The COVID-19 pandemic caused by nonstop infections of SARS-CoV-2 has continued to ravage many countries worldwide. Here we report that suramin, a 100-year-old drug, is a potent inhibitor of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and acts by blocking the binding of RNA to the enzyme. In biochemical assays, suramin and its derivatives are at least 20-fold more potent than remdesivir, the currently approved nucleotide drug for treatment of COVID-19. The 2.6 Å cryo-electron microscopy structure of the viral RdRp bound to suramin reveals two binding sites. One site directly blocks the binding of the RNA template strand and the other site clashes with the RNA primer strand near the RdRp catalytic site, thus inhibiting RdRp activity. Suramin blocks viral replication in Vero E6 cells, although the reasons underlying this effect are likely various. Our results provide a structural mechanism for a nonnucleotide inhibitor of the SARS-CoV-2 RdRp.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of coronavirus disease 2019 (COVID-19), with over 84.66 million infections and 1.83 million deaths as reported on 3 January 2021 (refs. 1,2). SARS-CoV-2 is a positive-sense, single-stranded RNA virus. SARS-CoV-2 and several related beta-coronaviruses, including SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), are highly pathogenic. Infections can lead to severe acute respiratory syndrome, loss of lung function and, in severe cases, death. Compared to SARS-CoV and MERS-CoV, SARS-CoV-2 has a higher capacity of human-to-human infections, which resulted in the rapidly growing pandemic3. Finding an effective treatment for COVID-19, potentially also through drug repurposing, is an urgent but unmet medical need.
Suramin (Fig. 1a) is a century-old drug that has been used to treat African sleeping sickness and river blindness4,5. It has also been shown to be effective in inhibiting the replication of a wide range of viruses, including enteroviruses6, Zika virus7, Chikungunya8 and Ebola viruses9. The viral inhibition mechanisms of suramin are diverse, including inhibition of viral attachment, viral entry and release from host cells in part through interactions with viral capsid proteins7,8,10,11. Recently, suramin has been shown to inhibit SARS-CoV-2 infection in cell culture by preventing cellular entry of the virus12. Here we report that suramin is also a potent inhibitor of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), an essential enzyme for the viral life cycle. The potency of suramin in biochemical RdRp inhibition assays is at least 20-fold more potent than remdesivir, the current Food and Drug Administration-approved nucleotide drug for the treatment of COVID-19. The activity of suramin in cell-based viral inhibition is similar to remdesivir because the highly negative charge of suramin prevents efficient cellular uptake. A cryogenic electron microscopy (cryo-EM) structure reveals that suramin binds to the RdRp active site, blocking the binding of both RNA template and primer strands. These results provide a structural template for the design of next generation suramin derivatives as SARS-CoV-2 RdRp inhibitors.
Inhibition of RdRp and anti-SARS-CoV-2 by suramin
The core RNA polymerase of SARS-CoV-2 is composed of nonstructural protein nsp12 with two accessary subunits nsp7 and nsp8 (refs. 13,14). Incubation of the purified nsp12–7–8 complex (Extended Data Figs. 1a,b) with a 30-base template and 20-base primer (poly-U in Fig. 1b) allowed primer extension to the same length as the template in the presence of saturated concentrations of ATP as illustrated in a gel-based assay (lane 1 in Fig. 1c). Addition of 8–32 µM suramin nearly abolished the elongation of the primer strand while it required 100–1,000 µM of remdesivir in its triphosphate form (RDV-TP) to achieve the same degree of inhibition under the same conditions15. Addition of 100 µM suramin completely blocked the formation of RdRp–RNA complex, while it required more than 5 mM of RDV-TP to inhibit the binding of RdRp to RNA (Fig. 1d and Extended Data Fig. 1c). Solution based assays of RdRp inhibition determined that the half-maximal inhibition concentration (IC50) of suramin is 0.26 µM (Fig. 1e), and the IC50 for RDV-TP is 6.21 µM under identical assay conditions (Extended Data Fig. 1d), suggesting that suramin is at least 20-fold more potent than RDV-TP. Cell-based experiments indicated that suramin was able to inhibit SARS-CoV-2 duplication in Vero E6 cells with a half-maximal effective concentration (EC50) of roughly 2.9 µM, which is about the same range as remdesivir in the same assay (Fig. 1f and Extended Data Fig. 1e)16. The apparent weaker inhibition of suramin in cell-based assays than in enzyme inhibition assays may be due to the highly negative charge of suramin that prevent its efficient uptake by the host cells. The CC50 (concentrations of drug required to reduce cell viability by 50%) of suramin is over 1,000 µM, indicating that its relatively low cytotoxicity that is lower than that of remdesivir (Fig. 1f and Extended Data Fig. 1e).
