key: cord-0973601-ll7e8ily
authors: Yan, Fangfang; Gao, Feng
title: An overview of potential inhibitors targeting non-structural proteins 3 (PLpro and Mac1) and 5 (3CLpro/Mpro) of SARS-CoV-2
date: 2021-08-24
journal: Comput Struct Biotechnol J
DOI: 10.1016/j.csbj.2021.08.036
sha: d46c1cc493c0108f7b0f70f8977ca69d46d94f12
doc_id: 973601
cord_uid: ll7e8ily

There is an urgent need to develop effective treatments for the coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid spread of SARS-CoV-2 has resulted in a global pandemic that has not only affected the daily lives of individuals but also had a significant impact on the global economy and public health. Although extensive research has been conducted to identify inhibitors targeting SARS-CoV-2, there are still no effective treatment strategies to combat the COVID-19. SARS-CoV-2 comprises two important proteolytic enzymes, namely, the papain-like proteinase, located within non-structural protein 3 (nsp3), and nsp5, both of which cleave large replicase polypeptides into multiple fragments that are required for viral replication. Moreover, a domain within nsp3, known as the macrodomain (Mac1), also plays an important role in viral replication. Inhibition of their function should be able to significantly interfere with the replication cycle of the virus, therefore these key proteins may serve as potential therapeutic targets. The functions of the above viral targets and their corresponding inhibitors have been summarized in the current review. This review provides comprehensive updates of nsp3 and 5 inhibitor development and would help advance the discovery of novel anti-viral therapeutics against SARS-CoV-2.

Since the end of 2019, a novel respiratory disease that manifests as pneumonia has spread across many countries, and has garnered significant attention worldwide. Deep whole-genome sequencing of the patient samples revealed that the pathogen responsible for this respiratory disease is a novel coronavirus (nCoV), tentatively named 2019-nCoV by the World Health Organization (WHO) . Similar to the severe acute respiratory syndrome coronavirus (SARS-CoV), 2019-nCoV belongs to the zoonotic β-coronaviruses family, and it shares a high degree of homology with SARS-CoV. On February 11, 2020, the International Committee on Taxonomy of Viruses (ICTV) renamed this coronavirus as SARS-CoV-2 [1] . The WHO termed the disease caused by SARS-CoV-2 as coronavirus disease 2019 (COVID-19) [2] . SARS-CoV-2 can achieve person-person transmission through a variety of ways, including prolonged close contact or via the inhalation of virus-containing aerosols [3, 4] . People who are infected with SARS-CoV-2 typically present a fever, cough and chest discomfort, and severe patients develop acute respiratory distress syndrome (ARDS), leading to death in some cases [5, 6] . SARS-CoV-2 has spread worldwide due to its high transmission rate, and the number of deaths caused by this disease continues to increase. The global COVID-19 pandemic not only brings panic or worry to people, but also poses a great threat to the global economy, public health and the daily livelihoods of many people globally [7, 8] . Considering the current grave situation, the development of reliable inhibitors to combat SARS-CoV-2 has become a critical task.

Currently, extensive efforts have been made to identify reliable inhibitors against SARS-CoV-2, and some inhibitors have even entered the stage of clinical trials. The results of some clinical trials have been briefly summarized in Table S1. Through the evaluation of possible inhibitors, it was found that some inhibitors failed to achieve the expected efficacy or showed obvious negative effects in clinical trials, which brings great difficulties to the treatment of COVID-19.

To identify effective viral inhibitors, it is necessary to have a comprehensive understanding of the potential therapeutic targets for SARS-CoV-2, and the study of the structural properties of SARS-CoV-2 can provide insights for the discovery of therapeutic targets and inhibitors. Recent developments in next-generation sequencing (NGS) technologies and related bioinformatics analysis methods can greatly assist in SARS-CoV-2 research. Within a month of the COVID-19 outbreak, the genome of SARS-CoV-2 was made available on public databases, such as the National Center for Biotechnology Information (NCBI) database [9] . SARS-CoV-2 is the largest genome among the known RNA viruses. The SARS-CoV-2 genome consists of 10 open reading frames (ORFs). Among them, ORF1a and ORF1b occupy approximately two-thirds of the whole SARS-CoV-2 genome, and they are used as templates to encode two large replicase polypeptides, pp1a and pp1ab, after the virus invades the host cell [10] . The pp1a and pp1ab can only initiate replication of their own genetic material after they are cleaved into various fragments that perform different functions [11] ; they can be separately cleaved into 11 non-structural proteins (nsps; nsp1-nsp11) and 16 nsps (nsp1-nsp16) by the virus-encoded 3-chymotrypsin-like proteinase (nsp5, also called 3CL pro or M pro ) and the papain-like proteinase (PL pro ) that is present in nsp3 ( Fig. 1A and 1C ).

Detailed information on the cleavage sites, cleavable octopeptides and the nsps of the replicase polypeptides in SARS-CoV-2 are shown in Fig. 1A and 1C, and they can be accurately predicted using our newly updated ZCURVE_CoV 2.0 database (http://tubic.tju.edu.cn/CoVdb) [12] .

According to the predicted results in Table S2 , it was found that three nsps (nsp1-nsp3) were produced via the cleavage by PL pro , while the others were produced by 3CL pro . Three-dimensional (3D) models of these nsps are shown in Fig. 1A and Fig. 1C and can be downloaded directly from https://zhanglab.ccmb.med.umich.edu/C-I-TASSER/2019-nCoV [13] . Some of the models are also available in the Protein Data Bank (PDB) database (http://www.rcsb.org) [14] . The 3D models of the cleavable octopeptides were also predicted using a suitable PEP-FOLD3 software with a lower limit of 5 amino acids (http://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3) [15] , as shown in Fig. 1 . As described by Chou et al., Gan et al. and Wei el al., the cleavable octapeptide of SARS-CoV should be able to bind to the active site of 3CL pro , and therefore, modified octapeptides could be used as ideal SARS-CoV inhibitors [16] [17] [18] . Similarly, modified octapeptides of SARS-CoV-2 are also expected to become candidate inhibitors for SARS-CoV-2, but there is a lack of relevant research in this respect. Apart from ORF1a and ORF1b, the remaining ORFs are distributed in the last third of the SARS-CoV-2 genome, and they encode at least four structural proteins: spike (S), envelope (E), membrane (M) and nucleocapsid (N), as well as some accessory proteins, including 3a, 6, 7a, 7b, 8, 9b, 9c, and 10 ( Fig. 1B) [19] .

