Invalidation of dieckol and 1,2,3,4,6-pentagalloylglucose (PGG) as SARS-CoV-2 main protease inhibitors and the discovery of PGG as a papain-like protease inhibitor

The COVID-19 pandemic spurred a broad interest in antiviral drug discovery. The SARS-CoV-2 main protease (Mpro) and papain-like protease (PLpro) are attractive antiviral drug targets given their vital roles in viral replication and modulation of host immune response. Structurally disparate compounds were reported as Mpro and PLpro inhibitors from either drug repurposing or rational design. Two polyphenols dieckol and 1,2,3,4,6-pentagalloylglucose (PGG) were recently reported as SARS-CoV-2 main protease (Mpro) inhibitors. With our continuous interest in studying the mechanism of inhibition and resistance of Mpro inhibitors, we report herein our independent validation/invalidation of these two natural products. Our FRET-based enzymatic assay showed that neither dieckol nor PGG inhibited SARS-CoV-2 Mpro (IC50 > 20 μM), which is in contrary to previous reports. Serendipitously, PGG was found to inhibit the SARS-CoV-2 papain-like protease (PLpro) with an IC50 of 3.90 μM. The binding of PGG to PLpro was further confirmed in the thermal shift assay. However, PGG was cytotoxic in 293T-ACE2 cells (CC50 = 7.7 μM), so its intracellular PLpro inhibitory activity could not be quantified by the cell-based Flip-GFP PLpro assay. In addition, we also invalidated ebselen, disulfiram, carmofur, PX12, and tideglusib as SARS-CoV-2 PLpro inhibitors using the Flip-GFP assay. Overall, our results call for stringent hit validation, and the serendipitous discovery of PGG as a putative PLpro inhibitor might worth further pursuing.


Introduction
COVID-19 is caused by the SARS-CoV-2, an enveloped, single-stranded, and positive-sense RNA virus [1]. Seven coronaviruses are known to infect humans including four common human coronaviruses OC43, 229E, NL63, and HKU1, and three highly pathogenic coronaviruses SARS-CoV, SARS-CoV-2 and MERS-CoV [2]. The COVID-19 pandemic is a timely call for the urgent need of orally bioavailable antivirals. Drug repurposing plays a pivotal role in advancing drug candidates to clinic [3]. For example, the rst FDAapproved COVID drug, remdesivir, was originally developed for Ebola virus [4], and was later found to have broad-spectrum antiviral activity against several viruses including SARS-CoV, MERS-CoV, and SARS-CoV-2 [5,6]. Similarly, molnupiravir was a clinical candidate for the in uenza virus before repurposed for SARS-CoV-2 [7,8]. The SARS-CoV-2 main protease (M pro ) and papain-like protease (PL pro ) are also highpro le drug targets for drug repurposing. Numerous virtual screenings and high-throughput screenings have been conducted, revealing structurally disparate inhibitors that are at different stages of preclinical and clinical development [9]. For example, boceprevir [10,11], calpain inhibitors [10], GC-376 [10,12], and masitinib [13] were among the rst hits reported as M pro inhibitors. GRL0617 [14,15], YM155 [16], 6thioguanine [17], SJB2-043 [18], and others were identi ed as PL pro inhibitors. Natural products are also a rich source of modern medicine [19], and multiple natural products have been reported as M pro and PL pro inhibitors [20]. For example, two polyphenols dieckol and 1,2,3,4,6-pentagalloylglucose (PGG) were recently reported as SARS-CoV-2 main protease (M pro ) inhibitors [21,22]. With our continuous interest in validation/invalidation of literature reported SARS-CoV-2 M pro and PL pro inhibitors [23][24][25][26], we report herein our independent validation of these two compounds using the established FRET enzymatic assay and cell-based Flip-GFP assay. In addition, we further con rmed that the previously reported promiscuous cysteine modi ers ebselen, disul ram, carmofur, PX12, and tideglusib [27] are not PL pro inhibitors, despite the claim from several publications that they act as PL pro inhibitors [28,29]. Interestingly, we serendipitously discovered PGG as a PL pro inhibitor and showed that PGG binds to PL pro and inhibited the enzymatic activity of PLpro in the FRET assay. Taken together, our results call for stringent hit validation, and the serendipitous discovery of PGG as a putative PL pro inhibitor might worth further investigation.

