key: cord-0754475-jqu9u5t5
authors: Legare, Scott; Heide, Fabian; Bailey-Elkin, Ben A.; Stetefeld, Jörg
title: Improved SARS-CoV-2 main protease high throughput screening assay using a 5-carboxyfluorescein substrate
date: 2022-02-17
journal: J Biol Chem
DOI: 10.1016/j.jbc.2022.101739
sha: 9c688c84d04ef148326fbeb7cca364d52481f055
doc_id: 754475
cord_uid: jqu9u5t5

The emergence of SARS-CoV-2 as a global threat to human health has highlighted the need for the development of novel therapies targeting current and emerging coronaviruses with pandemic potential. The coronavirus main protease (M(pro), also called 3CL(pro)) is a validated drug target against coronaviruses and has been heavily studied since the emergence of SARS-CoV-2 in late 2019. Here we report the biophysical and enzymatic characterization of native M(pro), then characterize the steady-state kinetics of several commonly used fluorescence resonance energy transfer (FRET) substrates, fluorogenic substrates, and 6 of the 11 reported SARS-CoV-2 polyprotein cleavage sequences. We then assessed the suitability of these substrates for high throughput screening. Guided by our assessment of these substrates, we developed an improved 5-carboxyfluorescein-based FRET substrate which is better suited for high throughput screening and is less susceptible to interference and false positives than existing substrates. This study provides a useful framework for the design of coronavirus M(pro) enzyme assays to facilitate the discovery and development of therapies targeting M(pro).

Coronaviruses are a family of viruses commonly found in wildlife, companion animals, livestock, and humans. Human coronaviruses include HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 continuously circulate in the population and mostly cause mild symptoms associated with the common cold. In contrast, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2 are highly pathogenic coronaviruses causing the SARS epidemic, MERS epidemic and most recently the coronavirus disease 2019 (COVID- 19) pandemic (1) . To date there have been over 220 million reported cases of COVID-19 and 4.6 million reported deaths. Despite the development of efficacious vaccines against COVID-19, SARS-CoV-2 transmission continues at high levels and case numbers continue to increase (2, 3) . As a result, there is an urgent need for effective antiviral drugs targeting SARS-CoV-2 that can be used to treat COVID-19.

SARS-CoV-2 is an enveloped positive-stranded RNA virus which infects cells of the upper and lower respiratory tract. Upon entry into the host cell, the viral RNA genome is translated into two polyproteins, pp1a and pp1ab. These polyproteins are proteolytically processed by two viral proteases, a papain-like protease (PL pro ) and a chymotrypsin-like main protease (M pro , also called 3CL pro ), releasing 16 nonstructural proteins (1) . M pro inhibitors can effectively block SARS-CoV-2 replication in cell culture, demonstrating M pro is a valid drug target (4) (5) (6) (7) (8) . SARS-CoV-2 M pro has been shown to preferentially recognize the A-X-L-Q-(A/S) consensus sequence (where X is any amino acid), with cleavage occurring after the glutamine (9, 10) . Interestingly, other coronaviruses including the related SARS-CoV and MERS-CoV share similar substrate specificity with SARS-CoV-2 M pro , suggesting that inhibitors of SARS-CoV-2 M pro could serve as broad spectrum antiviral drugs against future epidemic or pandemic causing coronaviruses (4, 11) .

Discovery of M pro inhibitors has relied heavily on the use of high throughput screens (HTS) using a fluorescence resonance energy transfer (FRET) based peptide substrate to monitor protease activity (5, 7, (12) (13) (14) (15) (16) (17) . Fluorogenic substrates which cleave an amino-coumarin based fluorophore attached to the carboxyl terminus of a peptide have also been used (14, 15, 18) . A number of M pro enzyme assays have been developed using different substrates, M pro constructs, and buffer conditions (14, 15, (19) (20) (21) (22) . As a result, there have been inconsistent findings regarding the identification of potential M pro inhibitors (20, 21, 23) . This has highlighted a clear need for an improved SARS-CoV-2 M pro assay that delivers better performance and improved consistency.