The structure of the RdRp–suramin complex
For the cryo-EM studies, we incubated the SARS-CoV-2 RdRp complex with tenfold molar excess of suramin (Methods). The structure was determined at a global resolution of 2.57 Å with 95,845 particles from over 8 million original particles auto-picked from 11,846 micrographs (Extended Data Fig. 2 and Table 1). The EM map reveals clear density for all key components of the RdRp–suramin complex, including one nsp12 (residues S6-C22, V31-I106, M110-L895 and N911-T929), one nsp7 (residues K2-G64), two nsp8 (residues D78-A191 for nsp8-1 and residues T84-A191 for nsp8-2, respectively) and two suramin molecules (Fig. 2a and Extended Data Fig. 3).
The overall structure of the RdRp–suramin complex is very similar to the apo–RdRp complex, with a root mean squared deviation (r.m.s.d.) of 0.465 Å for all Cα atoms between the two structures (Fig. 2b and Extended Data Fig. 4a). The core RdRp complex structure is also very similar to the recently solved core RdRp complex with nsp13 and nsp9 or nsp13 (refs. 17,18,19) (Extended Data Fig. 4b–d). Nsp12 adopts the same right-hand palm fingers configuration, with its catalytic active site composed of seven highly conserved motifs A–G (Fig. 3a). Two suramin molecules fit into the catalytic chamber (Figs. 2a,b and 3b).
The interactions of suramin with SARS-CoV-2 RdRp
One suramin molecule (suramin no. 1) is fit into a cavity formed by a conserved motif G and the N terminus of motif B (Figs. 3b and 4a). The chemical structure of suramin has a twofold symmetry with a urea linker at the center (Fig. 1a). The EM density map for suramin no. 1 is clearly defined but only for half of the suramin molecule without the urea linker (Fig. 4a,b). The key interactions of suramin no. 1 with RdRp were summarized in Fig. 4c and Supplementary Table 1, including hydrogen bonds, charge interactions and hydrophobic packing interactions with conserved RdRp residues, which restrain the naphthalene-trisulfonic acid head in a relative narrow cavity. Two out of the three sulfonates (positions 3 and 5) form hydrogen bonds with the side chains from N497, K500, R569 and Q573, and the main chain from N497, while the sulfonate at position 1 points toward the solvent and forms only one hydrogen bond with the side chain of N496. The K577 side chain forms cation–π stacking with the naphthalene ring, and also forms a hydrogen bond with the amide bond linker between the naphthalene and benzene rings. The amide bond linker between the benzene rings C and D forms a hydrogen bond with main chain NH of G590. In addition, suramin no. 1 is in van der Waals contact with several residues, including L576, A580, A685, Y689 and L758. The second suramin molecule (suramin no. 2) is fit into the cavity near the catalytic active site formed by conserved motifs A, C, E and F (Figs. 3b and 4b). Again, only half of the molecule was observed in the structure with clear EM density map. The key interactions of suramin no. 2 with RdRp are summarized in Fig. 4d and Supplementary Table 1, including hydrogen bonds, charge interactions and hydrophobic packing interactions. Different from suramin no. 1, the sulfonate at position 5 of suramin no. 2 points toward the solvent and forms only one hydrogen bond with the side chain of R555, while the other two sulfonates at positions 1 and 3 form hydrogen bonds with the side chains from K551, R553, R555 and R836, and the main chains from A550 and K551. Meanwhile the side chain of R555 also forms a hydrogen bond with the amide bond linker between the naphthalene and benzene rings. The R836 side chain forms cation–π stacking with the benzene ring C. The NH of the benzene ring D forms a hydrogen bond with the side chain of D865. In addition, suramin no. 2 is in van der Waals contact with several residues, including H439, I548, S549, A840, S861 and L862. Sequence alignment with RdRp from several coronaviruses indicated that these suramin-contacting residues are conserved (Supplementary Fig. 1), suggesting that suramin may be a general inhibitor of viral RdRp.