Results And Discussion
Invalidation of dieckol and PGG as SARS-CoV-2 M pro inhibitors.
Dieckol was reported as a SARS-CoV-2 M pro inhibitor through a uorescence polarization-based highthroughput screening [21]. In the assay design, the biotin-labeled M pro substrate was conjugated with a uorescein isocyanate (FITC) uorophore, resulting in a bifunctional probe FITC-AVLQ↓SGFRKK-Biotin (FITC-S-Biotin). Binding of this probe to avidin led to increased uorescence polarization. Upon M pro digestion, the uorophore-peptide conjugate FITC-AVLQ was released, which correlates with reduced millipolarization unit (mP) signal. Screening of a natural product library of 5,000 compounds identi ed dieckol as a potent M pro inhibitor with IC 50 values of 4.5 µM (no DTT) and 2.9 µM (1 mM DTT). The mechanism of action was characterized using the FRET assay and surface plasmon resonance binding assay, both of which showed consistent results as the FP assay. Enzymatic kinetic studies demonstrated that dieckol is a competitive M pro inhibitor. It is noted that dieckol was also previously reported as a SARS-CoV M pro inhibitor [30].
PGG was reported as an inhibitor for both SARS-CoV and SARS-CoV-2 M pro with IC 50 values of 6.89 and 3.66 µM, respectively [22]. In another study, PGG was found to bind to the SARS-CoV-2 spike protein receptor binding domain (RBD) with a K D of 6.69 µM in the bio-layer interferometry assay, while the binding of PGG to the ACE2 receptor was weaker with a K D of 22.2 µM [31]. PGG was further shown to block the RBD-ACE2 interactions in the ELISA assay with an IC 50 of 46.9 µM. In the SARS-CoV-2 pseudovirus assay, PGG dose-dependently inhibited the viral entry and replication.
To validate whether dieckol and PGG are M pro inhibitors, we repeated the FRET enzymatic assay using our standard FRET assay condition (20 mM HEPES, pH 6.5, 120 mM NaCl, 0.4 mM EDTA, 4 mM DTT, and 20% glycerol). Both dieckol and PGG were inactive (IC 50 > 20 µM) ( Table 1). To examine whether dieckol and PGG inhibited the intracellular protease activity of M pro , we characterized both compounds in the cellbased Flip-GFP M pro assay. Our previous results showed that there is generally a positive correlation between the Flip-GFP and antiviral assay results, while the correlation between the FRET enzymatic assay results and antiviral assay results is compound dependent [15]. In the Flip-GFP assay, the GFP is reconstituted upon cleavage of the engineered linker by M pro , and the normalized GFP/mCherry signal ratio is proportional to the M pro activity (mCherry serves as an internal control for the protein expression Page 4/14 level or compound toxicity) [32,33]. GC-376 was included as a positive control and it showed an EC 50 of 3.5 µM (Fig. 1A). The results showed that both compounds lacked the cellular M pro inhibitory activity at non-toxic drug concentrations (Fig. 1A). Dieckol was not active (IC 50 > 60 µM), while PGG was cytotoxic (CC 50 = 9.8 µM) (Fig. 1A), therefore the result was not conclusive. Taken together, dieckol and PGG were both invalidated as M pro inhibitors.
In parallel, we tested dieckol and PGG against SARS-CoV-2 PL pro in the FRET assay. While dieckol was not active (IC 50 > 20 µM), PGG was serendipitously found to inhibit SARS-CoV-2 PL pro with an IC 50 of 3.9 µM ( Fig. 1B and Table 1). To pro le the broad-spectrum activity, PGG was tested against SARS-CoV and MERS-CoV PL pro . PGG showed weak activity against SARS-CoV PL pro with an IC 50 of 12.3 µM, while it was inactive against the MERS-CoV (IC 50 > 60 µM) (Fig. 1B). These results suggest that the inhibition of SARS-CoV-2 PL pro by PGG might be speci c. We further characterized the binding of PGG to SARS-CoV-2 PL pro in the thermal shift assay and found that PGG increased the thermal stability of PL pro in a dose dependent manner (Fig. 1C). To determine whether PGG inhibits the intracellular protease activity of SARS-CoV-2 PL pro , we performed the Flip-GFP PL pro assay. Unfortunately, PGG was cytotoxic to the 293T cells used in the Flip-GFP PL pro assay (CC 50 = 7.7 µM), resulting in inconclusive results (Fig. 1D). Table 1 Validation and invalidation of SARS-CoV-2 M pro and PL pro inhibitors. IC 50 = 28.2 ± 9.5 (4 mM DTT) [25] IC 50 = 0.7 ± 0.1 (4 mM DTT) IC 50 > 60 (4 mM DTT) [25] Flip-GFP PL pro assay: IC 50 > 50 µM PX-12 IC 50 = 0.9 ± 0.2 (4 mM DTT) IC 50 > 60 (4 mM DTT) [25] IC 50 = 18.7 ± 2.6 (4 mM DTT) IC 50 > 60 (4 mM DTT) [25] Flip-GFP PL pro assay: To gain insights of the binding mode, we performed molecular docking of PGG with SARS-CoV-2 PL pro (PDB: 7JRN) [15] using the Schrödinger Glide extra-precision. The binding sites in PL pro were determined by the sitemap, which revealed the BL2 loop region as the highest-ranking binding site, therefore it was selected for PGG docking. The BL2 loop region is also the drug binding site of the known PL pro inhibitors GRL0617 [15]. Docking results showed that PGG ts snugly in the binding site with a Glide score of -10.024 ( Fig. 2A). PGG formed multiple hydrogen bonds with PL pro residues including the side chains of Tyr273, Asp302, Arg166, Lys157 and the main chain of Leu162 (Fig. 2B).