Here we provide the detailed biophysical and enzymatic characterization of SARS-CoV-2 M pro with native N-and C-termini, and asses the steady-state kinetic parameters of three commonly used SARS-CoV-2 M pro fluorescent substrates (4, 7, 18) . We measure the kinetic efficiency of 6 SARS-CoV2 M pro polyprotein cleavage sequences to determine the optimal substrate amino acid sequence. Guided by these results, an improved 5-carboxyfluorescein based FRET substrate was developed that is better suited for HTS and is less susceptible to interference and false positives than previously reported substrates. This study provides a useful framework for the design assays aimed at discovering and developing new coronavirus M pro inhibitors.

J o u r n a l P r e -p r o o f

Both fluorogenic and FRET based substrates were used in this work (Fig. 1) . The previously reported VKLQ -AMC fluorogenic substrate consists of a short peptide with a cleavable 7-amino-4-methylcoumarin (AMC) fluorophore at the P1' position ( Fig. 1A ) (18) . The FRET substrates consist of a fluorophore and quencher pair separated by a SARS-CoV-2 polyprotein cleavage sequence. The previously reported FRET nsp4-5-EDANS substrate uses an EDANS fluorophore and a DABCYL quencher (Fig. 1B) , while the nsp4-5-MCA substrate uses a MCA fluorophore with a lysine-2,4-dinitrophenyl quencher (Fig. 1C ) (4, 7) . The 5carboxyfluorescein (FAM) based FRET substrate developed in this work uses a DABCYL quencher (Fig. 1D ). Table 1 lists the amino acid sequence, cleavage site location, fluorophore, and quencher used for each substrate tested in this work. Biophysical characterization demonstrates excellent protein quality: Following the method developed by Xue et al. SARS-CoV-2 M pro with native N-and C-termini was expressed and purified to apparent homogeneity ( Fig. S1 ) for further biophysical and enzymatic characterization (24) . Nano-differential scanning fluorimetry (nanoDSF), dynamic light scattering (DLS), and circular dichroism (CD) spectroscopy were used to establish the quality of M pro used in this work. CD measurements confirmed that the M pro secondary structure is in agreement with what is expected based on the crystal structure (PDB ID: 6Y2E) ( Fig. 2A and Table S1 ). DLS was used to measure the hydrodynamic radius (RH) of M pro , and to measure the state of M pro self oligomerization (Fig. 2B ). Using the size distribution analysis model, the major intensity peak had an RH of 3.76 ± 0.14 nm with a polydispersity index of 0.18 ± 0.03. The measured RH of 3.76 ± 0.14 nm agrees with the expected RH for the M pro dimer based on the crystal structure (PDB ID: 6Y2E). The nanoDSF melt curve measured by the 350/330 nm fluorescence ratio showed a melting onset beginning at 51.1 o C and an inflection point or melting point (Tm) of 56.8 o C (Fig. 2C ). The baseline turbidity measurement is stable from 20 o C until the onset in turbidity increased beginning at 47.9 o C, indicating M pro was stable against aggregation until 3.2 o C before the onset of melting begins (Fig. 2D ). The inflection point of the turbidity measurement was 56.8 o C corresponding to the Tm of M pro . The measured Tm of 56.8 o C was slightly higher than previously reported values of between 48.5 to 54.2 o C (25, 26) . Taken together these results show M pro is highly pure, properly folded, thermodynamically stable, and monodisperse in solution with very little aggregation or higher order oligomerization present. Optimization of assay buffer conditions: The effects of different buffer conditions on M pro activity were assessed to determine optimal assays conditions. The AKLQ -AMC substrate was chosen for buffer optimization because it displayed good solubility up to a concentration of around 2.5 mM in all buffers tested. The optimal pH for M pro was found to be pH 7.0 (Fig. 3A) . M pro had a strong preference for NaPO4 as a buffering agent, followed by BIS-TRIS, HEPES and TRIS, when tested at 20 mM buffering agent, 150 mM NaCl, pH 7.0 ( Fig. 3B ), however this preference was lessened when tested at 20 mM buffering agent pH 7.0 without NaCl (Fig. 3C ). The highest enzyme activity was achieved when NaCl was omitted from the buffer while adding between 10 to 300 mM NaCl decreases the enzyme activity roughly the same degree (Fig. 3D ). Both glycerol and DMSO were found to have a negative effect on enzyme activity (Figs. 3E and 3F). Based on these results the optimized buffer found in this study consists of 20 mM NaPO4 at pH 7.0. It was found that the FRET substrates used in this work showed better solubility in buffers of low ionic strength, therefore 20 mM BIS-TRIS pH 7.0 was used instead of a NaPO4 based buffer. These optimized buffer conditions closely agree with other work showing that M pro is most thermodynamically stable at pH 7.0 in the absence of NaCl (26) . Measuring the reaction progress curve for the complete hydrolysis of substrate can help assess and identify non-optimal buffer conditions and loss of enzyme activity due to inactivation and inhibition. It is also helpful to verify that the measured initial rate corresponds to the true linear steady-state portion of the reaction progress curve (27) . When hydrolysing the FRET substrates, the M pro began to lose activity after about 400 seconds of reaction time in 20 mM BIS-TRIS pH 7.0 ( Fig. S2 ). Adding 2 mM EDTA and 2mM DTT to the assay buffer prevented this inactivation for the nsp4-5-FAM and nsp4-5-EDANS substrates ( Fig.  S2B -C), but not for the nsp4-5-MCA substrate ( Fig. S2D ) which was inactive in 20 mM BIS-TRIS pH 7.0, 2 mM EDTA. As previously discussed, the FRET substrates showed reduced solubility in buffers with higher ionic strength. The inactivity of the nsp4-5-MCA substrate in the 20 mM BIS-TRIS pH 7.0, 2 mM EDTA buffer may be caused by the increase in ionic strength of the buffer from the addition of 2 mM EDTA, reducing the solubility of the nsp4-5-MCA substrate. In contrast to the FRET substrates, M pro did not fully lose activity when hydrolysing the VKLQ -AMC substrate (Fig. S2A ), and addition of EDTA and DTT to the reaction buffer had a minimal effect on the enzyme's behaviour. The linear initial rate portion of the reaction progress curve could be measured for at least the first 600 seconds of reaction time with the VKLQ -AMC substrate, and about 200 seconds for the FRET substrates. This initial rate was unaffected by the addition of EDTA and DTT (Fig. S2 ).

