key: cord-1016970-9lanbq8z
authors: Li, Quanjie; Yi, Dongrong; Lei, Xiaobo; Zhao, Jianyuan; Zhang, Yongxin; Cui, Xiangling; Xiao, Xia; Jiao, Tao; Dong, Xiaojing; Zhao, Xuesen; Zeng, Hui; Liang, Chen; Ren, Lili; Guo, Fei; Li, Xiaoyu; Wang, Jianwei; Cen, Shan
title: Corilagin inhibits SARS-CoV-2 replication by targeting viral RNA-dependent RNA polymerase
date: 2021-02-15
journal: Acta Pharm Sin B
DOI: 10.1016/j.apsb.2021.02.011
sha: 4430d5cd48df7569e0487a989e4cd9143716994b
doc_id: 1016970
cord_uid: 9lanbq8z

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has become one major threat to human population health. The RNA-dependent RNA polymerase (RdRp) presents an ideal target of antivirals, whereas nucleoside analogs inhibitor is hindered by the proofreading activity of coronavirus. Herein, we report that corilagin (RAI-S-37) as a non-nucleoside inhibitor of SARS-CoV-2 RdRp, binds directly to RdRp, effectively inhibits the polymerase activity in both cell-free and cell-based assays, fully resists the proofreading activity and potently inhibits SARS-CoV-2 infection with a low 50% effective concentration (EC(50)) value of 0.13 μmol/L. Computation modeling predicts that RAI-S-37 lands at the palm domain of RdRp and prevents conformational changes required for nucleotide incorporation by RdRp. In addition, combination of RAI-S-37 with remdesivir exhibits additive activity against anti-SARS-CoV-2 RdRp. Together with the current data available on the safety and pharmacokinetics of corilagin as a medicinal herbal agent, these results demonstrate the potential of being developed into one of the much-needed SARS-CoV-2 therapeutics.

fundamental role in viral RNA synthesis. Moreover, RdRp has higher evolutionary stability and does not exist in mammalian cells, thus representing an extremely attractive drug target. Nucleos(t)ide inhibitors (NIs) such as remdesivir have been reported to suppress SARS-CoV-2 replication in vitro 9, 15, 16 . However, development of NIs that are incorporated into the product RNA chain is hampered by the proofreading activity of SARS-CoV-2 nsp14, which effectively decreases the incidence of mismatched nucleotides through its exoribonuclease (ExoN) activity, even that some of NIs exert antiviral activity through other mechanisms such as a chain terminator and exhibit less sensitive to the proofreading activity [17] [18] [19] . It is thus speculated that coronavirus may naturally have relatively high levels of resistance to nucleoside analogs. Unlike NIs that need to be incorporated into the growing viral RNA chain, non-nucleosides inhibitors (NNIs) usually exert antiviral activity by preventing RdRp conformational changes required for transcription 20 . It is thus proposed that NNIs may circumvent the resistance posed by the proofreading activity of coronavirus RNA transcription machinery. Unfortunately, no highly effective NNIs for SARS-CoV-2 currently exist.

Seeking for these NNIs has been facilitated by the rapid progress in the structural and enzymatic study of SARS-CoV-2 RdRp since the outbreak of COVID-19. Gao et al. 21 described the first near atomic resolution structure of nsp12 catalytic subunit in complex with cofactors nsp7-nsp8. Sharing a configuration similar to other RNA polymerases, SARS-CoV-2 RdRp shows a typical right-hand conformation, consisting of the palm, the thumb, and the finger subdomains. The primer-template entry site, the nucleoside triphosphate (NTP) entry channel, and the nascent product exit path converge in a central cavity that is formed by the conserved motifs (A-G) within the fingers and palm domain. The divalent cation-binding residue D618 in motif A and the catalytic residues S759, D760, and D761 in motif C are highly conserved in most viral RdRps. Compared with the apo complex, these residues undergo structural rearrangements to position the template and primer for an in-line attack on the approaching nucleotide. Later on, Yin et al. 22 reported the SARS-CoV-2 RdRp-RNA-remdesivir complex structure, in which the double-stranded RNA helix is inserted into the central channel of RdRp where the remdesivir monophosphate is covalently incorporated into the primer strand. Wang et al. 23 further proposed a transition model from primase to polymerase complex, revealing that the SARS-CoV-2 nsp7, nsp8, and nsp12 undergo structural rearrangements to adapt RNA binding. Importantly, they also elucidated the delayed-chain-termination mechanism of remdesivir through structural and kinetic analyses 23 . Compounds that interfere with these RdRp conformational changes are expected to impair RdRp function and hereby inhibit SARS-CoV-2 infection.

