key: cord-0740272-z2l1q67t
authors: Jin, Zhenming; Wang, Haofeng; Duan, Yinkai; Yang, Haitao
title: The main protease and RNA-dependent RNA polymerase are two prime targets for SARS-CoV-2
date: 2020-11-21
journal: Biochem Biophys Res Commun
DOI: 10.1016/j.bbrc.2020.10.091
sha: 4495d10cb5f2efe87bc71684f40dcd9454fef554
doc_id: 740272
cord_uid: z2l1q67t

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), posed an unprecedented global health crisis. It is particularly urgent to develop clinically effective therapies to contain the pandemic. The main protease (M(pro)) and the RNA-dependent RNA polymerase (RdRP), which are responsible for the viral polyprotein proteolytic process and viral genome replication and transcription, respectively, are two attractive drug targets for SARS-CoV-2. This review summarizes up-to-date progress in the structural and pharmacological aspects of the two key targets above. Different classes of inhibitors individually targeting M(pro) and RdRP have been discussed, which could promote drug development to treat SARS-CoV-2 infection.

can range from mild to fatal, which especially poses a higher risk for older adults or people with medical conditions [19, 20] . The WHO designated COVID-19 for this epidemic disease and subsequently declared it as a pandemic. Up to date, there have been over 30 million confirmed COVID-19 cases and over 1 million related deaths worldwide across more than 180 countries [21] . The mortality rate of COVID-19 is estimated to be about 3%, which is lower than SARS and MERS. Although the remaining undetected and asymptomatic infections may affect the precise value [22] , the value of basic reproduction number (R 0 ) of COVID-19 is estimated within 2-3 [23] , which means it is highly contagious.

Unfortunately, no approved effective vaccines or specific antiviral drugs are currently available to prevent and treat COVID-19. Due to a stark warning over the spread of COVID-19, it is urgently required to identify and characterize the drug targets for SARS-CoV-2 and to develop vaccines and effective drugs.

In this review, recent advances in structural and pharmacological studies of the SARS-CoV-2 main protease (M pro ) and RNA-dependent RNA polymerase (RdRP) will be summarized and discussed.

J o u r n a l P r e -p r o o f

SARS-CoV-2 belongs to the Betacoronavirus genus, which also includes SARS-CoV and MERS-CoV [16, 24] . The RNA genome of SARS-CoV-2 comprises ~30,000

nucleotides with 14 open reading frames (ORFs). The replicase gene (ORF 1ab) occupies two-thirds of the genome [16, 24] . In order to improve the efficiency of their own replication and transcription, a number of positive-sense, single-stranded RNA viruses encode large polyproteins, which are further hydrolyzed to produce essential subunits required for replication. The production of functional elements is an important regulatory mechanism for RNA viruses.

In the life cycle of SARS-CoV-2, the ORF 1ab encodes two long overlapping polyproteins, pp1a and pp1ab, which can be processed into 16 non-structural proteins (nsps) required for RNA transcription and genome replication [16, 25] (Figure 1 ). This extensive proteolytic processing is achieved by two cysteine proteases, the M pro and the papain-like protease (PL pro ). PL pro is responsible for releasing nsp1-3 from the Nterminal of polyproteins. M pro digests the polyprotein at the remaining 11 conserved cleavage sites (nsp4/nsp5, nsp5/nsp6, nsp6/nsp7, nsp7/nsp8, nsp8/nsp9, nsp9/nsp10, nsp10/nsp12, nsp12/nsp13, nsp13/nsp14, and nsp14/nsp15), starting with the autolytic cleavage of this enzyme itself (nsp5) from polyproteins pp1a and pp1ab. It is called the main protease because it plays a major role in processing replicase polyproteins and thus facilitating viral gene expression and replication. The M pro is a chymotrypsin-like cysteine protease (~33 kDa) [26] . The alternative name of the 3C-J o u r n a l P r e -p r o o f like (3CL) protease was assigned after the picornavirus 3C protease because of the similar substrate specificity and the identification of cysteine as a catalytic residue in the context of two β-barrel structures [27, 28] .

On February 5 th , 2020, the first crystal structure of SARS-CoV-2 M pro in complex with an inhibitor N3 (PDB 6LU7) was released to the public shortly after the outbreak [26] . Up to date, hundreds of structures for SARS-CoV-2 M pro have been reported for drug development [29] [30] [31] [32] [33] .