Substrate steady-state kinetic parameters: Measurements were performed with each substrate to determine their utility for characterizing M pro . kcat/Km was measured at low substrate concentrations where the initial rate increased linearly with substrate concentration. The results show the nsp4-5-MCA substrate had the highest kcat/Km, followed by the nsp4-5-FAM and nsp4-5-EDANS substrates, while the VKLQ -AMC substrate had the lowest kcat/Km value ( Fig. S3A -D and Table1). The FRET substrates suffered from poor solubility and large inner filter effects when used at high concentrations needed to reach saturating substrate concentrations (Vmax). These are commonly reported problems for FRET substrates in general, including FRET substrates used for SARS-CoV-2 M pro (18, (28) (29) (30) . As a result, a full Michaelis-Menten plot that reaches saturating substrate concentrations could only be measured using the VKLQ -AMC substrate ( Fig. 4 and Table 1 ). M pro recognizes and cleaves 11 sites on the viral pp1a and pp1ab during viral replication. The nsp4-5 cleavage sequence at the N-terminus of M pro is the sequence commonly used in M pro FRET substrates and is the sequence used in each of the nsp4-5-MCA, nsp4-5-EDANS and nsp4-5-FAM substrates (Table 1) . To test the kinetic efficiency of other SARS-CoV-2 polyprotein cleavage sequences, the kcat/Km of five additional FAM based FRET substrates utilizing the nps5-6, nsp6-7, nsp8-9, nsp10-12 and nsp14-15 cleavage sequences were tested and compared to the nsp4-5-FAM substrate ( Table 1 ). The kcat/Km values for these substrates shows that nsp4-5 has by far the highest kcat/Km value followed nsp5-6, nsp6-7 and nsp14-15, while nsp8-9 and nsp10-12 cleavage sites have by far the lowest kcat/Km values (Fig S3E-I and  Table 1 ). These results show that out of the substrates tested, the nps4-5 sequence has the most desirable kinetic properties for use in enzyme assays. Characterizing the improved nsp4-5-FAM substrate: To assess the suitability of each of the three FRET substrates for HTS, the Z'-factor for each substrate was determined. The Z'-factor is a statistical parameter used to evaluate the quality of a HTS (31) . The Z'-factor reflects two critical criteria which a good quality HTS must have. The first criteria is the signal dynamic range, which describes the difference in signal produced by a positive and negative control. When the assay signal dynamic range is large, the signal produced by an active compound can more confidently be distinguished from an inactive compound. The second criteria reflected in the Z'-factor is the variability or standard deviation of the signal produced by the positive and negative controls. When the positive and negative controls produce a consistent signal with low variability, the signal produced by an active compound can be more confidently differentiated from signal variability. To assess the Z'-factor for a FRET substrate, the mean and standard deviation of the initial rate measured for 16 positive and 16 negative controls was calculated. The signal dynamic range and Z'-factor were calculated as described in the experimental procedures. Baicalein is a non-covalent inhibitor of SARS-CoV-2 (32) and was used as a positive control, the negative control contained DMSO instead of baicalein. This assay was repeated in triplicate for each FRET substrate, with results reported in Table 2 . Of the FRET substrates tested in this work, the nsp4-5-EDANS substrate produces the smallest standard deviation in signal for the positive and negative controls, followed by the nsp4-5-FAM and nsp4-5-MCA substrates. The nsp4-5-FAM substrate produced the largest signal dynamic range followed by the nsp4-5-MCA and nsp4-5-EDANS substrates ( Table 2) . The large signal dynamic range produced by the nsp4-5-FAM substrate is largely attributed to the much higher brightness of the FAM fluorophore in comparison to the MCA and EDANS fluorophores (Fig. S4) .