Inhibition mechanism of suramin toward SARS-CoV-2 RdRp
Structural comparison of the RdRp–suramin complex with the remdesivir-bound RdRp complex reveals the mechanism of RdRp inhibition by suramin (Fig. 5a). If the base position of remdesivir was defined as a +1 position, then the first suramin molecule occupies the space of −1 to −3 positions of the RNA template strand (suramin no. 1 in Fig. 5b). The second suramin molecule at the active site occupies the space of the primer strand ranging from −4 to +1 positions (suramin no. 2 in Fig. 5c). The binding of these two suramin molecules thus blocks the binding of the RNA template–primer duplex to the active site as well as the entry of nucleotide triphosphate into the catalytic site, which would result in the direct inhibition of the RdRp catalytic activity. The direct inhibition mechanism of SARS-CoV-2 RdRp by suramin is different from the suramin-mediated inhibition of the norovirus RdRp, which also contained two binding sites for suramin20. In each site, only half of suramin molecule was seen. Structural comparison of the SARS-CoV-2 RdRp with the norovirus RdRp reveals that only one of the two suramin binding sites (suramin no. 2) partially overlapped (Extended Data Fig. 5a,c,d). The suramin binding sites in norovirus RdRp do not clash with the RNA strands but one of the suramin binding site overlapped with the proposed nucleotide entry channel, thus indirectly blocking RdRp polymerization activity. This mechanism is different from the direct block of the binding of the RNA template to the SARS-CoV-2 RdRp by suramin (Fig. 5). In addition, structural comparisons of the SARS-CoV-2 RdRp–suramin structure with the structures of the norovirus RdRp bound to suramin derivatives show that suramin and suramin derivatives bind to the RdRp with diverse conformations and orientations20,21 (Extended Data Figs. 5b,e and 6).
Inhibition SARS-CoV-2 RdRp by suramin derivatives
Suramin derivatives have been explored for diverse applications, including parasitic diseases and cancer10. To determine the structure–activity relationship for suramin derivatives, we screened a set of different ones using in vitro RdRp primer extension assays (Fig. 6a and Extended Data Fig. 1f). All eight tested suramin derivatives showed efficient inhibition of RdRp activity (Extended Data Fig. 1g). NF157, NF279 and NF449 are the most potent inhibitors with IC50 of 0.05 µM, about fivefold more potent than the parent drug (Fig. 6b). Cell-based assays showed that NF110 inhibited SARS-CoV-2 replication with an EC50 of 2.87 µM (Fig. 6c), while NF157 and NF279 inhibited SARS-CoV-2 replication with EC50 of roughly 10 µM. The CC50 values of all suramin derivatives are over 1,000 µM, indicating a good safety window. However, there is a 200-fold separation between their biochemical potency in inhibiting RdRp activity and their potency in inhibiting viral replication in cell-based assays. This is likely due to difficulties of these suramin derivatives to be taken up by host cells22. Future drug formulation, for example with glycol chitosan-based nanoparticles23, may improve their bioavailability to lung tissues and their potency in inhibiting viral replication.
The current COVID-19 pandemic evidences the need for effective vaccines and drug treatments for the disease. Suramin has been used to treat African sleeping sickness and that has also shown activity against a number of viruses in preclinical studies. In addition, suramin was shown to block SARS-CoV-2 at an earlier step of the replication cycle in time-of-addition assays12. Here, we demonstrate that suramin is a direct and potent inhibitor of the SARS-CoV-2 RdRp, an essential enzyme for the viral life cycle. The structure reveals that suramin binds to the active site of RdRp, blocking the binding of both strands of the template–primer RNA substrate and inhibiting the polymerase activity of the RdRp. Suramin derivatives also showed potent inhibition of RdRp activity and blocked viral replication in cell-based assays. Together, these results uncover the structural mechanism of a nonnucleotide inhibitor of the SARS-CoV-2 RdRp. The structure and biochemical results presented in this paper provide a rationale to develop suramin analogs and drug formulations that improve potency and efficacy of the drug. However, there are a number of limitations of suramin, including its high negative charge, which hinders its efficient entry into cell. In addition, there are potential risks of off-target effects on cellular polymerases and helicases. Nevertheless, suramin can serve as an interesting ‘tool compound’ for fundamental mechanistic studies into the viral RdRp, which could ultimately aid in drug development for COVID-19.
Constructs and expression of the RdRp complex
The RdRp complex was prepared according to same method reported14 as described below. The full-length gene of the SARS-CoV-2 nsp12 (encodes residues 1–932) was chemically synthesized with codon optimization (General Biosystems). The gene was cloned into a modified pFastBac baculovirus expression vector containing a 5′ ATG starting sequence and C-terminal tobacco etch virus (TEV) protease site followed by a His8 tag. The plasmid contains an additional methionine at the N terminus and GGSENLYFQGHHHHHHHH at the C terminus of nsp12. The full-length genes for nsp7 (encodes residues 1–83) and nsp8 (encodes residues 1–198) were cloned into the pFastBac vector containing a 5′ ATG starting sequence. All constructs were generated using the Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech) and verified by DNA sequencing. All constructs were expressed in Spodoptera frugiperda (Sf9) cells. Cell cultures were grown in ESF 921 serum-free medium (Expression Systems) to a density of 2–3 million cells per ml and then infected with three separate baculoviruses at a ratio of 1:2:2 for nsp12, nsp7 and nsp8 at a multiplicity of infection of about five. The cells were collected 48 h after infection at 27 °C and cell pellets were stored at −80 °C until use.
