Identification of Darunavir Derivatives for Inhibition of SARS-CoV-2 3CLpro

The effective antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed around the world. The 3C-like protease (3CLpro) of SARS-CoV-2 plays a pivotal role in virus replication; it also has become an important therapeutic target for the infection of SARS-CoV-2. In this work, we have identified Darunavir derivatives that inhibit the 3CLpro through a high-throughput screening method based on a fluorescence resonance energy transfer (FRET) assay in vitro. We found that the compounds 29# and 50# containing polyphenol and caffeine derivatives as the P2 ligand, respectively, exhibited favorable anti-3CLpro potency with EC50 values of 6.3 μM and 3.5 μM and were shown to bind to SARS-CoV-2 3CLpro in vitro. Moreover, we analyzed the binding mode of the DRV in the 3CLpro through molecular docking. Importantly, 29# and 50# exhibited a similar activity against the protease in Omicron variants. The inhibitory effect of compounds 29# and 50# on the SARS-CoV-2 3CLpro warrants that they are worth being the template to design functionally improved inhibitors for the treatment of COVID-19.


Introduction
The pandemic of COVID-19 caused by infection with SARS-CoV-2 has had an unprecedented impact on the health and economic development of the world, which has led to over 611 million cumulative cases as of 23 September 2022 (Coronavirus disease (COVID-19) (who.int)). SARS-CoV-2 is a positive-sense enveloped RNA virus with a singlestrand, which shares about 80% of its RNA genome identity with SARS-CoV of the genus Betacoronaviru [1,2]. According to the World Health Organization (WHO), there are no clinically effective vaccines and targeted therapeutics for SARS-CoV-2 infections. Up to now, although several medications are currently being evaluated in clinical trials, and some kinds of vaccines have been used to prevent the occurrence of SARS-CoV-2, it will take a long time to effectively cure patients or vaccinate the world population faced with viral mutants. Therefore, effective and curative treatment measures are still urgently needed for COVID-19.
SARS-CoV-2 is an RNA virus that is~30,000 nt in length. Its two overlapping open reading frames (ORF)1a and ORF1b, located at two-thirds of the viral genome from the 5 terminus, encode two polyproteins, pp1a and pp1ab [1,3]. These replicase polyproteins will be cleaved by two different proteins, the papain-like cysteine protease (PL pro ) and 3CL pro , encoded in the ORF1a to generate 16 nonstructural proteins (NSPs) for viral genome transcription and replication [4]. The 3CL pro , also referred to as the main protease (M pro ), plays an important role in the maturation of SARS-CoV-2, and the inhibition of the activity of the 3CL pro is expected to block the viral replication. Meanwhile, the sequence alignment shows that SARS-CoV-2 3CL pro is about 96% and 50% identical to the M pro of SARS-CoV

