key: cord-0909459-ribazjh9
authors: Wang, Zhonglei; Yang, Liyan; Zhao, Xian-En
title: Co-crystallization and structure determination: An effective direction for anti-SARS-CoV-2 drug discovery
date: 2021-08-19
journal: Comput Struct Biotechnol J
DOI: 10.1016/j.csbj.2021.08.029
sha: f9327d226e59eab42425f3278656dadaba31b739
doc_id: 909459
cord_uid: ribazjh9

Safer and more-effective drugs are urgently needed to counter infections with the highly pathogenic SARS-CoV-2, cause of the COVID-19 pandemic. Identification of efficient inhibitors to treat and prevent SARS-CoV-2 infection is a predominant focus. Encouragingly, using X-ray crystal structures of therapeutically relevant drug targets (PLpro, Mpro, RdRp, and S glycoprotein) offers a valuable direction for anti–SARS-CoV-2 drug discovery and lead optimization through direct visualization of interactions. Computational analyses based primarily on MMPBSA calculations have also been proposed for assessing the binding stability of biomolecular structures involving the ligand and receptor. In this study, we focused on state-of-the-art X-ray co-crystal structures of the abovementioned targets complexed with newly identified small-molecule inhibitors (natural products, FDA-approved drugs, candidate drugs, and their analogues) with the assistance of computational analyses to support the precision design and screening of anti–SARS-CoV-2 drugs.

The unprecedented coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread quickly across continents, with 4,180,161 deaths currently recorded on a global basis [1] . In view of the high virulence and mortality rates, the World Health Organization declared SARS-CoV-2 as the sixth public health emergency of international concern [2] . The pandemic is ongoing, and the effects of this highly contagious virus are widespread. Efforts are continuing to combat the SARS-CoV-2 outbreak with a variety of drugs, including remdesivir [3] , dalbavancin [4] , clofazimine [5] , mycophenolic acid [6] , plitidepsin In particular, PL pro plays an essential role in the cleavage and maturation of RNA virus polyproteins, dysregulation of the host inflammatory response, and disruption of host immune responses [26, 27] . M pro , also called 3C-like protease (3CL pro ), plays a crucial role in the polyprotein maturation process and may serve as a prime therapeutic target [28] [29] [30] . RdRp is another essential enzyme that regulates viral replication by catalyzing the RNA template-dependent development of phosphodiester bonds [31] . ACE2 is a host cell surface receptor necessary for SARS-CoV-2 infection [32] . The S protein plays a critical role in mediating membrane fusion and cell entry via interactions with the ACE2 cell surface receptor [33] . The immense therapeutic potential of targeting PL pro , M pro , RdRp, and the S protein in the development of effective drugs has been widely explored.

Innovative drug development constitutes an important research direction, and results to date have been outstanding, with several interesting articles already published from the perspectives of potential drugs and drug targets [34] [35] [36] [37] [38] , mechanisms of action and inhibition [26, 39, 40] , structurebased drug design [41] [42] [43] . Other articles were identified by virtual screening of the ZINC, TCMSP, ChEMBL, Pubchem, NPASS, and Drug bank databases [44] [45] [46] [47] [48] . However, to our knowledge, no systematic reviews of inhibitor-target co-crystal complexes of SARS-CoV-2 have been published.

In the current review, we focused on medicinal chemistry efforts based on analyses of X-ray cocrystal structures of the abovementioned targets in complex with newly identified small-molecule inhibitors as a means of supporting the precision design and screening of anti-COVID-19 agents through the characterization of ligand-bound targets. COVID-19 inhibitors of significance from the perspective of medicinal chemistry are prefaced by a brief introduction summarizing the relevant pharmacology, discovery process (drug design), preparation methods, and mechanisms of action.

The main objective of this report is to provide a 'co-crystal structure-guided drug discovery protocol' to facilitate the development of safe and effective drugs to combat COVID-19.

Natural products provide a rich and novel source for drug development and play an integral role as starting points in the development of therapeutic strategies for treating various complex diseases due to their tremendous structural diversity and unique chemical compositions [49] . In the current race to identify safe and efficacious drugs for COVID-19, natural products with a range of valuable bioactivities have attracted significant attention in terms of evaluating and validating their therapeutic effects [45] [46] [47] . As a result of continuing research, several natural products have been highlighted thus far as promising drug leads to combat COVID-19 [50] [51] [52] . As a result of continuing research, several natural products have been highlighted thus far as promising drug leads to combat COVID-19 [53] [54] [55] [56] [57] [58] .