S protein, as one of the four important structural proteins, plays a vital role in virus invasion and membrane fusion due to its two major regions (S1 and S2) [20] . S protein is regarded as an ideal target for vaccines and antibodies [20] [21] [22] , and related studies have been well summarized by different groups [23] [24] [25] [26] . Among all non-structural proteins, two viral proteases (nsp5 and nsp3) appear to be particularly important. Nsp5 and PL pro in nsp3 are responsible for cleaving the large replicase polypeptides into fragments that can perform multiple functions, thereby assisting the replication of the virus. Meanwhile, PL pro and nsp5 contain highly conserved and well-defined druggable binding sites, whereas no binding sites are found in some nsps, such as nsp2, nsp7 and nsp8 [27] . In addition to PL pro , nsp3 also contains another important domain (macrodomain) related to viral replication and to the host's innate immunity. Thus, nsp3 and nsp5 may potentially be good therapeutic targets for COVID-19. To promote a better understanding of the function of nsp3 and nsp5 in SARS-CoV-2 infection and the current state of inhibitor discovery, we conducted a comprehensive review of the function of these potential therapeutic targets and the development of corresponding inhibitors. This review is meant to provide a convenient resource of different viral targets and should assist with the development of specific inhibitors.

SARS-CoV-2 nsp3 is a multi-domain enzyme with 1,945 amino acids, and it is the largest protein among all nsps. Nsp3 is remarkable because of the existence of two functional domains.

One of the functional domains is a catalytically active PL pro domain. As shown in Fig. 1D , the PL pro can be divided into four distinct domains: the fingers (β4-β7), thumb (α2-α7), palm (β8-β13) and ubiquitin-like (UBL) (β1-β3), and the first three domains constitute the catalytic core of the protein [28] . The druggable substrate-binding pocket is mainly determined by nine residues (R166, L185, L199, V202-E203, M206-M208, and K232) [29] . PL pro participates in the efficient cleavage of the N-terminal replicase polyprotein to produce functional proteins; this process of functional protein production plays an essential role in maintaining the basic cellular processes of SARS-CoV-2, including viral replication [30] . The other important functional domain of nsp3 is the ADP-ribose phosphatase (ADRP) domain, also known as macrodomain (Mac1) or Macro X domain. Mac1 is mainly consists of seven β-sheets (β1-β7) and six α helices (α1-α6) (Fig. 1E ). Among them, four βsheets (β3 and β5-β7), α1, and two loops (β3-α2 and β6-α5) are involved in the formation of binding pocket [31] . Mac1 is a highly conserved and unique sequence in the genome of many viruses and organisms, including humans [32, 33] , and inhibitors targeting this enzyme may have broadspectrum antiviral activity. Notably, both PL pro and Mac1 can help the virus escape the antiviral immune response of the host [34] . Consequently, inhibiting the activity of PL pro and Mac1 will interfere with the replication cycle of the virus, maintain the innate immune pathways of the host and reduce the infection rate. In view of the importance of these two functional domains of nsp3, we summarized the inhibitor-related research that has been performed regarding these domains; moreover, we selected the best inhibitors from each study, as shown in Fig. 2 . To gain insight into the binding modes of inhibitors, the binding mechanisms of these selected inhibitors to proteins are summarized, as shown in Table S3 .

In April 2020, Osipiuk et al. first reported the crystal structure of SARS-CoV-2 PL pro , which is available in PDB (PDB ID: 6W9C). Based on this structure, studies have been performed to explore potential inhibitors of PL pro enzymatic activity. Alamri and colleagues conducted virtual screening (VS) of 6,968 protease inhibitors from Asinex library (https://www.asinex.com/protease) using AutoDock Vina software, and three compounds (ADM_13083841, LMG_15521745 and SYN_15517940) were identified as potential inhibitors of PL pro (Fig. 2A1 ) [35] . Kandeel et al. used the Maestro software to virtually screen 1,697 FDA-approved inhibitors from Selleckchem Inc. database (http://www.selleckchem.com) and obtained 26 compounds with lower negative docking scores (< -7 kcal/mol) [36] . After evaluating the binding free energies between compounds and PL pro , three compounds (phenformin, quercetin, and ritonavir) were considered as potential inhibitors (Fig.   2A2 ). Rut et al. performed a comprehensive location screening of the LKGG motif based on the Hybrid Combinatorial Substrate Library (HyCoSul), and the results indicated that the tetrapeptides VIR250 and VIR251 are expected to behave as potent peptide inhibitors (Fig. 2A3 ) [37] . Using the structure of apo PL pro as a reference, they predicted the structures of VIR250 and VIR251 bound to [38] . Based on the same molecular structure, a virtual screening study was carried out by Delre et al. to search SARS-CoV-2 PL pro inhibitors from 2,390 inhibitors used in clinical trials, as reported in the ChEMBL bank (https://www.ebi.ac.uk/chembl). Two covalent inhibitors (curcumin and afatinib) and several different types of non-covalent inhibitors (protein kinase inhibitors (dasatinib, pexidartinib and copanlisib), protease inhibitors (amprenavir, indinavir, anagliptin, boceprevir and semagacestat), adrenergic receptor modulators (vilanterol, arformoterol and atenolol), ACE inhibitors and direct oral anticoagulants (cilazaprilat, edoxaban and rivaroxaban) and inhibitors belonging to other groups (acotiamide, bentiromide, lymecycline, canagliflozin, darolutamide, lafutidine, vilazodone and methotrexate)) were obtained from this search [39] . Notably, covalent inhibitor bind irreversibly to the receptor through covalent bonds, while the binding of non-covalent inhibitor to the receptor is a reversible process. In general, the binding affinity of a covalent inhibitor to the target is stronger than that of the non-covalent inhibitors to the target [40, 41] .

In addition, naphthalene-based inhibitors of SARS-CoV PL pro are highly effective in reducing the activity of SARS-CoV-2 PL pro enzyme [42] . Based on the naphthalene scaffold structure, Bhati http://www.bindingdb.org/bind/index.jsp) was conducted, five compounds (ZINC43063883, ZINC387735, ZINC78808978, ZINC43071312 and ZINC993539) were selected as potential inhibitors of SARS-CoV-2 PL pro [45] , and ZINC43071312 showed the strongest binding ability to SARS-CoV-2 PL pro (Fig. 2B3 ).

