key: cord-305091-tfn2pyc6
authors: Tripathi, Praveen Kumar; Upadhyay, Saurabh; Singh, Manju; Raghavendhar, Siva; Bhardwaj, Mohit; Sharma, Pradeep; Patel, Ashok Kumar
title: Screening and evaluation of approved drugs as inhibitors of main protease of SARS-CoV-2
date: 2020-12-01
journal: Int J Biol Macromol
DOI: 10.1016/j.ijbiomac.2020.08.166
sha: 
doc_id: 305091
cord_uid: tfn2pyc6

The COVID-19 pandemic caused by SARS-CoV-2 has emerged as a global catastrophe. The virus requires main protease for processing the viral polyproteins PP1A and PP1AB translated from the viral RNA. In search of a quick, safe and successful therapeutic agent; we screened various clinically approved drugs for the in-vitro inhibitory effect on 3CL(Pro) which may be able to halt virus replication. The methods used includes protease activity assay, fluorescence quenching, surface plasmon resonance (SPR), Thermofluor® Assay, Size exclusion chromatography and in-silico docking studies. We found that Teicoplanin as most effective drug with IC(50) ~ 1.5 μM. Additionally, through fluorescence quenching Stern–Volmer quenching constant (K(SV)) for Teicoplanin was estimated as 2.5 × 10(5) L·mol(−1), which suggests a relatively high affinity between Teicoplanin and 3CL(Pro) protease. The SPR shows good interaction between Teicoplanin and 3CL(Pro) with K(D) ~ 1.6 μM. Our results provide critical insights into the mechanism of action of Teicoplanin as a potential therapeutic against COVID-19. We found that Teicoplanin is about 10–20 fold more potent in inhibiting protease activity than other drugs in use, such as lopinavir, hydroxychloroquine, chloroquine, azithromycin, atazanavir etc. Therefore, Teicoplanin emerged as the best inhibitor among all drug molecules we screened against 3CL(Pro) of SARS-CoV-2.

COVID-19 is an infectious disease caused by a newly discovered positive-sense single-stranded RNA virus called the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). The disease has emerged as a pandemic and causes respiratory complications in a majority of the cases and has caused lakhs of deaths worldwide owing to the lack of any treatment option. The family Coronaviridae under the order Nidovirales contains the members of Coronavirus (CoV) that are a potential health concern to human beings and possibly to other animals [1, 2] , having been responsible diseases such as Severe Acute Respiratory Syndrome (China) and Middle-East Respiratory Syndrome (MERS) [3] . Bats and other animals are natural reservoirs for CoVs, and SARS-CoV-2 [4] . However, the reported route of transmission till date is human to human that occurs by sneezing, coughing and spread of respiratory aerosols.

The symptoms manifested by infection of SARS-CoV-2 include the alteration in lung functioning, localized lesions, pneumonia, bronchiolitis and these are presented in a majority of the patients. The virus infects the lung endothelial cells and induces a pathological state like the lymphocytic endothelialitis and inflammatory cell invasion [5] . The SARS-CoV-2 infects multiple organs and recruits enormous numbers of immune cells and complexes in these organs [4] . It may also show central nervous system invasion can be presented in advanced stages of the CoV infection [6] . Other mild manifestations of the COVID-19 include fever, dry cough, dyspnoea, myalgia and fatigue. The hematological and serological examination reveals the augmentation in the levels of lactate dehydrogenase, serum amyloid A (SAA), and thrombocytopenia [7, 8] .

