ISG15 and ubiquitin.

2-phenylbenzisoselenazol-3(2H)-one (ebselen) is a small-molecule drug with a pleiotropic mode of action in cells.24 Ebselen is an excellent scavenger of ROS that acts as a mimic of glutathione peroxidase (GPx) and interacts with the thioredoxin (Trx) system by oxidation of reduced TrxR.25–27 Recently it was shown that ebselen inhibits both the SARS-CoV-2 proteases. Weglarz-Tomczyk et al. evaluated ebselen28 and a collection of its derivatives29 as inhibitors of PLpro, leading to identification of inhibitors with IC50 values in the nanomolar range. In another study, a library of approx. 10,000 drugs and drug candidates was screened for Mpro inhibitors. As a result, ebselen displayed the lowest IC50 among the substances tested (0.67 μM), furthermore it also displayed an antiviral effect in SARS-CoV-2 infected Vero cells.7

In this work, we used ebselen and a collection of 23 of its derivatives to evaluate their properties as SARS-CoV-2 PLpro and Mpro inhibitors. First, we screened the collection for inhibitors of both proteases. Next, we determined the half-maximum inhibitory concentration (IC50) values for the most promising hits. We show that ebselen may constitute a potential lead compound for development of novel antiviral agents. The results can be useful in the design of new active compounds targeting the proteases encoded by SARS-CoV-2, to be applied in COVID-19 treatment.

2. Results & Discussion

The efficacy of ebselen and other organoselenium compounds has been previously evaluated for HIV30,31, HSV232, HCV33, and Zika virus34 infections. Moreover, a recent report presents ebselen and its derivatives as potent inhibitors of SARS-CoV-2 PLpro.29 In order to find new inhibitors of proteases encoded by the new coronavirus, we screened a collection of ebselen and its 23 derivatives with mono- or disubstitutions within the phenyl ring (Tab. 1).

2.1. Compound library screening for SARS-CoV-2 PLpro inhibitors

First, we evaluated the inhibitory properties for the compounds at 1 μM inhibitor concentration and 100 nM SARS-CoV-2 PLpro. For the assay, we used the fluorogenic substrate Ac-LRGG-ACC with a structure based on the C-terminal epitope of Ub and ISG15 proteins as well as on the nsp1/2, nsp2/3, and nsp3/4 cleavage sites in the coronaviral polyprotein. PLpro screening resulted in identification of only one compound (7) with higher potency (84.4%) than ebselen (65.4%). However, 7 differed from the other compounds in the collection as it was the only investigated ebselen derivative with a 3-substituted pyridinyl moiety instead of a substituted phenyl ring. The results also show that electron-withdrawing groups (EWGs) at the ortho position of the phenyl ring hamper inhibition of the PLpro by the compounds. Derivatives with strong EWGs (trifluromethyl group for 5 and nitro group for 6) displayed the 2nd and 3rd lowest potency, while the other compounds displayed approx. 50% of PLpro inhibition. We did not observe any significant differences between inhibitory properties between mono- and disubstituted ebselen derivatives.

2.2. Compound library screening for Mpro inhibitors

Next, we screened the library at 100 nM inhibitors and 100 nM Mpro concentrations. For the assay, we used a novel tetrapeptide fluorogenic substrate for SARS-CoV-2 Mpro, QS1 (Ac-Abu-Tle-Leu-Gln-ACC; KM=207.3±12 μM, kcat/KM=859±57 M-1s-1).17 As a result, ebselen displayed 57.6% Mpro inhibition. The best hits were compounds 10 and 17 with >80% of Mpro inhibition. 10 represents monosubstituted derivatives with a nitro group at the para position. Other substitutions at the para position do not seem to have such a strong effect on inhibition of Mpro. On the other hand, our second best hit, 17, has a 5-chloro-2-fluoro disubstituted phenyl ring and represents a group of ebselen disubstituted derivatives. Analogically to 10, we did not identify any other 2,5-disubstitutions providing a similar effect on Mpro inhibition. Interestingly, the third best inhibitor (77.1%) has a 2,4-disubstituted phenyl ring. We also observed, that 2,4-dimethoxy derivative (16) displays potency towards both of the proteases close to ebselen’s, however, in comparison with ebselen, its toxicity evaluated in A549 human cell line was 10 times lower.35 In general, substitutions within the phenyl ring of ebselen boost inhibition of Mpro as we identified only 3 compounds (6, 7, 12) with a potency lower than for ebselen.

