key: cord-1005437-5nek9en9
authors: Zmudzinski, Mikolaj; Rut, Wioletta; Olech, Kamila; Granda, Jarosław; Giurg, Mirosław; Burda-Grabowska, Małgorzata; Zhang, Linlin; Sun, Xinyuanyuan; Lv, Zongyang; Nayak, Digant; Kesik-Brodacka, Malgorzata; Olsen, Shaun K.; Hilgenfeld, Rolf; Drag, Marcin
title: Ebselen derivatives are very potent dual inhibitors of SARS-CoV-2 proteases - PLpro and Mpro in in vitro studies
date: 2020-08-31
journal: bioRxiv
DOI: 10.1101/2020.08.30.273979
sha: 326ea036e0b7f53ad6e1d633ec4e991bb6b262a9
doc_id: 1005437
cord_uid: 5nek9en9

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.

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] [7] [8] [9] [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 nonstructural proteins (nsps), two of them being proteases. The SARS-CoV-2 main protease (M pro , 3CL pro , nsp5) and the papain-like protease (PL pro , 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 M pro generates 13 viral nsps, making it a key player in the process of virus replication and maturation. [13] [14] [15] M pro 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 M pro 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] [17] [18] [19] SARS-CoV-2 PL pro is a viral cysteine protease proposed as an excellent target for COVID-19 treatment due to its pathophysiological roles. PL pro 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] [21] [22] [23] Thus, PL pro 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] [26] [27] Recently it was shown that ebselen inhibits both the SARS-CoV-2 proteases.

Weglarz-Tomczyk et al. evaluated ebselen 28 and a collection of its derivatives 29 as inhibitors of PL pro , 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 M pro 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 PL pro and M pro 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.

The efficacy of ebselen and other organoselenium compounds has been previously evaluated for HIV 30, 31 , HSV2 32 , HCV 33 , and Zika virus 34 infections. Moreover, a recent report presents ebselen and its derivatives as potent inhibitors of SARS-CoV-2 PL pro . 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).

First, we evaluated the inhibitory properties for the compounds at 1 µM inhibitor concentration and 100 nM SARS-CoV-2 PL pro . 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. PL pro 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 PL pro by the compounds. Derivatives with 5 strong EWGs (trifluromethyl group for 5 and nitro group for 6) displayed the 2 nd and 3 rd lowest potency, while the other compounds displayed approx. 50% of PL pro inhibition. We did not observe any significant differences between inhibitory properties between mono-and disubstituted ebselen derivatives.

Next, we screened the library at 100 nM inhibitors and 100 nM M pro concentrations. For the assay, we used a novel tetrapeptide fluorogenic substrate for SARS-CoV-2 M pro , QS1 (Ac-Abu-Tle-Leu-Gln-ACC; KM=207.3±12 µM, kcat/KM=859±57 M -1 s -1 ). 17 As a result, ebselen displayed 57.6% M pro inhibition. The best hits were compounds 10 and 17 with >80% of M pro inhibition. 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 M pro inhibition. Interestingly, the third best inhibitor (77.1%) has a 2,4-disubstituted phenyl ring. We also observed, that 2,4dimethoxy 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 M pro as we identified only 3 compounds (6, 7, 12) with a potency lower than for ebselen. 

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 M pro (10, 17) or PL pro (7) in the screening assay; or b), displaying relatively high inhibition towards both the investigated proteases (3, 16, 20, 21) (Fig. 2) 

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 M pro 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 pathways 24,26 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] [37] [38] We utilized a collection of ebselen derivatives to find potent SARS-CoV-2 PL pro and M pro inhibitors. First, we screened a library of organoselenium compounds. Next, for selected compounds, we determined IC50 values. The most potent PL pro inhibitor, 2-(3-hydroxypyridin- 

SARS-CoV-2 PL pro 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 PL pro 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.

SARS-CoV-2 M pro was recombinantly produced as described. 13 Briefly, the gene of the M pro 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 

The synthesis of biologically active organoselenium compounds is of current interest to many research teams around the world. [39] [40] [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] [45] [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- 

Evaluation of the compound library for inhibitors of SARS-CoV-2 PL pro and SARS- 

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 PL pro , 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 M pro , 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%. 

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This work was supported by the Medical Research Agency in Poland through its Own Project