The Mac1 of SARS-CoV-2 nsp3 has the characteristic of binding to ADP-ribose [46] ; therefore, small molecules that can bind tightly to the binding sites of Mac1 and ADP-ribose are expected to be potential SARS-CoV-2 treatments. Based on an in-depth understanding of the interaction mechanism between Mac1 and ADP-ribose, investigations have been conducted on the inhibitors of this binding. Selvaraj [49] . After an in-depth assessment of the characteristics of inhibitors using various analyses, including MD analysis and absorption, distribution, metabolism, excretion and toxicity (ADMET) analysis, Folic acid, NA1 and MolPort-000-735-951 were determined to be the best among the inhibitors screened by the aforementioned research groups ( Fig.   2C2-C4) . Notably, the MD method applied by these researchers is a method commonly used for exploring the binding ability of ligands to receptors, which plays an important role in drug design [50] [51] [52] . Additionally, the Eppendorf Mastercycler ep Realplex Quantitative Realtime PCR System and AutoDock Vina software were separately used by Virdi et al. to conduct differential scanning fluorimetry (DSF) assays and virtual screening of 726 compounds, and steroids (estradiol valerate and flunisolide), β-lactams (cefaclor and cefatrizine) and benzimidazoles (rabeprazole and

Nsp5 is also described as the main protease (M pro ) or 3CL pro . It is a dimer structure composed of two monomers (residues 1-306) and each monomer has three domains (domians I, II and III), corresponding to residues 8-101, 102-184 and 201-303, respectively (Fig. 1F ) [54] . As reported by Jin et al., residues located between domains I and II form the binding pocket of 3CL pro . Although the monomer state of 3CL pro is inactive, the homodimer state formed by the dimerization of two monomers is active. Indeed, monomers and dimers exist simultaneously in the solution, and their equilibrium is affected by many aspects, including the binding of inhibitors and protein concentration [55] [56] [57] . 3CL pro is essential for replication and transcription of the virus due to its function in cleaving replicase polypeptides (pp1a and pp1ab). Therefore, inhibiting the activity of this enzyme can significantly affect the replication and transcription of SARS-CoV-2. In addition, there are no homologues of 3CL pro in humans [58] , 3CL pro inhibitors are likely to cause fewer side effects on humans. Therefore, 3CL pro is a potential drug target against COVID-19. A literature retrieval revealed that investigators are looking for inhibitors of 3CL pro from three main classes: natural compounds, approved or commercially available drugs and others.

Natural compounds have the advantages of cost-effectiveness, high efficiency and low toxicity.

Researchers are thus committed to mining inhibitors of COVID-19 from natural compounds. A literature retrieval revealed that the natural inhibitors currently under exploration are mainly derived from plants, marine organisms and microorganisms. The following is a summary of the natural inhibitors of SARS-CoV-2 3CL pro , and the binding mechanisms of these natural inhibitors to 3CL pro were listed in Table S4 . from Isatis indigotica (Fig. 3A1-A2) [60, 61] . Another study found that six polyphenolic compounds extracted from Rhus spp. could also potentially assist in the treatment of COVID-19, the structures of these compounds are shown in Fig. 3A3 [62] .

Alkaloids are a class of natural compounds found mainly in plants, particularly flowering plants. Alkaloids are considered to be one of the most pharmacologically active substances found in plants, including those that assist in the defense against pathogens [63] . Recently, computational analyses were performed on 10 public bioactive compounds and 20 alkaloid compounds, all of which have known antiviral activity [64, 65] . After evaluating the compounds from various aspects including physiochemical properties, three alkaloid compounds (caulerpin, thalimonine and sophaline D) were identified as potential inhibitors of SARS-CoV-2 3CL pro (Fig. 3B1-B3) . Similarly, a computational approach was applied by Gyebi et al. to explore inhibitors of SARS-CoV-2 3CL pro from 62 alkaloid compounds in African plants, which yielded two drug candidates: 10hydroxyusambarensine and cryptoquindoline (Fig. 3B4-B5 ) [66] .

Structurally, terpenoids contain one or more isoprene units, and their general structure is (C 5 H 8 ) n . Terpenoids can be classified into different types according to the number of isoprene unit, such as monoterpenes, sesquiterpenes and diterpenes [67] . They are involved in metabolic pathways of all living organisms and have a variety of pharmacological applications [68] . Importantly, previous studies have shown that some subtypes of terpenoids have strong antiviral activity against coronaviruses, such as CoV-229E and SARS-CoV [69, 70] . Thus, terpenoids and their derivatives may be helpful in the treatment of COVID-19.

In view of these findings, Diniz et al. reviewed 34 anti-coronavirus terpenoids from several studies, and identified three terpenoid compounds (methyl tanshinonate, sugiol and α-cadinol) that could bind tightly to SARS-CoV-2 3CL pro based on the binding free energies calculated using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method ( Fig. 3C1-C3 ) [71] . In addition, the work of Gyebi et al. not only explored the antiviral properties of alkaloids, but also discovered two potential SARS-CoV-2 3CL pro inhibitors (6-oxoisoiguesterin and 22-hydroxyhopan-3-one) from a set of 100 terpenoids extracted from African plants (Fig. 3C4-C5 ) [66] . Therefore, the aforementioned studies also suggested that terpenoids and their derivatives can be potentially developed into drugs against SARS-CoV-2.

Flavonoids are widely distributed in many parts of plants and are indispensable compounds for physiological processes in plants. Flavonoids, especially glycosylated flavonoids, are considered potential inhibitors with the ability to inhibit the activities of multiple proteases, including SARS-CoV 3CL pro [72] . In addition, a study has shown that almost all the annotated flavonoids extracted from Salvadora persica could stably bind to SARS-CoV-2 3CL pro [73] , suggesting that flavonoids may be suitable for the treatment of COVID-19.

Taking into account the sequence similarity between SARS-CoV and SARS-CoV-2 3CL pro , Jo et al. mined the flavonoid library using a proteolytic method, and three compounds (baicalin, pectolinarin and herbacetin) exhibited better inhibitory effects, with baicalin showing the greatest effect [74] . The measured IC50 values for these three compounds were 34.71, 51.64 and 53.90 μM, respectively ( Fig. 4A1-A3) . However, baicalin, as one of the four main ingredients (baicalein, baicalin, wogonin, and wogonoside) of Scutellaria baicalensis, was not the compound with the strongest SARS-CoV-2 3CL pro inhibitory activity. Experimental investigation by Liu and colleagues found that the flavonoid baicalein was the most promising inhibitor of SARS-CoV-2 among the four, with a corresponding IC50 of 0.39 μM. They then tested 10 analogues of baicalein obtained from suppliers, and 4 flavonoid compounds (scutellarein, dihydromyricetin, quercetagetin and myricetin) had the highest inhibitory activity against SARS-CoV-2 3CL pro (Fig. 4B1 and 4B2 ) [75] . It is worth noting that the high binding affinity between myricetin and SARS-CoV-2 3CL pro has been confirmed by other computational study [76] . Quercetin is also an analogue of baicalein, and it was Fig. 4B2 and 4B3 ) [78] . Furthermore, a large number of natural flavonoids obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov) were evaluated, and three compounds (quercetin 3rhamnoside, rutin and myricetin 3-rutinoside) were selected as drug candidates (Fig. 4C1-4C3 ).