In addition, the genes of nsp7 and nsp8 were cloned into a modified pET-32a(+) vector containing a 5′ ATG starting sequence and C-terminal His8 tag with a TEV cleavage site for expression in E. coli. Plasmids were transformed into BL21(DE3) (Invitrogen). Bacterial cultures were grown to an OD600 of 0.6 at 37 °C, and then the expression was induced with a final concentration of 0.1 mM of isopropyl β-d-1-thiogalactopyranoside and the growth temperature was reduced to 16 °C for 18–20 h. The bacterial cultures were pelleted and stored at −80 °C until use.
Purification of the RdRp complex
The purification of nsp7 and nsp8 expressed in bacteria BL21(DE3) was similar to the purification of nsp7 and nsp8 reported previously14. Briefly, bacterial cells were lysed with a high-pressure homogenizer operating at 800 bar. Lysates were cleared by centrifugation at 25,000g for 30 min and were then bound to Ni-NTA beads (GE Healthcare). After washing with buffer containing 50 mM imidazole, the protein was eluted with buffer containing 300 mM imidazole. The tag was removed with incubation of TEV protease overnight and protein samples were concentrated with 3 or 30 kDa molecular weight cut-off centrifuge filter units (Millipore Corporation) and then size-separated by a Superdex 75 Increase 10/300 GL column in 25 mM HEPES pH 7.4, 200 mM sodium chloride, 5% (v/v) glycerol. The fractions for the nsp7 or nsp8 were collected, concentrated to about 10 mg ml−1 and stored at −80 °C until use.
Sf9 cells containing the coexpressed RdRp complex were resuspended in binding buffer of 25 mM HEPES pH 7.4, 300 mM sodium chloride, 25 mM imidazole, 1 mM magnesium chloride, 0.1% (v/v) IGEPALCA-630 (Anatrace), 1 mM tris(2-carboxyethyl)phosphine (TCEP), 10% (v/v) glycerol with additional EDTA-free Protease Inhibitor Cocktail (Bimake) and then incubated with agitation for 20 min at 4 °C. The incubated cells were lysed with a high-pressure homogenizer operating at 500 bar. The supernatant was isolated by centrifugation at 30,000g for 30 min, followed by incubation with Ni-NTA beads (GE Healthcare) for 2 h at 4 °C. After binding, the beads were washed with 20 column volumes of wash buffer of 25 mM HEPES, pH 7.4, 300 mM sodium chloride, 25 mM imidazole, 1 mM magnesium chloride, 1 mM TCEP and 10% (v/v) glycerol. The protein was eluted with 3–4 column volumes of elution buffer of 25 mM HEPES pH 7.4, 300 mM sodium chloride, 300 mM imidazole, 1 mM magnesium chloride, 1 mM TCEP and 10% (v/v) glycerol.
The coexpressed RdRp complex was incubated with additional nsp7 and nsp8 from the bacterial expression in a 1:1:2 molar ratio and incubated at 4 °C for 4 h. Incubated RdRp complex was concentrated with a 100 kDa molecular weight cut-off centrifugal filter unit (Millipore Corporation) and then size-separated by a Superdex 200 Increase 10/300 GL column in 25 mM HEPES pH 7.4, 300 mM sodium chloride, 1 mM magnesium chloride and 1 mM TCEP. The fractions for the monomeric complex were collected and concentrated to up to 12 mg ml−1. Suramin sodium salt (purchased from MedChemExpress) was dissolved in water up to 50 mM. Suramin derivatives (purchased from TopScience) was dissolved in water at concentrations of 5 to 50 mM. For the suramin-bound complex, the concentrated RdRp complex at a concentration of 12 mg ml−1 were incubated with 0.8 mM suramin at 4 °C for 0.5 h for the next step of EM experiments.
Cryo-EM sample preparation and data acquisition
An aliquot of 3 μl of protein sample of suramin-bound complex (12 mg ml−1) containing 0.0035% DDM was applied onto a glow-discharged 200 mesh grid (Quantifoil R1.2/1.3), blotted with filter paper for 3.0 s and plunge-frozen in liquid ethane using a FEI Vitrobot Mark IV. Cryo-EM micrographs were collected on a 300 kV Titan Krios microscope (FEI) equipped with a Gatan image filter (operated with a slit width of 20 eV) (GIF) and K3 direct detection camera. The microscope was operated at a calibrated magnification of ×46,773, yielding a pixel size of 1.069 Å on micrographs. In total, 11,846 micrographs in total were collected at an electron dose rate of 22.7 e– Å−2 s−1 with a defocus range of −0.5 μm to −2.0 μm. Each video with an accumulated dose of 68 e– Å−2 on sample were fractionated into a video stack of 36 image frames.
