key: cord-0296018-zp2n6fc2
authors: Eberle, Raphael J.; Gering, Ian; Tusche, Markus; Ostermann, Philipp N.; Müller, Lisa; Adams, Ortwin; Schaal, Heiner; Olivier, Danilo S.; Amaral, Marcos S.; Arni, Raghuvir K.; Willbold, Dieter; Coronado, Mônika A.
title: Design of D-amino acids SARS-CoV-2 Main protease inhibitors using the cationic peptide from rattlesnake venom as a scaffold
date: 2021-11-10
journal: bioRxiv
DOI: 10.1101/2021.11.10.468025
sha: c7f27a582463c2d250a9b4a776952b6fe22c5fbd
doc_id: 296018
cord_uid: zp2n6fc2

The C30 Endopeptidase (3C-like protease; 3CLpro) is essential for the life cycle of SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) since it plays a pivotal role in viral replication and transcription and is hence a promising drug target. Molecules isolated from animals, insects, plants or microorganisms can serve as a scaffold for the design of novel biopharmaceutical products. Crotamine, a small cationic peptide from the venom of the rattlesnake Crotalus durissus terrificus has been the focus of many studies since it exhibits activities such as analgesic, in vitro antibacterial and hemolytic activities. The crotamine derivative L-peptides (L-CDP) that inhibit the 3CL protease in the low µM range were examined since they are susceptible to proteolytic degradation; we explored the utility of their D-enantiomers form. Comparative uptake inhibition analysis showed D-CDP as a promising prototype for a D-peptide-based drug. We also found that the D-peptides can impair SARS-CoV-2 replication in vivo, probably targeting the viral protease 3CLpro.

In Wuhan, Hubei Province, China, in December 2019, a rapid increase in the number of pneumonia suspect cases [1] quickly aroused interest, and it sounded the emergency call in the World Health Organization (WHO) in January 2020, as a "public health emergency of international concern". The resulting disease, Coronavirus Disease-2019 (COVID- 19) , exploded into a global pandemic within a few months, claiming lives in all continents. SARS-Coronavirus-2 (SARS-CoV-2) has resulted in over 248 million confirmed cases and over 5 million deaths worldwide reported by the WHO on 5th of November 2021 [2] . The worldwide vaccination campaign using clinical safe and efficient vaccines against SARS-CoV-2 (e.g. BioNTech-Pfizer, Moderna, Johnson & Johnson, and AstraZeneca vaccines) [3] [4] [5] [6] , and so far, more than 7 billion vaccine doses have been administered [2] .

Several candidate drugs that may inhibit SARS-CoV-2 infection and replication have been approved for emergency use (e.g., Remdesivir, dexamethasone, Favipiravir, Lopinavir/ritonavir, and Darunavir) [7] [8] [9] [10] [11] . Given a considerable limitation of direct-acting antivirals for COVID- 19 [12] , it remains a strategic priority to develop new candidates with minimal side effects and which are also targeted against new variants. Upon entering and uncoating the viral particles, the positivestranded RNA genome is rapidly translated into two polyproteins processed (pp1a and pp1ab) by 3CL and papain-like proteases into 16 nonstructural proteins (NSPs) [13, 14] . 3CL pro is a cysteine protease organised in three domains (domains I to III) with a chymotrypsin-like fold [15] . Its active form consists of two protomers (homodimer) containing a noncanonical Cys-His dyad located in the cleft between domains I and II [15] [16] [17] . The functional importance of 3CL pro in the viral life cycle combined with the absence of closely related homologues in humans indicates that this protease is an attractive target for developing antiviral drugs [18] .

In recent years, antimicrobial peptides (AMPs) have been considered to hold the promise as a viable solution to form the basis for the design of novel peptides to combat hazardous microorganism infections. The use of AMPs can be promising as a therapeutic tool to address increasing viral infections, for which no current or authorised medication or treatment is available [19] . AMPs are frequently used to treat viral-related diseases such as Zika (ZIKV), Dengue (DENV) [20] , and Influenza A virus infection (IAV) [21] .