SARS-CoV-2 comprising of about 30000 RNA, encodes for about 66 % nonstructural region. The nsP5 is chymotrypsin-like (CL) and possesses cysteine protease activity [9] . It is called the main protease or 3CL Pro. This protease is essential for the J o u r n a l P r e -p r o o f Journal Pre-proof processing of polyproteins PP1A and PP1B, translated from the RNA of the virus. The 3CL Pro is very important for virus to replicate and propagate and its inhibitors may therefore be able to halt the replication of the virus. It recognizes and cleaves the virus non-structural polyprotein at 12 sites. It generally acts on the sequence Leu-Gln*(Ser, Ala, Gly) (* denotes the cleavage site). Due to its role in initiation events of viral replication, it is an attractive drug target. Besides 3CL Pro , nucleocapsid protein (N), envelope protein (E), spike glycoprotein (S), membrane protein (M), and two isoforms of replicase polyprotein, namely 1a and 1ab are considered as potential drug/vaccine targets [10] . Its indispensable role in the initiation events of the replication cycle makes it an attractive drug target [11] . 3CL Pro is an attractive and relatively safer drug target because its recognition sequence is dissimilar to any of the proteases in the human body.

A number of clinically approved drugs are being tested for their potential to ameliorate the effects of the SARS-CoV-2 infection. We tested the different classes of drugs- Hydroxychloroquine, Chloroquine, Nitazoxanide, oseltamivir, amoxicillin, famciclovir, aciclovir, lopinavir, atazanavir, and azithromycin [12] have been screened in this study. Here, we present Teicoplanin as a potent inhibitor of main protease and speculate its role as to halt the replication of the virus.

SARS-CoV-2 3CL Pro gene was cloned between BamHI and XhoI sites in pET28a vector having N terminal 6X-His tag, FLAG tag and PreScission protease tag. Protein was transformed in BL-21DE3 Rosetta RIL cells and autoinduction method was used for expression and purification. The culture was grown at 37 °C for 2-3 hours and 18 °C for overnight. The cell pellet after centrifugation at 10000 x g for 30 minutes, was resuspended in 50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol, 10 mM Imidazole, 1mM DTT, 0.5 mM PMSF, 10 ug DNaseI and lysozyme (1mg/mL). After 30 minutes incubation, it was sonicated at 50 % duty cycles with 30 sec ON and 30 sec OFF for 20 minutes. The supernatant was applied on Ni-NTA columns in FPLC system (ÄKTA TM start, GE Healthcare). The bound proteins was eluted with 300 mM imidazole gradient. The protein fractions were applied on Q-FF ion-exchange chromatography, eluted with 500 mM NaCl gradient. The His tag was cleaved with PreScission protease and subjected to size exclusion chromatography (SEC) (Superdex TM 75 10/300 GL, GE Healthcare). Homogeneous fractions were pooled, dialyzed and kept at -80°C for all biochemical and biophysical studies.

3CL Pro amino acid sequence is highly conserved between SARS-CoV and SARS-CoV-2, it cleaves 11 sites in the polyproteins to result many functional proteins, including helicase, RNA-dependent RNA polymerase, protease, RNA-binding protein, exoribonuclease, endo-ribonuclease, and 2′-O-ribose methyltransferase etc. 3CL pro belongs to cysteine protease category and is excised from polyproteins by its own proteolytic activity, further exhibiting specificity for its own C-terminal auto processing. Various peptide cleavage sites has been reported in literature for the in-vitro 3CL Pro protease activity J o u r n a l P r e -p r o o f Journal Pre-proof measurement [13] . In this study cleavage sequence MYTPHTVLQ↓AVGACVLCN ( cleavage at nsp12/nsp13) has been used for FRET based protease assay.

A well-established method for assessment of protease activity by 3CL Pro of SARS-CoV has been described previously [15] . Thus, a custom synthesized fluorogenic peptide 

Through the sequence analysis, we found that casein substrate has some cleavage junction peptides matching with the consensus cleavage sequences of 3CL Pro of SARS-CoV-2. Hence, we have used the casein substrate also for the protease activity measurements. The protease activity of 3CL Pro was previously established by using casein substrate [14, 15] concentration for an hour followed by addition of 135 µM of casein in 400 µl phosphate buffer at pH 7.5, incubated at 37 °C for an hour before terminating the reaction with 200 µl of 1.7% trichloro-acetic acid and absorbance measurements was done as above. The enzyme inhibitory potential IC 50 value was calculated by fitting the curve of percentage residual activity with inhibitor concentration.

