key: cord-1010709-pb1twrtl
authors: Kuo, Chih-Jung; Chao, Tai-Ling; Kao, Han-Chieh; Tsai, Ya-Min; Liu, Yi-Kai; Wang, Lily Hui-Ching; Hsieh, Ming-Chang; Chang, Sui-Yuan; Liang, Po-Huang
title: Kinetic Characterization and Inhibitor Screening for the Proteases Leading to Identification of Drugs against SARS-CoV-2
date: 2021-03-18
journal: Antimicrob Agents Chemother
DOI: 10.1128/aac.02577-20
sha: 6f4e0412e7b7dca5c6287b6e3b11ac01d4fed1de
doc_id: 1010709
cord_uid: pb1twrtl

Coronavirus (CoV) disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has claimed many lives worldwide and is still spreading since December 2019. The 3C-like protease (3CL(pro)) and papain-like protease (PL(pro)) are essential for maturation of viral polyproteins in SARS-CoV-2 life cycle and thus regarded as key drug targets for the disease. In this study, 3CL(pro) and PL(pro) assay platforms were established, and their substrate specificities were characterized. The assays were used to screen collections of 1,068 and 2,701 FDA-approved drugs. After excluding the externally used drugs which are too toxic, we totally identified 12 drugs as 3CL(pro) inhibitors and 36 drugs as PL(pro) inhibitors active at 10 μM. Among these inhibitors, six drugs were found to suppress SARS-CoV-2 with the half-maximal effective concentration (EC(50)) below or close to 10 μM. This study enhances our understanding on the proteases and provides FDA-approved drugs for prevention and/or treatment of COVID-19.

PAGE) (Fig. 1A) . Using the fluorogenic peptide Dabcyl-KTSAVLQSGFRKME-Edans containing a fluorescence quenching pair as the substrate, the K m value was determined to be 10.5 6 3.2 mM and the k cat value was 2.2 6 0.3 s 21 (Fig. 1B) . The k cat /K m of SARS-CoV-2 3CL pro with this fluorogenic substrate was 0.21 mM 21 s 21 , twofold higher than that (0.11 mM 21 s 21 ) for SARS-CoV 3CL pro using the same substrate (40) .

PL pro was expressed with an N-terminal His tag to facilitate its purification using nickel-nitrilotriacetic acid (Ni-NTA) column as shown by the reducing SDS-PAGE (Fig.  1C ). Using the commercially available fluorogenic peptide substrate Z-Arg-Leu-Arg-Gly-Gly-AMC, the K m value was measured to be 6.9 6 1.4 mM and the k cat value was 0.11 6 0.03 s 21 (Fig. 1D) . The catalytic efficiency k cat /K m of 0.016 mM 21 s 21 is threefold higher than the 0.005 mM 21 s 21 previously reported for SARS-CoV PL pro using the same substrate (41) . The purified recombinant 3CL pro and PL pro were used to screen FDAapproved drugs to find their inhibitors as shown below.

Substrate specificities of 3CL pro and PL pro were then examined using the 12-aminoacid (aa) peptides derived from their cleavage sequences on the polyproteins. The sequences of the 11 and 3 cleavage sites of the 3CL pro and PL pro are shown in Fig. 2A . When the 11 peptides were used as the substrates, the rates of their cleavage by 3CL pro based on the high-performance liquid chromatography (HPLC) assay to monitor 48, 35, 28 , and 17 kDa. Lanes 1 and 2 show the cell lysate without and with IPTG induction to overexpress 3CL pro fused with thioredoxin and His tag, respectively. Lane 3 is the tagged protease after Ni-NTA column chromatography. Lane 4 represents the tagged 3CL pro treated with FXa to remove thioredoxin and His tag. Two extra bands, intact 3CL pro and the thioredoxin-His tag with lower molecular mass, appear in this lane. Lane 5 shows the purified tag-free 3CL pro after the Ni-NTA column. (B) Kinetic constants of 3CL pro . The reaction initial rates of the protease were plotted against different substrate concentrations to yield the V max and K m values. (C) SDS-PAGE analysis for purifying the recombinant PL pro . Lane M contains the molecular mass markers of 100, 75, 63, 48, 35, 28, and 17 kDa. Lanes 1 and 2 show the cell lysates without and with IPTG induction to overexpress PL pro fused with thioredoxin and His tag, respectively. Lane 3 is the tagged PL pro after Ni-NTA column chromatography. (D) Kinetic constants of PL pro . The reaction initial rates of PL pro were plotted against different substrate concentrations to yield the V max and K m values. the formation of two fragments from 100 mM each peptide and 0.75 mM 3CL pro at pH 7.0 are displayed in Fig. 2B and summarized in Table 1 . The highest rate is 1.6 mM/s, obtained from the peptide of the N-terminal cleavage site. This is also the sequence used in the fluorescence resonance energy transfer (FRET) peptide substrate for assaying the 3CL pro kinetics. The cleavage rates using 100 mM concentration of the three peptides as the substrates of 0.75 mM PL pro are shown in Fig. 2C and summarized in Table 1 . PL pro cleaved its own N-terminal cleavage site most efficiently at a rate of 0.09 mM/s, its own C-terminal site at 0.01 mM/s, while that of the site between NSP1 and NSP2 at 0.003 mM/s was the lowest.