Crotamine (Cro) , a small cationic polypeptide originally encountered in the venom of the South American rattlesnake Crotalus durissus terrificus [22, 23] , possess cell wall penetrating properties [24, 25] , and several biological functions of this polypeptide were described, including antimicrobial, antifungal and antitumoral activities [24] [25] [26] [27] . These properties were mainly considered to be determined by the overall positive net surface charge distribution of crotamine [24] [25] [26] [27] . Characterised as a novel cellpenetrating polypeptide (CPP) nanocarrier, Cro has biotechnological applications due to its peculiar specificity for highly proliferating cells. [24, 25, 28] . Similar to other CPPs, Cro showed a rapid translocation efficiency (within 5 min) into all cell types investigated to date [29] . A small peptide composed by 42 amino acid residues (YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGSG) [22, 23] containing two putative nuclear localization sequence (NLS) motifs Cro_2-18 (KQCHKKGGHCFPKEKIC) and Cro_27-39 (KMDCRWRWKCCKK) [29] . The sequence Crot_27-39 was selected and named L-CDP1 as the initial sequence for inhibition studies against SARS-CoV-2 3CL pro . Based on the first sequence, several

Crotamine Derivative Peptides (CDPs) in L-form were specifically modified, and D-forms were designed. They were tested concerning their inhibitory potential against the virus' main protease.

SARS-CoV-2 3CL pro _GST fusion protein was expressed in E. coli Lemo21 (DE3) cells and purified using a GSH-Sepharose column ( Supplementary Fig. S3A ). The relevant protein fractions were concentrated and prepared for PreScission protease cleavage to remove the GST-tag. The SDS gel ( Supplementary Fig. S1 ) indicates the cleavage efficiency and the purity of 3CL pro .

The L-CDP1 (wild type sequence Cro_27-39) includes three cysteine residues. The L-CDP2-9 are modified peptides by substituting the cysteine to serine residues in a different position (Table 1 and Supplementary Fig. S2 and S3) to efficiently achieve an optimised sequence aiming to inhibit the 3CLpro. In principle, the positively charged residues were maintained due to their membrane modify properties.

According to the procedure described earlier, the SARS-CoV-2 3CL pro activity assay was performed using DABCYL-KTSAVLQ/SGFRKME-EDANS (Bachem, Switzerland) as substrate [2] [3] [4] [5] . A primary inhibition test with Cro and the L-CDPs (30 µM) was performed to screen the best inhibitor peptide against the virus protease ( Fig. 1 ).

pro . Crotamine inhibits the virus protease activity by around 50%. L-CDP1, L-CDP2, L-CDP4, L-CDP7 and L-CDP8 inhibit the virus protease activity by more than 80%. Data shown are the mean ± SD from 3 independent measurements (n=3). Asterisks mean that the data differs from the control (0 µM inhibitor) significantly at p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***), level according to ANOVA and Tukey's test. The experimental model of Crotamine is shown in coulombic surfaces and cartoons, with the L-CDP1 sequence highlighted in blue (PDB entry: 4GV5).

The primary inhibition tests revealed a strong effect of L-CDP1, L-CDP2, L-CDP7 and L-CDP8

peptides against SARS-CoV-2 3CL pro activity assay, and those peptides inhibit the virus protease activity by more than 80%. All peptides were subject of the dose-dependent studies ( Supplementary Fig. S4) ;

however, the peptides that present more than 80% inhibition have been chosen for further studies.

The selected peptides were further analysed with respect to their actual potential to inhibit the catalytic activity of the 3CL pro in a biochemical assay. To gain insight into functional implications caused by the selected peptide (L-CDP1), we checked the minimum concentration of Cro required to inhibit 100% of the protease activity. The full-length polypeptide Cro was tested using a concentration range of 0-300 µM, and the polypeptide demonstrates 100% protease inhibition at a concentration of 300 µM ( Supplementary Fig. S4A ), presenting an IC50 value of 40 ± 3.1 µM ( Fig.2B ). The replacement of all cysteine residues of L-CDP1 by serine residues, a peptide named L-CDP2 (Table 1) , led to an increase in IC50 values (5.0 ± 0.8 µM), revealing that the cysteine residue can increase the inhibition rate of the protease ( Fig. 2C and Supplementary Fig. S4C ). The Crotamine derivative L-peptide-7, a peptide that presents an amino acid substitution, Cys36 (WT numbering) to Ser, and L-CDP8 (Cys37 to Ser) (see Table 1 ), inhibits the protease by 100% at 60 µM concentration (Supplementary Fig. S4HI ) with IC50 values of 1.5 ± 0.4 and 2.1 ± 0.4 µM ( Table 1 , Fig. 2DE ), respectively.