Among them, quercetin 3-rhamnoside had the lowest negative docking score (-9.7 kcal/mol), whereas the rutin-3CL pro complex was the most stable [79] . Another drug study on SARS-CoV-2 3CL pro also confirmed that rutin had the strongest inhibitory activity among 18 compounds extracted from Manilkara hexandra (Roxb.) [80] . Hence, rutin and its derivatives are expected to be potential Flavonoids can be divided into several sub-categories according to their chemical structure [82] . Anthocyanins represent one of the flavonoid groups, and have been extensively studied by Fakhar et al. [83] , who conducted a virtual screening of 3,435 anthocyanin-derived compounds from the PubChem database and selected two optimal drug candidates (compound IDs: 44256921 and 131751762) based on their docking score, physicochemical properties, structural stability and binding free energy. The basic structure of anthocyanins and of these two compounds are shown in Fig. S1 . Another class of flavonoids, flavan-3-ols, has also attracted attention. Zhu et al. performed docking simulations and experimental investigations on 10 main Flavan-3-ols compounds, and found that four of them (catechin gallate (CAG), epicatechin gallate (ECG), gallocatechin gallate (GCG) and epigallocatechin-3-gallate (EGCG)) had the potential to be utilized as inhibitors of SARS-CoV-2 3CL pro , with IC50 values of 2.98, 5.21, 6.38 and 7.51 μM, respectively (Fig. 4B4-4B5 ) [84] . These four compounds are abundant in tea plants, particularly green tea [85, 86] . The ingredients in green tea may be helpful in fighting SARS-CoV-2, owing to their multi-faceted functions [87] . In fact, a study of eight compounds with known antiviral activity in green tea also found that EGCG, ECG and GCG are the best drug candidates for inhibiting the activity of SARS-CoV-2 3CL pro [88] . Other related studies have also confirmed the inhibitory effect of EGCG against SARS-CoV-2 3CL pro [89] [90] [91] .

Previous experiments confirmed that four biflavonoids (amentoflavone, bilobetin, ginkgetin, sciadopitysin) and eight diterpenoids isolated from Torreya nucifera showed inhibitory activity against SARS-CoV [92] . Recently, the inhibitory effect of these compounds against SARS-CoV-2 was evaluated by Ghosh et al. [93] . Their computation results show that three of the four bioflavonoids: amentoflavone, bilobetin and ginkgetin, can bind stably to the binding sites of 3CL pro (PDB ID: 6LU7). The binding free energies of these three inhibitors to the protein calculated using the molecular mechanics generalized Born surface area (MM-GBSA) method were -59.57, -66.31 and -63.62 kcal/mol, respectively, and the corresponding Ki values were 0.17, 0.21 and 0.26 μM, respectively (5A1-5A3). As shown in Fig. 5A1-5A3 , the screened compounds share structural similarity and the differences among them originate from the different substitutions at positions a and b of the apigenin motif. Notably, biflavonoid sciadopitysin was also derived from the replacement of the apigenin motif at positions a, b, and c (Fig. 5A4) , but it was revealed to be a toxic molecule that is not suitable for use as an antiviral drug. Based on this result, substitutions on the structure of apigenin can be attempted to identify inhibitors with more favorable physical and chemical properties.

Flavonoids and biflavonoids are expected to behave as inhibitors of SARS-CoV-2 3CL pro .

Bharadwaj et al. elucidated that four best inhibitors screened from 653 natural compounds in the NP-lib database are flavonoids (rutin, quercimeritrin 6''-O-L-arabinopyranoside) (Fig. 4C2 and 4C6) and biflavonoids (2,3-dihydroamentoflavone and podocarpusflavon-B) (Fig. 5A5-5A6) [94] .

Therefore, there is an urgent need to design and optimize the structure of flavonoids and biflavonoids to develop powerful inhibitors of SARS-CoV-2.

Based on the above studies, it can be inferred that some medicinal plants may be good sources of compounds that could potentially behave as inhibitors targeting SARS-CoV-2. Due to the lack of effective drugs, some medicinal plants have been studied under emergency situations, and a few studies have attempted to extract antiviral medicinal ingredients from a single plant [95] [96] [97] [98] .

Potential inhibitors of SARS-CoV-2 3CL pro , including dehydroglyasperin C, licochalcone D and liquiritin, were found in Glycyrrhiza glabra, while other compounds including withanoside II, withanoside IV, withanoside V and sitoindoside IX were extracted from Withania somnifera (Ashwagandha). Moreover, calycin and rhizocarpic acid have been found in lichen. Efforts have also been made to identify the most effective inhibitors by screening compounds from multiple plants [99] [100] [101] [102] [103] . For example, Mahmud et al. identified three potential inhibitors (curcumin, gartanin and robinetin) that can stably bind to SARS-CoV-2 3CL pro by screening 3,063 compounds from more than 200 plants. For ease of reference, all prospective SARS-CoV-2 3CL pro inhibitors and their corresponding sources are shown in Fig. 5B .

The marine ecosystem is rich in resources and comprises a wide variety of organisms. Recently, an increasing number of natural medicines for preventing or treating many diseases have been extracted from marine organisms. According to previous studies, some marine-derived natural compounds have been demonstrated to possess antiviral and antibacterial activities [104, 105] .

Moreover, some marine-derived natural compounds have activities that are applied in treating nervous and immune systems, while others are used as diagnostic tools. Therefore, marine natural compounds represent a favorable source for the development of pharmaceuticals that may be used to alleviate COVID-19 pandemic. The marine natural product library (http://docking.umh.es/downloaddb) is a valuable database for mining marine drugs, which contains more than 10,000 compounds. Structure-based and ligand-based virtual screening studies were performed separately in this database, and 17 of 14,064 marine natural compounds were selected as putative inhibitors of SARS-CoV-2 3CL pro . Of these, heptafuhalol A has the lowest binding free energy [106] . Other computational studies demonstrated that four marine drugs (eribulin mesylate, plitidepsin, trabectedin and fostularin 3) also had excellent affinity for binding to SARS-CoV-2 3CL pro [107, 108] .