View this table:
Table 1.

The results of inhibitor screening for SARS-CoV-2 proteases. Assay conditions for SARS-CoV-2 PLpro: [E]=100 nM, [I]=1 μM, [S]=10 μM; for SARS-CoV-2 Mpro: [E]=100 nM, [I]=100 nM, [S]=50 μM.

2.3. IC50 determination

Based on the screening results, we selected ebselen and seven of its derivatives for further inhibitory property evaluation. We chose compounds: a), exhibiting the highest potency towards Mpro (10, 17) or PLpro (7) in the screening assay; or b), displaying relatively high inhibition towards both the investigated proteases (3, 16, 20, 21) (Fig. 2). During the assays, IC50 values for PLpro were in the micromolar range while for Mpro, they were in the low nanomolar range. For the reference inhibitor, ebselen, IC50 values were respectively 1.12μM ± 0.06 and 30.91 ± 2.67 nM. Compound 7, which was the best hit in the PLpro inhibitor screening assay, indeed had the lowest IC50 value (0.58 ± 0.04 μM) among the tested compounds. Despite ebselen being the second best PLpro inhibitor in the screening assay, we found that two other compounds (17, 21) displayed slightly lower IC50 values. As expected, 10 and 17, which were selected for the analysis as the best Mpro inhibitors, displayed lower IC50 values than ebselen. The most potent Mpro inhibitor with IC50=15.24 nM was compound 17, the second best hit from the screening experiment. Interestingly, the best hit (11) displayed an IC50 value similar to values determined for 3 and 21 (respectively 27.95, 25.69, and 27.37 nM). For 16 and 20, we observed that despite a higher potency in the screening assay, the IC50 values determined for these compounds were higher than for ebselen. In general, the screening results correlated well with the determined IC50 values (Tab. 2).

Figure 2.
Figure 2.

IC50 values of SARS-CoV-2 Mpro and PLpro inhibitors.

View this table:
Table 2.

Inhibitory properties for the selected compounds.

3. Conclusions

With increasing concerns about upcoming waves of new SARS-CoV-2 infections, the need for an effective and safe therapy against coronaviral diseases is growing. A promising strategy involves Mpro inhibition and this approach can lead to novel, broad spectrum anticoronaviral drugs.13 Recently, repurposing efforts enabled identification of ebselen as a potential drug against COVID-19, due to its action as a potent inhibitor of the SARS-CoV-2 main protease.7 Ebselen has a pleiotropic mode of action that is a result of its reactivity towards cysteine residues affecting many biological pathways24,26 but on the other hand is a well-known substance, the efficacy and safety in humans of which has been evaluated in various studies.36–38 We utilized a collection of ebselen derivatives to find potent SARS-CoV-2 PLpro and Mpro inhibitors. First, we screened a library of organoselenium compounds. Next, for selected compounds, we determined IC50 values. The most potent PLpro inhibitor, 2-(3-hydroxypyridin-2-yl)-1,2-benzoselenazol-3-one, displayed the highest potency in the screening assay and the lowest IC50 value (0.578 μM). IC50 determination enabled identification of two more compounds with inhibitory properties similar to ebselen. These compounds were 2,4- and 2,5-disubstituted derivatives of ebselen that displayed lower potency during screening, but also slightly lower IC50 parameters than for the reference inhibitor. A similar analysis for the Mpro enabled identification of four compounds displaying higher potency during screening and a lower IC50 parameter. Two of them had a monosubstituted phenyl ring at the para and ortho positions, and two were disubstituted ebselen derivatives. The best inhibitor with an IC50 value approx. 2 times lower than for ebselen was 2-(5-chloro-2-fluorophenyl)-1,2-benzoselenazol-3-one. In this work, we showed that ebselen derivatives with substitutions and other modifications within the phenyl ring generally possess good inhibitory properties against both the proteases encoded by the novel coronavirus. These compounds constitute a promising platform for novel therapeutics and we believe that the results obtained can be used to facilitate efforts towards new anticoronaviral drugs to be used for the treatment of COVID-19.

