key: cord-0992358-l1gkg1nj
authors: Sarkar, Arighna; Mandal, Kalyaneswar
title: Repurposing an Antiviral Drug against SARS‐CoV‐2 Main Protease
date: 2021-09-21
journal: Angew Chem Int Ed Engl
DOI: 10.1002/anie.202107481
sha: f1d8d861bce9f80dfb4f959a0f867a209c91261c
doc_id: 992358
cord_uid: l1gkg1nj

This article highlights recent pioneering work by Günther et al. towards the discovery of potential repurposed antiviral compounds (peptidomimetic and non‐peptidic) against the SARS‐CoV‐2 main protease (M(pro)). The antiviral activity of the most potent drugs is discussed along with their binding mode to M(pro) as observed through X‐ray crystallographic screening.[Image: see text]

19 has turned out to be amajor pandemic of the 21 st century. With nearly 200 million cases and 4million deaths worldwide to date,i th as created havoc in society and immense human suffering. In ar ace against time to stop the spread of the disease,concurrent efforts of the scientific community worldwide have brought forth several potential small-molecule inhibitors of SARS-CoV-2 infection within av ery limited span of time. [1] Reports have shown that the viral entry and replication within the host cells involve multiple molecular factors from both the host and the virus. [2, 3] One of those important molecular factors is the main protease (M pro ), also referred to as the 3C-like protease (3CL pro ). M pro is essential for the cleavage of the viral polyprotein pp1ab at 11 discrete sites, Leu-Gln-fl-Ser/Gly/Ala being the cleavage motif.A fter cleavage,the released non-structural proteins form areplicase complex, which in turn is responsible for the viral replication. [2] Inhibition of this proteolytic cleavage can prevent SARS-CoV-2 replication inside host cells.H ence,M pro is ap rime target for antiviral drug discovery against SARS-CoV-2.

Within ay ear of the first reported case of COVID-19, several candidate drug molecules targeting M pro appeared in the literature,which include both ab initio designed [2, 4] as well as repurposed drugs. [3, 5] Particularly notable is the report by Douangamath et al. that demonstrated electrophilic as well as noncovalent fragment screening against M pro using ac ombination of mass spectrometry and crystallographic techniques with hits found both in the active site as well as in the dimerization interface. [6] Recently,u sing av ery similar yet elegant drug-repurposing crystallographic screen, Günther et al. [7] identified several highly potent small-molecule inhibitors of SARS-CoV-2 M pro .T he all-inclusive nature of the work by Günther et al. is vindicated by the discovery of both active-site as well as allosteric inhibitors of M pro from the library of molecules which are either already approved drugs or are in the clinical trial. They had shown 37 compounds that bind to SARS-CoV-2 M pro ,a mong which seven exhibited exceptional antiviral properties at nontoxic concentrations in cell-based viral reduction assays.T hese seven compounds consist of six nonpeptidic and one peptidomimetic smallmolecule binders ( Figure 1A -G).

TheM pro enzyme consists of aC -terminal helical domain and ac atalytic duo consisting of Cys 145 and His 41 in its active site. [2] Most of the binders use the active site for binding to M pro ,but, very interestingly,some of the binders employ novel allosteric binding sites,providing interesting insights into drug development against SARS-CoV-2 ( Figure 1H ). Günther et al. selected 5953 compounds from two repurposing libraries,T he Fraunhofer IME Repurposing Collection and Safein-man library from DompØ Farmaceutici S.p.A, [8] forc ocrystallization and then crystallographically screened them against M pro .After X-ray diffraction data collection followed by structure refinement, cluster analysis, [9] and pan dataset H) As chematic of the M pro dimer structure (the two protomers are shown in grey and orange). The active site and allosteric binding sites 1a nd 2along with 29 unambiguous binders bound to them are shown in green. Catalytic residues His41 and Cys145 in one of the protomers are also highlighted. [7] [*] A. Sarkar 