Screening of 1,068 and 2,701 FDA-approved drugs to find inhibitors of SARS-CoV-2 3CL pro . After screening the 1,068 FDA-approved drugs by using the FRET assay, the compounds that inhibited 3CL pro are colored in yellow and that inhibited both 3CL pro and PL pro are colored in green (please see Data Set S1 in the supplemental material). After excluding the highly toxic externally used drugs, we found one drug, tolcapone, that could inhibit 3CL pro with a 50% inhibitory concentration (IC 50 ) of 7.9 mM (see Table S1 in the supplemental material and Fig. 3A ). However, tolcapone showed a weak antiviral activity only at 50 mM (figure not shown).

The 2,701 FDA-approved drugs were screened against 3CL pro and the compounds that inhibited 3CL pro are colored in yellow and the compounds inhibited both 3CL pro and PL pro are colored in green (Data Set S2). After excluding the externally used medicines, 11 hits (Table S2) were identified as active in inhibiting 3CL pro at 10 mM. Of these hits, only levothyroxine and manidipine-2HCl in addition to the previously reported 3CL pro inhibitors were shown to inhibit virus replication by plaque reduction assay (see below). Here, the dose-dependent 3CL pro inhibition curves were used to determine the IC 50 values of levothyroxine and manidipine-2HCl being 19.2 6 1.2 and 10.4 6 1.6 mM, respectively ( Fig. 3B and C) .

Screening on 1,068 and 2,701 FDA-approved drugs to find inhibitors of SARS-CoV-2 PL pro . Through screening the 1,068 FDA-approved drugs, we found 10 hits (Table S3 ) that could inhibit PL pro activity when 10 mM was used in the assay. All the drugs screened are listed in Data Set S1, and the compounds that inhibited PL pro are colored in blue and those inhibited both 3CL pro and PL pro are colored in green. However, only two drugs, maprotiline and reserpine, showed antiviral activity in the plaque reduction assay as shown later. The dose-dependent inhibition curves of maprotiline and reserpine were determined as shown in Fig. 4A and B, and the derived IC 50 values were 9.7 6 0.3 and 5.7 6 0.7 mM, respectively.