Scutellariae radix (Scutellaria baicalensis) is a well-known herbal medicine used to treat lung injury [59] and inflammatory diseases [60] . It is a widely used nutritional supplement with an impressive safety profile. Liu [61] . Baicalein is the major component of scutellariae radix and exhibits broad-spectrum antiviral effects ( Fig. 2A ) [62, 63] . As expected, baicalein was shown to be effective in inhibiting M pro activity (half-maximal inhibitory concentration [IC 50 ] of 0.39 μM) [61] . Du and co-workers reported that baicalein can be used to treat acute lung injury, as it improves respiratory function, inhibits inflammatory cell infiltration, and downregulates the release of cytokines [64] . A docking model showed that the 6-OH group of baicalein plays a pivotal role in terminating RNA replication via hydrogen bonding interactions with L141 [61] . To elucidate the underlying molecular mechanisms of baicalein, active sites were explored. Su and co-workers generated a crystal structure of baicalein-M pro at a resolution of 2.2 Å (PDB ID: 6M2N) [65] . Further analysis revealed that the non-peptidomimetic inhibitor baicalein was ensconced in the core of the substrate-binding pocket via multiple interactions. For example, hydroxyl groups of baicalein form hydrogen bond interactions with Leu141/Gly143 and Ser144/His163 of M pro to stabilize the tetrahedral transition state of the proteolytic reaction. The enone carbonyl group of baicalein forms hydrogen bond interactions with Glu166, and the free aromatic rings of baicalein form π-π interactions with Cys145 and His41 as well as hydrophobic interactions with Gln189, Arg188, Met49, Cys44, and His41 to prevent substrate access to the active site, rather than covalently blocking catalytic Cys145. These interactions could underlie the observed potent activity (IC 50 of 0.94 μM, EC 50 of 2.94 μM, and SI >212) of baicalein against SARS-CoV-2 M pro [65] . Shuanghuanglian oral liquids extracted from three types of traditional Chinese medicines (including Scutellaria baicalensis Georgi) effectively blocked the replication of SARS-CoV-2 in Vero E6 cells (IC 50 of 0.064 μM), highlighting the significant contributory role of this active component in the biological activity of Chinese herbal treatments [65] . Importantly, the crystal structure of the M pro -baicalein complex provided direct data that could facilitate elucidation of the molecular mechanisms underlying the beneficial effects of traditional medicines.

Myricetin, a flavonol monomer, is a natural product isolated from the "medicine food homology" species Chinese bayberry (Myrica rubra Sieb. et Zucc.) and exhibits a range of pharmacological effects against acute lung injury [66] , inflammatory diseases, and infections with pathogenic microbes [67] . Kuzikov and co-workers reported that myricetin inhibits the synthesis of SARS-CoV-2 M pro in vitro (IC 50 of 0.22 μM) [68] . To optimize precision drug design, Su and co-workers generated a crystal structure of the M pro -myricetin complex at a resolution of 2.1 Å (PDB ID: 7DPP) [68] (Fig. 2B) , which unambiguously revealed that myricetin is covalently bound to catalytic Cys145. Su et al. also showed unprecedented inhibitor-target binding patterns, specifically, a covalent bond between the Cys145 sulfur and the 2' position of myricetin, which was distinct from baicalein and other reported flavonoid structures [68] . In addition, Kuzikov and co-workers reported another crystal structure of the M pro -myricetin complex with the same covalent binding mode, at 1.77-Å resolution (PDB ID: 7B3E) [69] . Further analysis of the X-ray structure of the myricetin-M pro complex revealed that the binding pocket is only partially occupied by myricetin, presenting an important signal and ideal lead for structure-based drug design. Based on molecular dynamics (MD) simulations and molecular mechanics Poisson-Boltzmann surface area (MMPBSA) calculations, Sharma et al. [70] revealed that baicalein forms a stable combination with nonstructural protein 15 (Nsp15) with low binding energy (−65.663 kJ/mol). The natural naphthoquinone shikonin, derived from Lithospermum erythrorhizon Sieb. et Zucc ( Fig. 2C) , has numerous pharmacological properties that support its further development as a therapeutic agent [71] . This agent has been shown to exert anti-inflammatory, anti-fungal, and anti-HIV effects [72] . Yang et al. revealed that shikonin inhibits the replication of SARS-CoV-2 M pro in vitro (EC 50 of 15.75 μM) [73] . Li and co-workers [74] initially solved the crystal structure of the shikonin-M pro complex at 2.45 Å (Fig. 2C) . Crystallography data showed that the His41-Cys145 catalytic dyad undergoes large conformational changes upon complex formation, leading to significant differences with other reported structures [75] . Li and co-workers recently revealed that two novel hydrogen-bonding interactions (one with Gln189 and Thr190, another with Met165, His164, and Cys145) and a π-π stacking interaction with His41 play critical roles in the binding of shikonin with M pro [74] . These findings clearly indicate a distinct binding pattern suggestive of binding site diversity. However, Ma and co-workers recently reported that shikonin may not be a target-specific M pro inhibitor, as its activity declines markedly in the presence of reducing reagents [76] . Further elucidation of the different binding modes should provide a solid foundation for effective COVID-19 drug design.

The genome of SARS-CoV-2 contains ~29 800 bases encoding 16 nonstructural proteins (Nsp1-Nsp16) essential for viral replication [77] . For example, Nsp16 plays a critical role in immune evasion during virus replication [78] . The natural nucleoside antibiotic sinefungin is a structural analogue of S-adenosylmethionine (SAM), which serves as a key methyl group donor to numerous biomolecules [79] . SAM-dependent 2′-O-RNA methyltransferase (MTase) is a potential relevant drug target in COVID-19 chemotherapy [80] . The function of MTase is associated with Nsp16, which requires Nsp10 as a cofactor for activity [80, 81] . Mahalapbutr et al. [80] also reported that sinefungin exhibits very high susceptibility to Nsp16 (especially through electrostatic interactions with the 2'-OH and N3 of the RNA's adenosine moiety) based on atomistic MD simulations and free-energy calculations. To establish the mechanisms by which sinefungin inhibits the Nsp16 MTase at a molecular level, Krafcikova and co-workers [82] determined the crystal structure of SARS-CoV-2 Nsp10-Nsp16 complexed with sinefungin at a resolution of 2.4 Å (PDB ID: 6YZ1) (Fig. 2D ). The co-crystallization structure showed that sinefungin binds the SAMbinding pocket localized in a canyon within Nsp16 and forms several interactions with specific residues (nucleoside-binding pocket: Asp99, Asn101, and Asp114; methionine-binding pocket:

Asn43, Asp130, and Lys170). These data provide an important starting point for structure-based inhibitor discovery.