Establishing a High-Throughput Screening System with FRET Assay Targeting SARS-CoV-2 3CL pro
To identify candidate inhibitors accurately and quickly, we first established a highthroughput screening system with a FRET assay targeting SARS-CoV-2 3CL pro in vitro [9,23]. The full-length gene encoding SARS-CoV-2 3CL pro was codon-optimized and inserted into the pET-28a (+) vector, whereafter it was transformed into the BL21(DE3) Escherichia Coli strain and high-yieldingly expressed with a His-tag in its C-terminus. The recombinant 3CL pro was purified with a Ni-NTA column to high purity ( Figure 1). The enzymatic activity of the 3CL pro was then evaluated by a fluorescently labeled peptide substrate, MCA-AVLQ↓SGFR-Lys(Dnp)-Lys-NH2, based on the auto-cleavage sequence of the 3CL pro N-terminal. Additionally, the FRET assay had an obvious difference between the negative group (substrate + inactivating 3CL pro ) and workgroup (substrate + activating 3CL pro ). The assay was further optimized for high-throughput screening by the assessment of its statistical parameters, as previously described [24], to determine the performance and robustness of this assay. The results showed that all the Z factors (0.84), %CV (13%), and S/N (22.3), met the criteria required for the high-throughput screening (HTS) assay ( Figure 1). Together, we successfully established a high-sensitivity and high-throughput screening system with the FRET assay to find the inhibitors targeting the recombinant 3CL pro . and S/N (22.3), met the criteria required for the high-throughput screening (HTS) assay ( Figure 1). Together, we successfully established a high-sensitivity and high-throughpu screening system with the FRET assay to find the inhibitors targeting the recombinan 3CL pro .
The primary screening of these derivatives was conducted at a single concentration of 20 μM. We used nirmatrelvir, ebselen, and darunavir as comparisons, respectively, and DMSO-treated wells served as an intraplate negative control for the data normalization [8]. At least triplicate repeats were performed for each tested compound and the control The results show that nirmatrelvir and ebselen can strongly inhibit 3CL pro activity, as re ported( Figure 2). The DRV exhibited no inhibitory activity using this assay, which is con sistent with the efficacy of the DRV/c in clinics [22]. The initial hits were defined as thos active compounds that displayed cleaving efficiency with a significant difference com pared with that of the control group, which identified two compounds (29# and 50#) with significant inhibition against SARS-CoV-2 3CL pro . Among the hits, 29# contains a polyphe nol P2 ligand, and 50# contains a caffeine P2 ligand.
The primary screening of these derivatives was conducted at a single concentration of 20 µM. We used nirmatrelvir, ebselen, and darunavir as comparisons, respectively, and DMSO-treated wells served as an intraplate negative control for the data normalization [8]. At least triplicate repeats were performed for each tested compound and the control. The results show that nirmatrelvir and ebselen can strongly inhibit 3CL pro activity, as reported( Figure 2). The DRV exhibited no inhibitory activity using this assay, which is consistent with the efficacy of the DRV/c in clinics [22]. The initial hits were defined as those active compounds that displayed cleaving efficiency with a significant difference compared with that of the control group, which identified two compounds (29# and 50#) with significant inhibition against SARS-CoV-2 3CL pro . Among the hits, 29# contains a polyphenol P2 ligand, and 50# contains a caffeine P2 ligand.

The Inhibitory Activity of Hits against SARS-CoV-2 3CL pro In Vitro
To verify the screening results, we further characterized the inhibitory activities of these hits against SARS-CoV-2 3CL pro in a dose-response experiment to determine their IC 50 values. The FRET values were measured, and the inhibitory rates were calculated for the inhibitor treatments compared to the DMSO control, which was then plotted as dose-response curves for determining the inhibitor efficacies of the IC 50 values. The results showed that nirmatrelvir and ebselen could dose-dependently inhibit the enzymatic activity of the 3CL pro with an IC 50 value of 3.24 µM and 3.60 µM (Figure 3), which are comparable to the reported IC 50 value. We also observed a gradual decrease in the cleavage efficacy of the 3CL pro under the treatment of our hits (29# and 50#) with an IC 50 value of 6.30 µM and 3.50 µM, respectively. As a counter screen, we measured the activity of 29# and 50# against Cathepsin L, a member of the lysosomal cysteine protease [32]. The result showed that both the compounds exhibited a very limited inhibitory effect ( Figure S1), providing evidence to support the specific activity of 29# and 50# against the 3CL pro .      Figure 2. Results of the high-throughput screening. Twenty μM of the compounds was pre-incubated with 100 nM of SARS-CoV-2 3CL pro for 30 min at 30 °C, and then a 10 μM FRET substrate was added to the reaction mixture to initiate the reaction. The reaction was monitored for 2 h. The initial velocity was calculated by linear regression using the data points from the first 15 min of the reaction. The calculated initial velocity with each compound was normalized to DMSO control. The results are the average of three repeats.