In this study, the nsp4-5-MCA and nsp4-5-EDANS substrates produced a Z'-factor of between 0.72 to 0.79, while the nsp4-5-FAM substrate produces a considerably higher Z'-factor of between 0.82 to 0.85 (Table 2) . J o u r n a l P r e -p r o o f

The SARS-CoV-2 M pro is a validated drug, and M pro inhibitors have been shown to block viral replication in cell culture (4) (5) (6) (7) (8) . Additionally, M pro inhibitors could have broad spectrum antiviral activity against related coronaviruses because of the conserved features of the M pro recognition sequence (4, 11) . Florescent substrates are commonly used to study M pro enzymatic activity, identify inhibitors through HTS, and test inhibitor efficacy. In this study we perform a biophysical characterization of SARS-CoV-2 M pro and asses the steady-state kinetic parameters of three commonly used substrates, as well as 6 polyprotein cleavage sequences. We then develop the improved nsp4-5-FAM substrate that is better suited for HTS when compared to commonly used FRET substrates, resulting from the higher brightness of the FAM fluorophore. Additionally, the FAM fluorophore is less susceptible to interference and false positives due to the green-shifted absorption and emission spectra of the FAM fluorophore.

The protease used in this study was produced recombinantly in E.coli following a previously described method (24) . M pro produced by this method has been successfully used for structural and enzymatic studies (4, 7, 8, 18) . The primary advantage of this method is that it generates M pro with native N-and Ctermini which is known to be structurally different, and more catalytically active than M pro with non-native N-or C-termini (4, 24) . In addition, conflicting M pro enzymatic data has been published in the literature which has been in part attributed to the use of different M pro constructs with non-native termini (20, 21, 23) . Work on the closely related SARS-CoV M pro has recommended the standard adoption of native termini M pro for enzymatic and structural studies (30) . For these reasons, native SARS-CoV-2 M pro was used for the biophysical and enzymatic work done in this study.

Characterization of substrate kinetic parameters is critical for understanding the behaviour of both the substrate and enzyme, and can also help guide the development of a properly optimized enzyme assay. kcat/Km is an informative and useful parameter that gives a measure of substrate specificity and is the apparent second order rate constant for product formation. We found that the value of 14,190 ± 420 M -1 s -1 for the nsp4-5-MCA substrate was 6 to 7 times higher than the value of 2448 ± 85 M -1 s -1 and 1960 ± 190 M -1 s -1 measured for the nsp4-5-FAM and nsp4-5-EDANS substrates respectively, which is consistent with values reported in the literature(4, 7). The kcat/Km value of 18.5 ± 1.0 M -1 s -1 for the VKLQ -AMC substrate is far lower than the FRET substrates due to the shorter recognition sequence of the VKLQ -AMC substrate which lacks the residues C-terminal to the cleavage site (18) .