The earth is rich in microorganisms, and bioactive substances acquired from microorganisms are of great significance in the development of novel drugs. Therefore, natural inhibitors of microbial origin have attracted extensive attention in the pharmaceutical industry. Since the emergence of SARS-CoV-2, researchers have searched for inhibitors of microbial origin. There is currently a large microbial natural product database (https://www.npatlas.org/joomla/index.php) containing more than 20,000 compounds from bacteria and fungi [109] . To obtain high-efficiency inhibitors that target SARS-CoV-2 3CL pro activity, one research group conducted a layer-by-layer screening of 24,581 compounds from the database, and six of them (citriquinochroman, holyrine B, proximicin C, pityriacitrin B, (+)-anthrabenzoxocinone and penimethavone A) were identified as having high potential for inhibiting SARS-CoV-2 3CL pro ( Fig. 6 (1)-6(6)) [110] . Another study conducted computational screening of 100 fungal metabolites from PubChem using molecular docking and MD simulations. Among the 100 selected metabolites, pyranonigrin A was regarded as a potent inhibitor of SARS-CoV-2 3CL pro (Fig. 6(7) ) [111] . Based on similar computational approaches, hexadecanoic acid and deoxycylindrospermopsin were selected from the metabolites of Bacillus species and cyanobacteria, respectively ( Fig. 6 (8)-6(9)) [112, 113] .

Given the current critical public health situation, i.e. lack of effective drugs to control COVID-19, the repurposing of already-approved or commercially available drugs is a quick and desirable strategy to develop safe and effective treatments. Drug repositioning is a process of re-screening existing drugs for their new applications using related techniques, and therefore, it is also regarded as drug recycling, drug repositioning, etc [114] . Previously approved drugs have many undeniable advantages over newly developed drugs. For example, they have known safety, pharmacokinetics and toxicity profiles, which not only saves time and investments, but also reduces the possibility of negative effects on the human body [115] . Additionally, there are infrastructures available for the large-scale production of approved or commercially available drugs, which greatly improves the efficiency of drug production [116] . It is therefore a good strategy to screen inhibitors that can control the activity of SARS-CoV-2 3CL pro among the approved or commercially available drugs.

Of course, this strategy will also encounter many challenges including patent application, investment and unexpected negative effects [117] .

A literature retrieval revealed that the main sources of approved drugs are existing databases, 

The inhibitors summarized above are mainly for a single target (nsp3 or nsp5) of SARS-CoV-2.

Of course, the development of dual inhibitors for different targets of SARS-CoV-2 is also a more promising direction that cannot be ignored, which may greatly reduce the combination of drugs and improve the efficiency of treatment. To better understand the current development status of dual inhibitors, we have briefly summarized the studies on dual inhibitors related to nsp3 and nsp5, which are listed in Table 3 .

The paucity of relevant information on therapeutic targets and development of inhibitor against SARS-CoV-2 has hindered the treatment of COVID- 19 Although extensive effort has been made to explore and develop SARS-CoV-2 inhibitors, most of the current research is focused on in silico analysis, and there is a lack of relevant experimental confirmation or in vitro verification. Future research should be conducted with more in-depth experimental investigations and in vitro verification based on the existing computational data.

Moreover, studies of dual inhibitors for multiple targets of SARS-CoV-2 are relatively lacking. In addition to the development of single inhibitors against COVID-19, more attention should also be paid to the exploration of dual inhibitors in the future. Another issue that cannot be ignored is that the proteases of SARS-CoV-2 may undergo unpredictable mutations at any time, and some mutations may enhance the structural stability and drug resistance of the protein [122, 123] , which brings great challenge to the drug design targeting proteases. It is recommended to develop relevant technologies or software to predict high-risk or drug-resistant mutations, so as to find effective drugs to combat the mutant SARS-CoV-2 in advance. The last thing that needs to be pointed out is the importance of selective inhibitors, because some inhibitors of nsp3 and nsp5 may be related to the activity of the host protein, such as human cathepsins L and B [124] .

All authors claim no conflict of interest. 

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Genome composition and divergence of the novel coronavirus (2019-nCoV) originating in China

Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein

Structural features of coronavirus SARS-CoV-2 spike protein: Targets for vaccination

Analysis of the SARS-CoV-2 spike protein glycan shield reveals implications for immune recognition

Receptor-binding domain-specific human neutralizing monoclonal antibodies against SARS-CoV and SARS-CoV-2

A structural landscape of neutralizing antibodies against SARS-CoV-2 receptor binding domain

Structural basis of SARS-CoV-2 and SARS-CoV antibody interactions

Therapeutic antibodies and fusion inhibitors targeting the spike protein of SARS-CoV-2

Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods

Crystal structure of SARS-CoV-2 papain-like protease

Functional and druggability analysis of the SARS-CoV-2 proteome

Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity

Crystal structures of SARS-CoV-2 ADP-ribose phosphatase: from the apo form to ligand complexes

Example study of a highly conserved sequence motif in Nsp3 of SARS-CoV-2 as a therapeutic target

Viral macrodomains: unique mediators of viral replication and pathogenesis

Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity

Discovery of human coronaviruses pan-papain-like protease inhibitors using computational approaches

Repurposing of FDA-approved antivirals, antibiotics, anthelmintics, antioxidants, and cell protectives against SARS-CoV-2 papain-like protease

Activity profiling and crystal structures of inhibitor-bound SARS-CoV-2 papain-like protease: a framework for anti-COVID-19 drug design

Discovery of small molecule PLpro inhibitor against COVID-19 using structure-based virtual screening, molecular dynamics simulation, and molecular mechanics/Generalized Born surface area (MM/GBSA) calculation

Repurposing known drugs as covalent and non-covalent inhibitors of the SARS-CoV-2 papain-like protease

Targeting SARS-CoV-2 M3CLpro by HCV NS3/4a inhibitors: in silico modeling and in vitro screening

Covalent inhibitors in drug discovery: from accidental discoveries to avoided liabilities and designed therapies

Characterization and noncovalent inhibition of the deubiquitinase and delSGylase activity of SARS-CoV-2 papainlike protease

Structure-based drug designing of naphthalene based SARS-CoV PLpro inhibitors for the treatment of COVID-19

Structure of papainlike protease from SARS-CoV-2 and its complexes with non-covalent inhibitors

Structure-based screening to discover new inhibitors for papain-like proteinase of SARS-CoV-2: An in silico study

High-throughput screening and quantum mechanics for identifying potent inhibitors against Mac1 domain of SARS-CoV-2 Nsp3

Comparison of binding site of remdesivir and its metabolites with NSP12-NSP7-NSP8, and NSP3 of SARS CoV-2 virus and alternative potential drugs for COVID-19 treatment