SARS-CoV-2 RdRp (K D =0.54-220 μmol/L). Their inhibition of SARS-CoV-2 RdRp was measured with cell-free and cell-based polymerase activity assays. The most potent inhibitor, named RAI-S-37, binds to SARS-CoV-2 RdRp with a K D value of 0.54 μmol/L, and drastically inhibits the polymerase activity. Importantly, RAI-S-37 inhibits SARS-CoV-2 replication in cell culture with a 50% effective concentration (EC 50 ) value of 0.13 μmol/L, thus warrants being further developed into effective SARS-CoV-2 therapeutics.

Starting from the cryo-electron microscopy structure of SARS-CoV-2 RdRp (PBD ID: 6M71), we employed the AutoDockTools (ADT) (version 1.5.6) to prepare the protein and gird map parameters 24 . The PDB file of protein was converted to PDBQT format after being added polar hydrogens and Gasteiger charges. The central cavity of SARS-CoV-2 RdRp was designated as a box of 26 Å×26 Å×26 Å, centered near the active sites (x=123. 6 TargetMol Bioactive compounds Library (Catalog Nos. L4000 and L4150) were docked to SARS-CoV-2 RdRp central cavity defined above and ranked by their calculated binding free energies (ΔG ADV ). The protein-ligand interactions were visually inspected by using PyMOL (version 2.3.4) and Free Maestro (version 11.8.012). The top ranked hits with high structural diversity and favorable binding mode were chosen for further examination.

The generated SARS-CoV-2 RdRp/RAI-S-37 complex structure through molecular docking was further relaxed using molecule dynamic (MD) simulation 28 . Amber11 software package was used to conduct all the simulations and analyze the results SARS-CoV-2 RdRp was described using the Amber ff99SB protein force field 29 . The missing hydrogen atoms of protein residues were added using LEAP module. The force field and internal coordinate preparation files of RAI-S-37 was generated utilizing the general Amber force field (GAFF) through the Antechamber module 30, 31 . The restrained electrostatic potential charges of RAI-S-37 were fitted from quantum mechanism calculation at B3LYP/6-31G* level using Gaussian 09 program 32 . Total 6 Na + counterions were added to SARS-CoV-2 RdRp/RAI-S-37 complex for charge neutralization. The complex was then J o u r n a l P r e -p r o o f solvated in a box filled with 28,791 TIP3P water molecules that extended 10 Å outside the protein 33 .

Prior to MD simulation, the system was subjected to energy minimization as previously described 34 .

Followed by this, the system was heated from 0 to 300 K for a period of 50 ps under constant volume periodic boundary conditions (NVT), with protein fixed with a force constant 10 kcal/(mol·Å 2 ). Subsequently, the system was equilibrated for 500 ps at a constant pressure of 1 atm and temperature of 300 K (NPT). Finally, a production run of 10 ns simulations were performed with a time step of 2 fs under the same conditions. Throughout the simulation, the nonbonded interactions were evaluated with a cutoff of 10 Å, and the long-range electrostatic interactions were treated by Particle Mesh Ewald (PME) method. The SHAKE algorithm was applied to constrain the hydrogen bonds 35 .

The trajectories and presence of hydrogen bonds were analyzed using ptraj module in Amber. The representative structures of SARS-CoV-2 RdRp/RAI-S-37 complex were obtained through cluster analysis by using kclust module in MMTSB Tool Set 36 . The binding free energy decomposition were calculated with molecular mechanics/generalized born surface area (MM/GBSA) method as previously described 37 . All the simulation results were visualized using VMD (version 1.9.3) and

PyMOL (version 2.3.4).