The Inhibitory Activity of Hits against SARS-CoV-2 3CL pro In Vitro
To verify the screening results, we further characterized the inhibitory activities of these hits against SARS-CoV-2 3CL pro in a dose-response experiment to determine their IC50 values. The FRET values were measured, and the inhibitory rates were calculated for the inhibitor treatments compared to the DMSO control, which was then plotted as doseresponse curves for determining the inhibitor efficacies of the IC50 values. The results showed that nirmatrelvir and ebselen could dose-dependently inhibit the enzymatic activity of the 3CL pro with an IC50 value of 3.24 μM and 3.60 μM (Figure 3), which are comparable to the reported IC50 value. We also observed a gradual decrease in the cleavage efficacy of the 3CL pro under the treatment of our hits (29# and 50#) with an IC50 value of 6.30 μM and 3.50 μM, respectively. As a counter screen, we measured the activity of 29# and 50# against Cathepsin L, a member of the lysosomal cysteine protease [32]. The result showed that both the compounds exhibited a very limited inhibitory effect ( Figure S1), providing evidence to support the specific activity of 29# and 50# against the 3CL pro . Figure 2. Results of the high-throughput screening. Twenty µM of the compounds was pre-incubated with 100 nM of SARS-CoV-2 3CL pro for 30 min at 30 • C, and then a 10 µM FRET substrate was added to the reaction mixture to initiate the reaction. The reaction was monitored for 2 h. The initial velocity was calculated by linear regression using the data points from the first 15 min of the reaction. The calculated initial velocity with each compound was normalized to DMSO control. The results are the average of three repeats. The asterisk indicates 29# contains a polyphenol P2 ligand, and 50# contains a caffeine P2 ligand.

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6 of 1 Figure 3. Dose-response inhibition curves of selected hits against the 3CL pro . The hits with differen concentrations were pre-incubated with 100 nM of SARS-CoV-2 3CL pro for 30 min at 30 °C, and the a 10 μM FRET substrate was added to the reaction mixture to initiate the reaction. The reaction wa monitored for 2 h. The initial velocity was calculated by linear regression using the data points from the first 15 min of the reaction. The calculated initial velocity with each compound was normalize to the DMSO control. The results are an average ±standard deviation of three repeats.

Binding of Hits to SARS-CoV-2 3CL pro
We next performed a bio-layer interferometry (BLI) kinetic binding assay to asses the binding capacity of these hits to SARS-CoV-2 3CL pro in vitro in expectation of explor . Dose-response inhibition curves of selected hits against the 3CL pro . The hits with different concentrations were pre-incubated with 100 nM of SARS-CoV-2 3CL pro for 30 min at 30 • C, and then a 10 µM FRET substrate was added to the reaction mixture to initiate the reaction. The reaction was monitored for 2 h. The initial velocity was calculated by linear regression using the data points from the first 15 min of the reaction. The calculated initial velocity with each compound was normalized to the DMSO control. The results are an average ±standard deviation of three repeats.

Binding of Hits to SARS-CoV-2 3CL pro
We next performed a bio-layer interferometry (BLI) kinetic binding assay to assess the binding capacity of these hits to SARS-CoV-2 3CL pro in vitro in expectation of exploring the possible mechanisms of these hits against SARS-CoV-2 3CL pro . The purified SARS-CoV-2 3CL pro was immobilized on the biosensor surface and then incubated with different concentrations of the hits. Meanwhile, a binding assay tested the fitting association and dissociation response curves of the inhibitors after different concentration hits and made a blank control containing only the sample buffer for reference.
The BLI results revealed that 29# and 50# bind to SARS-CoV-2 3CL pro in a dosedependent manner (Figure 4 and Table 2). The kinetic affinity constant values (K D ) of the hits were determined. 29# and 50# exhibited high affinity with the K D values of 52 µM and 62 µM, respectively. These results further support and explain the indirect function of the nominated hits that were provided by the enzymatic inhibition assay in vitro on the anti-SARS-CoV-2 3CL pro activity. To rule out the possibility of non-specific binding, we measured the binding affinity of 29# and 50# with the Norovirus RNA-dependent RNA polymerase (RdRp) using the BLI assay ( Figure S2). The results showed either a large K D value or a not-detected K D value, suggesting the specific binding of 29# and 50# to SARS-CoV-2 3CL pro .