The Supplementary Fig. S4 shows the inhibition effect for all modified L-CDP against the 3CL protease and the Supplementary Fig. S5 the dose-response curves for IC50 determination for the remaining modified peptides (L-CDP3, 4, 5, 6 and 9). Regarding residues substitution (Table 1) , the secondary structure of L-CDP1, L-CDP2, L-CDP7 and L-CDP8 was investigated by Circular dichroism (CD), demonstrating that the substitution of the cysteine residue will results in conformational changes ( Supplementary Fig. S6 ). As expected, difference in uptake behavior was observed with the replacement of only one cysteine residue, as observed by IC50 values (Table 1 and Fig. 2C ). However, the substitution at position 36 (WT numbering) increases the inhibitory efficiency of the peptide L-CDP7 (Table 1 and Fig. 2D ). Table 1 summarises the experiments performed with the modified L-CDP peptides. 

Detailed mechanistic studies using a fluorescence-based protease assay demonstrated that L-CDP1, L-CDP7, and L-CDP8 peptides are competitive inhibitors (Fig. 3) . The results indicate that these peptides interact directly with amino acid residues located in the active site or with amino acids located in the substrate-binding region of the protease, preventing substrate entry to the active site. Since L-CDP1 and L-CDP7 present IC50 values < 2 µM, they were considered for profound evaluation. [S] is the substrate concentration; v is the initial reaction rate. A: Lineweaver-Burk plot for L-CDP1 inhibition of SARS-CoV-2 3CL pro . B: Lineweaver-Burk plot for L-CDP7 inhibition of SARS-CoV-2 3CL pro . C: Lineweaver-Burk plot for L-CDP8 inhibition of SARS-CoV-2 3CL pro . Data shown are the mean ± SD from 3 independent measurements (n=3).

We further investigated the binding affinity of L-CDP1 and L-CDP7 molecules using Surface

Plasmon Resonance (SPR) ( Table 2 Table 2 . From the KD value obtained from SPR, it was clearly observed that 3CL pro had a higher binding efficacy for the L-CDP1 receptor. The observed changes in the dissociation process can be attributed to a distinct aspect that the substitution of a sulfur-containing amino acid (Cysteine) by a hydroxyl group (Serine) change the secondary structure (as described before), as well as the mode of interaction of the peptide with the target protease. Jha et al. described in an internalisation study using a Cro derivative peptide (CyLoP-1) that the substitution or deletion of the cysteine residue reduces cellular uptake and cytosolic distribution [30] . Both studies show the importance of the cysteine residue, not only for internalisation but also for inhibiting the SARS-CoV-2 protease.

In order to conserve all the essential biological properties of the L-enantiomers peptides against human protease degradations, CDP1 and CDP7 were synthesised in D-enantiomers form. D-peptides, when compared to their L-enantiomeric equivalents, possess several therapeutic advantages. As shown previously, the proteolytic stability of D-peptides is superior to L-peptides, which can dramatically increase serum half-life. [42, 43] , resulting also in reduced immunogenicity and increased bioavailability of D-peptides [44] . Welch [S] is the substrate concentration; v is the initial reaction rate. F: Lineweaver-Burk plot for D-CDP7 inhibition of SARS-CoV-2 3CL pro . Data shown are the mean ± SD from three independent measurements (n=3).

The determined IC50 values for D-CDP1 (4.9 ± 1.7 µM) and D-CDP7 (1.9 ± 0.3 µM) are slightly higher than that in L-peptides with an increase of two-fold for D-CDP1 (Table 3) . Like the L-peptides, D-CDP1 and D-CDP7 reveal the same as before, a competitive inhibition mode (Fig. 5 E and F) .

SPR experiments of the D-enantiomers with 3CL pro showed no evaluable results. Therefore, the KD values for the D-CDP1 and -D-CDP7 interaction with the 3CL pro was determined using microscale thermophoresis (MST) (Supplementary Fig. S8 ), which we did not compare with the results for L-CDPs (SPR) as we modified the method to determine the KD.

It is well described that a structural conversion or steric incompatibility in D-enantiomers can negatively influence the inhibition and binding behaviour of the D-peptides with the target proteins [46, 47] or the method of choice to determine the KD, which was also observed for D-CDP1 and D-CDP7.

However, we have shown that both L-enantiomers (CDP1, CDP7) could be significantly modified without altering their function. The D-CDP1 interaction with 3CL pro is around ten times stronger than D-CDP7; this tendency was also observed for the L-enantiomers (Table 3 and Supplementary Table S1 ). 

