Author of summary: Rocco Stirparo, Livia Tepshi; Reviewer: Valeria Montis, Giulia Poggi
Mpro is a key SARS-CoV-2 enzyme, which plays a pivotal role in mediating viral replication and transcription, making it an attractive drug target. Currently, there are no targeted therapeutics against SARS-CoV-2 and effective treatment options remain very limited. In order to rapidly discover lead compounds for clinical use, the authors of this mini-review performed a different set of virtual screening, such as combined structure-assisted drug design, virtual drug screening and high-throughput screening, to identify new or existing drugs targeting the CoViD-19 virus main protease.
Mpro is a crucial protease in SARS-CoV-2, responsible for viral replication and transcription. Mpro has three domains: Domains I (residues 8–101), II (residues 102–184) and III (residues 201–303). Mpro has a Cys–His catalytic dyad, and the substrate-binding site is located in a cleft between Domain I and II. X-ray crystallography structure of SARS-CoV-2 Mpro shows that this protease can be found in monomeric and dimeric states and only the latter represent the functional state in the biological context (Fig. 1).
Crystal structural and docking data have shown that N3 inhibitor, a Michael acceptor that act as a mechanism-based inhibitor, can bind to the substrate-binding pocket of SARS-CoV-2 Mpro, which seems to be highly conserved among all CoV Mpros (MERS and SARS). The structure of Mpro in complex with N3 provides a model for identifying lead inhibitors to target this protease through in silico screening.
Using virtual screening, the 5-HT2A and 5-HT2C serotonin receptor antagonist Cinanserin was found to fit in the substrate-binding pocket of Mpro. FRET assay-based screening, performed on a library of ~10,000 compounds (including approved drugs, clinical trial drug candidates and natural products) suggests Ebselen as the strongest inhibitor of SARS-CoV-2 Mpro activity . This organoselenium compound presents with anti-inflammatory, antioxidant and cytoprotective properties and is currently in phase II clinical trial for treating bipolar disorders and hearing loss.
An independent computational ligand screening identified the target site of four pharmacophores with potential activity against SARS-CoV-2 Mpro protease (i.e. OEW, Remdesivir, Hydroxychloroquine and N3) and employed it for screening 50,000 new potentially active compounds from the ZINC Database . The most promising and newly identified molecules belong to the β-carboline, Alkaloids and Polyflavonoids classes. As well as the known ligands, these compounds interact with the Cys–His dyad of the protease pocket and are therefore suitable candidates to be tested in vitro and in vivo for pharmacological applications.
Previously acquired knowledge on Coronaviridae Mpro structure has also been employed to devise pharmacological approaches against the new SARS-CoV-2. In fact, it has been observed that backbone and binding site conformations of Coronaviridae Mpro are conserved, despite variation in some of the residues. This allows for repurposing of already available viral protease inhibitors . Joshi et al. screened a custom-made library of ~7100 molecules, including active ingredients present in the Ayurvedic antitussive medicines, antiviral phytochemicals and synthetic antivirals,. Natural compounds as δ-Viniferin, Myricitrin, Taiwanhomoflavone A, Lactucopicrin 15-oxalate, Nympholide A, Biorobin and Phyllaemblicin B were shown to strongly bind SARS-CoV-2 MPro and to target also RdRp and hACE-2.
Docking approaches have been also employed to repurpose commercially available drugs. MD simulations coupled with the MM/GBSA approach provided insights on the binding efficiency and inhibitory activity of 15 FDA-approved drugs towards the monomeric and dimeric state of SARS-CoV-2 Mpro . With this approach, Bello  revealed the structural and energetic basis of the inhibitory properties of Lopinavir and Ritonavir and identified new (and potentially more efficacious) inhibitors of dimeric SARS-CoV-2 Mpro, namely Perampanel, Praziquantel and Nelfinavir.
Also, structural similarities among the Hepatitis C virus (HCV) NS3/4A protease, the HIV-1 protease and SARS-CoV2 protease Mpro have been reported (Fig. 2). Therefore, HCV or HIV-1 protease inhibitors could fit well into the active-site cleft of the SARS-CoV2 protease Mpro and show comparable inhibitory activity .
Indeed, virtual docking predictions and comparison of AutoDock docking scores indicate that 8 HCV protease inhibitors (Nelfinavir, Saquinavir, Indinavir, Ritonavir, Darunavir, Tipranavir, Atazanavir, Lopinavir, Amprenavir) may effectively bind to the SARS-CoV-2 Mpro active site.
The functional importance of Mpro in the viral life cycle, together with the absence of closely related homologues in humans, identify the Mpro as an attractive target for antiviral drug design and drug repurposing could be evaluated as CoViD-19 therapeutics.
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 Joshi R, Jagdale S, Bansode S, et al. Discovery of Multi-Target-Directed Ligands by Targeting Host-specific SARS-CoV-2’s Structurally Conserved Main Protease. Preprints.org; 2020.
 Martiniano Bello. Prediction of potential inhibitors of the dimeric SARS-CoV-2 main proteinase through the MM/GBSA approach, 06 April 2020, PREPRINT (Version 1).
 Bafna, Khushboo; Krug, Robert M.; Montelione, Gaetano (2020): Structural Similarity of SARS-CoV2 Mpro and HCV NS3/4A Proteases Suggests New Approaches for Identifying Existing Drugs Useful as COVID-19 Therapeutics. ChemRxiv. Preprint.