Human 293T (HEK293T, Cat# CRL-11268, ATCC), Huh-7 (Cat# CCL-185, ATCC), and Vero (Cat# CCL-81, ATCC) cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) at 37 °C in a humidified atmosphere of 5%

The SARS-CoV-2 used in this study was isolated from respiratory samples of confirmed COVID-19 patients and was as propagated in Vero cells as previously described 38, 39 Selected compounds from virtual screening procedure and remdesivir (Cat# T7766) were purchased from Target Molecule Corp (Target Mol). All compounds were of a purity>95%.

The binding affinities between tested compounds and SARS-CoV-2 RdRp were measured using BLI-based Octet Platform (Octet Red 96, Fortebio). A temperature of 30 °C and a stirring speed of J o u r n a l P r e -p r o o f the experiment. 1×phosphate-buffered saline (PBS) (pH=7.4) with 0.01% Tween-20, 0.1% bovine serum albumin (BSA), and 2% DMSO was used as assay buffer for all measurements. The purified His-tagged SARS-CoV-2 RdRp were captured via Ni-NTA (NTA) biosensors (Cat# 18-5101, ForteBio) at a concentration of 150 μg/mL, resulting in a saturation response of 5 to 6 nm after 300 s.

Subsequently, the loaded biosensors were washed for 3 min in buffer to clear up loose nonspecifically bound SARS-CoV-2 RdRp and to establish a stable baseline. For binding kinetic measurements, the association of SARS-CoV-2 RdRp and tested compounds (3.125 to 100 μmol/L in assay buffer) was measured for 60-180 s and the dissociation of them was measured for 120 s in assay buffer. Reference wells that utilized buffer instead of tested compounds were also included to correct the baseline shift. A parallel set of Ni-NTA sensors that were incubated in buffer-only were prepared as the negative reference controls to correct the non-specific binding of the compounds to the biosensor surface. Raw kinetic data were analyzed using a double reference subtraction approach in which both the background and non-specific binding were subtracted. The binding affinity constant K D (K D =k dis /k on ; k on is the association rate constant, k dis is the dissociation rate constant) values were calculated using 1:1 binding model through global fitting of multiple kinetic traces. Data Analysis 9.0 software was used to analyze the real-time monitoring data. All the measurements were performed in three independent experiments.

The genes of SARS-CoV-2 nsp7 and nsp8 (GenBank: MN908947.3) were synthesized and cloned into pET-21a (+) vector (Genewiz Tec, Suzhou, China). The nsp7-6His-nsp8 gene, in which a 6×histidine was introduced between the two subunits, was also prepared in the same manner. The SARS-CoV-2 nsp12 expression vector was kindly provided by Zihe Rao (Shanghai Tech University, Shanghai, China). Specifically, the genes of nsp12 (GenBank: MN908947) were cloned into a modified pET-22b vector with a 10×His tag added at the C-terminus 21, 23 . For purification of nsp7, nsp8, and nsp7-6His-nsp8, the target proteins were first purified through Ni-NTA affinity chromatography by loading onto the Histrap excel column (GE Healthcare, USA) and further purified by passing through a Hitrap Q ion-exchange column (GE Healthcare, USA). The final products were concentrated to more than 10 mg/mL and store at −80 °C.

The in vitro measurement of SARS-CoV-2 polymerase activity was performed as previously described with slight modifications 7 . The activity assays were performed in the reaction buffer containing 20 mmol/L Tris-HCl (pH 8.0), 10 mmol/L KCl, 1 mmol/L DTT, and 4 mmol/L MgCl 2 .