4. Materials and methods

4.1. SARS-CoV-2 PLpropreparation

SARS-CoV-2 PLpro was prepared as described.22 In brief, pGEX6P-1-SARS-CoV-2PLpro was transformed into BL21 (DE3) codon plus E. coli cells and induced with 0.1 mM IPTG and 0.1 mM ZnSO4 at 18°C overnight. GST-fusion SARS-CoV-2 PLpro was purified using standard protocol. The fusion protein was cleaved using GST-PreScission protease at 4°C overnight followed with desalting and passing through fresh glutathione beads to remove cleaved GST and GST-PreScission protease. The sample was further purified using Superdex 200 pg sizeexclusion columns (GE) equilibrated with 20 mM Tris-Cl pH 8.0, 40 mM NaCl and 2 mM DTT. The peak fractions were pooled and concentrated to ~10 mg/ml and snap frozen in liquid nitrogen for later use.

4.2. SARS-CoV-2 Mpro preparation

SARS-CoV-2 Mpro was recombinantly produced as described.13 Briefly, the gene of the Mpro was cloned into the PGEX-6p-1 vector, which has a Nsp4-Nsp5 and a PreScission cleavage site at the N- and C-termini to generate the authentic target protein, respectively. The gene of the target protein was expressed in the E. coli of the BL21-Gold (DE3) (Novagen) strain. The recombinantly produced Mpro was purified by employing HisTrap FF (GE Healthcare) and ionexchange chromatography (Q FF, GE Healthcare), respectively. Finally, the target protein with high purity was subjected to a buffer exchange (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.8) for further experiments.

4.3. Synthesis of ebselen derivatives

The synthesis of biologically active organoselenium compounds is of current interest to many research teams around the world.39–41 Ebselen and other benzisoselenazol-3(2H)-ones have been previously prepared by several ways.42,43 In this work, we successfully synthesized ebselen and benzisoselenazol-3(2H)-ones 1–23 functionalized at the N-2 position of the aryl ring, using a four-step procedure previously described in literature, starting with anthranilic acid and elemental selenium.35,44–46 The diazotation of previously protonated anthranilic acid, selenentylation with freshly prepared disodium diselenide gave 2,2’-dicarboxydiphenyl diselenide which was isolated before the reaction with thionyl chloride (SOCl2) to form 2-(chloroseleno)benzoyl chloride, which is a key substrate in the synthesis of the benzisoselenazol-3(2H)-one unit. Tandem selenenylation/acylation reaction of appropriate anilines used in excess or in stoichiometric amounts in the presence of anhydrous triethylamine base in anhydrous MeCN or DCM provided ebselen and final products 1–23. As a key aniline reagents we used 2-amino-3-hydroxy-pyridine, aniline and its mono- and disubstituted derivatives. The aniline substituents used in different positions of the benzene ring were Me, Ac, NHAc, NO2, CF3, OH, OMe and F, Cl, Br and I halides. Ebselen and compounds 1–23, of which eight are new, were obtained. Purity of all new compounds was characterized.