Linoleic acid, an essential free fatty acid, is an important modulator of the inflammatory response [83] . Toelzer and co-workers [84] . As for SARS-CoV-2, Wang et al. [88] confirmed that calpain inhibitor II is highly effective against M pro (IC 50 of 0.97 μM).

Wang and co-workers [89] also reported the crystal structure of calpain inhibitor II-M pro at 1.65-Å resolution (PDB ID: 6XA4). Further analysis of the complex structure revealed that stabilized thiohemiketal with the (S) configuration occupies the oxyanion hole, which is formed by Gly143, Cys145, and, in part, Ser144 (Fig. 3) . Additionally, the structure exhibited several other interactions with active site residues, including multiple hydrogen bonds of the amide group with His164, Met165, and Glu166 (Fig. 3) . Interestingly, a weak hydrogen bond between the sulfur group and His163 was detected, which could explain the stronger activity of calpain inhibitor II (IC 50 = 0.97 μM) relative to calpain inhibitor I (IC 50 = 8.60 μM) [89] . The introduction of X-ray crystallography technology has provided a highly successful strategy for exploration of natural products and their analogues, improving our understanding of drug interactions at the molecular level based on structures of complexes, to facilitate the design of effective COVID-19 therapies. While natural products have demonstrated potential effectiveness, clinical trial evidence of their utility as anti-COVID-19 agents is currently lacking. Future research will therefore focus on obtaining stronger evidence to support the clinical utility of natural products.

In this scenario, strategies for the large-scale manufacture of natural products and analogues, such as shikonin, sinefungin, and calpain inhibitor II, are urgently required.

Remdesivir, an RdRp inhibitor, was the first and only drug against COVID-19 approved by the U.S. Food and Drug Administration (FDA) in 2020 [90] [91] [92] [93] . The highly potent 1'-CN-substituted GS-441524, inspired by the natural product tubercidin, is important in the design of anti-SARS-CoV-2 agents [94] . Triphosphate GS-443902 is established as the active form of GS-441524.

However, monophosphate conversion of GS-441524 to GS-441524 monophosphate is extremely difficult [95] , highlighting the necessity of developing a prodrug that can overcome this delivery bottleneck. To achieve better cellular uptake in vivo, monophosphorylated remdesivir was developed by Gilead Sciences (Fig. 4A ) [96] . The GS-5734 1′-CN group plays a pivotal role in terminating RNA replication by disrupting the exoribonuclease proofreading activity and inhibiting the RdRp polymerization activity [97] . To increase the efficiency of remdesivir, researchers at Gilead Science initially developed strategies for preparation of the P-stereogenic prodrug using a scalable process through chiral preparative high-performance liquid chromatography (HPLC) or chiral resolution [96] , which inevitably led to waste of resources. For further improvement of efficiency, an elegant asymmetric strategy (Fig. 4B ) for producing remdesivir via chiral bicyclic imidazole-catalyzed dynamic kinetic asymmetric transformation (DyKAT) with excellent stereoselectivity (S P :R P = 22:1) was recently developed by Zhang and colleagues [98] . In the clinical context, development of a large-scale synthesis method for remdesivir is urgently needed. As remdesivir is a phosphoramidate prodrug, conversion into the triphosphate GS-443902 is required. Similar to many prodrugs, the postulated activation pathway of remdesivir is divided into four sequential steps (Fig. 5) : cell entry (via increased hydrophobicity with the aid of a phosphorylated group), removal of the masking group (via enzymatic and chemical demasking), phosphorylation (via NMP kinases), and incorporation into the growing SARS-CoV-2 RNA strand. In cells, the triphosphate form, GS-443902, can block SARS-CoV-2 replication by evading the "proofreading" of viral RNA sequences [99] .

Wakchaure et al. [100] showed that remdesivir has high binding energy (−29.7 kCal/mol) with respect to RdRp, and MD simulations also revealed that remdesivir binds in the catalytic site via the formation of hydrogen bond interactions. Xu et al. [101] reported the cryo-EM structure of RdRpremdesivir (using its triphosphate metabolite GS-443902) at 2.5-Å resolution (PDB ID: 7BV2). The cryo-EM structure unambiguously demonstrated that remdesivir monophosphate (IV), positioned at the center of the catalytic site of the primer RNA, covalently binds to the 1+ position of the template strand, thus terminating chain elongation (Fig. 4A) . Three strong H-bonds with active site residues (ribose -OH groups: Asp623, Ser682, and Asn691; sugar 2′-OH: Asn691) were identified ( Fig. 4A) . Notably, the implicit, cryptic, and allosteric binding sites provide a versatile platform for designing high-potency inhibitors with kinetic selectivity [102] [103] [104] . Based on thermodynamic profiling, Srivastava et al. found that triphosphate GS-443902 inactivates RdRp by not only interfering with binding in the initial catalytic site but also by significantly blocking the nucleoside 5'-triphosphate entrance site [105] . Based on MMPBSA calculations, Khan et al. [106] proposed that remdesivir inhibits SARS-CoV-2 via a multi-target mechanism, as it binds to M pro (−7.8 kCal/mol), membrane protein (−7.4 kCal/mol), and RDRP (−7.1 kCal/mol). μM, and 0.024 μM, respectively [107] [108] [109] [110] [111] . Moreover, remdesivir reportedly protects rhesus monkeys from MERS-CoV infection [112] and displays clinical safety and efficacy against EBOV infection [113] . Although remdesivir does not appear to be highly effective in patients with COVID-19 [114] , it has the obvious advantage of rapid clinical translation to a rational prodrug strategy against SARS-CoV-2. In addition, multiple lines of evidence suggest that combinations of remdesivir and other agents (such as baricitinib [115] and dexamethasone [116] ) exert synergistic therapeutic effects against COVID-19.