For assembling stable nsp7-nsp8 complex, purified nsp7 was incubated with nsp8 at a molar ratio of We established a cell-based SARS-CoV-2 RdRp reporter assay system by modifying the previously developed system 40, 41 . The plasmids nsp12, nsp7, nsp8, nsp10, and nsp14 were used to express codon-optimized Flag-nsp12, Flag-nsp7, Flag-nsp8, Flag-nsp10, and Flag-nsp14, respectively, all of which contain a Flag tag at the C-terminus. To generate plasmid pCoV-Gluc, which produces positive-strand of vRNA encoding Gaussia-luciferase (Gluc), 5ʹ untranslated region (UTR)-Gluc-3ʹ

UTR was first synthesized (Sangon Biotech), then inserted into the BamH I and Not I sites of pRetroX-tight-Pur vector (kindly provided by Dr. Guo Fei). The primers are (5ʹ-GGCGGATCCATTAAAGGTTTATAC-3ʹ (forward) and

5ʹ-TTAGCGGCCGCGTCATTCTCCTAAGAA-3ʹ (reverse).

HEK293T cells were transfected with CoV-Gluc, nsp12, nsp7, and nsp8 plasmids at the ratio of 

Cell viability was examined with Cell Counting kit-8 (CCK-8, Beyotime), which is a water-soluble tetrazolium salt-8 (WST-8) reagent. HEK293T cells were seeded in 96-well plates with a density of 4×10 4 cells per well. 1 μL of each tested compound was added to cells and incubated for 24 h. Then 10 μL of CCK-8 reagent was added into each well and incubated for 90 min at 37 °C. The absorbance at 450 nm was measured using the Enspire 2300 Multiable reader (PekinElmer). 

Huh-7 cells were seeded into 12-well plates at 1×10 5 

To identify candidate inhibitors of SARS-CoV-2 RdRp, we first performed structure-based virtual screening using molecular docking calculation (Fig. 1A) . Table S1 (Supporting Information).

Insert Fig. 1 

To assess the ability of the selected 50 compounds to interact with SARS-CoV-2 RdRp, we developed a biosensor-based compound screening assay using BLI technology that can directly monitor the binding kinetics between compounds and the target protein in vitro. The NTA biosensor loaded with his-tagged SARS-CoV-2 RdRp were flowed through wells containing compounds.

Meanwhile, the BLI response signals that reflect the binding interaction between tested compounds and SARS-CoV-2 RdRp were generated. We first performed a primary screen of the 50 compounds at a single concentration of 50 μmol/L. As shown in Fig. 2 and Table S1, RdRp. The detected BLI K D , k on , and k dis values were shown in Table 1 . K D is a ratio of k dis /k on . The association and dissociation curves and chemical structures were presented in Fig. 3 Insert Fig. 2 Insert Table 1 Insert Fig. 3 

Having found that compounds of RAI-S-11, RAI-S-35, RAI-S-36, RAI-S-37, RAI-S-45, and RAI-S-47 bind directly to SARS-CoV-2 nsp12 (Table 1) , we established the primer-dependent RNA elongation reaction assay as previously described 7 , in order to assess the inhibitory effects of these compounds on RdRp and the contribution of compound-protein interaction to their anti-RdRp activity. Since cofactors nsp7 and nsp8 were reported to form hexadecameric primase complex in the early stage of reaction 23 , we purified recombinant nsp7, nsp8, and the fusion protein nsp7-6His-nsp8, in which a 6×histidine linker was inserted between nsp7 and nsp8 sequences (Fig. 4A ). In the cell-free polymerase activity assay, a 40 nt template RNA mimicking the 3ʹ UTR of SARS-CoV-2 RNA genome without the polyA tail and a 20 nt primer labeled with FAM were used to monitor the polymerase activity of RdRp, which generated the 40 nt FAM-labeled product.

As shown in Fig. 4B and C, nsp12 itself was capable of RNA synthesis albeit with low efficiency.

Including the nsp7 and nsp8 proteins enhanced the polymerase activity of nsp12 by ~1.9-fold.

Stronger RdRp activity of nsp12 was observed with the addition of nsp7-6His-nsp8 fusion protein, which increased the polymerase activity by ~1.3-fold compared to addition of nsp7 and nsp8.