4.4. Inhibitor screening

Evaluation of the compound library for inhibitors of SARS-CoV-2 PLpro and SARS-CoV-2 Mpro was carried out in Corning 96-wells plates. For PLpro, 1 μL of each compound in DMSO solution was added to the wells. Next, 79 μL of enzyme preincubated for 10 min at 37°C in assay buffer (50 mM Tris, 5 mM NaCl, 0.075% BSA, pH 7.5) was added to each well. The enzyme was incubated with the compounds at 37°C for 30 min. Next, 20 μL Ac-LRGG-ACC substrate in assay buffer was added to the wells. Final concentrations were: 100 nM enzyme, 10 μM substrate and 1 μM tested compounds. In the assay for Mpro, 1 μL of each compound in DMSO solution was added to the wells. Next, 79 μL of enzyme in assay buffer (50 mM Tris, 1 mM EDTA, pH 7.3)47 was added to each well and the plate was incubated at room temperature for 2 min. Next, 20 μL of QS1 substrate in assay buffer was added to the wells. Final concentrations were: 100 nM enzyme, 50 μM substrate and 100 nM tested compounds. Measurements were carried out at 37°C using a Molecular Devices Spectramax Gemini XPS spectrofluorometer. ACC fluorophore release was monitored for 30 min (λex=355 nm, λem=460 nm). For the further analysis, the linear range of the progress curves was used. Measurements were performed at least in duplicate. Results were presented as mean values of relative enzyme inhibition (%, compared to the control measurement without inhibitor) with standard deviations. During the assays, the DMSO concentration in the wells was <2%.

4.5. IC50 determination

To determine IC50, the relative activity of investigated proteases was assessed in at least 11 different concentrations of selected inhibitors. Initial compound concentrations were found experimentally. Serial dilutions of inhibitors in assay buffers (described above) were prepared on 96-well plates (20 μL of each dilution in wells). For SARS-CoV-2 PLpro, 60 μL enzyme preincubated for 10 min at 37°C in assay buffer was added to the wells. The enzyme was incubated with inhibitors for 30 min at 37°C. Next, 20 μL substrate (Ac-LRGG-ACC) in assay buffer was added to the wells. Final concentrations were 100 nM enzyme and 10 μM substrate.

For SARS-CoV-2 Mpro, 60 μL enzyme was added with no preincubation. The enzyme was incubated with inhibitor for 2 min at room temperature. Next, 20 μL of substrate (QS1) in the assay buffer was added to the wells. Final concentrations were 100 nM for the enzyme and 50 μM for the substrate. Measurements were carried out at 37°C using a Molecular Devices Spectramax Gemini XPS spectrofluorometer. ACC fluorophore release was monitored for 30 min (λex=355 nm, λem=460 nm). IC50 values were determined with GraphPad Prism software using non-linear regression (dose-response - Inhibition equation) and presented as relative enzyme activity vs. inhibitor concentration. Measurements were performed at least in triplicate. Results are presented as mean values with standard deviations. During the assays, the DMSO concentration in wells was <2%.

Author contributions

M. Z. and M. D. designed the research; M. Z. and W. R. performed the research and collected data; K. O., J. G., M. G. and M. B.-G. synthesized and provided the collection of compounds, M. K.-B., L. Z., X. S. and R. H. provided SARS-CoV-2 Mpro enzyme; Z. L., D. N. and S. K. O. provided SARS-CoV-2 PLpro enzyme, M. Z. and W. R. analyzed and interpreted the inhibitory data and M. Z. wrote the manuscript; all authors critically revised the manuscript.

Acknowledgements

This work was supported by the Medical Research Agency in Poland through its Own Project (grant 2020/ABM/SARS/1) and by the National Science Centre in Poland (2020/01/0/NZ1/00063). The Drag laboratory is supported by the “TEAM/2017-4/32” project, which is conducted within the TEAM programme of the Foundation for Polish Science cofinanced by the European Union under the European Regional Development Fund. Work in the Hilgenfeld laboratory was supported by the SCORE project of the European Union (grant agreement # 101003627), by the German Center for Infection Research (DZIF), and by the Government of Schleswig-Holstein through its Structure and Excellence Fund, as well as by a close partnership between the Possehl Foundation (Lübeck) and the University of Lübeck. L.Z. is supported by a stipend from the German Center for Infection Research (DZIF). We are thankful to Wroclaw University of Science and Technology for support for the Department of Organic and Medicinal Chemistry – K20 (Statute Founds 82013902).