Then 2,701 FDA-approved drugs were screened against PL pro and 26 additional hits (Table S4) were identified. All the 2,701 drugs screened are listed in Data Set S2, and the compounds that inhibited PL pro are colored in blue and those that inhibited both 3CL pro and PL pro are colored in green. Among these 26 hits, levothyroxine, loperamide, manidipine-2HCl, and proanthocyanidin were shown to inhibit virus replication by the plaque reduction assay as shown later. The dose-dependent inhibition curves of these four drugs were determined ( Fig. 4C to F). Based on the data, the IC 50 values for levothyroxine, loperamide, manidipine-2HCl, and proanthocyanidin were 15.3 6 4.2, 33.5 6 5.8, 14.2 6 1.7, and 2.4 6 0.3 mM, respectively. Antiviral EC 50 measurements of the inhibitors. As mentioned above, a total of 46 drugs from the two drug collections were identified as either 3CL pro or PL pro inhibitors and 2 drugs inhibited both at the concentration of 10 mM. The antiviral activities of these 46 drugs (10 mM) were tested using plaque reduction assay, and the active com- pounds are colored red in supplemental tables. Initially, to maximize the chances to identify antivirals, we pretreated the cells with each drug, added each drug during the virus infection and after virus removal. By this full-time treatment as shown in Fig. 5A , we identified two 3CL pro -inhibiting drugs, levothyroxine and manidipine-2HCl, as well as six PL pro -inhibiting drugs, levothyroxine, loperamide, manidipine-2HCl, maprotiline, reserpine, and proanthocyanidin, effective in reducing viral plaques. Next, two other treatment methods were performed to distinguish the stages for the drugs against SARS-CoV-2. As shown in Fig. 5A , "entry" was to pretreat the cells and virus during infection with each drug but without adding the drug after removal of virus, and "postentry" means each drug was added to the cells only after virus infection. By using these treatment methods, we suspected loperamide, manidipine-2HCl, and maprotiline antagonized viral replication by mainly inhibiting the viral protease(s) because postentry treatment could still inhibit the virus. In contrast, levothyroxine and proanthocyanidin suppressed the viral plaques probably by blocking the virus entry because postentry treatment could not inhibit the virus. Therefore, the measurements of EC 50 were performed with "postentry" treatment for loperamide, manidipine-2HCl, and maprotiline and "entry" treatment for levothyroxine and proanthocyanidin as shown in Fig. 5A . For reserpine, apparent antiviral activity was observed only when full-time treatment was applied, and the EC 50 was measured using full-time treatment (entry plus . The EC 50 values for these drugs were determined accordingly using plaque reduction assay (see Fig. S1 in the supplemental material), and the plots of concentration-dependent virus plaque reduction under serially diluted drug concentrations are shown in Fig. 5B to G. The derived EC 50 values for loperamide, manidipine-2HCl, maprotiline, levothyroxine, proanthocyanidin, and reserpine were 11.4 6 1.6, 14.5 6 0.8, 9.3 6 0.1, 7.0 6 2.2, 2.5 6 0.4, and 6.6 6 1.5 mM, respectively. The 50% cytotoxic concentration (CC 50 ) values for loperamide and maprotiline were 56.4 6 0.7 and 31.8 6 0.4 mM, respectively, and those for other drugs were all .100 mM. The derived selectivity index (SI) for reserpine (SI . 15.2), levothyroxine (SI . 14.3), and proanthocyanidin (SI . 40) were higher than 10, while those for loperamide (SI = 4.9), manidipine-2HCl (SI . 6.9), and maprotiline (SI = 3.4) were below 10.

As demonstrated here, we have purified the recombinant 3CL pro and PL pro and monitored their activities using the fluorogenic peptide substrates (Fig. 1A to D) . The kinetic efficiencies (k cat /K m ) of two SARS-CoV-2 proteases are higher than that of SARS-CoV proteases. The cleavage rates of the examined peptides derived from their respective cleavage site sequences reveal that the N-terminal autocleavage reactions are the fastest for both 3CL pro and PL pro ( Fig. 2A to C). This is probably due to the fact that the maturation of NSPs in two polyproteins requires first cleavage of the proteases' N termini. After cleavage of their N termini, the proteases become more flexible to undergo C-terminal cleavage toward fully active 3CL pro and PL pro which subsequently cleave other sites on the polyproteins. Our data also reveal the most preferred recognition sequences of Leu(P2)-Gln(P1);Ser(P1') and Lys(P3)-Gly(P2)-Gly(P1);Ala(P1') for SARS-CoV-2 3CL pro and PL pro , respectively.