SARS-CoV-2 M pro is composed of three domains [26] (Figure 2A ). Domain I (residues 8-101) and domain II (residues 102-184) contain antiparallel β-barrels structure, which is reminiscent of the chymotrypsin family. SARS-CoV-2 M pro possesses a catalytic dyad (Cys-His), and the substrate-binding site is located in a cleft between domain I and domain II. Domain III (residues 201-306) contains five αhelices which arrange into a large antiparallel globular cluster. Domain III has a unique topology in CoVs, which is required for homodimer formation. Domain III is connected to domain II through a long loop (residues 185-200).

The crystal structures show that SARS-CoV-2 M pro forms a homodimer with two protomers (denoted as "A" and "B") which are nearly perpendicularly to each other, which is consistent with its active form in solution [34] . The dimer has a contact interface of ~1394 Å 2 , predominantly between domain II of protomer A and the Nterminal residues of protomer B. In each protomer, the N-terminal finger (residues 1-7) inserts between domains II and III of its neighbor protomer, promoting the J o u r n a l P r e -p r o o f formation of the dimer and stabilizing the activity site of each protomer. Additionally, Domain III regulates dimerization mainly through a salt-bridge interaction between Glu290 of one protomer and Arg4 from its neighbor [29] .

The substrate-binding pocket of SARS-CoV-2 M pro is located in a cleft between domain I and domain II. According to the nomenclature of substrate for protease [35] , with the cleavage site as the center, the positions for the residues from the cleavage site to the N-terminal are named P1, P2, P3, P4, and P5..., respectively. The positions for the residues from the cleavage site to the C-terminal are named P1′, P2′, P3′, P4′, and P5′… respectively. The subsites in the substrate-binding pocket, which correspondingly accommodate each residue of the substrate, are named S1, S2, S3, S4, S5…and S1′, S2′, S3′, S4′, S5′…. The CoV M pro s are cysteine proteases, which are analogs to picornavirus 3CL proteases. Although their structural similarities are limited, the two classes of viral proteases share common features in the specificity for their substrates. It has been reported that SARS-CoV M pro prefers Leu-Gln-Ser or Leu-Gly-Ala at P2-P1-P1′ sites [36] . Consistently, the SARS-CoV-2 M pro cleaves polyproteins at least 11 sites which contain a conserved Leu-Gln↓(Ser, Ala, Asn) sequence.

In contrast with common serine and cysteine proteases, which adopt a catalytic triad of His-Asp(Glu)-Ser(Cys) [37, 38] , the CoV M pro s only possesses a catalytic dyad of His-Cys (His41-Cys145 for SARS-CoV-2) and completely lacks a third catalytic residue at the active site. In the apo structure of SARS-CoV-2 M pro , there is a J o u r n a l P r e -p r o o f stable water molecule in the oxyanion hole, corresponding to the position of the missing third catalytic residue [39] . This water molecule forms a hydrogen bond with catalytic residue His41, indicating that its possible role is to protonate histamine during proteolytic cleavage, like the aspartic acid residue in classic serine proteases or glutamic acid in picornavirus 3CL proteases [27, 28, 40] . It is believed that the proteolytic process follows a multi-step mechanism. After the imidazole of histidine deprotonates the side chain of cysteine, the resulting nucleophilic thiolate attacks the carbonyl carbon of the substrate amide bond. The C-terminal portion of the peptide substrate is then released, and the histidine is deprotonated. Thereafter, the thioester is hydrolyzed to release the N-terminal portion of the peptide substrate, and the catalytic dyad is restored and ready for another round of catalytic reaction [36, 41] .