Reference

  1. 1.↵
    Wang, C., Horby, P. W., Hayden, F. G. & Gao, G. F. A novel coronavirus outbreak of global health concern. Lancet 395, 470–473 (2020).
    OpenUrlCrossRefPubMed
  2. 2.↵
    Wu, F. et al. A new coronavirus associated with human respiratory disease in China. Nature 579, 265–269 (2020).
    OpenUrlCrossRefPubMed
  3. 3.↵
    Andersen, K. G., Rambaut, A., Lipkin, W. I., Holmes, E. C. & Garry, R. F. The proximal origin of SARS-CoV-2. Nat. Med. 26, 450–452 (2020).
    OpenUrlCrossRefPubMed
  4. 4.↵
    Dong, E., Du, H. & Gardner, L. An interactive web-based dashboard to track COVID-19 in real time. Lancet Infect. Dis. 20, 533–534 (2020).
    OpenUrlCrossRefPubMed
  5. 5.↵
    Zumla, A., Chan, J. F. W., Azhar, E. I., Hui, D. S. C. & Yuen, K. Y. Coronaviruses-drug discovery and therapeutic options. Nat. Rev. Drug Discov. 15, 327–347 (2016).
    OpenUrlCrossRefPubMed
  6. 6.↵
    Khodadadi, E. et al. Study of combining virtual screening and antiviral treatments of the Sars-CoV-2 (Covid-19). Microb. Pathog. 146, 104241 (2020).
    OpenUrl
  7. 7.↵
    Jin, Z. et al. Structure of Mpro from COVID-19 virus and discovery of its inhibitors. Nature 582, (2020).
  8. 8.
    Elfiky, A. A. Ribavirin, Remdesivir, Sofosbuvir, Galidesivir, and Tenofovir against SARS-CoV-2 RNA dependent RNA polymerase (RdRp): A molecular docking study. Life Sci. 253, (2020).
  9. 9.
    Kandeel, M. & Al-Nazawi, M. Virtual screening and repurposing of FDA approved drugs against COVID-19 main protease. Life Sci. 251, 117627 (2020).
    OpenUrl
  10. 10.↵
    Rameshrad, M., Ghafoori, M., Mohammadpour, A. H., Nayeri, M. J. D. & Hosseinzadeh, H. A comprehensive review on drug repositioning against coronavirus disease 2019 (COVID19). Naunyn. Schmiedebergs. Arch. Pharmacol. 2019, (2020).
  11. 11.↵
    Joshi, S., Joshi, M. & Degani, M. S. Tackling SARS-CoV-2: proposed targets and repurposed drugs. Future Med. Chem. 2, (2020).
  12. 12.↵
    Beigel, J. H. et al. Remdesivir for the Treatment of Covid-19 — Preliminary Report. N. Engl. J. Med. 1–12 (2020) doi:10.1056/nejmoa2007764.
    OpenUrlCrossRef
  13. 13.↵
    Zhang, L. et al. Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved a-ketoamide inhibitors. Science (80-.). 368, 409–412 (2020).
    OpenUrlAbstract/FREE Full Text
  14. 14.
    Maier, H. J., Bickerton, E. & Britton, P. Coronaviruses: Methods and protocols. Coronaviruses Methods Protoc. 1282, 1–282 (2015).
    OpenUrl
  15. 15.↵
    Hilgenfeld, R. From SARS to MERS: crystallographic studies on coronaviral proteases enable antiviral drug design. FEBS J. 281, 4085–4096 (2014).
    OpenUrlCrossRefPubMed
  16. 16.↵
    Anand, K., Ziebuhr, J., Wadhwani, P., Mesters, J. R. & Hilgenfeld, R. Coronavirus Main Proteinase (3CLpro) Structure: Basis for Design of Anti-SARS Drugs. Science (80-.). 300, 1763–1767 (2003).
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    Rut, W. et al. Substrate specificity profiling of SARS-CoV-2 main protease enables design of activity-based probes for patient-sample imaging. PREPRINT bioRxiv 2020.03.07.981928 (2020) doi:10.1101/2020.03.07.981928.
    OpenUrlAbstract/FREE Full Text
  18. 18.
    Goyal, B. & Goyal, D. Targeting the Dimerization of the Main Protease of Coronaviruses: A Potential Broad-Spectrum Therapeutic Strategy. ACS Comb. Sci. 22, 297–305 (2020).