Drug repurposing, a time-saving process, is a viable strategy to quickly, safely, and successful discover clinically approved drugs for COVID-19 treatment [117] [118] [119] [120] . Favipiravir, a promising repurposed prodrug, was identified by Toyama Chemical Co., Ltd., and approved in Japan in 2014

for the treatment of influenza virus infections [121] . To date, widespread research attention has focused on the utility of favipiravir as a specific anti-COVID-19 agent [122] . Favipiravir is an orally bioavailable prodrug that exhibits potent activity against COVID-19 (EC 50 interactions (side-chains and two catalytic Mg 2+ ions) to terminate chain extension (Fig. 6 ). Peng and colleagues [131] reported an alternative structural snapshot (PDB ID: 7CTT; EMD ID: 30469) distorted in one direction, suggesting another productive conformation at the catalytic site. These distinct cryo-EM states may account for the slow, weak, and inefficient incorporation of the inhibitor into the RNA primer strand, leading to the high EC 50 Similar to the prodrug remdesvir, favipiravir must be converted into the active form, favipiravir-RTP. The activation mechanism of favipiravir involves four sequential steps (Fig. 6 ):

cell entry, phosphoribosylation, phosphorylation, and non-covalent incorporation into the growing SARS-CoV-2 RNA strand [132] . Notably, favipiravir-RTP binds non-covalently to the polymerase in a manner distinct from that of remdesivir-RTP. Furthermore, Celik et al. [133] used MD simulations to highlight the binding site differences of favipiravir and its active triphosphate metabolites (favipiravir-RTP) with RdRp. Their results demonstrated that favipiravir-RTP forms a more-stable complex than favipiravir. This finding indicates an unusual binding mode, which provides an important basis for non-covalent RdRp inhibitor discovery.

mechanics represents a promising approach to guide structural modifications in drug discovery research [134] [135] [136] . Based on this method, Jorgensen's group [137] recently showed that the noncovalent SARS-CoV-2 M pro inhibitor perampanel analogue 5 effectively treats SARS-CoV-2 infection by inhibiting M pro activity (IC 50 of 0.14 μM) and inducing significant suppression of viral replication in Vero E6 cells (EC 50 of 1.5 μM), with moderate cytotoxicity (SI = 14.7). Perampanel, a poorly effective inhibitor of M pro with an IC 50 of 100-250 μM, was an initial hit compound selected for redesign and further optimization based on its relatively simple structure and FEP modeling studies [138] . As shown in Figure 7A , perampanel analogue 2 was identified as a highly potent target via initial docking analyses using FEP calculations [138, 139] . To increase the efficiency of target perampanel analogues 2, 4, and 5, Jorgensen et al. [137] further developed a general route for synthesis, as shown in Figure 7C . Briefly, Suzuki crosscoupling of 5-bromo-2-methoxypyridine with (2-cyanophenyl) boronic acid yielded S1, followed 

A wide range of drug candidates may be effective against COVID-19 via broad-spectrum effects, both in vitro and in vivo [140, 141] . These candidate drugs provide a basis to further explore [20] . To improve its plasma half-life, lead 11r was modified by hiding the P3-P2 amide bond within a pyridone ring. As expected, the plasma half-life in mice increased from 18 min to 1 h by preventing the cellular protease-mediated cleavage of the amide bond. In addition, to enhance solubility and antiviral activity, compound 13b (EC 50 of 0.67 μM) was produced by replacing the hydrophobic cinnamyl and cyclohexyl moieties with a lowhydrophobic Boc group and smaller cyclopropyl group, respectively. The researchers reported total synthesis of compound 13r on the ~50-mg scale [20] . The key components of the strategy were nucleophilic substitution (SN 2 ) and nucleophilic addition of isocyanides to aldehydes (Fig. 8) . Fig. 8 . Structure-based design and synthesis of the peptidomimetic inhibitor 13b.

Notably, compound 13b exhibited significantly improved plasma protein binding (reduced from the 99% of 11r to 90%) and plasma half-life in mice (increased from the 18 min of 11r to 1.8 h). Compound 13b exhibited favorable pharmacokinetic properties in mice, including good tropism to the lungs after subcutaneous administration, suitability for direct administration via inhalation, long-term efficacy, and good tolerability without adverse reactions. The hydrophobic Boc group appeared to play an essential role in crossing of the cell membrane and binding to viral M pro , without which the compound was almost inactive [20] . The above studies offer a platform for research into more-effective pyridone-containing anti-SARS-CoV-2 drugs to suppress disease progression and support the importance of structure-based approaches in the design and optimization of SARS-CoV-2 inhibitors. Rungrotmongkol et al. [143] reported that compound 13b was a suitable template for structural modifications at the P1' and P4 sites to enhance interactions.