Frames in each video stack were aligned for beam-induced motion correction using the program MotionCorr2 (ref. 24). CTFFIND4 (ref. 25) was used to determine the contrast transfer function (CTF) parameters. From this, 10,241 good micrographs were selected for further data processing. Auto-picking program of Relion 3.0 (ref. 26) was used to pick the particles with the model of the apo–RdRp complex of COVID-19 (PDB ID 7BV1)15 as a reference, yielding a total of 8,557,180 picked particles. Then, the extracted particle stack was transferred to software Cryosparc v.2 (ref. 27) and a round of reference-free 2D classification was performed. Next, 3,159,808 particles were selected from classes representing projections of suramin-bound RdRp complex in different orientations and were subjected to two rounds of heterogenous refinement using a reconstruction of the apo–RdRp complex of COVID-19 (EMD-30209)15 as a starting map. One converged three-dimensional (3D) class with a high-resolution feature contains one nsp12, one nsp7 and two copies of nsp8. The particles from that 3D class were then imported back into Relion 3.0 and subjected to a round of focused alignment with a mask including the whole protein components. Finally, 95,845 particles from a 3D class showing the highest resolution feature were selected for a round of 3D refinement. After a round of CTF refinement and Bayesian polishing of particles, another round of 3D refinement was performed, yielding a final reconstruction at a global resolution of 2.57 Å based on the gold-standard Fourier shell correlation (FSC) = 0.143 criterion28. The local resolution was calculated with Relion 3.0.
The model of suramin-bound RdRp complex was built by docking the model of apo structure of COVID-19 RdRp (PDB ID 7BV1) into the density map using UCSF Chimera29, followed by ab initio model building of the N-terminal NiRAN domain of nsp12 and one copy of nsp8 in COOT30, and real space refinement using real_space_refine program in PHENIX31. The model statistics were calculated with MolProbity32 and listed in Table 1. Structural figures were prepared in Chimera or ChimeraX33.
Preparation of template–primer RNA for polymerase assays
For the poly-A template–primer RNA, a short RNA oligonucleotide with sequence of 5′-FAM-GCUAUGUGAGAUUAAGUUAU-3′ (Sangon Biotech) was used as the primer strand and a longer RNA oligonucleotide with a sequence of 5′-AAAAAAAAAAAUAACUUAAUCUCACAUAGC-3′ (Sangon Biotech) was used as template strand. To anneal the RNA duplex, both oligonucleotides were mixed at equal molar ratio in annealing buffer (10 mM Tris-HCl, pH 8.0, 25 mM NaCl and 2.5 mM EDTA), denatured by heating to 94 °C for 5 min and then slowly cooled to room temperature. The poly-U template–primer RNA was prepared similar to poly-A with the sequences of 5′-FAM-GCUAUGUGAGAUUAAGUUAU-3′ and 5′-UUUUUUUUUUAUAACUUAAUCUCACAUAGC-3′.
Gel mobility shift assay to detect RNA–RdRp protein binding
A gel mobility shift assay was performed to detect the effect of tested compounds on RNA binding by the RdRp complex. The binding reaction contained 20 mM Tris-HCl 8.0, 10 mM KCl, 6 mM MgCl2, 0.01% Triton-X100, 1 mM DTT, 1.14 U μl−1 RNase inhibitor (Vazyme Biotech), 9 μg of RdRp complex protein with 1 μg of poly-A template–primer RNA and increasing amounts of corresponding compounds (0, 1, 10, 100, 1,000 and 5,000 μM for suramin, and 0, 1, 10, 100, 1000, 5,000 and 10,000 μM for RDV-TP). Binding reactions were incubated for 30 min at room temperature and resolved on 4–20% native polyacrylamide gel (Thermo Fisher) running in 1× TBE buffer at 90 V for 1.5 h in a 4 °C cool room. The gel was imaged with a Tanon-5200 Multi Fluorescence Imager according to the manufacturer’s protocol.
