key: cord-1045449-yw8hikop
authors: Soulère, Laurent; Barbier, Thibaut; Queneau, Yves
title: Docking-based virtual screening studies aiming at the covalent inhibition of SARS-CoV-2 M(Pro) by targeting the cysteine 145
date: 2021-02-20
journal: Comput Biol Chem
DOI: 10.1016/j.compbiolchem.2021.107463
sha: cdb75764ed3f51f1440c1c677c7a953ab1b7e051
doc_id: 1045449
cord_uid: yw8hikop

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 which has infected millions of people worldwide. The main protease of SARS-CoV-2 (M(Pro)) has been recognized as a key target for the development of antiviral compounds. Taking advantage of the X-ray crystal complex with reversible covalent inhibitors interacting with the catalytic cysteine 145 (Cys145), we explored flexible docking studies to select alternative compounds able to target this residue as covalent inhibitors. First, docking studies of three known electrophilic compounds led to results consistent with co-crystallized data validating the method for SARS-CoV-2 M(Pro) covalent inhibition. Then, libraries of soft electrophiles (overall 41 757 compounds) were submitted to docking-based virtual screening resulting in the identification of 17 molecules having their electrophilic group close to the Cys145 residue. We also investigated flexible docking studies of a focused approved covalent drugs library including 32 compounds with various electrophilic functional groups. Among them, the calculations resulted in the identification of four compounds, namely dimethylfumarate, fosfomycin, ibrutinib and saxagliptin, able first, to bind to the active site of the protein and second, to form a covalent bond with the catalytic cysteine.

COVID-19 is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). (Lai et al., 2020) In November 2020, this disease has infected more than 55 300 000people worldwide, including more than 1 300 000 deaths (https://covid19.who.int/).

Consequently, there is an urgent need to identify new anti-viral drugs targeting this virus. Several strategies for identifying coronavirus anti-viral drugs have been described in the literature and they have been recently reviewed by Thanigaimalai Pillaiyar and co-workers. (Pillaiyar et al., 2020) Among them, an important approach consist in inhibiting the SARS-CoV-2 main protease (M pro ) by peptide mimics or other types of compounds. (Lu et al., 2006; Pillaiyar et al., 2016) Several studies have been devoted to computational determination of potential inhibitors of the SARS-CoV-2 main protease such as computational drug repurposing studies, (Arun et al., 2020; Wang, 2020) structure-based virtual screening studies (Gahlawat et al., 2020; Ton et al., 2020) and docking studies of natural compounds. Ngo et al., 2020) A strategy of achieving irreversible inhibition of this protease has also been addressed by the design of compounds to create a covalent bond with the cysteine 145 residue (Cys145) of the catalytic dyad. (Pillaiyar, et al., 2020) While classical docking studies are widely reported in the literature, (Kitchen et al., 2004) docking studies for covalent protein inhibition are less common, (Kumalo et al., 2015; Sotriffer, 2018) in particular with SARS-CoV-2 main protease. Paul et al., 2020) Despite covalent inhibition approaches are less studied because the requirement of a nucleophilic residue is a structural limitation and they can be considered as harmful, the resurgence of covalent drugs encourage to also consider covalent inhibition. (Dalton et al., 2020; Ghosh et al., 2019; Singh et al., 2011) Irreversible specific protein inhibitors are now reported, such as in the case of the Ras protein possessing a G12C mutation, a J o u r n a l P r e -p r o o f 5 promising example of potential anticancer strategy. (Goody et al., 2019) Recent studies describe the inactivation of the SARS-CoV-2 M Pro with either the N3 inhibitor, (Jin et al., 2020) originally discovered for SARS-CoV, (Yang et al., 2005) or the alpha-ketoamide inhibitor 1 (Figure 1 ).(L. Zhang et al., 2020) These studies show the importance of the catalytic Cys145 residue to design covalent inhibitors. Taking advantage of the X-ray crystal structure of the complex M Procompound 1,(L. Zhang, et al., 2020) we report herein flexible docking studies to identify potential irreversible inhibitors using electrophilic compounds libraries.

J o u r n a l P r e -p r o o f Zhang, et al., 2020) 

The main protein of SARS-CoV-2 M Pro was obtained from the protein data bank (PDB code 6Y2F, 5RHF, 5REN, 5REK).

Compounds 2-4 were generated as 3D mol file using Arguslab (Thompson, 2004) and they were then docked within the M Pro active site (PDB codes 5RHF, 5REN, 5REK respectively) using Arguslab software with the Argusdock engine with default parameters. The obtained binding modes were compared to crystallographic data to validate the method. The distance between the sulfur and the methylene group of the chloroacetamide group was monitored to establish the structural bases of the M pro covalent inhibition (distance < 4 Å). The distance between the sulfur and the electrophilic center was monitored and compounds were selected when a distance < 4 Å was measured. Binding modes were examined with PyMOL and hydrogen bonds networks were generated using LigPlot +. (Laskowski et al., 2011) Docking studies of the approved covalent drugs library