Disul ram was previously reported as a PL pro inhibitor of both SARS-CoV and MERS-CoV [28]. Enzymatic
kinetic studies showed that disul ram acts as an allosteric inhibitor of MERS-CoV PL pro and a competitive inhibitor of the SARS-CoV PL pro . In contrary, our previous study revealed that the inhibition of SARS-CoV-2 PL pro by ebselen in the FRET-based enzymatic assay is reducing reagent dependent [25].
Ebselen inhibited SARS-CoV-2 PL pro with an IC 50 of 6.9 µM in the absence of DTT but was not active in the presence of DTT (IC 50 > 60 µM) (  [29]. Disul ram and ebselen were also proposed to inhibit SARS-CoV-2 PL pro through ejecting zinc from the zinc-binding domain [34]. Given the debate whether reducing reagent should be added to the cysteine protease assay buffer, coupled with the controversy FRET assay results of ebselen in the presence of DTT, we were interested in further characterizing the inhibition of SARS-CoV-2 PL pro by these compounds in a native cellular environment. For this, we employed our recently established cellular Flip-GFP PL pro assay [15] to test the intracellular activity of these compounds. It was found that none of the compounds tested reduced the GFP/mCherry ratio at non-cytotoxic concentrations (Fig. 3), suggesting they lack the intracellular target engagement and PL pro inhibition. Collectively, our data suggest that disul ram, ebselen, carmofur, PX-12, and tideglusib should not be classi ed as PL pro inhibitors.

Conclusion
In conclusion, our data suggested that dieckol and PGG are not M pro inhibitors as shown from the FRET and Flip-GFP M pro assays. Furthermore, the previous reported promiscuous cysteine modi ers ebselen, disul ram, carmofur, PX-12, and tideglusib were also invalidated as PL pro inhibitors by the Flip-GFP PL pro assay. Taken together with our previous efforts in invalidating these compounds as M pro inhibitors, it can be concluded that M pro and PL pro enzymatic assay results obtained in the absence of reducing reagents have no correlation with their cellular activity. Among the list of compounds examined, ebselen was previously shown to inhibit SARS-CoV-2 viral replication in cell culture [27,35]. Coupled with the results presented here, it appears that the antiviral mechanism of action of ebselen is independent of either M pro or PL pro inhibition.
Since the FRET assay conditions used in different labs vary, it might be challenging to directly compare the results. Nonetheless, the cell-based Flip-GFP assay is a valuable tool in evaluating the intracellular protease activity and is a close mimetic of virus-infected cells.
In summary, the results presented herein call for stringent hit validation before investing resources for lead optimization and translational antiviral development. The discovery of PGG as a PL pro inhibitor provides another starting point for further optimization.

Materials And Methods
All compounds were purchased from commercial source without further puri cation. PGG was ordered from Toronto Research Chemical with the Cat # P270450.
FRET-Based Enzymatic Assay. For the IC 50 measurement with the FRET-based assay, the reaction was carried out in 96-well format with 100 µL of 200 nM PL pro protein in a PL pro reaction buffer (50 mM HEPES (pH 7.5), 5 mM DTT, and 0.01% Triton X-100); 1 µL of testing compounds at various concentrations was added to each well and was incubated at 30°C for 30 min. The reaction was initiated by adding 1 µLof1 mM FRET substrate and was monitored in a Cytation 5 image reader with lters for excitation at 360/40 nm and emission at 460/40 nm at 30°C for 1 h. The initial velocity of the enzymatic reaction was calculated from the initial 10 min enzymatic reaction. The IC 50 was calculated by plotting the initial velocity against various concentrations of testing compounds using a four-parameter variable slope dose − response curve in Prism 8 software. IC 50 values for the testing compounds against SARS-CoV-2 M pro was determined as previously described [10].
Flip-GFP M pro and PL pro assay. Plasmid pcDNA3-TEV-FlipGFP-T2A-mCherry was ordered from Addgene The mixture was then transfected to each well according to manufacturer's instructions. After 2.5-3 hours of incubation in 37ºC, 1 µL of testing compound was added into each well directly and mixed by gentle plate shaking. 48 hours post transfection, uorescence was quanti ed using SpectraMax iD3 plate reader (Molecular Devices) and images were taken using BZ-X800E uorescence microscope (Keyence) in GFP and mCherry channels at 4X objective lens.
Differential Scanning Fluorimetry (DSF). The thermal shift binding assay (TSA) was carried out using a Thermo Fisher QuantStudio 5 Real-Time PCR system as described previously [10].