RdRp enzymatic activity assay and its inhibition by suramin
The purified SARS-CoV-2 RdRp complex from insect cell at final concentration of 1 μM was incubated with 3.0 μM poly-A template–primer RNA and 10 mM UTP(Macklin) in the presence of 1.14 U μl−1 RNase inhibitor in reaction buffer containing 20 mM Tris, pH 8.0, 10 mM KCl, 6 mM MgCl2, 0.01% Triton-X100 and 1 mM DTT, which were prepared with DEPC-treated water. The total reaction volume was 20 μl. After incubation for 60 min in a 37 °C water bath, 40 μl of quench buffer (94% formamide, 30 mM EDTA, prepare with DEPC-treated water) was added to stop the reaction. A sample of 18 μl of reaction was mixed with 2 μl of 10× DNA loading buffer (Solarbio). Half of the sample (10 μl) was loaded onto a 10% urea–PAGE denatured gel, run at 120 V for 1 h, and imaged with a Tanon-5200 Multi Fluorescence Imager. The setup for the inhibition assays of the RdRp by suramin is identical to the above for the RdRp enzymatic assays, except that suramin was added to final concentrations of 0, 1, 2, 4, 8, 16 and 32 μM for 60 min before the addition of 10 mM UTP.
Fluorescence-based activity assay for SARS-CoV-2 RdRp
The detection of RNA synthesis by SARS-CoV-2 RdRp were established based on a real-time assay with the fluorescent dye SYTO 9 (Thermo Fisher), which binds double-stranded but not single-stranded RNA template molecules. The fluorescence emitted was recorded in real-time using a TECAN F200 with excitation and emission filters at 485 and 520 nm, respectively. The assay records the synthesis of dsRNA in a reaction using a poly-U molecule as a template and ATP as the nucleotide substrate, which has been adapted from methods previously documented for the detection of Zika virus polymerase activity34. Reactions were performed in individual wells of black 384-well low volume round bottom plates. The standard reaction contained 20 mM Tris-HCl, pH 8.0, 10 mM KCl, 6 mM MgCl2, 180 μM ATP, 0.2 μM poly-U template–primer RNA, 0.01%Trition-X100,1 mM DTT,0.025 U ml−1 RNase inhibitor (Vazyme Biotech) and 0.25 μM SYTO 9 (50 μM stock solution in TE buffer pH 7.5). The assay was initiated by the addition of 5 μg ml−1 SARS-CoV-2 RdRp and the fluorescence was recorded over 30 min at room temperature. The reaction with equivalent of dimethylsulfoxide (DMSO) was set as a maximum control, while the reaction with no SARS-CoV-2 RdRp was set as a minimum control. The reactions were carried out in the presence of 0.2 μM poly-U template–primer RNA and 180 μM ATP, and increasing concentrations of each inhibitor. Fluorometric results were expressed as mean ± s.d. Statistical significance was analyzed by two-way analysis of variance (ANOVA) using GraphPad Prism, v.8, as specified in the figure legends. Km determinations were obtained by plotting the velocity of the reaction as a function of nucleotide or ssRNA template concentrations using nonlinear regression. IC50 values were obtained by fitting the velocity data to a four-parameter logistic equation. Kinetic parameters and IC50 values were calculated using Sigmaplot v.11.
Vero E6 cell-based antiviral assay for suramin and suramin derivatives
African green monkey kidney Vero E6 cell line was obtained from American Type Culture Collection (no. 1586) and maintained in Dulbecco’s Modified Eagle Medium (Gibco Invitrogen) supplemented with 10% fetal bovine serum (Gibco Invitrogen), 1% antibiotic/antimycotic (Gibco Invitrogen), at 37 °C in a humidified 5% CO2 incubator. A clinical isolate of SARS-CoV-2 (nCoV-2019BetaCoV/Wuhan/WIV04/2019) was propagated in Vero E6 cells that were tested free of mycoplasma contamination, and viral titer was determined by 50% tissue culture infective dose using immunofluorescence assay16. All the infection experiments were performed at biosafety level-3 (BSL-3).
Preseeded Vero E6 cells (5 × 104 cells per well) were incubated with the different concentrations of the indicated compounds for 1 h, and then were infected with SARS-CoV-2 at a multiplicity of infection of 0.01. Two hours later, the virus–drug mixture was removed and cells were further cultured with a fresh compound containing medium. At 24 h post infection, we measured viral RNA copy number in cell supernatant using real-time PCR16. Briefly, the viral RNA was extracted from the cell culture supernatant using the MiniBEST Viral RNA/DNA Extraction Kit (Takara, catalog no. 9766) according to the manufacturer’s instructions. Then 3 μl total RNA was digested with genomic DNA eraser to remove contaminated DNA. In a 20 μl reaction system, the first-strand complementary DNA was synthesized, from which 2 μl of cDNA was used as a template for the next step of quantitative PCR. The primers used for quantitative PCR were RBD-qF1: 5′-CAATGGTTTAACAGGCACAGG-3′ and RBD-qR1: 5′-CTCAAGTGTCTGTGGATCACG-3′. The PCR amplification was performed as follows: 95 °C for 5 min followed by 40 cycles consisting of 95 °C for 15 s, 54 °C for 15 s and 72 °C for 30 s. DMSO was used in the controls. At least two independent experiments were carried out for each compound.