    OpenUrl
  19. 19.↵
    Yang, H. et al. Design of wide-spectrum inhibitors targeting coronavirus main proteases. PLoSBiol. 3, (2005).
  20. 20.↵
    Shin, D. et al. Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity. Nature (2020) doi:10.1038/s41586-020-2601-5.
    OpenUrlCrossRef
  21. 21.
    Freitas, B. T. et al. Characterization and Noncovalent Inhibition of the Deubiquitinase and deISGylase Activity of SARS-CoV-2 Papain-Like Protease. ACS Infect. Dis. 15, (2020).
  22. 22.↵
    Rut, W. et al. Activity profiling and structures of inhibitor-bound SARS-CoV-2-PLpro protease provides a framework for anti-COVID-19 drug design. PREPRINT bioRxiv 2020.04.29.068890 (2020) doi:10.1101/2020.04.29.068890.
    OpenUrlAbstract/FREE Full Text
  23. 23.↵
    Klemm, T. et al. Mechanism and inhibition of SARS-CoV-2 PLpro. PREPRINT bioRxiv 2020.06.18.160614 (2020) doi:10.1101/2020.06.18.160614.
    OpenUrlAbstract/FREE Full Text
  24. 24.↵
    Mugesh, G., Du Mont, W. W. & Sies, H. Chemistry of biologically important synthetic organoselenium compounds. Chem. Rev. 101, 2125–2179 (2001).
    OpenUrlCrossRefPubMedWeb of Science
  25. 25.↵
    Sarma, B. K. & Mugesh, G. Antioxidant activity of the anti-inflammatory compound ebselen: A reversible cyclization pathway via selenenic and seleninic acid intermediates. Chem. - A Eur. J. 14, 10603–10614 (2008).
    OpenUrl
  26. 26.↵
    Azad, G. K. & Tomar, R. S. Ebselen, a promising antioxidant drug: Mechanisms of action and targets of biological pathways. Mol. Biol. Rep. 41, 4865–4879 (2014).
    OpenUrlCrossRefPubMed
  27. 27.↵
    Antony, S. & Bayse, C. A. Modeling the mechanism of the glutathione peroxidase mimic ebselen. Inorg. Chem. 50, 12075–12084 (2011).
    OpenUrlPubMed
  28. 28.↵
    Węglarz-Tomczak, E., Tomczak, J. M., Talma, M. & Brul, S. Ebselen as a highly active inhibitor of PLProCoV2. PREPRINT bioRxiv 2020.05.17.100768 (2020) doi:10.1101/2020.05.17.100768.
    OpenUrlAbstract/FREE Full Text
  29. 29.↵
    Weglarz-Tomczak, E., Tomczak, J. M., Giurg, M., Burda-Grabowska, M. & Brul, S. Discovery of potent inhibitors of PLproCoV2 by screening libraries of selenium-containing compounds. PREPRINT bioRxiv 2020.05.20.107052 (2020) doi:10.1101/2020.05.20.107052.
    OpenUrlAbstract/FREE Full Text
  30. 30.↵
    Thenin-Houssier, S. et al. Ebselen, a small-molecule capsid inhibitor of HIV-1 replication. Antimicrob. Agents Chemother. 60, 2195–2208 (2016).
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    Sancineto, L. et al. Design and Synthesis of DiselenoBisBenzamides (DISeBAs) as Nucleocapsid Protein 7 (NCp7) Inhibitors with anti-HIV Activity. J. Med. Chem. 58, 9601–9614 (2015).
    OpenUrlCrossRefPubMed
  32. 32.↵
    Sartori, G. et al. Antiviral Action of Diphenyl Diselenide on Herpes Simplex Virus 2 Infection in Female BALB/c Mice. J. Cell. Biochem. 117, 1638–1648 (2016).
    OpenUrl
  33. 33.↵
    Mukherjee, S. et al. Ebselen inhibits hepatitis C virus NS3 helicase binding to nucleic acid and prevents viral replication. ACS Chem. Biol. 9, 2393–2403 (2014).
    OpenUrlCrossRefPubMed
  34. 34.↵
    Simanjuntak, Y. et al. Ebselen alleviates testicular pathology in mice with Zika virus infection and prevents its sexual transmission. PLoS Pathog. 14, 1–23 (2018).