Using these assay platforms, we have screened the collections of 1,068 and 2,701 FDA-approved drugs and found 1 hit from 1,068 drugs and additional 11 hits from 2,701 drugs, which were active at 10 mM against 3CL pro . Of the hits, three drugs, disulfirum, ebselen, and boceprevir, have been previously reported (11, 33) , and one drug, tolcapone, was previously found active in inhibiting feline CoV (42) . Moreover, 10 hits from 1,068 drugs and additional 26 hits from 2,701 drugs, which displayed PL pro -inhibiting activity at 10 mM were obtained from the screening. The inhibition concentrationdependent curves against the 3CL pro (Fig. 3) and PL pro (Fig. 4) for those drugs showing antiviral activities were determined. Note that although levothyroxine and manidipine-2HCl inhibit both 3CL pro and PL pro in the in vitro assay, their antiviral mechanisms vary. For example, levothyroxine exerts its antiviral activities at the entry step (EC 50 =7.0 6 2.2 mM) (Fig. 5E ) instead of the postentry stage (EC 50 .20 mM), while manidipine-2HCl exerts its antiviral activities only at the postentry stage (EC 50 =14.5 6 0.8 mM) (Fig. 5C) . Interestingly, of the three PL pro inhibitors showing anti-SARS-CoV-2 activities, proanthocyanidin, like levothyroxine, also showed strong antiviral activity at the entry step with the EC 50 value of 2.5 6 0.4 mM (Fig.  5F ). Therefore, levothyroxine and proanthocyanidin might block the entry of virus, which requires further experiments to prove. Another two PL pro -only inhibitors, loperamide and maprotiline, showed antiviral activities at the postentry stage with the EC 50 values of 11.4 6 1.6 and 9.3 6 0.1 mM, respectively ( Fig. 5B and D) , but not the entry step, implying that they certainly inhibit virus replication inside the host cells. In contrast, reserpine exerts its antiviral activity only when it was given twice (entry plus postentry) (full time), suggesting that both its PL pro -inhibiting and virus entry-inhibiting activities might contribute to its antivirus effect (EC 50 = 6.6 6 1.5 mM) (Fig.  5G) . Therefore, the use of full-time, entry, and postentry treatments allows determination of the stages where the antiviral drugs exert their inhibitory effects.

Loperamide, sold under the brand name of Imodium, is a medication used to decrease the frequency of diarrhea. It is an opioid receptor agonist and acts on the m-opioid receptors in the myenteric plexus of the large intestine to decrease the tone of the longitudinal and circular smooth muscles of the intestinal wall (43), thereby allowing more water to be absorbed from the fecal matter. Manidipine-2HCl is a calcium channel blocker (dihydropyridine type) that is used clinically as an antihypertensive (44) . Maprotiline, sold under the brand name Ludiomil, is a tetracyclic antidepressant that is used in the treatment of depression. It is known to function as a norepinephrine reuptake inhibitor and also a strong antagonist of the H1 receptor (45) . Reserpine is one of dozens of indole alkaloids isolated from the plant Rauvolfia serpentine. It is used for the treatment of high blood pressure by irreversibly blocking the H 1coupled vesicular monoamine transporters, VMAT1 and VMAT2. It is the blockade of neuronal VMAT2 by reserpine that inhibits uptake and reduces stores of the monoamine neurotransmitters norepinephrine, dopamine, serotonin, and histamine in the synaptic vesicles of neurons (46) . Reserpine was identified by our previous study (47) to inhibit SARS-CoV replication, but the target was not known until now to be PL pro and virus entry. Its EC 50 values against SARS-CoV was measured to be 3.4 mM (47). Six natural derivatives of reserpine were previously shown to exhibit activities toward SARS-CoV at less than 100 mM (47), and their activities against SARS-CoV-2 could be further examined in the future. Levothyroxine, also known as L-thyroxine, is a manufactured form of the thyroid hormone thyroxine (T4), used to treat thyroid hormone deficiency, including the severe form known as myxedema coma (48) . Proanthocyanidins, a class of polyphenols found in a variety of plants, can function as antioxidants to remove harmful free oxygen radicals from cells. Therefore, proanthocyanidins have been known to confer significant health benefits as reported by several studies using human and animal models (49) .

In summary, by using the 3CL pro and PL pro fluorescence assays, we identify six FDA-approved drugs which could suppress SARS-CoV-2. Considering the frequent uptake of loperamide and proanthocyanidins in some populations, it will be interesting to know the effectiveness of these drugs in prophylactic prevention of SARS-CoV-2 infection. We thus provide here candidates for prevention and/or treatment of COVID-19.