Chemie density analysis, [10] Thea dvantage of utilizing compounds from drug-repurposing libraries is that these molecules have already proven cell permeability and bioactivity. [11] Günther et al. harnessed this advantage very effectively to target M pro .F rom the hits obtained from the X-ray screen, they examined several compounds for antiviral activities in SARS-CoV-2 cell-based assays.Surprisingly,nine compounds were able to reduce viral RNAreplication by afactor of 100. Tw oofthem (AT7519 and Ifenprodil) showed reduction in viral RNAr eplication to aslightly lesser extent than that but had different binding sites outside the active site,m aking them worth further investigation. These 11 compounds were then further examined to evaluate the effective concentration at which 50 %o ft he SARS-CoV-2 infectious particles were reduced (EC 50 ). This evaluation finally resulted seven compounds (AT7519, Calpeptin, Ifenprodil, Tolperisone,P elitinib,M UT056 399, Tr iglycidyl isocyanurate;F igure 1A-G) that were antivirally active with nearly 100-fold reduction of infectious particles and aselectivity index (CC 50 /EC 50 )greater than 5, representing no cytotoxicity in tested concentration range.H ere CC 50 stands for 50 %c ytotoxic concentration.

Among these seven antiviral candidates,t he two most potent ones are Calpeptin (EC 50 = 72 nM, CC 50 > 100 mM) and Pelitinib (EC 50 = 1.25 mM, CC 50 = 13.96 mM), which are suitable for preclinical testing and deserve more discussion in this context. Calpeptin is ap eptidomimetic inhibitor that binds to the active-site residue Cys 145 ,forming athiohemiacetal through its aldehyde warhead. Thebackbone is involved in hydrogen bonding with His 164 and Glu 166 ,w hereas the norleucine side chain is engaged in van der Waals interaction with Phe 140 ,L eu 141 ,a nd Asn 142 (Figure 2A ). Thei nhibitory effect of this molecule on SARS-CoV-2 replication in Vero E6 cells is shown ( Figure 2C ). Calpeptin also inhibits Cathepsin L [12] and thus this dual targeting of M pro and Cathepsin Lcan provide an ew avenue to drug discovery.P elitinib,a n anticancer drug [13] with the second highest anti SARS-CoV-2a ctivity,o nt he other hand, is unique because it does not bind to the active site of M pro .I tr ather makes use of the hydrophobic pocket in the C-terminal dimerization domain which forms the first allosteric binding site.Pelitinib inserts its halogenated aromatic moiety into the hydrophobic groove formed by Ile 213 ,L eu 253 ,G ln 256 ,V al 297 ,a nd Cys 300 .A lthough Pelitinib is aM ichael acceptor,n oc ovalent linkage with the active-site Cys 145 was observed in the electron density maps, as it binds far away from the active site.T he function of M pro depends on the structure of the active site and the correct orientation of the subdomains.T he ethyl ether substituent of Pelitinib pushes the Ty r 118 and Asn 142 residues of the opposing protomer within the dimer and most likely hampers the dimerization process as well as the correct structural orientation of the pocket necessary for M pro function ( Figure 2B ). Hence,b yb inding to the first allosteric site it is capable of stopping viral replication. Theinhibitory effect of Pelitinib on SARS-CoV-2 replication in Vero E6 cells is shown in Figure 2D .

Thep ioneering work by Günther et al. provides us valuable insights on functional antiviral drugs against the deadly SARS-CoV-2 obtained from high-throughput crystallographic screening,several of which are suitable for preclinical studies.T he two allosteric sites identified during the screening could be exploited further as additional targets to accelerate drug discovery efforts.T he potential of peptidomimetic drugs has also been excellently demonstrated along with non-peptidic drugs,h ighlighting the fact that screening against such repurposing drug libraries has great potential and can be used to target other regions of the coronavirus like the spike receptor binding domain and/or N-terminal domain. Thec ovalent binding strategy would be an added advantage for the same.A lso,t he beautiful example of allosteric inhibition as well as dual inhibition provides an ew vista to the science of drug discovery.T he study by Günther et al. is apromising start to the production of even more potent drug candidates against this deadly coronavirus and may have opened the door to ad eeper perception in drug discovery against any virus that might have the power to create future pandemics. . The viral RNA (v-RNA) yield (filled circles), viral titers (half-filled circles), and cell viability (empty circles) for Calpeptin (C) and Pelitinib (D);f or each, the EC 50 for viral titer reduction is given. [7] Portions of this figure are reprinted with permissionf rom ref. [7] ,C opyright 2021, American Association for the Advancement of Science.

2020, 12, eabc5332;c )

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Thea uthors acknowledge the intramural funds at TIFR Hyderabad from the Department of Atomic Energy (DAE) to K.M.

Theauthors declare no conflict of interest.