To measure the direct inhibition of the polymerase activity of SARS-CoV-2 RdRp by the selected compounds, nsp12 was pre-incubated with different concentrations (2.5, 10, and 40 μmol/L) of these compounds prior to polymerization reaction. Results in Fig. 4D and E showed that, at 40 μmol/L, still exhibited 73% inhibition of the polymerization reaction ( Fig. 4F and G) . Even at 2.5 μmol/L, RAI-S-37 reduced the activity of SARS-CoV-2 RdRp by 24% ( Fig. 4H and I) . Given the strong binding affinity of RAI-S-37 to SARS-CoV-2 RdRp, these functional data suggest that RAI-S-37 is a potentially effective SARS-CoV-2 RdRp inhibitor.

To further measure the efficiency of RAI-S-37 against SARS-CoV-2 RdRp, we developed a cell-based reporter assay with Gluc as the reporter to evaluate the anti-SARS-CoV-2 RdRp activity.

The luciferase expression reporter plasmid pCoV-Gluc was generated by inserting the coding sequence of Gluc between the 5ʹ UTR and 3ʹ UTR of SARS-CoV-2, such that pCoV-Gluc expresses Gluc under the control of SARS-CoV-2 UTRs. Upon expression of SARS-CoV-2 RdRp, the Gluc RNA is further amplified by viral RdRp, resulting in the increase of Gluc mRNA and Gluc protein.

Therefore, the increased Gluc activity represents the activity of SARS-CoV-2 RdRp. Using this system, we measured SARS-CoV-2 RdRp activity in cells that were exposed to different doses of the selected compounds. The RdRp inhibitor remdesivir was used as a positive control. As shown in corroborating that the non-nucleoside inhibitor RAI-S-37 is resistant to the proofreading activity.

Because remdesivir and RAI-S-37 inhibit SARS-CoV-2 RdRp activity through different mechanism, we also tested whether the combination treatment using both could increase the inhibitory effect. As shown in Fig. 5A , the combination of RAI-S-37 with remdesivir can enhance the inhibitory effects (EC 50 =1.25 μmol/L). The additive interaction was also observed in the presence of nsp10-nsp14 (Fig. 5B) . The dose-response curve for drugs remdesivir and RAI-S-37 administered together is very close to the sum of the two-individual dose-response curves.

To determine whether RAI-S-37 inhibits the replication of SARS-CoV-2, we infected Vero cells with 

To Insert Table 2 Sequence alignment of SARS-CoV-2 and HCoV-OC43 RdRps showed 72% homology (Supporting Information Fig. S5 ), suggesting that RAI-S-37 may inhibit HCoV-OC43 RdRp through interactions similar to those with SARS-CoV-2 RdRp, which is supported by the dose-dependent inhibition of HCoV-OC43 by RAI-S-37 (Fig. 6B) . To shed more light on these interactions, we computed the 3D structure of HCoV-OC43-RdRp starting from SARS-CoV-2 nsp12 and then docked RAI-S-37 to the central cavity of the predicted structure. The ligand-polymerase interaction diagram is depicted in Supporting Information Fig. S6 . The calculated binding free energy (ΔG ADV =−8.6 kcal/mol) was equivalent to that of SARS-CoV-2 RdRp (ΔG ADV =−8.8 kcal/mol). Superposition of these two complexes showed similar structural features at the RAI-S-37 binding sites (Fig. 7E) . Notably, RAI-S-37 forms hydrogen bonds with D756, W796, and E807, which are equivalent to D760, W800, and E811 in SARS-CoV-2 RdRp. Furthermore, the top 10 amino acids identified for SARS-CoV-2

RdRp/RAI-S-37 binding are conserved in HCoV-OC43 RdRp ( Fig. 7F and Supporting Information Fig. S5 ). This further supports a similar mechanism behind the binding RAI-S-37 to RdRps of SARS-CoV-2 and HCoV-OC43, which leads to the inhibition of both viral RdRps.