The Hilgenfeld group [20] determined the crystal structure of 13b-M pro at 1.95-Å resolution (PDB ID: 6Y2F), which unambiguously revealed the presence of a stabilized thiohemiketal generated via nucleophilic attack of Cys145 over the α-carbonyl of the ketoamide warhead.

Moreover, several interactions with active site residues (such as the amide oxygen of 13b forming hydrogen bond interactions with Gly143, Cys145, and partly Ser144 to form the "oxyanion hole";

the α-keto group of 13b forming a covalent bond with catalytic Gly145 to yield the thiohemiketal group) were identified. Apparently, there is a large space (distance >3.6 Å) between Thr190, Gln189, and the pyridone ring of compound 13b, suggesting that a group larger than the pyridone ring could be designed based on the P3 moieties. In addition, a small model of the thiol moiety (active center of M pro ) with inhibitor 13b was generated (Fig. 9 ). In this model, the C-S bond can be formed directly (without deprotonation of the thiol group), and water molecules play an important role in the kinetics, as the energy barrier decreases by 9.0 kCal/mol for the intermediate when assisted with an explicit water molecule, which was facilitated via a 6-membered transition state instead of a constrained 4-membered state. Furthermore, Kumari et al. [144] used MD simulations to highlight the binding site differences of 13b with monomeric and dimeric M pro . Their results showed that dimeric M pro exhibits higher affinity for 13b via residue interactions between S1 and F140, E166, and H172. Chirality is one of the most crucial attributes for a vast majority of pharmaceutical compounds in the natural world and chiral bioactive substances play a key role in disease control [145] . Chiral agents have attracted significant research attention due to enantiomers often possessing differential binding affinities for SARS-CoV-2 enzymes and proteins.

Kitamura and co-workers [146] recently demonstrated that the non-covalent SARS-CoV-2 M pro inhibitor 23R can be used to treat the virus infection. The compound inhibited M pro (IC 50 of 0.31 μM) and exerted significant suppressive effects in Vero E6 cells (EC 50 of 1.27 μM, SI >78.7).

Initial research on 23R began with structural modification of the SARS-CoV M pro agent ML188 (R), which was separated using chiral stationary-phase supercritical fluid chromatography from ML188 enantiomers (S/R). In 2013, Jacobs and co-workers [147] demonstrated that the lead compound ML188 (S/R) and its single stereoisomer ML188 (R) effectively inhibited replication of SARS-CoV M pro with IC 50 values of 4.8 μM and 1.5 μM, respectively. However, the single stereoisomer ML188 (S) was inactive and had no inhibitory effect on SARS-CoV replication. The X-ray crystal structure of the M pro -ML188 (R) complex (PDB ID: 3V3M) revealed that an optimal shape complementary to SARS-CoV was formed through several key interactions (Fig. 10A) [147] .

Among these, the 3-pyridyl group occupied the S 1 -subpocket via a critical hydrogen bond with the His163 side chain. Knowledge of the structure of this non-covalent SARS-CoV M pro inhibitor could further guide effective inhibitor design. Based on the high-affinity binding (between the hydrogen bond and His-163 side chain) strategy, 39 compounds (a mixture of enantiomers or diastereomers) were prepared using the Ugi four-component reaction (Ugi-4CR), which provides an expeditious strategy for lead optimization [146] . Among these compounds, 23 (S/R) significantly inhibited viral replication, with an IC 50 of 0.66 μM and low toxicity (SI >303). This elegant process of 23 (S/R) synthesis is depicted in Figure   10B . After separation of the single stereoisomers, 23R and 23S, via reverse-phase HPLC, IC 50 values were determined as 0.31 μM for the diastereomer 23R and 5.61 μM for the less-active 23S diastereomer [146] . To further elucidate the binding forces, Kitamura and co-workers [146] generated a crystal structure of 23R complexed with M pro at 2.6 Å resolution (PDB ID: 7KX5). The P1 pyridinyl ring occupied the S1 pocket, forming a close (2.9 Å) hydrogen bond with the His163 side chain. Another point of particular interest is the location of the terminal α-methylbenzyl group between the S2 and S4 sites, which represents a previously unreported mechanism of binding pocket formation. The S4 pocket remained mainly unoccupied by 23R, indicating the presence of sufficient space that could be explored for further drug development. The COVID-19 pandemic has highlighted the urgent need for rapid structure-based drug discovery and development, and this strategy achieved expedited design of a non-covalent M pro inhibitor through coupling of co-crystal structures, structure-based design, and Ugi-4CR methodologies.

Numerous small-molecule inhibitors have shown promising results as efficient therapeutic agents for SARS-CoV-2. Earlier, Hoffman and co-workers [148] at anti-SARS-CoV-2 activity. Additionally, the Dittmann group [149] reported that statistically, PF-00835231 is a more-potent inhibitor than remdesivir. In addition to the abovementioned active compounds, several other small-molecule inhibitors with known crystal structures have been shown to exhibit significant anti-SARS-CoV-2 activity (Table 1) . Further research is required to establish the clinical safety and efficacy of these inhibitor candidates, however. [114] . Over the past year, the scientific community has made remarkable inroads into developing promising anti-COVID-19

agents by employing multiple techniques and strategies. Among these approaches, the generation of X-ray crystal structures of relevant drug targets has provided effective guidance for anti-SARS-CoV-2 drug discovery and lead optimization.