CoVs M pro s mainly recognizes P4 to P1′ positions of the substrate, and the substrate specificity is dominantly determined by S1, S2, S4 and S1′. Among them, the S1 subsite possesses the strongest selection and has an absolute requirement for glutamine at the P1 site of the substrate. The structure of SARS-CoV-2 M pro revealed that the side chains of Phe140, Asn142, His163, Glu166, His172, along with the main chains of Phe140, Leu141constitue the S1 subsite. Interestingly, the neighbor protomer also contributes to stabilize the S1 site by extending its N-terminus close to the S1 site, allowing its Ser1 to interact with Glu166, which helps maintain the conformation of the S1 subsite. The side chains of His41, Met49, Tyr54, Met165, as well as the alkyl portion of the side chain of Asp187 are involved in the formation of the deep hydrophobic S2 subsite, which can accommodate the relatively bulky side J o u r n a l P r e -p r o o f chain of Leu or Phe at the P2 site of SARS-CoV-2 M pro . The side chain of the P3 is solvent-exposed, which implies that this site could tolerate a wide range of functional groups. The side chains of Met165, Leu167, Phe185, Gln192, and the main chain of Gln189 form a small hydrophobic S4 subsite of SARS-CoV-2 M pro , which suggests that only small amino acid residues such as Ala, Val, Thr, or Pro could be held at the P4 position. The S1′of SARS-CoV-2 M pro is a shallow subsite, which can tolerate a small group of residues, such as Ser, Ala, or Asn.

Previous studies indicate that M pro s have a highly conserved substrate binding pocket across the species, which serve as a drug target for the design of broadspectrum inhibitors [42, 43] . 

Compound 1 (N3) is the first inhibitor whose structure in complex with SARS-CoV-2 M pro has been deposited to the Protein Data Bank (PDB) [26] . This peptidomimetic inhibitor contains a Michael acceptor as a warhead that can irreversibly modify the catalytic residue Cys145. The strategy of designing this inhibitor involves replacing the amide bond at the cleavage site of the substrate with a Michael acceptor. The cysteine residue and the Michael acceptor group (warhead) of inhibitor undergoes 1,4-addition. In this process, the protonation of the α-carbanion from catalytic His-H + results in the covalent bond formation. The structure shows that replacing the glutamine which is absolutely required at P1 site, with a lactam group, can result in strong binding for the inhibitor at the S1 subsite. Compound 1 displayed inhibition against SARS-CoV-2 with a half-maximal effective concentration (EC50) value of 16.77 μM in the plaque-reduction assay. 

The repurposing of approved pharmaceutical drugs and drug candidates provides an alternative approach that allows the rapid identification of potential drug leads to Among them, ebselen (9) exhibited promising antiviral activity in the plaquereduction assay (EC 50 = 4.67 μM). Ebselen (9) is an organoselenium compound that has previously been investigated for the treatment of multiple diseases, including bipolar disorders [55] and hearing loss [56] . Its low cytotoxicity in humans has been evaluated in clinical trials [55] [56] [57] . Ebselen (9) has been approved by the U.S. Food and Drug Administration (FDA) to enter phase II clinical trials (NCT04484025 and NCT04483973) to treat COVID-19. Carmofur (11) is a derivative of 5-fluorouracil (5-FU) and an approved antineoplastic agent. The X-ray crystal structure of SARS-CoV-2 M pro in complex with carmofur (11) reveals that the carbonyl reactive group of carmofur (11) is covalently bound to catalytic Cys145, whereas its fatty acid tail occupies the hydrophobic S2 subsite [31] . Carmofur (11) inhibits viral replication in cells (EC50 = 24.30 μM) and provides a basis for the rational design of analogs with enhanced inhibitory efficacy to treat COVID-19.

Virtual screening identified cinanserin (compound 15) as an inhibitor targeting SARS-CoV-2 M pro . It is a well-characterized serotonin, as a potential inhibitor [26] . It has previously been shown to inhibit SARS-CoV M pro with an IC 50 value of 5 µM [58] .

It has displayed anti-SARS-CoV-2 activity with an EC 50 value of 20.6 µM, which potential for further optimization as an antiviral drug lead. (17) revealed that this inhibitor non-covalently binds to the substrate-binding pocket of M pro through interacting with the catalytic dyad of His41-Cys145, the crucial S1/S2 subsites, and the oxyanion loop. It may acts as a "shield" to effectively prevent the substrate from accessing to the catalytic dyad [33] . [59] . Furthermore, it has been reported that dipyridamole (18) showed therapeutic improvement against COVID-19 in a small-scale clinical trial [59] .

