key: cord-0993952-u11naoa9 authors: Xu, Yunxia; Chen, Ke; Pan, Juanli; Lei, Yingshou; Zhang, Danting; Fang, Lipei; Tang, Jinle; Chen, Xin; Ma, Yanhong; Zheng, Yi; Zhang, Bao; Zhou, Yaoqi; Zhan, Jian; Xu, Wei title: Repurposing clinically approved drugs for COVID-19 treatment targeting SARS-CoV-2 papain-like protease date: 2021-08-06 journal: Int J Biol Macromol DOI: 10.1016/j.ijbiomac.2021.07.184 sha: fc714d1422df9d105b2054f5867a95a49d83532b doc_id: 993952 cord_uid: u11naoa9 COVID-19 is a disease caused by SARS-CoV-2, which has led to more than 4 million deaths worldwide. As a result, there is a worldwide effort to develop specific drugs for targeting COVID-19. Papain-like protease (PLpro) is an attractive drug target because it has multiple essential functions involved in processing viral proteins, including viral genome replication and removal of post-translational ubiquitination modifications. Here, we established two assays for screening PLpro inhibitors according to protease and anti-ISGylation activities, respectively. Application of the two screening techniques to the library of clinically approved drugs led to the discovery of tanshinone IIA sulfonate sodium and chloroxine with their IC50 values of lower than 10 μM. These two compounds were found to directly interact with PLpro and their molecular mechanisms of binding were illustrated by docking and molecular dynamics simulations. The results highlight the usefulness of the two developed screening techniques for locating PLpro inhibitors. Coronavirus Disease 2019 (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In March 2020, the World Health Organization announced that the COVID-19 epidemic has constituted a global pandemic. COVID-19 has many similarities with severe acute respiratory syndrome (SARS) that broke out in China in 2003. Although the fatality rate of COVID-19 is far lower than SARS and Middle East respiratory syndrome (MERS), its infection rate and death toll far exceed the previous two diseases. Until now, few drugs are available for COVID -19 treatment. SARS-CoV-2 is a linear single-stranded RNA virus with a full-length genome of about 30kb. SARS-CoV-2 encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely PLpro. PLpro is responsible for cutting the three N-terminal cleavage sites on ORF1a to yield product nsp1, nsp2 and nsp3 [1] . The cleavage specificity of PLpro corresponds to the pattern (R/K)L(R/K)GG↓X [2] . After protease digestion, these non-structural proteins participate in the assembly of the viral replicase complex, which initiates the replication and transcription of the viral genome [1, 3, 4] . Interferon Stimulated Gene 15 (ISG15) is a ubiquitin-like protein, which performs ISGylation by covalently binding to the target protein. As one of the strongest interferon-stimulating proteins, ISG15 can bind to lysine residues to induce the host antiviral response [5] . However, some viruses have evolved a variety of mechanisms to escape the host antiviral response by inhibiting or removing ISGylation. ISG15 has the unique sequence LRLRGG at the C-terminus, consistent with the coronavirus PLpro recognition motif [2] . Thus, PLpro should be able to effectively remove ISGylation and ubiquitylation J o u r n a l P r e -p r o o f Journal Pre-proof 300mM NaCl and 5%(w/w) glycerol with pH 8.5. The protein fractions were concentrated by 10 kDa MWCO concentrator (Amicon Millipore) and stored at -80℃. The cloning, expression, and purification of the PLpro C111S mutant (PLpro-C111S) and human ISG15 were the same as the procedure for PLpro because they have a similar size and a similar predicted isoelectric point (PI=6.9 for PLpro-WT, 6.9 for PLpro-C111S and 7.5 for ISG15). Moreover, they were all tagged by six of histidine residues for purification. Thus, we can purify those three proteins with the same protocol (the Ni-NTA resin, Q ion-exchange chromatography and gel filtration column on Superdex 200). A fluorogenic peptide, ALKGG-AMC, was designed based on the substrate sequence of the SARS-CoV-2 nsp3-nsp4 cleavage site. After cleavage, a free AMC will be released, and its fluorescence signal can be monitored to measure the protease activity of SARS-CoV-2 PLpro. This fluorogenic peptide was synthesized and purified to a > 95% purity by GenScript. The fluorogenic peptide was dissolved in Buffer A (50 mM HEPES, pH 7.5, 5 mM DTT and 0.1 mg/ml BSA) to a final concentration of 10 mM and stored at -20℃. 