The novel coronavirus SARS-CoV-2 poses a severe threat to public health. There is an urgent need for the development of COVID-19 therapeutics. Virtual screening has become an important tool in drug development for its knowledge-driven, cost-effective, and time-saving advantages. Using this technique, we successfully identified a strong RdRp inhibitor of SARS-CoV-2. Since the three-dimensional structure of target protein is available, we applied structure-based virtual screening of commercial chemical databases (total 15,220 compounds) against the cavity center of SARS-CoV-2 RdRp. A small set of 50 compounds were selected and further examined using in vitro BLI binding assays. At the end, 6 out of these 50 compounds were identified for direct binding to SARS-CoV-2 RdRp with micromolar range affinities. In particular, RAI-S-37, which showed the strongest binding affinity to RdRp (K D =0.54 μmol/L), effectively inhibited SARS-CoV-2 RdRp in Vero cells with an EC 50 value of 0.13 μmol/L. It has been reported that compounds of high docking scores may fail to bind the target protein in the in vitro binding assay 42 . For example, we did not detect the binding of RAI-S-38, a compound that was predicted to bind to SARS-CoV-2 RdRp with the most favorable binding energy (ΔG ADV =−10.1 kcal/mol). It is known that the driving forces that dictate protein-ligand binding are the sum of diverse interactions and energy exchanges among the protein, ligand, water, and buffer ions 43, 44 . The ADV docking procedure did not consider the energy of solvated sate, thus provides an estimate, not an exact value of binding affinity. The most precise J o u r n a l P r e -p r o o f method to compute relative binding energy is the use of free-energy perturbation techniques [45] [46] [47] .

However, due to the high computational cost, we did not include the solvent factors, such as liquid water and buffer ions in the primary virtual screening procedure.

RAI-S-37 differs from the existing SARS-CoV-2 RdRp inhibitors, such as ribavirin and remdesivir, because it is a NNI, thus can circumvent the proofreading activity of coronavirus. The nsp14 of SARS-CoV-2 plays a pivotal role in decreasing the incidence of mismatched nucleotides through its ExoN activity 17, 18 . It is thus speculated that coronavirus may generally have a relatively high level of resistance to nucleoside analogs. For example, the NI ribavirin poorly inhibits SARS-CoV-2 in vitro with a high EC 50 value of 109.5 μmol/L 9 . Although remdesivir has been shown to be more effective than other NIs, it is also sensitive to the proofreading activity of ExoN and thus requires rigorous clinical trial evaluation before approved for regular clinical use 48 . Data in our study confirm that expression of nsp10-nsp14 leads to a 2.1-fold increase in EC 50 value of remdesivir in cell-based RdRp activity assay (Fig. 5 ). In contrast, the EC 50 values of RAI-S-37 remain the same in the absence and presence of the nsp10-nsp14, suggesting that the NNI RAI-S-37 is resistant to the coronavirus proofreading activity. Given that RAI-S-37 and remdesivir use different mechanisms to inhibit the viral activity, it was hypothesized that using both of them may reduce the chances of drug resistance. In this regard, we examined the inhibitory effect of remdesivir provided in combination with RAIS-S-37 on SARS-CoV-2 RdRp activity. The results showed that when remdesivir is combined with RAI-S-37, it has additive inhibitory effect against SARS-CoV-2 RdRp (Fig. 5) . We also evaluated the combinational effect of these two drugs against HCoV-OC43 infection in cells.

Interestingly, RAI-S-37 showed a moderate synergistic effect with remdesivir on inhibiting

HCoV-OC43 infection, with maximal ZIP-scores of 11.7 (Supporting Information Fig. S7 ). Further studies are warranted to evaluate the anti-SARS-CoV-2 efficacy of RAI-S-37 alone, and/or in combination with remdesivir. Combined, these results indicate that the non-nucleoside inhibitor RAI-S-37 provides a complementary treatment strategy for COVID-19.