Fluorescence quenching is routinely used to ascertain the effect of a particular drug on the target protein. The impact of the drug on the protein can be dynamic due to collisions between the fluorophore (protein) and the quencher (drug), or it may be static, owing to the protein and the quencher forming a complex at ground-state [16, 17] . An intense quenching of the fluorescence marked an unvarying local dielectric environment. The tertiary structure perturbations post-interaction to ligands were assessed using the principle of intrinsic fluorescence of 3CL Pro which has three tryptophan residues which contribute to its hydrophobicity and fluorescence properties. In the fluorescence quenching experiment 1 µM of 3CL Pro was allowed to interact with varying concentrations of drugs. Fluorescence signals were monitored at 295 nm excitation with emission scan between 310 to 400 nm using Cary Eclipse fluorescence spectrophotometer (Agilent technologies).

The binding between 3CL Pro and Teicoplanin was measured using surface plasmon resonance spectroscopy (SPR) Biacore 3000 (GE Healthcare.). For our SPR experiments, 3CL Pro protein (10mg/mL) solution in PBS buffer pH 7.4 was first immobilized to a CM5 gold chip using EDC-NHS coupling and the binding of varying concentrations of Teicoplanin was tested. The resultant sensorgram was analysed using BIA evaluation software to determine the binding constants. 

Bimolecular interactions of 3CL Pro and Teicoplanin were measured by Thermofluor® assay [18] . 3CL Pro enzyme was incubated with Teicoplanin and 20 X SYPRO® Orange Protein Gel Stain (S5692) in buffer and thermal scanning was performed from 25 ˚C to 99 ˚C at an interval of 0.5 ˚C/min with hold of 0.5 minute at each measurement. The shift in melting curve was plotted and analyzed.

The backbone chain without water or other drugs of 3CL Pro (PDB: 6LU7) was used for docking studies using ParDOCK + . The best prediction coordinates were in agreement with the reported active sites i.e. His41 and Cys145. The coordinate near the active site was fixed and subjected to site-specific docking by ParDOCK +. The 3-Dimensional structure of Teicoplanin (Pubchem ID: 16152170) was used as a ligand for docking. The partial charge model used was AM1BCC with minimization cycles of 2500. The four best poses were received and analyzed.

The protein 3CL Pro was cloned in pET28a vector and purified using affinity, ion exchange and SEC (Fig.1) . The relative molecular weight of the protein was observed ~ 34kDa on 12% SD-PAGE. The protein identity was confirmed with western blot using FLAG antibody (DYKDDDDK , # 2368 Cell Signaling Technology). Table 1 ). The effect of selected 11 inhibitors on the enzymatic activity of 3CL Pro was monitored by peptide substrate and casein substrate (Fig. 2, A-D) . Teicoplanin emerged as the best inhibitor in our studies with an IC 50 value of 1.61 ± 0.09 µM and 1.46 ± 0.05 µM with peptide and casein substrate respectively (Fig. 2E, 2F) . Teicoplanin was found to be J o u r n a l P r e -p r o o f more effective among the currently used/ proposed anti-COVID19 drugs such as Lopinavir, Hydroxychloroquine, Chloroquine, Atazanavir, Azithromycin.

The fluorescence quenching spectra of 3CL Pro protease titrated to increasing concentrations of various molecules are shown in Fig. 3 . The overall structural perturbations on the 3CL Pro with ligands were assessed by fluorescence quenching. Upon titration with Teicoplanin there was a gradual decrease in fluorescence intensity with concentration (Fig.   3A) . A significant quenching of the 3CL Pro fluorescence emission was observed with

Teicoplanin. This observation indicates the probable involvement of Trp residues of protein in the conformational dynamics. The concentration-dependent quenching was also observed in case of lopinavir, hydroxychloroquine and chloroquine (Fig. 3 B-D) . The interaction of 3CL Pro with hydroxychloroquine and chloroquine led to protein conformation changes from native state to unfolded state (332 nm to 360 nm peak transitions). This depicts that three tryptophan residues in 3CL Pro are exposed more in unfolded state with hydroxychloroquine and chloroquine. The other drugs like aciclovir, famciclovir, atazanavir, and azithromycin displayed some quenching (Fig. 3 E-H) . Rest of the drugs were not able to display any observable changes in the protein (Fig. 3 I-K) .