Materials. Fluorogenic peptide substrate Dabcyl-KTSAVLQSGFRKME-Edans of 3CL pro was synthesized by Yuan Yu Ltd. (Taiwan). The fluorogenic substrate peptide (Z-Arg-Leu-Arg-Gly-Gly-AMC) for PL pro was purchased from Bachem (Bubendorf, Switzerland). The collections of 1,068 and 2,701 FDA-approved drugs were purchased from Target Molecule Corp. (Boston, MA, USA) and Selleck Chemicals (Houston, TX, USA), respectively. After screening, the active compounds in bulk size (e.g., 25 mg) were purchased from the companies for IC 50 , EC 50 , and CC 50 measurements. The plasmid miniprep kit, DNA gel extraction kit, and Ni-NTA resin were purchased from Qiagen (Hilden, Germany). FXa and the protein expression kit, including pET32a and pET16b vectors as well as competent Escherichia coli JM109 and BL21(DE3) cells were obtained from Novagen (Merck KGaA, Darmstadt, Germany). The SARS-CoV-2 virus used in this study was SARS-CoV-2/NTU13/TWN/human/2020 (accession identifier [ID] EPI_ISL_422415) as previously described (50) . All commercial buffers and reagents were of the highest grade.

Expression and purification of SARS-CoV-2 3CL pro . The gene encoding SARS-CoV-2 3CL pro was synthesized by Genewiz Co. (South Plainfield, NJ, USA) and cloned into the pET32a vector by using PCR with the forward primer 59-CAT GCC ATG GCT ATC GAG GGA AGG AGT GGT TTT AGG AAA ATG GC-39 containing FXa and NcoI cleavage sequences and the reverse primer 59-CCG CTC GAG TTA TTG GAA GGT AAC ACC AGA G-39 containing XhoI site sequence (the underlined sequences encode the N and C termini of 3CL pro , respectively). The vector pET32a also encodes thioredoxin and His tag at the N terminus of the target protein. The recombinant 3CL pro plasmid was then used to transform the E. coli JM109 competent cells and the transformed cells were streaked on a Luria-Bertani (LB) agar plate containing 100 mg/ml ampicillin. Ampicillin-resistant colonies were selected from the agar plate and sequenced to detect the 3CL pro gene. The correct construct was subsequently transformed to E. coli BL21(DE3) for protein expression. The 5-ml overnight culture of a single transformant was used to inoculate 500 ml of fresh LB medium containing 100 mg/ml ampicillin. The cells were grown to an optical density at 600 nm (OD 600 ) of 0.6 and induced with 1 mM isopropyl-b-thiogalactopyranoside. After 4 to 5 h, the cells were harvested by centrifugation at 7,000 Â g for 15 min.

The 3CL pro purification was conducted at 4°C. The cell paste obtained from 1-liter cell culture was suspended in 40 ml lysis buffer containing 25 mM Tris-HCl (pH 7.5) and 150 mM NaCl. A French press instrument (AIM-AMINCO spectronic instruments; Cambridge Scientific Products, MA, USA) was used to disrupt the cells at 12,000 lb/in 2 . The lysis solution was centrifuged, and the debris was discarded. The cell extract was loaded onto a 10-ml Ni-NTA column equilibrated with the same buffer containing 5 mM imidazole. The column was washed with 5 mM imidazole followed by 30 mM imidazole-containing buffer. The His-tagged 3CL pro was eluted with the buffer containing 300 mM imidazole. The protein solution was dialyzed against 1 liter of lysis buffer two times. After overnight dialysis, the tagged 3CL pro was treated with FXa protease to remove thioredoxin and His tag, and the mixture was loaded onto a Ni-NTA column equilibrated with the buffer containing 5 mM imidazole. The tag-free 3CL pro in flowthrough was dialyzed against the buffer without imidazole (12 mM Tris-HCl [pH 7.5], 120 mM NaCl, 0.1 mM EDTA, and 2 mM dithiothreitol [DTT] ) and stored at 270°C. The enzyme concentrations used in experiments were determined based on the absorbance at 280 nm.