Modes of Interaction for SARS-CoV-2 3CL pro with Hits
To explore the binding mode of the identified inhibitors, molecular docking studies of the DRV and its two derivatives (29# and 50#) against SARS-CoV-2 3CL pro were performed by AutoDock Vina. The calculated binding energies (DG ADV ) of the DRV, 29#, and 50# were −7.1 kcal/mol, −7.8, and −7.6 kcal/mol, respectively. Due to their common structural skeleton, all the compounds presented a similar arrangement within the binding cavity of SARS-CoV-2 3CL pro ( Figure 5). As shown in Figure 5A, the binding pocket of the DRV is located in the catalytic cavity of SARS-CoV-2 3CL pro . The benzene ring of the DRV formed a π-π interaction with H41, one of the catalytic dyad residues that are important for the proteolytic activity of SARS-CoV-2 3CL pro . Compared with the DRV, 29# formed two additional H-bonds with the 3CL pro active site residues, T25 and N142 ( Figure 5B), respectively. As for inhibitor 50#, after the introduction of the o-dihydroxybenzene group ( Figure 5C), 50# formed a H-bond with E166, a key residue for the dimerization of the 3CL pro [8]. Additionally, another H-bond interaction was observed between 50# and the active site residue, Q192. This may account for the stronger inhibitory activity of DRV derivatives 29# and 50#. Taken together, the data suggest a direct interaction between the hits and SARS-CoV-2 3CL pro .
In addition, in the predicted binding poses of compounds 50# and 29#, the phenolic hydroxyl groups at the P2 position formed H-bonds with the 3CL pro active site residues, such as N142, T25, and E66. This phenolic hydroxyl group may be largely absent in those series of compounds that show no activity against SARS-CoV-2 3CL pro . Thus, we speculated that the introduction of the phenolic hydroxyl group at the P2 position may improve the inhibitory effect of the compounds.

Modes of Interaction for SARS-CoV-2 3CL pro with Hits
To explore the binding mode of the identified inhibitors, molecular docking studies of the DRV and its two derivatives (29# and 50#) against SARS-CoV-2 3CL pro were performed by AutoDock Vina. The calculated binding energies (DGADV) of the DRV, 29#, and 50# were −7.1 kcal/mol, −7.8, and −7.6 kcal/mol, respectively. Due to their common structural skeleton, all the compounds presented a similar arrangement within the binding cavity of SARS-CoV-2 3CL pro ( Figure 5). As shown in Figure 5A, the binding pocket of the DRV is located in the catalytic cavity of SARS-CoV-2 3CL pro . The benzene ring of the DRV formed a π-π interaction with H41, one of the catalytic dyad residues that are important for the proteolytic activity of SARS-CoV-2 3CL pro . Compared with the DRV, 29# formed two additional H-bonds with the 3CL pro active site residues, T25 and N142 ( Figure 5B), respectively. As for inhibitor 50#, after the introduction of the o-dihydroxybenzene group ( Figure 5C), 50# formed a H-bond with E166, a key residue for the dimerization of the 3CL pro [8]. Additionally, another H-bond interaction was observed between 50# and the active site residue, Q192. This may account for the stronger inhibitory activity of DRV derivatives 29# and 50#. Taken together, the data suggest a direct interaction between the hits and SARS-CoV-2 3CL pro .
In addition, in the predicted binding poses of compounds 50# and 29#, the phenolic hydroxyl groups at the P2 position formed H-bonds with the 3CL pro active site residues, such as N142, T25, and E66. This phenolic hydroxyl group may be largely absent in those series of compounds that show no activity against SARS-CoV-2 3CL pro . Thus, we speculated that the introduction of the phenolic hydroxyl group at the P2 position may improve the inhibitory effect of the compounds. Here, the color code is that dark blue is positively charged, red is negatively charged, cyan is polar, and green is hydrophobic. Hydrogen bonding is depicted as the magenta arrows, π-cation interactions are depicted as the red line, and π-π interactions are depicted as the green line.

The Inhibitory Activity of Hits against SARS-CoV-2 3CL pro at Cellular Level
To further confirm and characterize the inhibitory activities of these hits against SARS-CoV-2 3CL pro at the cellular level, we performed a cell-based bioluminescence resonance energy transfer (BRET) assay, as previously described [33]. HEK293T cells were Figure 5. Predicted binding poses for darunavir (A) and its two derivatives (B,C). Residues within 5 Å of the compounds were shown on the 2D ligand interaction diagram. For clarity, only polar hydrogen atoms were shown. Here, the color code is that dark blue is positively charged, red is negatively charged, cyan is polar, and green is hydrophobic. Hydrogen bonding is depicted as the magenta arrows, π-cation interactions are depicted as the red line, and π-π interactions are depicted as the green line.