Theoretically, components associated with each stage of the SARS-CoV-2 replication cycle could be comprehensively explored as promising therapeutic targets. Current anti-SARS-CoV-2 drug discovery and development strategies primarily focus on preventing viral replication via the targeting of M pro , PL pro , or RdRp and blocking binding of the S protein to ACE2 receptors. To improve the precision design and screening of anti-SARS-CoV-2 drugs, we focused on all available X-ray co-crystal structures of the abovementioned targets in complex with newly identified smallmolecule inhibitors, including natural products, FDA-approved drugs, and candidate drugs. The determined co-crystal structures offer a direct molecular-level perspective that could help elucidate the underlying mechanisms of action and provide guidance for future structure-based drug design.

In the case of compound 13b, for example, a larger group at the P3 moiety could be designed to further improve the anti-SARS-CoV-2 activity in view of the space between Thr190, Gln189, and the pyridone ring of 13b. On the other hand, examination of the structures revealed no strong or durable effects for some non-covalent inhibitors (such as favipiravir, 23R, and perampanel analogue 5), supporting the need for further research focusing on covalent inhibitors to reduce "off-target"

risks. In addition, combinations of several small molecules (such as linoleic acid and PF-00835231)

with remdesivir have shown significant additive/synergistic anti-SARS-CoV-2 activities in vitro,

presenting candidate drug combinations that may be effective in treating COVID-19 patients.

Furthermore, the high mutation rate of SARS-CoV-2, resulting in the generation of novel variants, has led to increased pathogenicity [178, 179] . This may be one of the reasons for the failure of inhibitors against SARS-CoV-2. In this case, we suggest that collaborating groups should pay close attention to conserved binding sites available near the binding pocket during structure-based drug development, as this highly conserved region has promising druggable value but often goes unnoticed [180] [181] [182] . Despite substantiation of in vitro effectiveness and extensive clarification of associated mechanisms and interactions, direct clinical evidence of therapeutic efficacy for many of these agents is lacking at present. Nevertheless, we suggest that small-molecule inhibitors (including drug combinations) represent useful agents to prevent and control the COVID-19 pandemic. Conceptualization, Writing -review & editing.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 

Note from the editors: World Health Organization declares novel coronavirus (2019-nCoV) sixth public health emergency of international concern

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Targeting SARS-CoV-2 proteases and polymerase for COVID-19 treatment: State of the art and future opportunities

Potential SARS-CoV-2 main protease inhibitors

RNA-dependent RNA polymerase (RdRp) inhibitors: The current landscape and repurposing for the COVID-19 pandemic

The development of Coronavirus 3C-Like protease (3CL pro ) inhibitors from 2010 to 2020

COVID-19: Inflammatory responses, structure-based drug design and potential therapeutics

What coronavirus 3C-like protease tells us: From structure, substrate selectivity, to inhibitor design

Targeting the RdRp of emerging RNA viruses: The structure-based drug design challenge

Screening of world approved drugs against highly dynamical spike glycoprotein of SARS-CoV-2 using caverdock and machine learning

Chinese herbal compounds against SARS-CoV-2: Puerarin and quercetin impair the binding of viral S-protein to ACE2 receptor

Resources and computational strategies to advance small molecule SARS-CoV-2 discovery: Lessons from the pandemic and preparing for future health crises

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

Exploring phytochemicals of traditional medicinal plants exhibiting inhibitory activity against main protease, Spike glycoprotein, RNA-dependent RNA polymerase and non-structural proteins of SARS-CoV-2 through virtual screening

Strategies to diversify natural products for drug discovery

The strategies and techniques of drug discovery from natural products

Natural products in drug discovery: Advances and opportunities

The re-emergence of natural products for drug discovery in the genomics era

Turning the tide: Natural products and natural-product-inspired chemicals as potential counters to SARS-CoV-2 infection

Natural products with potential to treat RNA virus pathogens including SARS-CoV-2

Natural products, alone or in combination with FDA-approved drugs, to treat COVID-19 and lung cancer

Current prevention of COVID-19: Natural products and herbal medicine

Chinese herbal medicine: Fighting SARS-CoV-2 infection on all fronts

Screening for natural and derived bioactive compounds in preclinical and clinical studies: One of the frontlines of fighting the coronaviruses pandemic

An untargeted metabolomics approach to investigate the wineprocessed mechanism of Scutellariae radix in acute lung injury

Differences in anti-inflammatory effects between two specifications of Scutellariae Radix in LPS-induced macrophages in vitro

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

Baicalein attenuates cardiac hypertrophy in mice via suppressing oxidative stress and activating autophagy in cardiomyocytes

Protective effects and molecular mechanisms of baicalein on thioacetamide-induced toxicity in zebrafish larvae

The comprehensive study on the therapeutic effects of baicalein for the treatment of COVID-19 in vivo and in vitro

Anti-SARS-CoV-2 activities in vitro of Shuanghuanglian preparations and bioactive ingredients

Myricetin ameliorates sepsis-associated acute lung injury in a murine sepsis model