The VKLQ -AMC substrate was the only substrate that could be used at concentrations needed reach Vmax. By measuring the Michaelis-Menten kinetics of the VKLQ -AMC substrate, we report a kcat/Km of 24.5 ± 5.0 M -1 s -1 which agrees with the value of 18.5 ± 1.0 M -1 s -1 obtained using low substrate concentrations. This demonstrates that the behaviour of the VKLQ -AMC substrate is consistent at low and high concentrations, therefore the VKLQ -AMC substrate is relatively unaffected by the inner filter effect. Others have also found that these fluorogenic substrates are better suited for use at high concentrations than FRET substrates (18) . A chromogenic substrate very similar to the fluorogenic VKLQ -AMC substrate was more useful for catalytic mechanism studies of SARS-CoV M pro than the nsp4-5-EDANS FRET substrate (33) .

Of the M pro polyprotein cleavage sequences tested, we found that the nsp4-5 cleavage sequence has by far the highest kcat/Km and therefore is best suited for use in enzyme assays. This result is consistent with measurements done on the SARS-CoV M pro which also show that the nsp4-5 cleavage sequence has the highest kcat/Km of the 11 polyprotein cleavage sequences (34) . N-terminomics studies have identified the preferred cleavage sequence of SARS-CoV-2 M pro to be A-X-L-Q↓(A/S)(9, 10). Of the cleavage sequences we tested, the nsp4-5 sequence is the only one which strictly represents this consensus sequence.

By characterizing the properties of the previously published nsp4-5-EDANS, nsp4-5-MCA, and VKLQ -AMC substrates, we recognized that an improved substrate for HTS could be developed. Because the kcat/Km of the VKLQ -AMC substrate is low, high concentrations of substrate and enzyme are needed to generate a measurable fluorescent signal. This makes the VKLQ -AMC substrate undesirable for HTS due to the larger amounts of enzyme and substrate that would be consumed. We also recognized that both the nsp4-5-MCA and nsp4-5-EDANS substrates use fluorophores with relatively low brightness and undesirable excitation and emission spectra that makes them susceptible to interference from assay compounds (35) .

To develop an improved FRET substrate for HTS, we chose to use a FAM fluorophore because of its higher brightness, and spectral properties that are less prone to interference. Additionally, FAM is an inexpensive and readily available fluorophore that can easily be incorporated into the peptide substrate by most custom peptide synthesis companies.

To assess whether our new nsp4-5-FAM substrate is better for HTS than existing nsp4-5-MCA and nsp4-5-EDANS substrates, we characterized the Z'-factor for these substrates. An ideal assay produces a Z'factor of 1, however an experimental assay could never achieve this value. A Z'-factor of greater than 0.5 is usually considered an excellent quality assay (31) . We found that both the nsp4-5-MCA and nsp4-5-EDANS substrates produced a Z'-factor of 0.75, indicating our assay conditions are well optimized. However, because of the low brightness of the MCA and EDANS fluorophores, these substrates produce a low signal dynamic range which limits the Z'-factor. The most effective way to develop a substrate that is better suited for HTS is to use a brighter fluorophore. We found that by using the brighter FAM fluorophore, we were able to greatly increase the signal dynamic range of the assay and increase the Z'factor of the assay to 0.84. This demonstrates that the improved nsp4-5-FAM substrate is better suited for HTS.

False positives are another common issue encountered in HTS assays and are especially problematic when screening large libraries of compounds (35) . Fluorescent based assays are especially susceptible to false positives caused by compounds which interfere with the measured fluorescent signal. Additionally, the vast majority of compounds tested in HTS absorb and fluoresce at wavelengths in the ultraviolet and blue region of the spectrum (< 490 nm) (36) . This makes the nsp4-5-MCA and nsp4-5-EDANS substrates (ex/em 320/405 nm, and 350/480 nm respectively) especially susceptible to interference and false positives. In contrast, the nsp4-5-FAM substrate absorbs and emits green light (ex/em 490/530 nm) and is therefore largely unaffected by this source of interference, reducing the potential for false positives (35) .

The biophysical and enzymatic characterization of the native SARS-CoV-2 M pro described in this work will serve as a valuable reference for future studies investigating the activity of SARS-CoV-2 M pro . Using optimized assay conditions, we were able to compare properties of commonly used M pro substrates and develop an improved nsp4-5-FAM substrate that is better suited for HTS. When compared to commonly used M pro FRET substrates, this substrate generates a better-quality HTS assay because of the higher brightness of the FAM fluorophore, and is less susceptible to interference from assay compounds due to its green-shifted absorbance and emission spectra. This substrate will thus serve as a valuable tool in the development and design of future HTS assays aimed at identifying and characterizing novel direct-acting antivirals targeting the SARS-CoV-2 M pro .