To accelerate the development of antiviral drugs against COVID- 

After entry into the cells, SARS-CoV-2 employs a multi-subunit replication-andtranscription complex (RTC) to accomplish the replication and transcription of its RNA genome [61] . RTC is assembled by an array of non-structural proteins (nsps) released from polyprotein pp1ab by viral protease cleavage [62] . In this multi-subunit machinery, the core component is nsp12, which is the catalytic subunit and also called RNA-dependent-RNA polymerase (RdRP). Nsp12 is responsible for the catalysis of elongation of a new RNA chain from RNA templates [63] . However, nsp12 itself only displays little activity in RNA synthesis, and two other accessory factors nsp7 and nsp8 are required to increase nsp12's binding to the template and subsequent RNA synthesis [64, 65] .

The RdRp core consists of the thumb, palm, and fingers subdomains forming an encircled human right hand architecture, which is involved in template binding, polymerization, nucleoside triphosphate (NTP) entry, and other related functions [66, 67] . In addition, SARS-CoV-2 nsp12 contains a nidovirus-specific N- [69] [70] [71] , such as remdesivir (RDV) and favipiravir. In comparison with other viral RdRPs , SARS-CoV-2 nsp12 shows structural similarity. Multiple key amino acid residues at the active site are conserved [72] , indicating that antivirals targeting RdRPs may provide first lines of defense against future CoV-associated diseases.

A series of cryo-electron microscopy (cryo-EM) structures has recently revealed that SARS-CoV-2 nsp12 (RdRP) forms a complex with nsp7 and nsp8, as previously observed for RdRP complex from SARS-CoV. The viral RdRP interacts with an nsp7-nsp8 pair through its thumb subdomain and with another molecule of nsp8 through its finger subdomain [66] [67] [68] [72] [73] [74] [75] . These structures show that although the SARS-CoV-2 polymerase shares a similar structural architecture with that of SARS-CoV, it has been found that an N-terminal β hairpin of SARS-CoV-2 nsp12 inserts into the groove clamped by the NiRAN domain and the palm subdomain, resulting in a cluster of tight contacts to stabilize the overall complex ( Figure 4B ). The most significant hit for NiRAN domain identified by the DALI webserver search is P.

syringae SelO [74] , a pseudokinase harboring a kinase fold without key catalytic residues for canonical kinases [76] . Interestingly, the structural analysis shows that the residues involved in ADP-Mg 2+ binding at the N-terminal domain of SARS-CoV nsp12 are very similar to those involved in AMP-PNP binding in SelO. [74] .

Although whether the NiRAN domain possesses nucleotidylation activity has yet to J o u r n a l P r e -p r o o f be determined, it has been found that it plays an essential role in viral replication for SARS-CoV [77] , suggesting it may serves as a new druggable site.

The structures of nsp12 in complex with nsp7, nsp8, and RNA template-product duplex provides detailed information for the interactions between RdRP and RNA.

Cryo-EM structure of the RdRP complex shows that RdRP engages with over two turns of duplex RNA and identifies a long protruding RNA and extended protein regions in nsp8 [73] . The active-site cleft of nsp12 binds to the first turn of RNA, and two copies of nsp8 bind to opposite sides of the cleft and the second turn of RNA ( Figure 4C ).

It has been proposed that the RdRP is able to cooperate with other nsp proteins for viral genomic replication and transcription. SARS-CoV-2 Nsp13 belongs to a superfamily 1B helicases that unwinds RNA in an NTP-dependent manner [78] [79] [80] .

Another cryo-EM structure of RdRP complex demonstrates that nsp12, nsp8, nsp7, and nsp13 are able to form a larger complex [74] . This complex contains two molecules of nsp13, each of which binds to the N-terminal extension of each copy of nsp8. One nsp13 molecule also contacts the thumb subdomain of nsp12 ( Figure 5C ).

A possible backtracking mechanism was proposed for the sub-genomic RNA transcription.

Remdesivir, an inhibitor of nucleotide analogue which has been developed targeting RdRP from ebola virus , has demonstrated broad-spectrum inhibitory effect against a wide range of RNA viruses, including filoviruses, arenaviruses, and J o u r n a l P r e -p r o o f CoVs [81] [82] [83] . Remdesivir is a prodrug which can be metabolized into its active form of triphosphate in the cells (Figure 5B ), which could interfere viral RNA synthesis and inhibit the elongation of the viral RNA product [81] . Red, residues are entirely identical among M pro s from all coronaviruses; orange, conserved substitution in M pro s of more than one of the coronaviruses. S1, S2, S4, and S1′ subsites are indicated. 

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