Identification of FDA approved drugs and nucleoside analogues as potential SARS-CoV-2 A1pp domain inhibitor: An in silico study

In silico identification of potential inhibitors of ADP-Ribose phosphatase of SARS-CoV-2 nsp3 by combining E-pharmacophore-and receptorbased virtual screening of database

Role of molecular dynamics and related methods in drug discovery

Comparison of the binding characteristics of SARS-CoV and SARS-CoV-2

RBDs to ACE2 at different temperatures by MD simulations

Effect of mutations on binding of ligands to guanine riboswitch probed by free energy perturbation and molecular dynamics simulations

Discovery of drug-like ligands for the Mac1 domain of SARS-CoV-2 Nsp3

Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors

The dimer-monomer equilibrium of SARS-CoV-2 main protease is affected by small molecule inhibitors

Impact of dimerization and N3 binding on molecular dynamics of SARS-CoV and SARS-CoV-2 main proteases

Biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3C-like proteinase

Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors

Polyphenols, oral health and disease: a review

Identification of polyphenols from Broussonetia papyrifera as SARS CoV-2 main protease inhibitors using in silico docking and molecular dynamics simulation approaches

Depicting the inhibitory potential of polyphenols from Isatis indigotica root against the main protease of SARS CoV-2 using computational approaches

Phytochemicals of rhus spp. as potential inhibitors of the SARS-CoV-2 main protease: molecular docking and drug-likeness study

A review on the alkaloids an important therapeutic compound from plants

In silico analysis of selected alkaloids against main protease (Mpro) of SARS-CoV-2

The inhibitory effect of some natural bioactive compounds against SARS-CoV-2 main protease: insights from molecular docking analysis and molecular dynamic simulation

Potential inhibitors of coronavirus 3-chymotrypsin-like protease (3CL pro ): an in silico screening of alkaloids and terpenoids from African medicinal plants

Herbal extracts and phytochemicals: plant secondary metabolites and the enhancement of human brain function

Medically useful plant terpenoids: biosynthesis, occurrence, and mechanism of action

Anti-human coronavirus (anti-HCoV) triterpenoids from the leaves of euphorbia neriifolia

Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus

Bioactive terpenes and their derivatives as potential SARS-CoV-2 proteases inhibitors from molecular modeling studies

Inhibition of SARS-CoV 3CL protease by flavonoids

Molecular docking reveals the potential of Salvadora persica flavonoids to inhibit COVID-19 virus main protease

Flavonoids with inhibitory activity against SARS-CoV-2 3CL pro

Scutellaria baicalensis extract and baicalein inhibit replication of SARS-CoV-2 and its 3C-like protease in vitro

Molecular docking and dynamics study of natural compound for potential inhibition of main protease of SARS-CoV-2

Structural stability of SARS-CoV-2 3CLpro and identification of quercetin as an inhibitor by experimental screening

Identification of potential inhibitors of SARS-CoV-2 main protease and spike receptor from 10 important spices through structurebased virtual screening and molecular dynamic study

Potential bioactive glycosylated flavonoids as SARS-CoV-2 main protease inhibitors: a molecular docking and simulation studies

Inhibition of SARS-CoV-2 main protease by phenolic compounds from Manilkara hexandra (Roxb.) Dubard assisted by metabolite profiling and in silico virtual screening

Structure-based lead optimization of herbal medicine rutin for inhibiting SARS-CoV-2's main protease

Flavonoids: an overview

Anthocyanin derivatives as potent inhibitors of SARS-CoV-2 main protease: an in-silico perspective of therapeutic targets against COVID-19 pandemic

Docking characterization and in vitro inhibitory activity of flavan-3-ols and dimeric proanthocyanidins against the main protease activity of SARS-Cov-2

Discovery and characterization of tannase genes in plants: roles in hydrolysis of tannins

Functional demonstration of plant flavonoid carbocations proposed to be involved in the biosynthesis of proanthocyanidins

Evaluation of medicinal herbs as a potential therapeutic option against SARS-CoV-2 targeting its main protease

Evaluation of green tea polyphenols as novel corona virus (SARS CoV-2) main protease (Mpro) inhibitors -an in silico docking and molecular dynamics simulation study

The inhibitory effects of PGG and EGCG against the SARS-CoV-2 3C-like protease

Epigallocatechin-3-gallate, an active ingredient of traditional chinese medicines, inhibits the 3CLpro activity of SARS-CoV-2

Tea polyphenols EGCG and theaflavin inhibit the activity of SARS-CoV-2 3CL-Protease in vitro

Biflavonoids from Torreya nucifera displaying SARS-CoV 3CL pro inhibition

Computer aided identification of potential SARS CoV-2 main protease inhibitors from diterpenoids and biflavonoids of Torreya nucifera leaves

Exploration of natural compounds with anti-SARS-CoV-2 activity via inhibition of SARS-CoV-2 Mpro

Glossary of phytoconstituents: can these be repurposed against SARS CoV-2? A quick in silico screening of various phytoconstituents from plant Glycyrrhiza glabra with SARS CoV-2 main protease

Identification of bioactive molecule from Withania somnifera (Ashwagandha) as SARS-CoV-2 main protease inhibitor

Structure-based screening of novel lichen compounds against SARS Coronavirus main protease (Mpro) as potentials inhibitors of COVID-19

Identification of bioactive molecules from tea plant as SARS-CoV-2 main protease inhibitors

Virtual screening and molecular dynamics simulation study of plant-derived compounds to identify potential inhibitors of main protease from SARS-CoV-2

Moroccan medicinal plants as inhibitors against SARS-CoV-2 main protease: computational investigations

Structural basis of SARS-CoV-2 3CL pro and anti-COVID-19 drug discovery from medicinal plants

Unravelling lead antiviral phytochemicals for the inhibition of SARS-CoV-2 Mpro enzyme through in silico approach

In silico drug discovery of major metabolites from spices as SARS-CoV-2 main protease inhibitors

Marine-derived pharmaceuticals -challenges and opportunities

Marine pharmacology in 2007-8: Marine compounds with antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiprotozoal, antituberculosis, and antiviral activities; affecting the immune and nervous system, and other miscellaneous mechanisms of action

Putative inhibitors of SARS-CoV-2 main protease from a library of marine natural products: a virtual screening and molecular modeling study

Field-template, QSAR, ensemble molecular docking, and 3D-RISM solvation studies expose potential of FDAapproved marine drugs as SARS-CoVID-2 main protease inhibitors