The library was constructed based on the list of covalent drugs established by Vasudevan and coworkers, (Vasudevan et al., 2019) from which β-lactam derivatives, drugs withdrawn from market and mechanism based covalent drugs were excluded. For five of the 29 remaining compounds, their metabolites which are the active covalent binders (Shin et al., 2013) were used in the library. Overall, 32 compounds were used for the docking studies. When available, the SDF file for each compound was obtained from PubChem and was converted to PDB file using Accelrys Visualizer 2.0. For omeprazole, lansoprazole, pantoprazole and rabeprazole active metabolites, they were drawn using Vega ZZ (Pedretti et al., 2002 (Pedretti et al., , 2004 and were saved as PDB files. The 32 compounds were then docked within the M Pro active site centered on the β-keto amide 1 using Arguslab software (Thompson, 2004) with the Argusdock engine with default parameters. The distance between the sulfur and the electrophilic center was monitored and compounds were selected when a distance < 4 Å was measured. Binding modes were examined with PyMOL and hydrogen bonds networks were generated using LigPlot +. (Laskowski and Swindells, 2011) 

When applicable, the search for covalent inhibitors by docking can be carried out according to the scheme depicted in Figure 2 by targeting a nucleophilic residue such as a cysteine residue. It is then necessary that the electrophilic center is placed at the vicinity of the thiol function of this residue. This approach can be investigated with the M Pro protein that contains a catalytic cysteine residue (Cys145). In order to explore the potentialities to covalently inhibit M Pro , a flexible docking of compounds was performed to establish the structural bases for binding recognition with high affinity to improve the selectivity to the molecular target to form the complex M Pro + I. 

In order to explore the potentialities to covalently inhibit SARS-CoV-2 M Pro , we first studied docking of the covalent inhibitors 2-4 co-crystallized within the protease to establish the J o u r n a l P r e -p r o o f 9 structural bases for the covalent inhibition of this protein. (Douangamath et al., 2020) In particular, the location of the electrophilic moiety of compounds i.e. the distance between the electrophilic center and the thiol of Cys145 was investigated. Docking experiments were thus conducted on these three compounds; the results are depicted in Figure 3 . These experiments show consistent results with the crystallographic data (PDB codes 5RHF, 5REN, 5REK respectively) and show that the electrophilic center of compounds i.e. the methylene group of the chloroacetamide functional group is located at the vicinity of Cys145 with distance values between the carbon and the thiol atoms ranging from 2.83 Å to 3.19 Å.

J o u r n a l P r e -p r o o f Based on these docking experiments of compounds 2-4, we defined the structural basis for the covalent inhibition of Mpro as 1) binding the protein with a good affinity and 2) having an adequate orientation of the electrophilic moiety towards Cys145 with a distance inferior to 4 Å with the sulfur (Figure 2 ).

We then investigated docking-based virtual screening studies for covalent inhibition of M Pro using libraries of electrophilic inhibitors (Table 1) 

Binding mode of each compound was carefully examined in terms of hydrogen bonds with the protein M Pro , and the distance between the thiol and the electrophilic center was also measured (Table 2) . Hydrogen bonds numbers were ranging from one to six for the compound 134294169.

The docking score was found to be variable depending of the compounds library and the structure of the compounds. These values were ranging from -7.22 to -10.41 kcal.mol -1 for the compound 1102141. As example, we chose to depict the binding modes of two compounds with high docking score values. The binding modes of the compound 2011299, which exhibits a low distance between the thiol and the electrophilic center and the one of the compound 134294169 with six hydrogen bonds, are described in Figure 5 . 

Based on the approach described in Figure 2 , we investigated a library of known approved covalent drugs. For this, we used a list of 52 FDA (Food and Drug administration) approved drugs described by Anil Vasudevan and colleagues in 2019. (Vasudevan, et al., 2019) Among these, we excluded some molecules for our study, such as drugs withdrawn from the market and drugs with a mechanism-based inhibition, resulting in a small library of 32 compounds. On this library, the workflow described in Figure 2 was applied by means of flexible docking J o u r n a l P r e -p r o o f experiments and subsequent distance measurement between the Cys145 residue and the electrophilic center. Following this experiment, four compounds showed interesting results and they are presented in Table 3 . showing their potentialities to act as covalent inhibitors. The 2D-representation is described in Figure 6 for the four compounds, showing the hydrogen bonds networks.

J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f These four compounds are approved drugs for a designated pathology (Table 4 ) and some literature data can be found about relationships between COVID-19 and these compounds. First, Vittorio Mantero and colleagues reported that the use of dimethylfumarate for patients suffering from multiple sclerosis seems to have a positive impact against COVID-19. (Mantero et al., 2020) For ibrutinib, two studies shows that this compound seems to have a protective role against COVID-19, although they hypothesize that it may be due to the anti-inflammatory effect of the Bruton's tyrosine kinase pathway inhibition. (Thibaud et al., 2020; Treon et al., 2020 

None to declare.

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The authors are grateful to CNRS, MESRI for financial support and to the MESRI for a scholarship to Thibaut Barbier.