The Inhibitory Activity of Hits against SARS-CoV-2 3CL pro at Cellular Level
To further confirm and characterize the inhibitory activities of these hits against SARS-CoV-2 3CL pro at the cellular level, we performed a cell-based bioluminescence resonance energy transfer (BRET) assay, as previously described [33]. HEK293T cells were transfected with plasmids expressing pEYFP-linker-Rluc and SARS-CoV-2 3CL pro before being treated with increasing doses of the inhibitors. The BRET values were then measured, and the inhibitory rates were calculated for the inhibitor treatments compared to the DMSO control, which was then plotted as dose-response curves for determining the inhibitor efficacies of the EC 50 values ( Figure 6A). From the results, the EC 50 values of the two hits ranged from 8.9 to 13.5 µM, with the positive control, ebselen, at 9.2 µM. Meanwhile, we also evaluated the cytotoxicity of the inhibitor hits using a Cell Counting Kit-8 (CCK-8) assay. The results show that the CC 50 values of the two hits are higher than 24 µM (Figure 6C), which generally rules out the false positives due to significant cell toxicity. Ebselen has extremely low cytotoxicity (the median lethal dose in rats is >4600 mg kg −1 when taken orally), and darunavir is the latest protease inhibitor approved by the FDA; both are safe in humans and have been evaluated in a number of clinical trials [7,34]. It should be noted that the relatively low selectivity indexes of 29# and 50# suggest that their activity might be in part due to cytotoxicity; therefore, further optimization is clearly needed for future development. transfected with plasmids expressing pEYFP-linker-Rluc and SARS-CoV-2 3CL pro before being treated with increasing doses of the inhibitors. The BRET values were then measured, and the inhibitory rates were calculated for the inhibitor treatments compared to the DMSO control, which was then plotted as dose-response curves for determining the inhibitor efficacies of the EC50 values ( Figure 6A). From the results, the EC50 values of the two hits ranged from 8.9 to 13.5 μM, with the positive control, ebselen, at 9.2 μM. Meanwhile, we also evaluated the cytotoxicity of the inhibitor hits using a Cell Counting Kit-8 (CCK-8) assay. The results show that the CC50 values of the two hits are higher than 24 μM ( Figure 6C), which generally rules out the false positives due to significant cell toxicity. Ebselen has extremely low cytotoxicity (the median lethal dose in rats is >4600 mg kg −1 when taken orally), and darunavir is the latest protease inhibitor approved by the FDA; both are safe in humans and have been evaluated in a number of clinical trials [7,34]. It should be noted that the relatively low selectivity indexes of 29# and 50# suggest that their activity might be in part due to cytotoxicity; therefore, further optimization is clearly needed for future development. Next, we explored a mutation, P132H, in the 3CL pro , which was included in the SARS-CoV-2 Omicron variant. Based on the predicted binding model, this mutation most unlikely affected the binding of the two hits to the 3CL pro ; therefore, no difference in the susceptibility was expected. Whereafter, we performed a Western blot assay to validate the inhibition of the two hits on the SARS-CoV-2 3CL pro enzymatic activity. In line with it, we found a similar effect of the hits on the cleavage efficiency of the 3CL pro containing the mutation P132H ( Figure 6D).

Discussion
The Coronaviruses such as SARS, MERS, and SARS-CoV-2 that broke out in 2019 have caused three large-scale pandemics in human history during the past 20 years. With the ongoing outbreak of SARS-CoV-2, scientists and researchers around the world are facing finding effective antiviral drugs or vaccines with different strategies in a short time Next, we explored a mutation, P132H, in the 3CL pro , which was included in the SARS-CoV-2 Omicron variant. Based on the predicted binding model, this mutation most unlikely affected the binding of the two hits to the 3CL pro ; therefore, no difference in the susceptibility was expected. Whereafter, we performed a Western blot assay to validate the inhibition of the two hits on the SARS-CoV-2 3CL pro enzymatic activity. In line with it, we found a similar effect of the hits on the cleavage efficiency of the 3CL pro containing the mutation P132H ( Figure 6D).