Drug repurposing is a promising strategy to develop antiviral agents as new therapeutics 49 Unlike some other viral RdRps, such as poliovirus 3Dpol, that can synthesize RNA by themselves, SARS-CoV-2 nsp12 requires viral cofactors including nsp7 and nsp8 to prompt its polymerase activity. Hillen et al. 52 reported that the helical extensions of nsp8 protrude along the exiting RNA and can be regarded as a positively charged "sliding poles", which is required for replicating the long SARS-CoV-2 RNA genome. Wang et al further showed that the N-terminal helical extension of nsp8 undergoes structural arrangement and forms a stable platform that may enable progressive replication of viral RNA 23 . In agreement with these biochemical studies, our study further demonstrates the essential roles of the nsp7 and nsp8 in stimulating the polymerase activity of nsp12.

As shown in Fig. 4B and C, SARS-CoV-2 nsp12 itself is capable of conducting RNA polymerization with low efficiency, whereas inclusion of nsp7 and nsp8 boosted the polymerase activity of nsp12 by ~1.9-fold. Notably, nsp7-6His-nsp8 further enhanced the efficiency of RNA synthesis by ~1.3-fold,

suggesting that the nsp7-nsp8 complex enhances processivity of nsp12-mediated RNA synthesis.

Inserting a linker of six histidine between nsp7 and nsp8 was used to better assimilate the association of nsp7 and nsp8, which is believed to help achieve optimal RNA polymerase activity. These data are consistent with the reported transition model, in which the nsp7-nsp8 hexadecameric complex operates as a primase before transition to the nsp12-nsp7-nsp8 catalytic complex 23 .

The predicted binding sites of RAI-S-37 in SARS-CoV-2 RdRp are located at the palm subdomain.

Specifically, the pocket consists of G616 to Y619 in motif A, D761 to V763 in motif C, K798 in motif D, E811 to S814 in motif E, and I548 to K551 in motif F. RAI-S-37 forms a strong hydrogen bond with D761, a key catalytic residue in the SDD motif (residues 759-761). In addition, D618, the classic divalent cation-binding residue, is also in close contact with RAI-S-37. It has been reported that D618, D760, and D761 are involved in coordinating two magnesium ions at the catalytic center 21 . Given the close interactions between RAI-S-37 and these residues, it is posited that the binding of RAI-S-37 may block the conformational change that is needed to coordinate the divalent cations during the catalytic process. Multiple interactions of RAI-S-37 with motif F residues may block the entry of NTP to the active side, thus contributing to the inhibition of viral RNA synthesis.

Of note, corilagin is reported to be a potential inhibitor of SARS-CoV-2 main protease (Mpro) via in silico molecular docking studies. For this reason, we performed SARS-CoV-2 Mpro enzyme activity inhibition assay to assess the efficacy of corilagin. However, even at a high concentration of 10 μmol/L, corilagin did not show any inhibitory effect on SARS-CoV-2 Mpro (Supporting Information   Fig. S8 ). Together, these results suggest that RAI-S-37 exerts its antiviral activity by preventing conformational changes of RdRp which are obligatory for nucleotide incorporation into the growing viral RNA chain, while the detailed mechanism awaits further investigation.

We have identified a novel NI of SARS-CoV-2 RdRp, called corilagin (RAI-S-37), through structure-based virtual screening and experimental validation. We were able to show that RAI-S-37 J o u r n a l P r e -p r o o f binds directly to SARS-CoV-2 RdRp, effectively inhibits the polymerase activity in both cell-free and cell-based polymerase activity assays, and that RAI-S-37 exhibits does-dependent inhibition of SARS-CoV-2 infection with a very low EC 50 value of 0.13 μmol/L. We further predicted the potential binding modes of RAI-S-37 to viral RdRp and found that RAI-S-37 may function by preventing the conformational change of RdRp which is essential for synthesizing the long viral RNA genome. In addition, combination of RAI-S-37 with remdesivir exhibits additive activity against anti-SARS-CoV-2 RdRp. Combined, these results indicate that RAI-S-37 holds great promise of becoming a new and effective anti-SARS-CoV-2 drug to treat COVID-19 patients.