The term "promiscuous" inhibitors describe compounds whose inhibition mechanism involves interacting aggregates of many molecules with the target protein. Classified also as "promiscuous" are redox cycling compounds (RCCs) that generate µM concentrations of hydrogen peroxide (H2O2) in the presence of strong reducing agents, which is presented in the assay buffer in order to maintain the catalytic activity of cysteine proteases, like 3CL [12] . H2O2 generated by RCCs can indirectly inhibit the catalytic activity of proteins by oxidising accessible cysteine and or tryptophan that are present in CDP1

and CDP7 in L-and D-enantiomers.

To exclude the capacity of both D-peptides to behave as a RCC, we performed a hydrogen peroxide (H2O2) assay under the influence of TCEP. Our results demonstrated that L/D-CDP1 and L/D-CDP7 do not produce H2O2 under the influence of 1 mM TCEP and can be excluded as RCCs ( Supplementary Fig. S9 ). Furthermore, a detergent-based control was performed to exclude peptide inhibitors that possibly act as an aggregator of 3CL pro , and the experiment was performed by adding 0.001%, 0.01% and 0.1% Triton X-100 to the reaction. Supposed that a molecule would exhibit significant inhibition of 3CL pro , which is diminished by detergent, it is almost certainly acting as an aggregationbased inhibitor, as described before [11] , which was not observed for L/D-CDP1 and L/D-CDP7

( Supplementary Fig. S10 ), discarding the possible aggregation properties of the peptides.

Many peptide-based inhibitors lose the inhibitory effect over the time. The stability of the Land D-peptides (CDP1 and CDP7) selected in this survey was tested over 24 h. The results demonstrated a constant inhibition of SARS-CoV-2 3CL pro over 24 h by both peptides, demonstrating their stability over time and is not prone to digestion by the protease. (Supplementary Fig. S11 ).

A critical factor in evaluating the eligibility of potential lead peptides is cytotoxicity. showed that both molecules have no cytotoxic effect at the calculated IC50 concentrations described above ( Supplementary Fig. S12) . The low cytotoxicity of both D-peptides agrees with the results described before for the L-peptides [30] .

To further substantiate the enzyme inhibition results, we evaluated the ability of these peptides to inhibit SARS-CoV-2. To test whether the two SARS-CoV-2 3CL pro inhibitors, D-CDP1 and D-CDP7, were able to inhibit SARS-CoV-2 replication in cell culture, an African green monkey cell line that supports productive SARS-CoV-2 replication was used. These Vero cells were pre-treated with the two inhibitors for 1 h; subsequently, the cells were infected at an MOI of 0.05. Viral replication was analysed by determining the SARS-CoV-2 RNA within the cell culture supernatant 2 dpi (days post-infection).

Pre-treatment at a non-toxic concentration (50 µM) resulted in a significant decrease in viral RNA after 2 dpi (Fig. 6) , suggesting that both SARS-CoV-2 3CL pro inhibitors, D-CDP1 and D-CDP7, contribute to impaired SARS-CoV-2 replication through their anti-3CL pro activity.

This experiment demonstrates that both D-peptides can impair SARS-CoV-2 replication, most likely by explicitly targeting the viral protease 3CL pro . While a twofold reduction in viral RNA, as seen after treatment with D-CDP1, may not seem much regarding the high replication rate, it clearly shows that our compounds exert antiviral activity within SARS-CoV-2-infected cells. These results provide the first proof that the peptides target the inhibition of the SARS-CoV-2 replication. Whether this effect is due to the anti-3CLpro activity and whether the inhibitory effect observed can be optimised by additional modifications is subject to further investigation.

Future work should aim to reveal the mechanism behind the action of both D-peptides and the optimisation of the amino acid composition. The graph shows individual data points with mean ± SD (n = 6). The results were calculated from MD simulation and was selected with the parameter value below -1 kcal/mol. B) Left side, surface representation of the 3CL pro in complex with the L-CDP1 peptide (sticks), the substrate binding sites are colored according: S1 cyan; S1´orange; S2 magenta; S3 red. Right side, cartoon representation of the protease in complex with the peptide (sticks), the amino acids of the catalytic site are shown in sticks (orange). C) Left side, surface representation of the 3CLpro in complex with the D-CDP1 peptide (sticks), the substrate binding sites are colored according: S1 cyan; S1´orange; S2 magenta; S3 red. Right side, cartoon representation of the protease in complex with the peptide (sticks), the amino acids of the catalytic site are shown in sticks (orange).