Myricetin: A review of the most recent research

Identification of pyrogallol as a warhead in design of covalent inhibitors for the SARS-CoV-2 3CL protease

Identification of inhibitors of SARS-CoV-2 3CL-Pro enzymatic activity using a small molecule in vitro repurposing screen

An in-silico evaluation of different bioactive molecules of tea for their inhibition potency against non structural protein-15 of SARS-CoV-2

Phytochemical constituents, distributions and traditional usages of Arnebia euchroma: A review

Shikonin, a component of chinese herbal medicine, inhibits chemokine receptor function and suppresses human immunodeficiency virus type 1

Structure of M pro from SARS-CoV-2 and discovery of its inhibitors

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

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

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

A comprehensive analysis of cis-acting RNA elements in the SARS-CoV-2 genome by a bioinformatics approach

Structural basis of RNA cap modification by SARS-CoV-2

Genomewide analysis of mode of action of the s-adenosylmethionine analogue sinefungin in leishmania infantum

Structural insight into the recognition of s-adenosyl-l-homocysteine and sinefungin in SARS-CoV-2 Nsp16/Nsp10 RNA cap 2'-O-methyltransferase

In silico identification of novel SARS-COV-2 2′-O-methyltransferase (nsp16) inhibitors: Structure-based virtual screening, molecular dynamics simulation and MM-PBSA approaches

Structural analysis of the SARS-CoV-2 methyltransferase complex involved in RNA cap creation bound to sinefungin

The need for precision nutrition, genetic variation and resolution in Covid-19 patients

Free fatty acid binding pocket in the locked structure of SARS-CoV-2 spike protein

The fatty acid site is coupled to functional motifs in

GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease

SARS-CoV-2 main protease reveal strategy for developing dual inhibitors against Mpro and cathepsin L

Remdesivir metabolite GS-441524 effectively inhibits SARS-CoV-2 infection in mouse models

Mechanism of SARS-CoV-2 polymerase stalling by remdesivir

GS-5734: a potentially approved drug by FDA against SARS-CoV-2

Remdesivir against COVID-19 and other viral diseases

Practical synthesis of 1′-substituted tubercidin Cnucleoside analogs

Therapeutic Efficacy of The Small Molecule GS

Against Ebola virus in rhesus monkeys

Discovery and synthesis of a phosphoramidate prodrug of a pyrrolo[2,1-f][triazin-4-amino] adenine C-nucleoside (GS-5734) for the treatment of Ebola and emerging viruses

1′-Ribose cyano substitution allows Remdesivir to effectively inhibit nucleotide addition and proofreading during SARS-CoV-2 viral RNA replication

Catalytic asymmetric synthesis of the anti-COVID-19 drug remdesivir

Advantages of the parent nucleoside GS-441524 over remdesivir for Covid-19 treatment

Revealing the inhibition mechanism of RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 by remdesivir and nucleotide analogues: A molecular dynamics simulation study

Structural basis for inhibition of the RNA-dependent RNA polymerase from SARS-CoV-2 by remdesivir

Elucidation of cryptic and allosteric pockets within the SARS-CoV-2 main protease

SARS-CoV-2 simulations go exascale to predict dramatic spike opening and cryptic pockets across the proteome

Uncovering cryptic pockets in the SARS-CoV-2 spike glycoprotein

Molecular dynamics simulations reveal the interaction fingerprint of remdesivir triphosphate pivotal in allosteric regulation of SARS-CoV-2 RdRp

Remdesivir strongly binds to RNA-dependent RNA polymerase, membrane protein, and main protease of SARS-CoV-2: Indication from molecular modelling and simulations

Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses

Comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against MERS-CoV

Broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent RNA dependent RNA polymerase

Recombinant RNA-dependent RNA polymerase complex of Ebola virus

Characterisation of infectious Ebola virus from the ongoing outbreak to guide response activities in the Democratic Republic of the Congo: A phylogenetic and in vitro analysis

Prophylactic and therapeutic remdesivir (GS-5734) treatment in the rhesus macaque model of MERS-CoV infection

Late Ebola virus relapse causing meningoencephalitis: A case report

Remdesivir: A pendulum in a pandemic

Baricitinib plus remdesivir for hospitalized adults with Covid-19

Effectiveness of remdesivir with and without dexamethasone in hospitalized patients with COVID-19

Transcriptomic signatures and repurposing drugs for COVID-19 patients: Findings of bioinformatics analyses

Repurposing anticancer drugs for the management of COVID-19

Drug repurposing: New strategies for addressing COVID-19 outbreak

Discovery of the FDA-approved drugs bexarotene, cetilistat, diiodohydroxyquinoline, and abiraterone as potential COVID-19 treatments with a robust two-tier screening system

Favipiravir, an anti-influenza drug against life-threatening RNA virus infections

Favipiravir and the need for early ambulatory treatment of SARS-CoV-2 infection (COVID-19)

Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro

Favipiravir antiviral efficacy against SARS-CoV-2 in a hamster model

The combined treatment of molnupiravir and favipiravir results in a marked potentiation of antiviral efficacy in a SARS-CoV-2 hamster infection model

Efficacy and safety of favipiravir, an oral RNAdependent RNA polymerase inhibitor, in mild-to-moderate COVID-19: A randomized, comparative, open-label, multicenter, phase 3 clinical trial

A prospective, randomized, open-label trial of early versus late favipiravir in hospitalized patients with COVID-19