Discussion
The Coronaviruses such as SARS, MERS, and SARS-CoV-2 that broke out in 2019 have caused three large-scale pandemics in human history during the past 20 years. With the ongoing outbreak of SARS-CoV-2, scientists and researchers around the world are facing finding effective antiviral drugs or vaccines with different strategies in a short time [35]. One of the quickest and relatively safest ways to find therapeutic drugs is to repurpose the clinical drug. Remdesivir, the viral polymerase inhibitor, shows the greatest treatment effect and is being evaluated in several clinical trials [36], and it also obtained emergencyuse authorization from the FDA. Lopinavir and Ritonavir, which are HIV drugs, were also reported in some research. However, the combination of the two HIV drugs failed in a clinical trial for the SARS-CoV-2 infection, as no significant therapeutic efficacy was observed [37].
To address this urgent medical need, we designed and initiated a drug repurposing screening to identify effective inhibitors against SARS-CoV-2 3CL pro from the structurally improved protease inhibitors that were approved by the FDA. The M pro has been shown to be a validated antiviral drug target for SARS-CoV and MERS-CoV [38]. As SARS-CoV-2 3CL pro (also referred to as M pro ) shares high sequence similarity with SARS-CoV and, to a lesser extent, with MERS-CoV, studies have also reasoned that inhibiting the enzymatic activity of SARS-CoV-2 3CL pro will similarly prevent viral replication [6]. Herein, we designed and synthesized a series of compounds improved from several kinds of HIV-1 protease inhibitors that had been used in clinical. Through testing with a high-throughput screening system with a recombinational 3CL pro , we found that compounds 29# and 50# exhibited favorable anti-SARS-CoV-2 3CL pro potency, with IC 50 values of 6.30 and 3.50 µM and EC 50 values of 8.90 and 13.50 µM. Therefore, we successfully designed two hits that are worthy of being functionally improved for the treatment of COVID-19.
The known SARS-CoV-2 3CL pro inhibitors in the existing studies mainly include two types: peptide inhibitors and small molecule compounds. The reported small molecule inhibitors targeting the 3CL pro are significantly less than the peptide inhibitors, possibly because the latter are designed on the peptidomimetic substrates with known and various chemical warheads, such as α-ketoamide, aldehyde, aldehyde prodrug bisulfite, chloromethyl ketones, and others [6]. Herein, we designed compounds based on these chemical warheads. The 29# and 50# compounds have similar activity to the recently reported SARS-CoV-2 3CL pro inhibitors (ebselen, N3, and 13b), which also represent the potent and selective peptidomimetic hits designed and renovated based on the chemical warheads.
In summary, this study identified two potent SARS-CoV-2 3CL pro inhibitors with convincing enzymatic inhibition in vitro, as well as cellular antiviral activity. Further improvement based on the structure of these hits might produce clinically therapeutic drugs for SARS-CoV-2 infection.
Coelenterazine-h was purchased from Promega. Compounds were designed and synthesized in our laboratory and prepared in DMSO. The DYKDDDDK (Flag) Tag antibody (8146) was purchased from Cell Signaling Technology, Inc (CST, Danvers, MA, USA), and the β-actin antibody (ab8224, Cambridge, UK) was purchased from Abcam.

In Vitro Enzymatic Inhibition Assay
The enzyme activity and inhibition assays have been described previously [9]. The recombinant SARS-CoV-2 3CL pro (1 µM at the final concentration) was mixed with serial dilutions of each compound in a 50 µL assay buffer (50 mM Tris-HCl, pH 7.3, 1 mM EDTA) and incubated for 5 min. The reaction was initiated by adding a 50 µL fluorogenic substrate with a final concentration of 20 µM and then incubated for 30 min with gentle shaking at room temperature. After that, the fluorescence signal at 320 nm (excitation)/405 nm (emission) was measured every 5 min and 10 times with a PerkinElmer plate reader. The Vmax of the reactions added with the compounds at various concentrations compared to the reaction added with the DMSO was calculated and used to generate IC50 curves. For each compound, IC50 values against SARS-CoV-2 3CL pro were measured at nine concentrations, and three independent experiments were performed. All experimental data were analyzed using GraphPad Prism software(San Diego, CA, USA).