Marine natural compounds as potents inhibitors against the main protease of SARS-CoV-2-a molecular dynamic study

The natural products atlas: an open access knowledge base for microbial natural products discovery

Microbial natural products as potential inhibitors of SARS-CoV-2 main protease (Mpro)

Reckoning a fungal metabolite, Pyranonigrin A as a potential Main protease (Mpro) inhibitor of novel SARS-CoV-2 virus identified using docking and molecular dynamics simulation

Bacillus species; a potential source of anti-SARS-CoV-2 main protease inhibitors

Cyanobacterial metabolites as promising drug leads against the M pro and PL pro of SARS-CoV-2: an in silico analysis

Drug repositioning: identifying and developing new uses for existing drugs

Drug repurposing: progress, challenges and recommendations

Drug repurposing from the perspective of pharmaceutical companies

Challenges and opportunities of drug repositioning

ETCM: an encyclopaedia of traditional Chinese medicine

Potent noncovalent inhibitors of the main protease of SARS-CoV-2 from molecular sculpting of the drug perampanel guided by free energy perturbation calculations

2-Pyridone natural products as inhibitors of SARS-CoV-2 main protease

Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease

Structural and evolutionary analysis indicate that the SARS-CoV-2 Mpro is a challenging target for smallmolecule inhibitor design

Structure-guided design of a perampanel-derived pharmacophore targeting the SARS-CoV-2 main protease

Challenges for targeting SARS-CoV-2 proteases as a therapeutic strategy for COVID-19

Drug repurposing against SARS-CoV-2 using E-pharmacophore based virtual screening, molecular docking and molecular dynamics with main protease as the target

Identification of 3-chymotrypsin like protease (3CLPro) inhibitors as potential anti-SARS-CoV-2 agents

Structure-based virtual screening and biochemical validation to discover a potential inhibitor of the SARS-CoV-2 main protease

Reprofiling of approved drugs against SARS-CoV-2 main protease: an in-silico study

Targeting SARS-CoV-2 main protease: a computational drug repurposing study

Computational drug repurposing for the identification of SARS-CoV-2 main protease inhibitors

SARS-CoV-2 Mpro inhibitors: identification of anti-SARS-CoV-2 Mpro compounds from FDA approved drugs

Profiling SARS-CoV-2 main protease (MPRO) binding to repurposed drugs using molecular dynamics simulations in classical and neural network-trained force fields

Virtual screening of approved drugs as potential SARS-CoV-2 main protease inhibitors

Virtual screening of approved clinic drugs with main protease (3CL pro ) reveals potential inhibitory effects on SARS-CoV-2

In silico prediction of potential inhibitors for the main protease of SARS-CoV-2 using molecular docking and dynamics simulation based drug-repurposing

Novel cyclohexanone compound as a potential ligand against SARS-CoV-2 main-protease

Discovery of potent inhibitors for SARS-CoV-2's main protease by ligand-based/structure-based virtual screening, MD simulations, and binding energy calculations

FDA-approved antiviral and anti-infection agents as potential inhibitors of SARS-CoV-2 main protease: an in silico drug repurposing study

Virtual screening and repurposing of FDA approved drugs against COVID-19 main protease

In silico drug repurposing for SARS-CoV-2 main proteinase and spike proteins

Prediction of novel inhibitors of the main protease (M-pro) of SARS-CoV-2 through consensus docking and drug reposition

Repurposing existing drugs: identification of SARS-CoV-2 3C-like protease inhibitors

Predicting commercially available antiviral drugs that may act on the novel coronavirus (SARS-CoV-2) through a drug-target interaction deep learning model

Screening and evaluation of approved drugs as inhibitors of main protease of SARS-CoV-2

Revisiting activity of some glucocorticoids as a potential inhibitor of SARS-CoV-2 main protease: theoretical study

Computational insights on the potential ofsSome NSAIDs for treating COVID-19: priority set and lead optimization

Molecular docking, molecular dynamics, and in vitro studies reveal the potential of angiotensin II receptor blockers to inhibit the COVID-19 main protease

Structure-based discovery of novel nonpeptide inhibitors targeting SARS-CoV-2 Mpro

Identification of potential inhibitors of 3CL protease of SARS-CoV-2 from ZINC database by molecular docking-based virtual screening

In-silico pharmacophoric and molecular docking-based drug discovery against the Main Protease (M pro) of SARS-CoV-2, a causative agent COVID-19

Potential covalent drugs targeting the main protease of the SARS-CoV-2 coronavirus

Potential inhibitors for novel coronavirus protease identified by virtual screening of 606 million compounds

1,2,4 triazolo 1,5-a pyrimidin-7-ones as novel SARS-CoV-2 Main protease inhibitors: in silico screening and molecular dynamics simulation of potential COVID-19 drug candidates

Rational design of potent anti-COVID-19 main protease drugs: An extensive multi-spectrum in silico approach

Computational prediction of potential inhibitors of the main protease of SARS-CoV-2

Probing CAS database as prospective antiviral agents against SARS-CoV-2 main protease

Theoretical insights into the anti-SARS-CoV-2 activity of chloroquine and its analogs and in silico screening of main protease inhibitors

An in silico approach for identification of novel inhibitors as potential therapeutics targeting COVID-19 main protease

A molecular modelling approach for identifying antiviral selenium-containing heterocyclic compounds that inhibit the main protease of SARS-CoV-2: an in silico investigation

Identification of high-affinity inhibitors of SARS-CoV-2 main protease: towards the development of effective COVID-19 therapy

Virtual screening, ADMET prediction and dynamics simulation of potential compounds targeting the main protease of SARS-CoV-2

Computational discovery of small drug-like compounds as potential inhibitors of SARS-CoV-2 main protease

Structure-based virtual screening to discover potential lead molecules for the SARS-CoV-2 main protease

In-silico drug repurposing and molecular dynamics puzzled out potential SARS-CoV-2 main protease inhibitors

Drugs repurposing using QSAR, docking and molecular dynamics for possible inhibitors of the SARS-CoV-2 Mpro protease

Drug repurposing and polypharmacology to fight SARS-CoV-2 through inhibition of the main protease

Potential anti-SARS-CoV-2 drug candidates identified through virtual screening of the ChEMBL database for compounds that target the main coronavirus protease

Repurposing of known anti-virals as potential inhibitors for SARS-CoV-2 main protease using molecular docking analysis

COVID-19: rational discovery of the therapeutic potential of Melatonin as a SARS-CoV-2 main protease inhibitor

Identify potent SARS-CoV-2 main protease inhibitors via accelerated free energy perturbation-based virtual screening of existing drugs