Favipiravir versus arbidol for COVID-19: A randomized clinical trial. medRxiv

Experimental treatment with favipiravir for COVID-19: An open-label control study

Structure of the SARS-CoV-2 RNA-dependent RNA polymerase in the presence of favipiravir-RTP

Structural basis of SARS-CoV-2 polymerase inhibition by favipiravir

Role of favipiravir in the treatment of COVID-19

In silico evaluation of potential inhibitory activity of remdesivir, favipiravir, ribavirin and galidesivir active forms on SARS-CoV-2 RNA polymerase

Alchemical binding free energy calculations in AMBER20: Advances and best practices for drug discovery

Relative binding free energy calculations in drug discovery: Recent advances and practical considerations

Accurate and reliable prediction of relative ligand binding potency in prospective drug discovery by way of a modern free-energy calculation protocol and force field

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

Identification of 14 known drugs as inhibitors of the main protease of SARS-CoV-2

Potent SARS-CoV-2 direct-acting antivirals provide an important complement to COVID-19 vaccines

Candidate drugs against SARS-CoV-2 and COVID-19

Underlying mechanisms and candidate drugs for COVID-19 based on the connectivity map database

α-Ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication: Structure-based design, synthesis, and activity assessment

Computational study on peptidomimetic inhibitors against SARS-CoV-2 main protease

Binding mode characterization of 13b in the monomeric and dimeric states of SARS-CoV-2 main protease using molecular dynamics simulations

The exploration of chirality for improved druggability within the human kinome

Expedited approach toward the rational design of noncovalent SARS-CoV-2 main protease inhibitors

Discovery, synthesis, and structure-based optimization of a series of N-(tert-Butyl)-2-(N-arylamido)-2-(pyridin-3-yl) acetamides (ML188) as potent noncovalent small molecule inhibitors of the severe acute respiratory syndrome coronavirus (SARS-CoV) 3CL protease

Discovery of ketone-based covalent inhibitors of coronavirus 3CL proteases for the potential therapeutic treatment of COVID-19

A comparative analysis of SARS-CoV-2 antivirals in human airway models characterizes 3CL pro inhibitor PF-00835231 as a potential new treatment for COVID-19

Malleability of the SARS-CoV-2 3CL M pro activesite cavity facilitates binding of clinical antivirals

Repurposing the HCV NS3-4A protease drug boceprevir as COVID-19 therapeutics

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

Development of a cell-based luciferase complementation assay for identification of SARS-CoV-2 3CLpro inhibitors

Dual inhibition of SARS-CoV-2 and human rhinovirus with protease inhibitors in clinical development

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease

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

Repurposing low-molecular-weight drugs against the main protease of severe acute respiratory syndrome coronavirus 2

Inhibition mechanism of SARS-CoV-2 main protease by ebselen and its derivatives

Identification of ebselen and its analogues as potent covalent inhibitors of papain-like protease from SARS-CoV-2

Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication

Efficacy of GC-376 against SARS-CoV-2 virus infection in the K18 hACE2 transgenic mouse model

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

The complex structure of GRL0617 and SARS-CoV-2 PLpro reveals a hot spot for antiviral drug discovery

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

Structural insights into plasticity and discovery of remdesivir metabolite GS-441524 binding in SARS-CoV-2 macrodomain

Masitinib is a broad coronavirus 3CL inhibitor that blocks replication of SARS-CoV-2

In silico characterization of masitinib interaction with SARS-CoV-2 main protease

SARS-CoV-2 M pro inhibitors with antiviral activity in a transgenic mouse model

Crystal structure of SARS-CoV-2 main protease in complex with the non-covalent inhibitor ML188

A quick route to multiple highly potent SARS-CoV-2 main protease inhibitors

Mechanism of inhibition of SARS-CoV-2 M pro by N3 peptidyl michael acceptor explained by QM/MM simulations and design of new derivatives with tunable chemical reactivity

Discovery of ketone-based covalent inhibitors of coronavirus 3CL proteases for the potential therapeutic treatment of COVID-19

Structural basis for inhibition of the SARS-CoV-2 RNA polymerase by suramin

Suramin inhibits SARS-CoV-2 infection in cell culture by interfering with early steps of the replication cycle

A small molecule compound with an indole moiety inhibits the main protease of SARS-CoV-2 and blocks virus replication

Multiscale simulations of SARS-CoV-2 3CL protease inhibition with aldehyde derivatives. Role of protein and inhibitor conformational dynamics in the reaction mechanism

Peptidomimetic α-acyloxymethylketone warheads with six-membered lactam P1 glutamine mimic: SARS-CoV-2 3CL protease inhibition, coronavirus antiviral activity, and in vitro biological stability

SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity

SARS-CoV-2 D614G spike mutation increases entry efficiency with enhanced ACE2-binding affinity

Unlocking COVID therapeutic targets: A structure-based rationale against SARS-CoV-2, SARS-CoV and MERS-CoV Spike

Mutations in the SARS-CoV-2 Spike RBD are responsible for stronger ACE2 binding and poor anti-SARS-CoV mAbs cross-neutralization

Dissecting nucleotide selectivity in viral RNA polymerases

This work was supported by the project of the PhD research start-up funds of Qufu Normal University, China (Grant No. 614901, and 615201) . The authors, therefore, gratefully acknowledge the Qufu Normal University for the financial supports.