250 μM ALKGG-AMC was loaded onto PLpro at concentrations ranging from 420 to 13.125 nM with 4-fold dilution in a black flat-bottom 96 well plate The compound library, purchased from APExBio, consists of 1971 compounds of clinically approved drugs. Each well was added 50 μl of PLpro in Buffer A at a concentration of 100 nM, followed by 1 μl of each compound (~10 mM in DMSO). After 10 min of pre-incubation, an addition of 50 μl of 125 μM fluorogenic peptide was made to the plates. Then, after 10 min of incubation at room temperature, the reaction was stopped after adding 50 μl of 0.2 M sodium acetate and the end-point fluorescence signals were read by the plate reader. One negative control (compound-negative control) was made with no compounds added. Another negative control (PLpro-negative control) was made with no PLpro added. All the controls were performed in triplicates. We employed the percentage of effect and a Z score to select the compounds for further studies. The percentage of effect is the inhibition rate of the sample against the PLpro activity at a certain concentration. The following formula was used to calculate the percentage of effect (Eq. (1)): where μ c1-is the mean value of compound-negative controls and μ c2-is the mean value of PLpro-negative controls. We calculated Z' factor to evaluate the quality of the assay. The Z' factor can be calculated as follows (Eq. (2)). standard deviations from the mean. Thus, Z score >3, indicating a statistically significant finding. Z score is calculated by using the equation below (Eq. (3)). where x is the fluorescence of a sample, x̅ is the mean of all samples on each plate and standard deviation is denoted as . Protein topologies were prepared under Amber99SB-ildn [15] [16] [17] forcefield with Ambertools20 [18] and converted to GROMACS formats with ParmED [19] . ZAFF parameters [20] were applied to the zinc finger of PLpro. Topologies for all ligands were prepared with ACPYPE [21] . Protein structures complexed with a ligand were selected from the representative docking results. The system was then solvated with TIP3P water in a dodecahedron box extending 2 nm from the solute. Na + and Clions were added to neutralize the system's net charge and to reach a salt concentration of 150 mM. All simulations were set up with the running parameters described in previous study [22] and with GROMACS 2019.3 [23] on the high performance cluster of Shenzhen Bay Laboratory. Each simulation was performed for at least 200 ns. In the luciferase-based IFN-β reporter assay, the effect of a compound on suppression of IFN- promoter activity by SARS-CoV2 PLpro was investigated. HEK293T cells grown to 80% confluency in Journal Pre-proof PLpro and PLpro-C111S were expressed by E.coli BL21(DE3) and purified by nickel column affinity, anion exchange, and gel filtration chromatography as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Fig. 1a ). PLpro-C111S is a PLpro mutant, in which the catalytic cysteine is replaced by serine to inactivate the protease activity but retain the binding capacity. ISG15 was also expressed and purified by nickel column (Fig. 1a ). 3.2 Establishing two assays for high-throughput drug screening targeting SARS-CoV-2 PLpro. We established two assays for inhibitor screening. The first one is based on the protease activity of SARS-CoV-2 PLpro. We designed a fluorogenic peptide ALKGG-AMC as the substrate. PLpro will recognize the peptide and specifically cleave the peptide bond after ALKGG. After cleavage, the AMC fluorophore will be released to its free state and emit fluorescence (Fig. 1b) . If a compound binds to the corresponding active site of PLpro, the fluorogenic peptide ALKGG-AMC will not be cleaved by PLpro, thus, a lower fluorescence signal will be observed (Fig. 1b) . The second screening assay is based on fluorescence polarization. The binding activity between PLpro and ISG15 can be determined by monitoring the change of fluorescence polarization signal of confirming the reliability of the assay as 0.5 is considered as the threshold for a reliable assay [26] . There were 10 compounds whose inhibitory-effect values are greater than 80% against PLpro and Z scores are lower than -3 (Fig. 2a) . We performed dose-dependent inhibition assays on PLpro to determine the IC50s of these 10 compounds. Only tanshinone IIA sulfonate sodium showed a lower than 10 μM IC50, with an IC50 of 1.65±0.13 μM. In fluorescence polarization-based assay, the Z'-factor values ranged from 0.7-0.95. There were 20 compounds whose inhibitory effect values are greater than 80% against PLpro-C111S, Z scores are lower than -3 and fluorescence polarization lower than the positive control. (Fig. 2c) . For further studies, we selected these compounds to determine their IC50s. Only chloroxine has an IC50 lower than 10 μM (7.24±0.68 μM). We confirmed the interactions between PLpro and the above two compounds by biolayer