Identification of LASSBio-1945 as an inhibitor of SARS-CoV-2 main protease (M-PRO) through in silico screening supported by molecular docking and a fragment-based pharmacophore model

Targeting the SARS-CoV-2 main protease using FDA-approved Isavuconazonium, a P2-P3 α-ketoamide derivative and Pentagastrin: An in-silico drug discovery approach

Targeting the main protease of SARS-CoV-2: from the establishment of high throughput screening to the design of tailored inhibitors

Structural basis for the inhibition of SARS-CoV-2 main protease by antineoplastic drug carmofur

Molecular characterization of ebselen binding activity to SARS-CoV-2 main protease

Ebselen, disulfiram, carmofur, PX-12, tideglusib, and shikonin are nonspecific promiscuous SARS-CoV-2 main protease inhibitors

A computational evaluation of targeted oxidation strategy (TOS) for potential inhibition of SARS-CoV-2 by disulfiram and analogues

Crystal structure of SARS-CoV-2 main protease in complex with the natural product inhibitor shikonin illuminates a unique binding mode

Prospective role of peptide-based antiviral therapy against the pain Protease of SARS-CoV-2

Dual targeting of 3CL pro and PL pro of SARS-CoV-2: a novel structure-based design approach to treat COVID-19

Dual inhibitors of SARS-CoV-2 proteases: pharmacophore and molecular dynamics based drug repositioning and phytochemical leads

Virtual high throughput screening: Potential inhibitors for SARS-CoV-2 PL PRO and 3CL PRO proteases

Ginkgolic acid and anacardic acid are specific covalent inhibitors of SARS-CoV-2 cysteine proteases

Identification of a novel dual-target scaffold for 3CLpro and RdRp proteins of SARS-CoV-2 using 3D-similarity search, molecular docking, molecular dynamics and ADMET evaluation

Targeting 3CLpro and SARS-CoV-2 RdRp by amphimedon sp. metabolites: a computational study

In silico study of natural compounds from sesame against COVID-19 by targeting M pro , Pl pro and RdRp

Promising anti-SARS-CoV-2 drugs by effective dual targeting against the viral and host proteases

Molecular docking and dynamics simulation study of hyrtios erectus isolated scalarane sesterterpenes as potential SARS-CoV-2 dual target inhibitors

Docking of fda approved drugs targeting nsp-16, n-protein and main protease of sars-cov-2 as dual inhibitors

Dual inhibition of SARS-CoV-2 spike and main protease through a repurposed drug, rutin

In silico screening of natural compounds as potential inhibitors of SARS-CoV-2 main protease and Spike RBD: targets for COVID-19

Dual inhibition of COVID-19 spike glycoprotein and main protease 3CLpro by Withanone from Withania somnifera

FDA-approved drugs [128] Hyaluronic acid; Acarbose; Lopinavir 2,800 FDA-approved drugs [129] Tipiracil; Aprepitant DrugBank Commercially available drugs [130] Leuprolide; Nelfinavir; Ritonavir; Teniposide

Ziprasidone 4,384 approved drugs [133] Ergotamine; Bromocriptine; Meclocycline; Amrubicin ZINC 5,903 approved clinical drugs [134] Viomycin; Capastat; Carfilzomib; Saquinavir 77 FDA-approved drugs

MD simulations ChEMBL275592; Montelukast

Kd=17.5nM) Selleckchem 487 FDA-approved drugs [139] Ribavirin; Telbivudine; Vitamin B12

Targetmol 3,118 FDA-approved drugs [140] VS Angiotensin II; GHRP-2; Indinavir; Polymyxin B; Fexofenadine

Reaxys 6,466 approved drugs [141] Perampanel; Carprofen; Celecoxib; Alprazolam; Trovafloxacin

Experimental evaluation Manidipine (IC50=4.8μM)

Efonidipine (IC50=38.5μM)

Screen-Well 774 FDA-approved drugs [143] HTVS

Molecular docking Ethacrynic acid (IC50=1.11μM)

Butenafine hydrochloride (IC50=5.40μM)

Tranylcypromine hydrochloride (IC50=8.64μM)

Saquinavir mesylate (IC50=9.92μM) DTC

FDA-approved glucocorticoids

FDA-approved non-steroidal anti-inflammatory drugs [147] Sulfinpyrazone

Z1759961356(IC50=0.69μM)

Note: 1. The words in bold indicate that the inhibitor has been confirmed through different studies. 2. The binding mechanisms of all inhibitors to 3CL pro are

SKS-02

SKS-03

MCULE-9349798441

MD simulations Levothyroxine; Amobarbital

Saquinavir; Faldaprevir; Brecanavir; Grazoprevir; Lopinavir Protein Data Bank

Chloroquine (Ki=0.56μM)

Experimental evaluation LASSBio-1945 (IC50=15.97μM)

Antivirus Drug Library~ 10,000 [54] HTVS; VS

Experimental evaluation Ebselen (IC50=0.67μM)

PX-12 (IC50=21.39μM) Note: These inhibitors have been further confirmed

AVPdb database 88 [180] Molecular docking

Note: The binding mechanisms of all inhibitors to 3CL

485 Anti-SARS-CoV-2 or antiviral compounds in the Life Chemicals database and 8,722 antiviral compounds in Asinex database. h 2,454 FDA approved drugs from DrugBank, 144 coronavirus M pro inhibitors from published literature and 138 natural compounds from an in-house database. j 74 ligand-M pro complexes and 59 analogues. k 6 analogs of SARS-CoV-2 3CL pro inhibitor (13b) [58] in DrugBank and 5,010 analogs of 11 antiviral agents (Atazanavir, Darunavir, Fosamprenavir, Indinavir, Lopinavir, Ritonavir, Saquinavir, Tipranavir, Delavirdine, Nevirapine and Remdesivir) in PubChem. Dual inhibitors against multiple SASR-CoV-2 targets including nsp3 and/or nsp5. Promising dual inhibitor Source Target Naloxone

Withanone PubChem database [192,193] Nsp5; S protein a NPASS: Natural compounds from Natural Product Activity and Species Source b RdRp: RNA-dependent RNA polymerase

 Table 2 Potential inhibitors of SARS-CoV-2 3CL pro selected from existing databases and published literature.

Total Method Potential inhibitor 2,000 [150] ZINC32960814/12006217/03231196/33173588 1,500 a [151] VS ZINC20291569/90403206/95480156 5,811 b [152] SCAR protocol Telcagepant; Vidupiprant; Poziotinib; Fostamatinib. 606 million [153] ( − )-Taxifolin; Rhamnetin