Expression and purification of SARS-CoV-2 PL pro . The gene encoding SARS-CoV-2 PL pro was synthesized by Genewiz Co. (South Plainfield, NJ, USA) and cloned into the pET16b vector by using PCR with the forward primer 59-CAT GCC ATG GCC CAT CAT CAT GAA GTG AGG ACT ATT AAG GTG-39 containing NcoI cleavage sequence and reverse primer 59-CCG CTC GAG TTA TAT GGT TGT GTA ACT GTT TTC TTT GTA G-39 containing XhoI site sequence (the underlined sequences encode the N and C termini of PL pro , respectively). The forward primer also encodes extra MAHHHHHH at the N terminus of the target protein. The recombinant PL pro plasmid was then used to transform E. coli JM109 competent cells, and the transformed cells were streaked on a Luria-Bertani (LB) agar plate containing 100 mg/ml ampicillin. Ampicillin-resistant colonies were selected from the agar plate and sequenced for the PL pro gene. The correct construct was subsequently transformed to E. coli BL21(DE3) for protein expression. The 5ml overnight culture of a single transformant was used to inoculate 500 ml of fresh LB medium containing 100 mg/ml ampicillin. The cells were grown to an OD 600 of 0.6 and induced with 1 mM isopropylb-thiogalactopyranoside. After 4 to 5 h, the cells were harvested by centrifugation at 7,000 Â g for 15 min.

The PL pro purification was conducted at 4°C. The cell paste obtained from 1-liter cell culture was suspended in 40 ml lysis buffer containing 25 mM Tris-HCl (pH 7.5) and 150 mM NaCl. A French press instrument (AIM-AMINCO spectronic instruments; Cambridge Scientific Products, MA, USA) was used to disrupt the cells at 12,000 lb/in 2 . The lysis solution was centrifuged, and the debris was discarded. The cell extract was loaded onto a 10-ml Ni-NTA column equilibrated with the same buffer containing 5 mM imidazole. The column was washed with 5 mM imidazole followed by 30 mM imidazole-containing buffer. The His-tagged PL pro was eluted with the buffer containing 300 mM imidazole. The His-tagged PL pro in flowthrough was dialyzed into a buffer containing 12 mM Tris-HCl (pH 7.5), 120 mM NaCl, 0.1 mM EDTA, and 2 mM DTT for storage at 270°C. The enzyme concentrations used in all experiments were determined from the absorbance at 280 nm.

Kinetics of 3CL pro and PL pro measured using the fluorogenic substrates. The kinetic measurements were performed in the buffer (20 mM Bis-Tris [pH 7.0]) at 25°C. Enhanced fluorescence due to cleavage of the fluorogenic substrate peptide (Dabcyl-KTSAVLQSGFRKME-Edans) for 3CL pro or (Z-Arg-Leu-Arg-Gly-Gly-AMC) for PL pro was monitored at 538 and 460 nm, respectively, with excitation at 355 nm using a fluorescence plate reader (Fluoroskan Ascent; ThermoLabsystems, Sweden). The enzyme concentrations used for measuring K m and V max values were 35 nM for 3CL pro and 75 nM for PL pro , and the substrate concentrations were from 0.5-to 5-fold K m values. 3CL pro substrate concentration was determined by using the extinction coefficients 5,438 M 21 cm 21 at 336 nm (Edans) and 15,100 M 21 cm 21 at 472 nm (Dabcyl). PL pro substrate concentration was determined by using the extinction coefficient of 17,800 M 21 cm 21 at 354 nm (AMC). The initial rates within 10% substrate consumption under different substrate concentrations were used to calculate the kinetic parameters by fitting with the Michaelis-

where S is substrate) using KaleidaGraph computer program. For each data point, the measurements were repeated three times to yield the average number and standard deviation.

Peptide substrate specificity measurements. Peptides for testing as the substrates were chemically synthesized by Mission Biotech (Taipei, Taiwan) with a solid-phase methodology based on the sequences of cleavage sites on viral polyproteins ( Fig. 2A) . Reaction mixtures (100 ml) contained 0.75 mM protease and 100 mM each peptide in the buffer of 20 mM Bis-Tris (pH 7.0) for 3CL pro or the buffer of 20 mM HEPES (pH 7.0) for PL pro . Following incubation at 37°C for 0, 2.5, 5, and 10 min, reactions were stopped by adding 5 ml trifluoroacetic acid and applied to a C 18 reverse-phase HPLC column (250 Â 4.6 mm; Hichrom, Berkshire, UK). The column was eluted at room temperature with a linear acetonitrile gradient in the presence of 0.1% (vol/vol) trifluoroacetic acid at a flow rate of 1.0 ml/min. The areas of the product peaks monitored at 214 nm were integrated to calculate the cleavage rate of each peptide substrate under the catalysis of 3CL pro or PL pro .