MD simulation revealed a possible mode of interaction of the CDP1 peptides with SARS-CoV-2 3CL pro . Therefore, His41, the amino acid residue of the active site interacts through hydrogen bond with one of the residues (Lys31) of the L-CDP1, and the other residues are accommodated in the substrate binding regions ( Figure 7B ), confirming the competitively mode of interaction, as already described by experimental results (Fig. 3A, 5E and Table 3 ). However, D-CDP1 does not interact with the amino acids residues of the catalytic dyad; nevertheless, the peptide is seated in the substrate binding region blocking the interaction of the substrate with the active site of the protease (Fig. 7B ).

Using PDBsum platform the interaction type and amino acid residues involved in the interaction between 3CL pro with the L-or D-CDP1 was identified (Table 4 and Supplementary Fig. S14 ). 

The purification of Crotamine has been described previously [31] . Briefly, Crotamine from crude Crotalus durissus terrificus venom obtained from CEVAP (Center for the Study of Venoms and Venomous Animals), Botucatu, Brazil, was isolated by a single cation-exchange chromatography step by utilising a MonoS HR 10/10 column (Amersham Biosciences).

Synthetic Crotamine derivative peptides (CDP) in the L-and D-enantiomeric conformations were synthesised by Genscript (Leiden, NL), with a purity of > 95% (Supplementary Fig. S15 ). The peptides were acetylated at the N-terminus and methylated at the C-terminus. Essential information about the CDPs used in this study is summarised in Supplementary Fig. S2, S3 and Table S2 .

SARS-CoV-2 3CL pro (Uniprot entry: P0DTD1, virus strain: hCoV-19/Wuhan/WIV04/2019) was cloned, expressed and purified as described previously [32] .

SARS-CoV-2 3CL pro activity assay was performed using a fluorogenic substrate DABCYL- 

Inhibition of SARS-CoV-2 3CL pro activity by Cro, L-and D-CDPs was investigated using the activity assay described above. 30 µM of the peptides was used for a preliminary screening test. 

The inhibition mode was determined using different final concentrations of the inhibitors and substrate. Briefly, 0.5 µM SARS-CoV-2 3CL pro was incubated with the inhibitor in various concentrations for 30 minutes at RT. Subsequently, the reaction was initiated by the addition of the corresponding concentration series of the substrate. The data were analysed using a Lineweaver-Burk plot; therefore, the reciprocal of velocity (1/V) vs the reciprocal of the substrate concentration (1/[S]) was compared [36, 37] . All measurements were performed in triplicate, and data are presented as mean ± SD.

Stable inhibition of SARS-CoV-2 3CL pro by L-/D-CDP1 and L-/D-CDP7 was monitored via a 24h inhibition experiment. Briefly, 0.5 µM SARS-CoV-2 3CL pro was incubated with 5 µM of the peptide and incubated for 1/2h, 1h, 2h, 3h, 4h, 5h and 24h at RT. The control was performed with the 3CL pro in the absence of the peptide and measured together after each time point. Subsequently, the reaction was initiated by the addition of the substrate. All measurements were performed in triplicate, and data are presented as mean ± SD.

A detergent-based control assay was performed to exclude inhibitors that possibly act as aggregators of the 3CL pro by adding 0.001%, 0.01%, and 0.1% of Triton X-100 to the reaction [38] . Four concentrations of L-CDP1 and D-CDP1 (0.5 µM, 1 µM, 5 µM and 10 µM) and L-CDP7 and D-CDP7 (0.25 µM, 0.5 µM, 1 µM and 5 µM), were tested. All measurements were performed in triplicate, and data are presented as mean ± SD.

Redox cycling compounds (RCCs) generate H2O2 in the presence of potent reducing agents (e.g.

DTT or TCEP). We performed a colourimetric assay to exclude L-CDP1, D-CDP1 and L-CDP7, D-CDP7 as a compound that induces redox cycling in reducing environments. 

CD measurements were carried out with a Jasco J-1100 Spectropolarimeter (Jasco, Germany).

Far-UV spectra were measured in 190 to 260 nm using a peptide concentration of 30 µM in H2O. The secondary structure of L-CDP1, L-CDP2, L-CDP7 and L-CDP8 and D-CDP1 and D-CDP7 was checked.