    OpenUrlCrossRef
  35. 35.↵
    Giurg, M. et al. Reaction of bis[(2-chlorocarbonyl)phenyl] diselenide with phenols, aminophenols, and other amines towards diphenyl diselenides with antimicrobial and antiviral properties. Molecules 22, 974 (2017).
    OpenUrl
  36. 36.↵
    Lynch, E. & Kil, J. Development of ebselen, a glutathione peroxidase mimic, for the prevention and treatment of noise-induced hearing loss. Semin. Hear. 30, 47–55 (2009).
    OpenUrlCrossRef
  37. 37.
    Yamaguchi, T. et al. Ebselen in Acute Ischemic Stroke. Stroke 29, 12–17 (1998).
    OpenUrlAbstract/FREE Full Text
  38. 38.↵
    Masaki, C. et al. Effects of the potential lithium-mimetic, ebselen, on impulsivity and emotional processing. Psychopharmacology (Berl). 233, 2655–2661 (2016).
    OpenUrl
  39. 39.↵
    Sands, K. N. & Back, T. G. Key steps and intermediates in the catalytic mechanism for the reduction of peroxides by the antioxidant ebselen. Tetrahedron 74, 4959–4967 (2018).
    OpenUrl
  40. 40.
    Azeredo, J. B., Schwab, R. S. & Braga, A. L. Synthesis of Biologically Active Selenium-Containing Molecules From Greener Perspectives. Curr. Green Chem. 3, 51–67 (2016).
    OpenUrl
  41. 41.↵
    Kumar, S. et al. An ebselen like catalyst with enhanced GPx activity via a selenol intermediate. Org. Biomol. Chem. 12, 1215–1219 (2014).
    OpenUrl
  42. 42.↵
    Balkrishna, S. J., Bhakuni, B. S. & Kumar, S. Copper catalyzed/mediated synthetic methodology for ebselen and related isoselenazolones. Tetrahedron 67, 9565–9575 (2011).
    OpenUrl
  43. 43.↵
    Balkrishna, S. J., Bhakuni, B. S., Chopra, D. & Kumar, S. Cu-catalyzed efficient synthetic methodology for ebselen and related se-N heterocycles. Org. Lett. 12, 5394–5397 (2010).
    OpenUrlPubMed
  44. 44.↵
    Lesser, R. & Weiß, R. Über selenhaltige aromatische Verbindungen (VI). Ber. Dtsch. Chem. Ges (A B Ser. 57, 1077–1082 (1924).
    OpenUrl
  45. 45.
    Młochowski, J., Kloc, K., Syper, L., Inglot, A. D. & Piasecki, E. Aromatic and Azaaromatic Diselenides, Benzisoselenazolones and Related Compounds as Immunomodulators Active in Humans: Synthesis and Properties. Liebigs Ann. der Chemie 1993, 1239–1244 (1993).
    OpenUrl
  46. 46.↵
    Pietka-Ottlik, M. et al. Synthesis of new alkylated and methoxylated analogues of ebselen with antiviral and antimicrobial properties. Arkivoc 2017, 546–556 (2017).
    OpenUrl
  47. 47.↵
    Xue, X. et al. Production of Authentic SARS-CoV Mpro with Enhanced Activity: Application as a Novel Tag-cleavage Endopeptidase for Protein Overproduction. J. Mol. Biol. 366, 965–975 (2007).
    OpenUrlCrossRefPubMed
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Posted August 31, 2020.
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Ebselen derivatives are very potent dual inhibitors of SARS-CoV-2 proteases - PLpro and Mpro in in vitro studies
Mikolaj Zmudzinski, Wioletta Rut, Kamila Olech, Jarosław Granda, Mirosław Giurg, Małgorzata Burda-Grabowska, Linlin Zhang, Xinyuanyuan Sun, Zongyang Lv, Digant Nayak, Malgorzata Kesik-Brodacka, Shaun K. Olsen, Rolf Hilgenfeld, Marcin Drag
bioRxiv 2020.08.30.273979; doi: https://doi.org/10.1101/2020.08.30.273979
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