Inhibition assay. The collections of drugs at 10 mM were screened for inhibiting the proteases, and the active inhibitors were confirmed three times. Only the IC 50 values of the inhibitors which showed antiviral activity were measured in reaction mixtures containing 2.5 nM 3CL pro with 6 mM fluorogenic substrate in a buffer of 20 mM Bis-Tris (pH 7.0) or 75 nM PL pro with 10 mM fluorogenic substrate in a buffer of 20 mM Bis-Tris (pH 7.5) in the presence and absence of various concentrations of the inhibitors. The fluorescence changes that resulted from the reactions were followed over time at 538 nm with excitation at 355 nm for 3CL pro or at 460 nm with excitation at 355 nm for PL pro using the fluorescence plate reader. The initial velocities of the inhibited reactions were plotted against the different inhibitor concentrations to yield the IC 50 value by fitting with the equation: A(I) = A(0) Â{1 2 [I/(I 1 IC 50 )]}. In this equation, A(I) is the enzyme activity with inhibitor concentration I, A(0) is the enzyme activity without inhibitor, and I is the inhibitor concentration. For each data point, the measurements were repeated three times to yield the average number and standard deviation.

Cell-based assay. For the SARS-CoV-2 plaque reduction assay, Vero E6 cells were seeded to a 24well culture plate in Dulbecco modified Eagle medium (DMEM) with 10% FBS and antibiotics 1 day before infection. VeroE6 cells were infected by SARS-CoV-2 virus (50 to 100 PFU) for 1 h at 37°C. After removal of virus inoculum, the cells were washed once with phosphate-buffered saline (PBS) and overlaid with 1 ml overlay medium containing 1% methylcellulose for 5 days at 37°C. After 5 days, the cells were fixed with 10% formalin overnight. After removal of overlay media, the cells were stained with 0.5% crystal violet, and the plaques were counted. The percentage of inhibition was calculated as [1 2 (V D /V C )] Â 100%, where V D and V C refer to the virus titer in the presence and absence of the inhibitors, respectively. The minimal concentrations of compounds required to reduce plaque numbers 50% (EC 50 ) were calculated by regression analysis of the dose-response curves generated from plaque assays. For each data point, the measurements were repeated three times to yield the average number and standard deviation. To determine the stages where the drugs exert its antiviral effects, the drugs can be added before infection, during infection, or after infection as shown in Fig. 5A .

Cytotoxicity of the inhibitors was determined by using the acid phosphatase (ACP) assay. Briefly, Vero E6 cells were seeded onto a 96-well culture plate at a concentration of 2 Â 10 4 cells per well. The next day, medium was removed, and each well was washed once with PBS before adding DMEM containing 2% FBS and different concentrations of inhibitors. Next, DMEM containing 2 mg/ml tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-trypsin was added. After 1 h of incubation at 37°C, medium was removed, and cells were washed by PBS. Then, DMEM containing 2% FBS and different concentrations of each compound were added. After 3 days of incubation at 37°C, medium was removed, and each well was washed once with PBS. Next, buffer containing 0.1 M sodium acetate (pH 5.0), 0.1% Triton X-100, and 5 mM p-nitrophenyl phosphate was added. After incubating at 37°C for 2 h, 1 N NaOH was added to stop the reaction. The absorbance was then determined by an enzyme-linked immunosorbent assay (ELISA) reader (VERSAmax; Molecular Devices, Sunnyvale, CA) at a wavelength of 405 nm. The percentage of cytotoxicity was calculated using the following formula: cytotoxicity (%) = [1 2 (At/As) Â 100], where At and As refer to the absorbance of a tested substance and solvent control, respectively. The 50% cytotoxicity concentration (CC 50 ) was defined as the concentration reducing cell viability 50%. For each data point, the measurements were repeated three times to yield the average number and standard deviation.

Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.9 MB. SUPPLEMENTAL FILE 2, XLSX file, 0.1 MB. SUPPLEMENTAL FILE 3, XLSX file, 0.1 MB.

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This work was supported by grants (MOST109-2327-B-002-009, MOST109-2745-B-001-001, MOST109-3114-Y-001-001, and MOST109-2327-B-007-002) from the Ministry of Science and Technology, Taiwan. We also thank the support from Academia Sinica.We declare that we have no conflicts of interest.