Construct design, enzyme expression and storage: Design of the expression vector followed previously reported methods (4, 24) . The codon optimized SARS-CoV-2 M pro open reading frame was inserted at the BamHI and XhoI restriction sites of a PGEX-6p-1 expression vector. The M pro open reading frame contained the N-terminal autocleavage site AVLQ↓SGFRK (↓ denotes cleavage site) and a modified version of the C-terminal autocleavage site VTFQ↓GP followed by a His6-tag. Auto-cleavage occurs during protein expression to produce a native N-terminus. The modified C-terminal autocleavage site is not cleaved by M pro but can be cleaved by human rhinovirus 3C protease (HRV-3C) to produce the native M pro C-terminus during protein purification. This SARS-CoV-2 M pro expression vector was synthesized by GenScript.

The SARS-CoV-2 M pro expression vector was transformed into E.coli strain BL21-Gold (DE3) (37) . A single colony was used to inoculate a 50 mL culture of Miller's LB broth containing 100 µg/mL ampicillin overnight at 30 o C with shaking. 10 mL of overnight culture was used to inoculate 600 mL of LB broth containing 100 µg/mL ampicillin. This culture was grown at 37 o C until an OD600 of around 0. Circular dichroism (CD) spectroscopy: CD measurements of M pro were performed at a concentration of 0.5 mg/mL in 10mM Na2HPO4, pH 8.0. CD measurements were taken with a Jasco J-810 (JASCO corporation) spectropolarimeter at 20°C in a 0.05 cm path length quartz cuvette. Raw data were converted to mean residue ellipticity and secondary structure deconvolution was done using the CDSSTR algorithm and the SMP180 reference set on the DichroWeb server (39, 40) . Experimental secondary structure fractions were compared to the protease crystal structure (PDB ID: 6Y2E) using PDBsum (41) .

Fluorescent substrates: Amino acid sequences of the substrates used in this study can be found in Table  1 and Figure 1 . The nsp4-5-MCA substrate was purchased from CanPeptide Inc, all other substrates were purchased from GenScript. All substrates had a purity greater than 95% confirmed by HPLC and the molecular weight confirmed by mass spectrometry (testing done by supplier . Readings were taken every 20 seconds for 600 seconds to measure initial reaction rates, and up to 1.5 hours to measure complete hydrolysis. Measurements were done at ambient temperature. Initial rates were fit to the linear portion of the reaction progress, usually the first 200 seconds corresponding to less than 10% substrate hydrolysis. Fluorescence units were converted to concentration using a standard curve generated using a fluorophore standard in 20mM BIS-TRIS pH 7.0. 7-Methoxycoumarin-4-acetic acid (MCA) and 5-(2-Aminoethylamino)-1-naphthalenesulfonic acid (EDANS) were purchased from Fisher Scientific, 5-carboxyfluorescein (FAM) was purchased from Cayman Chemical Company and 7-amino-4-methylcoumarin (AMC) was purchased from Sigma-Aldrich. All parameter fitting by linear and non-linear regression was done in QtiPlot. All measurements were performed in triplicate, final values are expressed as the mean ± 1 standard deviation of the three measurements. High throughput screening assessment: The Z'-factor was assessed by measuring enzyme activity of 16 positive and 16 negative controls, and was repeated in triplicate for each FRET substrate. Baicalein (CAS # 491-67-8, Sigma-Aldrich) a non-covalent inhibitor of SARS-CoV-2 M pro was used as a positive control, the negative control contained DMSO instead of baicalein. The reaction contained 100µL of 10µM substrate, 40nM enzyme and either 50µM baicalein or DMSO as the positive and negative controls respectively. The final buffer composition was 20 mM BIS-TRIS pH 7.0, 1.2 % v/v DSMO. For each assay the mean and standard deviation of the initial rate for positive and negative controls was calculated. The signal dynamic range was calculated according to the following, where ̅ and ̅ are the mean of the negative and positive controls respectively.

The Z'-factor was calculated according to Zhang et al.(31) where and are the standard deviation of the positive and negative controls respectively. J o u r n a l P r e -p r o o f

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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All data are contained within the article and in the supporting information.

This article contains supporting information

All authors were involved in study design. S.L. produced M pro , produced and analysed nanoDSF, DLS and enzymatic data. 

The authors declare that they have no conflicts of interest with the contents of this article.