The cytotoxicity assay
The cytotoxicity of the tested drugs on Vero E6 were determined by CCK8 assays (Beyotime).
The IC50 values were expressed as mean ± s.d. from two independent experiments. The EC50 values were expressed as mean ± s.d. from three independent experiments. All values were determined via the nonlinear regression analysis using GraphPad Prism software v.8.0 (GraphPad Software).
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
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The cryo-EM data were collected at the Cryo-Electron Microscopy Research Center, Shanghai Institute of Materia Medica. This work was partially supported by the National Key R&D Program of China (grant no. 2020YFC0861000), CAMS Innovation Fund for Medical Sciences grant no. 2020-I2M-CoV19-001 and Tsinghua University-Peking University Center for Life Sciences grant no. 045-160321001 to S.Z.; the National Key R&D Programs of China grant no. 2018YFA0507002; Shanghai Municipal Science and Technology Major Project grant no. 2019SHZDZX02 and the Strategic Priority Research Program of Chinese Academy of Sciences XDB37030103 to H.E.X.; the CAS Young Innovator Association award to W.Y.; the 100 Talents Program of the Chinese Academy of Sciences, Chinese Academy of Sciences (grant no. XDA12010317), Natural Science Foundation of Shanghai (grant no. 18ZR1447700) to X.Y.; the National Natural Science Foundation of China (grant no. 31900869) and Shanghai Sailing Program (grant no. 19YF1456800) to Z.L.; Science and Technology Commission of Shanghai Municipal grant no. 20431900100 and Jack Ma Foundation grant no. 2020-CMKYGG-05 to H.J.; National Natural Science Foundation grant no. 31770796, National Science and Technology Major Project grant no. 2018ZX09711002, and K.C. Wong Education Foundation to Y.J., the National Natural Science Foundation of China (grant no. 31970165) to L.Z. and by the National Natural Science Foundation of China (grant no. 81903433) to J.S.
The authors declare no competing interests.
Peer review information Nature Structural and Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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a, Gel filtration profile of the RdRp complex with nsp7 and nsp8. b, SDS gel of the purified RdRp complex with nsp7 and nsp8. c, Gel mobility shift of the RdRp-RNA complex and the effect of RDV-TP. d, IC50 of RDV-TP for the RdRp complex, determined by two independent experiments and error bars means s.d. of the data. e, EC50 of remdesivir for SARS-CoV-2 inhibition and CC50 of remdesivir for cell-based toxicity, determined by three independent experiments and error bars means s.d. of the data. f, The structures of PPADS and iso-PPADS. g, Elongation of partial RNA duplex by the purified RdRp complex and its inhibition by 9 suramin derivatives at 0.5-5.0 mM concentrations depending on the solubility of each compound. Source data
a, Representative cryo-EM micrograph of the RdRp-suramin complex. b, Representative 2D class averages of the RdRp-suramin complex. c, Fourier shell correlation curves of cryo-EM map for the suramin-RdRp complex. d, Euler angle distribution of particles used in the final reconstruction. e, Flowchart of cryo-EM works of the suramin-RdRp complex with maps colored by local resolution (Å).
a-g, Cryo-EM map and model of nsp12 (residues 111-116, contour σ level: 4σ) (A), nsp8-1 (residues 127-132, contour σ level: 6σ) (B), nsp12 (residues 169-199, contour σ level: 6σ) (C), nsp7 (residues 26-40, contour σ level: 6σ) (D), nsp8-2 (residues 85–95, contour σ level: 5σ) (E), and the two bound suramin molecules (F and G, suramin#1, contour σ level: 5.5σ; suramin#2, contour σ level: 3.5σ).
Extended Data Fig. 4 Structural comparisons of the SARS-CoV-2 RdRp-suramin structure with the apo–RdRp structure and other structures of replication/transcription complex (RTC).