We report that RAI-S-37 (corilagin) acts as a non-nucleoside inhibitor of SARS-CoV-2 RdRp, binds directly to RdRp, effectively inhibits the polymerase activity in both cell-free and cell-based assays, fully resists the proofreading activity and potently inhibits SARS-CoV-2 infection in vitro.

J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f Table 1 The molecule name (MolName), CAS, molecule weight (MW, g/mol), calculated binding energies (∆G ADV , kcal/mol), and BLI binding kinetics (K D , k on , k dis , and R 2 ) of compounds selected from primary in vitro binding screen. 

A pneumonia outbreak associated with a new coronavirus of probable bat origin

The species Severe acute respiratory syndrome-related coronavirus: Classifying 2019-nCoV and naming it SARS-CoV-2

First case of 2019 novel coronavirus in the United States

Epidemiology and cause of severe acute respiratory syndrome (SARS) in Guangdong, Peopleʹs Republic of China

Coronaviruses post-SARS: Update on replication and pathogenesis

Aerosol and surface stability of SARS-CoV-2 as compared with SARS-CoV-1

Structural and biochemical characterization of the nsp12-nsp7-nsp8 core colymerase complex from SARS-CoV-2

Remdesivir in adults with severe COVID-19: A randomised, double-blind, placebo-controlled, multicentre trial

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

Efficacy of remdesivir in COVID-19

Compassionate use of remdesivir for patients with severe Covid-19

Coronaviridae and SARS-associated coronavirus strain HSR1

The coronavirus replicase

The nonstructural proteins directing coronavirus RNA synthesis and processing

An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice

Infidelity of SARS-CoV nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing

One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities

Nucleoside, nucleotide, and non-nucleoside inhibitors of hepatitis C virus NS5B RNA-dependent RNA-polymerase

Structure of the RNA-dependent RNA polymerase from COVID-19 virus

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

Structural basis for RNA replication by the SARS-CoV-2 polymerase

Using AutoDock 4 with AutoDocktools: A tutorial

Open Babel: An open chemical toolbox

Merck molecular force field. I. Basis, form, scope, parameterization, and performance of MMFF94

AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading

Combining docking and molecular dynamic simulations in drug design

Improved side-chain torsion potentials for the Amber ff99SB protein force field

Antechamber: An accessory software package for molecular mechanical calculations

Development and testing of a general amber force field

Gaussian 09, Revision d. 01

Structure and dynamics of the TIP3P, SPC, and SPC/E water models at 298 K

Identification of a broad-spectrum viral inhibitor targeting a novel allosteric site in the RNA-dependent RNA polymerases of dengue virus and norovirus

Settle: An analytical version of the SHAKE and RATTLE algorithm for rigid water models

MMTSB Tool Set: Enhanced sampling and multiscale modeling methods for applications in structural biology

The MM/PBSA and MM/GBSA methods to estimate ligand-binding affinities

Identification of a novel coronavirus causing severe pneumonia in human: A descriptive study

Activation and evasion of type I interferon responses by SARS-CoV-2

A cell-based high-throughput approach to identify inhibitors of influenza A virus

A cell-based reporter assay for screening inhibitors of MERS coronavirus RNA-dependent RNA polymerase activity

Recent development and application of virtual screening in drug discovery: An overview

Calculation of protein-ligand binding affinities

Insights into protein-ligand interactions: Mechanisms, models, and methods

A comparison of perturbation methods and Poisson-Boltzmann electrostatics calculations for estimation of relative solvation free energies

Ab initio quantum mechanics-based free energy perturbation method for calculating relative solvation free energies

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

Remdesivir and SARS-CoV-2: Structural requirements at both nsp12 RdRp and nsp14 exonuclease active-sites

Drug repurposing: progress, challenges and recommendations

A natural small molecule inhibitor corilagin blocks HCV replication and modulates oxidative stress to reduce liver damage

Corilagin in cancer: A critical evaluation of anticancer activities and molecular mechanisms

Structure of replicating SARS-CoV-2 polymerase

The residues are ranked by total energy. Only residues with binding energy greater than 0.5 kcal/mol