interferometry (BLI) assay. In this assay, a protein (PLpro or PLpro-C111S) labeled with biotin was loaded on the SA sensor, and different concentrations of the testing compound was placed in a 96-well J o u r n a l P r e -p r o o f Journal Pre-proof plate for association and dissociation. We found that K D is 145±8.5 μM between PLpro and tanshinone IIA sulfonate sodium (Fig. 2e) and 4.6±0.29 μM between PLpro-C111S and chloroxine (Fig. 2f) . We performed the thermal shifting assay to further examine the interaction between PLpro and these two compounds. In this assay, SYPRO Orange, a fluorogenic dye binding to hydrophobic residues, was utilized to monitor the denaturation of PLpro as the temperature increases and calculate the melting temperature (Tm) [27] . When a compound binds to the protein, the stability of the protein may change (i.e. a shift of Tm). For tanshinone IIA sulfonate sodium, the thermal shifting assay was conducted with a mixture of 25 μM PLpro and tanshinone IIA sulfonate sodium at a molar ratio of 1:1 under six different pH and using the buffer without tanshinone IIA sulfonate sodium as a control. PLpro and PLpro/tanshinone IIA sulfonate sodium exhibited the highest stability at pH = 7.0. Tm was increased by 1 °C for PLpro mixed with tanshinone IIA sulfonate sodium (Fig. 2g) . PLpro-C111S and PLpro-C111S/chloroxine also exhibited the highest stability at pH = 7.0. Mixing PLpro with chloroxine increases Tm by 2.5 °C (Fig. 2h) . Interestingly, the Tm value (47℃) of PLpro-C111S is 2 ℃ higher than that of PLpro (45 ℃). kcal/mol, marked as chloroxine-d). The above-selected binding conformations were subjected to MD simulations for 200 ns. Among all the simulations, the ones started from tanshinone IIA sulfonate sodium-a and chloroxine-c reached stable binding. The stable binding of tanshinone IIA sulfonate sodium (Fig. 3b, c) attributes to a stable conformation in the active pocket ( Fig. 3a) with the hydrophilic sulfonate group exposed to the solvent and the hydrophobic end inserted into the pocket. The stability is further strengthened by the interaction between the aromatic rings of tanshinone IIA sulfonate sodium and nearby residues (Fig. 3c) : π-π stacking to Tyr268 side chain and cation-π interaction on the other side to Arg166 side chain. These aromatic-related interactions clamp the ligand in a relatively rigid conformation (Fig. 3b) . To further confirm the stability of this binding mode, we extended the simulation to 300 ns and the binding binding site (Fig. 4a, b) . Chloroxine can either pack in the hydrophobic pocket (State 1) or be anchored by Arg65 through cation-π interaction (State 2). In both states, binding of chloroxine can cause significant change in the orientation of the helix where Arg65 resides (Fig. 4c ). This helix is critical for the binding of the N-terminal domain of ISG15 (Fig. 4c) . Additionally, residues Arg65, Pro77, Thr75 and Phe69 are all important interface residues for ISG15 binding. These residues can be re-orientated to interact with chloroxine in State 2. Binding of chloroxine could have a direct impact on interrupting the PLpro-ISG15 binding interface. To investigate the inhibitory effects of tanshinone IIA sulfonate sodium and chloroxine on the deISGylation activity of PLpro inside cells, two cell-based methods, luciferase-based IFN-β reporter assay and anti-ISG15 immunoblotting, were employed to observe whether the two compounds can recover the cellular ISGylation level inhibited by PLpro. Consistent with their activities in the two drug-screening assays, tanshinone IIA sulfonate sodium and chloroxine can recover the cellular ISGylation level with a dose-dependent manner in both cell-based methods (Fig. 5 , also See Supplementary Fig. S2, S3 and Table S1 , S2 for the original data), indicating that both compounds can enter cells to inhibit SARS-CoV-2 PLpro. Meanwhile, chloroxine showed a higher potency to recover J o u r n a l P r e -p r o o f Journal Pre-proof the cellular ISGylation level than tanshionone IIA sulfonate sodium, suggesting that chloroxine directly targets the PLpro-ISG15 binding interface, as shown from the protease activity assay, the binding assay, the docking results and the MD simulations. Drug development is a time-consuming and costly process because of unknown toxicity and efficacy of chemical compounds. As a result, repurposing existing drugs for new applications becomes increasingly attractive. This is particularly true as there is an urgent need for COVID-19 therapeutics. Here, we established two screening techniques for locating potential inhibitors of PLpro protease and ISG15-binding activities from clinically approved drugs. Two inhibitors (tanshinone IIA sulfonate sodium and chloroxine) were found with IC50<10M in our assays. Other studies also found several PLpro inhibitors with IC50 values at 1-6 M [28] . Those inhibitors provided potential drugs or lead compounds for treating COVID-19. Recently, Klemm et al[4] also performed a fluorescence polarization-based assay to screen potential PLpro inhibitors from 5576 compounds for inhibiting the protease cleavage function. They did not find any hits as genuine PLpro inhibitors. In our study, we screened potential PLpro inhibitors by developing two different assays based on the protease activity (Assay I) and the deISGylation activity via interacting with ISG15 (Assay II), respectively. Our Assay II is a new assay. Assay I differs from Klemm et al in the substrates used for the functional assay. Different substrates may have different sensitivities and specificities on the PLpro protease activity, which may lead to different screening results. We also noted that tashinone IIA sulfonate sodium, the hit from our Assay I, is not J o u r n a l P r e -p r o o f contained in the 5576 compounds screened by Klemm et al. That is, the ability to discover PLpro inhibitors in this work is due to a different compound library and new assays. Two PLpro-ligand systems were also previously investigated by Huynh et al [29] . Ligand rac5c was reported to stably bind to the active pocket of PLpro in MD simulations, while ergotamine, although having a good docking score, dissociated from the PLpro pocket during the MD simulation. To confirm our docking and MD protocols employed in our work, we utilized the same protocols on these two PLpro-ligand systems. Our docking results found the best score of -7.9 kcal/mol for rac5c and -9.2 kcal/mol for ergotamine, respectively. Considering the fact that our docking protocol is rigid docking to the crystal structure of PLpro, the score for ergotamine was consistent with the reported rigid docking score (-9.2 kcal/mol) in the previous work. The docking conformation of ergotamine was not compared because such a conformation was not provided in the previous work. For rac5c, our best-score docking conformation was similar to the docking structure of PLpro-rac5c complex in the previous work, excepting the flipped naphthalene orientation due to different ligand chirality. The best scored structures were then subjected to MD simulations following the same setup as of tanshinone IIA sulfonate and chloroxine, with the exception of resetting the temperature to 300 K in order to be consistent with the previous work. During the simulation, ergotamine escaped from the pocket after 23.6 ns, whereas the binding conformation of rac5c was initially distorted during heating, but restored in 22 ns and the conformation remained stable throughout the rest of the simulation. The RMSD of rac5c is shown in Supplementary Fig S1a. Thus, although different initial protein conformations (an experimentally determined structure versus a hypothetical structure) and different forcefields (Amber99SB-ildn versus CHARMM36 for PLpro and GAFF versus SwissParam generated forcefield for rac5c) were employed in J o u r n a l P r e -p r o o f this and previous work, the two resulting trajectories displayed binding features essentially the same: the naphthalene fragment (with the attached methyl group) is stably surrounded by P247, P248, T301, Y268 and the more distant M208 (Supplementary Fig S1b) ; the piperidine fragment interacts with nearby Y264, Y273 and Y268 (Supplementary Fig S1c) and the pyridine fragment interacts with Q269 and L162 in a flexible manner (Supplementary Fig S1d) . The hydrogen bond between the peptide-backbone of rac5c and the backbone oxygen of Y268 is also formed and persist throughout the simulation ( Supplementary Fig S1a) . The sidechain of Y268 is found repeatedly flipping up and down in a two-state manner Tanshinone IIA sulfonate sodium discovered here is a derivative of tanshinone IIA, which is very promising in the development of cardioprotective drugs. Chloroxine is an analogue of chloroquine. Since the SARS-CoV outbreak in 2003, chloroquine was reported to have an anti-SARS-CoV activity [30, 31] . SARS-CoV could enter a host cell through the pH-dependent mechanism and the low pH of lysosome could trigger the fusion between the virus and cell membrane [32] . It was suggested that IC50 with 7.24±0.68 μM targeting PLpro, compared to EC50 of 5.47 μM for chloroquine in vitro [34] . Thus, the potential usefulness of chloroxine as a drug should be further investigated by performing antiviral activity experiments at the cellular and animal levels. 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