Equations [19] . 

The maximum fluorescence intensity at 333 nm was used for all K SV calculations.

The Stern-Volmer plots for fluorescence quenching by all inhibitors are shown in Fig. 3 L.

The magnitude of K SV Stern-Volmer quenching constant for Teicoplanin was estimated 2.5 × 10 5 L·mol −1 , ( Table 2 ) which suggests a relatively high affinity between Teicoplanin and 3CL Pro protease. The strongest effect was observed with Teicoplanin while the others show a diminished effect.

Surface plasmon resonance was employed to monitor the molecular interactions in real-time between 3CL Pro and the identified ligand molecule. SPR is generally used to determine the binding specificity of molecules, including rates of association and dissociation between drug molecules and target proteins. The effect of Teicoplanin on the 3CL Pro protein was analysed using Thermofluor assay® which relies upon the binding of SYPRO® dye to exposed hydrophobic patches on heating. The melting temperature (T M ) of native protein was recorded ~57 ˚C, whereas the same for the Teicoplanin-3CL Pro complex was found ~ 49 ˚C (Fig. 4B) . The interaction of inhibitors shifts the melting temperature which is a signature of protein stability. The binding doesn't alter the monomeric conformation of 3CL Pro as depicted by SEC (Fig. 4C) .

Our docking analysis using PARDock revealed that the ligand drug Teicoplanin fits perfectly in the active site pocket (Fig.4D ) and the binding energy obtained for this binding is about -8 kcal/mol. The cavity is lined by the active site amino acids i.e. histidine 41 and cysteine 145 (Fig. 4E) . The residues His41 and Cys145 form the catalytic dyad, form the substrate binding region and are located at the cleft of domain I and II in which His acts as a proton acceptor while Cys behaves as a nucleophile. Previously, it is reported that Teicoplanin is well docked with SARS-CoV-2 main protease using AutoDock4, AutoDock

Vina and Dock6 docking programs, which also revealed that docked Teicoplanin is well J o u r n a l P r e -p r o o f accommodated within the inhibitor binding cavity in a manner similar a Michael acceptor inhibitor-known as N3 which is a potent and irreversible inhibitor of SARS-CoV-2 [20] .

Teicoplanin displays a bonding with the protein at the molecular level, via hydrophobic interactions, hydrogen bonding and halogen bonding. The hydrophobic interactions are seen with Asp187 and Glu166 (Fig. 4F) . These two residues are also involved in the formation of hydrogen bonds. The involvement of H-bond donors and acceptors around hydrophobic sites compel the Teicoplanin molecule to interact within the inhibitor binding pocket of the 3CL Pro protease. The formation of extensive hydrogen bonds with active site residues has been observed previously with Arg40 and Gly170 residues. In a recent MD simulation study, it was reported that Teicoplanin in complex with SARS-CoV-2 main protease has stable ligand-protein complex and intermolecular interactions during the simulated trajectory [21] .

Further our careful analysis revealed that the Leu141 and Ser144 residues of 3CL Pro form a halogen bond with the Teicoplanin molecule. It has been established that the halogen bond interactions are critical for the potential activity of the protease inhibitors. The halogen bond interaction characteristic of an inhibitor compound makes it more promising candidate as a novel anti-viral therapeutic [22] . We observed quite significant and different kinds of interactions between the Teicoplanin molecule and the 3CL Pro protein suggesting that Teicoplanin can act as a good inhibitor of 3CL Pro . These interactions around and at the active site amino acids most probably hinder the proton transfer and binding of substrate to the active site, leading to disruption in the protease activity.