BRET Assay
For the BRET assay, transfected cells were seeded in 96-well white plates with a clear bottom. After overnight incubation, the DMSO (Sigma-Aldrich, Burlington, MA, USA) or compounds were added to each well at the designated concentration. After further incubation for 48 h at 37 • C, the supernatant was discarded. A stock of coelenterazineh was dissolved in absolute ethyl alcohol to a concentration of 1.022 mmol/L. For the luminescence assay, 100 µL of 10 µM coelenterazine-h was injected, and the luminescence was measured using a VICTOR TM X5 2030 Multilabel Reader (PerkinElmer, USA).

Western Blot
HEK293T cells were seeded at a density of 2 × 10 5 cells per well in a 6-well plate. After overnight incubation, the DMSO (Sigma-Aldrich) or compounds were added to each well at the designated concentration. After further incubation for 48 h at 37 • C, transfected HEK293T cells were collected and lysed in a RIPA buffer containing 50 mM of Tris (pH 7.4), 150 mM of NaCl, 1% NP-40, 0.1% SDS, 1 mM of EDTA, and 0.5% sodium deoxycholate. Cell lysates were mixed with an SDS-loading buffer and boiled for 20 min, followed by electrophoresis in 10% polyacrylamide-SDS gels. Proteins were transferred onto the PVDF membrane. After blocking with 5% skim milk, the membrane was blotted with anti-flag (1:1000) or anti-β-actin (1:5000) antibodies. After incubation with HRPconjugated secondary antibodies (1:5000), protein signals were detected with enhanced chemiluminescence.

Cell Viability Assay
Cell viability was performed by a Cell Counting Kit-8 (CCK-8) (Beyotime, China). Typically, HEK293T cells were seeded in the wells of a 96-well plate at 100 µL/well and grown for 24 h. Then, 10 µL of CCk-8 reagent and incubated for 1.5 h at 37 • C with 5% CO 2 . The absorbance at 450 nm was subsequently measured using an EnSpire 2300 Multilabel Reader (PerkinElmer).

Bio-Layer Interferometry (BLI) Binding Assay
All binding affinity and kinetic profiles were conducted using a ForteBio Octet RED96 instrument (ForteBio, Inc., CA, USA) equipped with super streptavidin biosensor chips (ForteBio). Purified His-tagged nsp12 (50 µg/mL) were captured via Ni-NTA biosensors (960 s, at 25 • C, with 1000 rpm). Ligand biosensors and reference biosensors were dipped into multiple concentrations of the test compounds for 60 s (kon,1/Ms), then dissociated for 60 s (kdis,1/s). The blank binding using a buffer was used to correct the baseline shift during the analysis. Data analysis on the ForteBio Octet RED instrument was performed using reference well subtraction in the ForteBio data analysis software.

Docking (or Modeling)
To explore the binding mode of the identified inhibitors, a molecular docking study of simeprevir against SARS-CoV-2 3CL pro (PDB-ID: 6M2N) was performed by AutoDock Vina. Protein structures were prepared for docking using AutoDockTools (ADT, version: 1.5.6) by removing co-crystalized water molecules, adding polar hydrogens, and merging Gasteiger charges. The active site of the 3CL pro was defined as a box of 22 Å × 22 Å × 22 Å, centered near the ligand binding region (x = −32.169 Å, y = −64.8 Å, z = 41.176 Å). The grid step size for each docking volume was set to 1 Å. Ligands of the DRV, 29#, and 50# were prepared using an Open Babel toolkit version. Auto Dock Vina software package was used for docking, as previously described [39].

Quantification and Statistical Analysis
Data are presented as the mean ± standard deviation (SD) from at least three independent experiments unless otherwise indicated and were analyzed with GraphPad Prism.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms232416011/s1, Figure S1: The inhibitory activity of hits against Cathepsin L in vitro; Figure S2: Binding of hits to Norovirus RdRp.