Overall views of the RdRp-suramin structure overlapped with the apo RdRp structure (PDB ID: 7BV1) in panel a; the nsp132-RTC structure (PDB ID: 6XEZ) in panel b; the cap (-1)’-RTC structure (PDB ID: 7CYQ) in panel c; and the form 1 mini RTC structure (PDB ID: 7CXM) in panel d. For clarity, only the polymerase domains are shown. The thumb, palm and fingers domains of the RdRp of the SARS-CoV-2 RdRp-suramin structure are in blue, orange and red, respectively, with the two suramin molecules in green. The other RdRp complex structures are in light gray.
Extended Data Fig. 5 Comparison of SARS-CoV-2 RdRp-suramin complex with MNV (Murine Noroviruses) RdRp-suramin complex and MNV RdRp-NF023.
a, Superimposition of the SARS-CoV-2 RdRp-suramin structure with the MNV RdRp-suramin structure (PDB ID: 3UR0) based on the polymerase domain. Only the polymerase domain of SARS-CoV-2 RdRp is shown. RdRp in MNV RdRp-suramin structure is in light gray with the two suramin molecules in yellow. The thumb, palm and fingers domains in the RdRp of SARS-CoV-2 RdRp-suramin structure are in blue, orange and red, respectively, with the two suramin molecules in green. b, Superimposition of SARS-CoV-2 RdRp-suramin structure with MNV RdRp-NF023 structure (PDB ID: 3URF) based on the polymerase domain. The RdRp in MNV RdRp-NF023 structure is in light gray with the NF023 molecule in magenta. The thumb, palm and fingers domains in the RdRp of SARS-CoV-2 RdRp-suramin structure are in blue, orange and red, respectively, with the two suramin molecules in green. c, A close view of the suramin within the catalytic site in the MNV RdRp-suramin structure, color code as in panel a. d, A close view of the suramin within the catalytic site in the SARS-CoV-2 RdRp-suramin structure, color code as in panels a and b. e, A close view of the NF023 within the catalytic site in the MNV RdRp-NF023 structure, color code as in panel b.
Extended Data Fig. 6 Comparison of SARS-CoV-2 RdRp-suramin complex with MNV RdRp-suramin derivative 6 complex and HNV (Human Noroviruses) RdRp-suramin derivative 6.
a, Superimposition of SARS-CoV-2 RdRp-suramin structure with MNV RdRp-suramin derivative 6 structure (PDB ID: 4NUR) based on the polymerase domain. Only the polymerase domain of SARS-CoV-2 RdRp is shown. The RdRp in MNV RdRp-6 structure is in light gray with the suramin derivative 6 molecule in light blue. The thumb, palm and fingers domains in the RdRp of SARS-CoV-2 RdRp-suramin structure are in blue, orange and red, respectively, with the two suramin molecules in green. b, Superimposition of SARS-CoV-2 RdRp-suramin structure with HNV RdRp-suramin derivative 6 structure (PDB ID: 4NRT) based on the polymerase domain. The RdRp in HNV RdRp-6 structure is in light gray with suramin derivative 6 in cyan. The thumb, palm and fingers domains in the RdRp of SARS-CoV-2 RdRp-suramin structure are in blue, orange and red, respectively, with the two suramin molecules in green. c, A close view of the suramin derivative 6 within the active site in the MNV RdRp-suramin derivative 6 structure, the color code is used as in panel a. d, A close view of the suramin within the active site in the SARS-CoV-2 RdRp-suramin structure, the color code is used as in panels a and b. e, A close view of the suramin derivative 6 within the active site in the HNV RdRp-suramin derivative 6 structure, the color code is used as in panel b.
Supplementary Fig. 1. Sequence alignment of 12 coronavirus nsp12. Sequence alignment of nsp12 from eight β-CoVs (SARS-CoV-2, SARS-CoV, MERS-CoV, RaTG13, PCoV, MHV, HKU1, HCoV-OC43), one γ-CoV (infectious bronchitis virus) and three α-CoVs (HCoV-NL63, HCoV-229E, transmissible gastroenteritis virus). Black arrows mark the residues interacting with suramin no. 1, blue arrows mark the residues interacting with suramin no. 2, while the red box sign the residues interacting with suramin molecules by hydrogen bonds. Supplementary Table 1. Interactions between suramin and nsp12 in the RdRp–suramin structure.
Unprocessed gel for the Fig. 1c,d.
Statistical source data for Fig. 1e,f.
Statistical source data for Fig. 6b,c.
Statistical source data for Extended Data Fig. 1a,d,e.
Unprocessed gel for Extended Data Fig. 1b,c,g.
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Yin, W., Luan, X., Li, Z. et al. Structural basis for inhibition of the SARS-CoV-2 RNA polymerase by suramin. Nat Struct Mol Biol 28, 319–325 (2021). https://doi.org/10.1038/s41594-021-00570-0
Current Opinion in Virology (2021)