The relative efficacy of the drugs to inhibit the protease activity of main protease of SARS-CoV-2 was analysed using protease assay. Teicoplanin emerged as the most potent inhibitor in our involving 23 drugs (Table 1) . As compared to Teicoplanin with IC 50 value at 1.5 µM as 100 percent, lopinavir displayed only 9.12% activity inhibition and hydroxychloroquine showed 4.56% inhibition (Fig. 5) . The other drugs showed less than 5 % inhibition.

In this study, we have used a wide variety of drugs indicated in different infectious diseases such as retroviral HIV infections, bacterial infections, etc. and have used biochemical and biophysical analysis to compare these 23 drugs in terms of activity on the casein substrate, FRET peptide substrate as well as drug-protein interactions using fluorescence quenching. We have found that under similar laboratory conditions, Teicoplanin was found to be a much stronger inhibitor of 3CL Pro as compared to the other drugs like Hydroxychloroquine, Lopinavir, Chloroquine, Azithromycin, Atazanavir etc.

The COVID-19 pandemic has become a major challenge for healthcare workers and There are many approved drugs for other viral infections such as Ebola, HIV and Influenza suggested for tackling COVID-19. The secondary infections resulting from the COVID-19 infection is also contributing to fatality [23] . Therefore, searching for drugs with antiviral properties with minimum side effects would be an ideal solution. Teicoplanin is an effective glycopeptide antibiotic with reported anti-MERS CoV activity [24, 25] . We report Teicoplanin as an effective drug against 3CL Pro which works at a micromolar concentration of 1.5 µM (Fig. 2) and acts by blocking the active site of the protease (Fig. 4F) .

We studied protein-drug interactions in order to decipher the working of the drug and mechanism of inhibition at the molecular level. The protein-drug interaction shows fluorescence quenching by monitoring the intrinsic fluorescence, Teicoplanin interaction is gradual however the interaction of hydroxychloroquine and chloroquine is perturbing the conformational dynamics as depicted by wavelength shifts (Fig. 3) . The SPR finding suggests that the good affinity of Teicoplanin with 3CL Pro (Fig. 4A) . Lopinavir is another antiretroviral drug that acts by targeting HIV protease. It was under clinical trial for COVID-19 which concluded that its beneficial effects were minimal [26] . Hydroxychloroquine is in an ongoing drug trial and is currently included in the prime regimen of drugs used in the management of COVID-19. The proposed mechanism of action of the drug is the alteration in the pH of endosome thereby preventing the uncapping of capsid and genome release [27, 28] .

We found that the relative potency of lopinavir and hydroxychloroquine to inhibit the SARS-CoV-2 main protease was 9.12% and 4.56%, respectively (Fig. 5) . The activity of other drugs was found negligible or undetectable. These drugs have already proven their potential in the treatment of different DNA and RNA virus diseases which further supports their anti-viral capabilities.

In our study, Teicoplanin showed significant reduction of the proteolytic activity of 3CL Pro . As this protein is essential to the replication cycle of the virus, due to its irreplaceable role in the processing of viral polyproteins, which the virus needs to complete its life cycle, we propose that this reduction may contribute to its anti-SARS-CoV-2 effect via inhibition of the viral replication. We also observed some inhibitory activity in other drugs that we tested but as Teicoplanin was found most potent among them. Further experiments in higher validation systems are required to establish the efficacy of the molecule. We advocate the expedited systematic investigation in primate models and trials in terminally ill patients suffering from COVID-19.

Recently, the effect of Teicoplanin with respect to SARS-CoV-2 has been observed by other groups as well. In a nutshell, all these studies support each other that Teicoplanin might be a potential therapeutic option against COVID-19.

The approved drug Teicoplanin has already been tested for toxicity in human beings over a period of time. We propose Teicoplanin as an effective drug against SARS CoV-2. The study paves the path for an expedited research to an effective treatment of COVID-19. We also propose immediate attention for the randomized clinical trials for this drug to reduce infections and to help the society, as it has a proven mechanism of action and efficacy as compared to other recommended drugs for COVID-19. 

Authors declare no conflict of interest. J o u r n a l P r e -p r o o f

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