A 1 mm path length cell was used for the measurements; 15 repeat scans were obtained for each sample, and five scans were conducted to establish the respective baselines. The averaged baseline spectrum was subtracted from the averaged sample spectrum. The results are presented as molar ellipticity [θ], according to the equation (1):

[θ]λ = θ/(c*0.001*l*n) (1) where θ is the ellipticity measured at the wavelength λ (deg), c is the peptide concentration (mol/L), 0.001 is the cell path length (cm), and n is the number of amino acids. Secondary structure prediction based on the CD data was performed with the BeStSeL online tool [40] .

Cell viability assay was performed using the reduction of [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide -MTT] to investigate the cytotoxicity of D-CDP1 and D-CDP7. Therefore, (T/C) × 100 % (2) in which T and C represented the optical density of the treated well and control groups, respectively. The MTT assays for D-CDP1 and D-CDP-7 were performed as triplicates, and the results are shown as mean ± SD. 

All data are expressed as the mean ± the standard deviations (SDs). The statistical significance of the mean values' differences was assessed with one-way analyses of variance (ANOVA), followed by

Tukeys' multiple comparison test. Significant differences were considered at p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***). A standard two-tailed unpaired t-test was used to analyse the inhibitory effect of the D-CDP1 and D-CDP7 on SARS-CoV-2 replication. All statistical analyses were performed with GraphPad Prism software version 8 (San Diego, CA, USA).

The main protease (mpro) was retrieved from pdb databank (PDB code: 6M2Q), and the L-CDP1 was created using the crystal structure of the white type crotamie (PDB ID. 4GV5), the D-CDP1

was generated using the tleap in Amber. Docking was performed using HADDOCK webserver [48] .

The top four structures were downloaded and viewed by PyMOL. The H++ web server [49] was used to assign the correct lateral chain protonation of the amino acids to pH 7.0 for the simulated systems.

After that, the complex (mpro + L or D-peptide) was placed in an octahedral TIP3P water box with water extended 10 Å away from any solute atom and Cl-ions were added to neutralize the systems.

All the MD simulations were carried out by Amber18 [50] . Atomic interactions were represented by the FF19SB [51] force field. Starting structures were submitted in a two-step minimization process to remove bad contacts. In the first stage, the complex was restricted and minimized during 5000 steepest descending steps, followed by 5000 conjugated gradient steps, with a force constant of 10.0 kcal/mol-Ų. A second round of unconstrained energy minimization was performed during 10,000 steps. The system was slowly heated from 0 to 298 K for 0.5 ns under constant atom number, volume, and temperature (NVT) ensemble, with the complex constrained with a constant force of 10 kcal/mol-Ų.

Equilibration was divided into six steps of decreasing constant force for the complex atoms from 10 to 0 kcal/mol-Ų, and performed under constant atom number, pressure, and temperature (NPT) ensemble. Lastly, the production run was performed for 50 ns in the NVT ensemble without any restriction. In order to allow a 2 fs dynamic time interval the SHAKE restrictions were applied to all bonds with hydrogen atoms. The Particle mesh Ewald (PME) [52] method, with a 8 Šcutoff, was used to calculate the long-range electrostatics interactions. Temperature (298 K) was controlled by Langevin coupling and pressure (1 atm) was controlled by Berendsen barostat.

MD results were analyzed using CPPTRAJ [53] tools for the AmberTools19 package [54] . The Supplementary Materials: Figure S1 . Purification of SARS-Cov-2 3CL pro . Figure S2 . Primary structure

Abbreviation used: 3CL pro , C30 Endopeptidase/3C-like protease

SARS-CoV-2, severe acute respiratory syndrome-coronavirus-2

crotamine derivative D-enantiomeric peptide; WHO, World Health Organization; NSP, Nonstructural proteins; AMP

Influenza A virus

Cell-penetrating polypeptide

Surface plasmon resonance; KD, Dissociation constant; HIV-1, Human immunodeficiency virus 1

Redox cycling compound; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride

Minimum Inhibitory Concentration; dpi, Days post-infection

Root-mean-square deviation; RMSF, Root-meansquare flutuation

Radius of gyration

Center for the Study of Venoms and Venomous Animals; HRP-PR, Horseradish peroxidase-phenol red

Microscale thermophoresis; PBS, Phosphate buffered saline

4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

Fetal calf serum; DMEM, Dulbecco′s Modified Eagle′s Medium

Multiplicity of infection; NVT, Number, volume, and temperature

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