Potential inhibitors for 2019-nCoV coronavirus M protease from clinically approved medicines

Abstract
Starting from December 2019, a novel coronavirus, named 2019-nCoV, was found to cause Severe Acute Respiratory (SARI) symptoms and rapid pandemic in China.
With the hope to identify candidate drugs for 2019-nCoV, we adopted a computational approach to screen for available commercial medicines which may function as inhibitors for the M pro of 2019-nCoV. Up to 10 commercial medicines that may form hydrogen bounds to key residues within the binding pocket of 2019-nCoV M pro were identified, which may have higher mutation tolerance than lopinavir/ritonavir and may also function as inhibitors for other coronaviruses with similar M pro binding sites and pocket structures.
Coronavirinae, are enveloped positive-stranded RNA viruses which have spikes of glycoproteins projecting from their viral envelopes, thus exhibit a corona or halo-like appearance (Masters and Perlman, 2013; Cui et al., 2019) Coronaviruses are the causal pathogens for a wide spectrum of respiratory and gastrointestinal diseases in both wild and domestic animals, including birds, pigs, rodents, etc (Dhama et al., 2014). Previous studies have found that six strains of coronaviruses are capable to infect human, author/funder. All rights reserved. No reuse allowed without permission.

including four strains circulating yearly to cause common cold, and other two strains which the source for severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS-CoV), respectively (Cui et al., 2019; Dhama et al., 2014) .
Starting from December 2019, a novel coronavirus, which was later named 2019-nCoV ('n' stands for novel), was found to cause Severe Acute Respiratory (SARI) symptoms, including fever, dyspnea, asthenia and pneumonia among people in Wuhan, China (Zhu et al., 2020; Lu et al., 2020; Hui et al., 2020) . The first batch of patients infected by 2019-nCoV were almost all connected to a seafood market in Wuhan, which also trades wild animals. Later, contact transmission of 2019-nCoV among humans was confirmed, and the number of infected patients increased rapidly in Wuhan as well as other major cities in China. A series of actions have taken by the Chinese government to control the pandemic of the virus, and effective medical methods are in urgent needs to prevent 2019-nCoV infection and cure the disease.
Among all know RNA viruses, coronaviruses have the largest genomes ranging from 26 to 32 kb in length (Regenmortel et al., 2000; Schoeman and Fielding, 2019) .
Besides encoding structural proteins, majority part of the coronavirus genome is transcribed and translated into a polypeptide, which encodes proteins essential for viral replication and gene expression (Lai and Holmes, 2001) . The ~306 aa long main protease (M pro ), a key enzyme for coronavirus replication, is also encoded by the polypeptide and responsible for processing the polypeptide into functional proteins (Lai and Holmes, 2001) . The M pro has similar cleavage-site specificity to that of picornavirus 3C protease (3C pro ), thus is also known as 3C-like protease (3CL pro ) (Gorbalenya et al., 1989) . Studies have shown that M pro s of difference coronaviruses are highly conserved in terms of both sequences and 3D structures (Xue et al., 2008) . These features, together with its functional importance, have made M pro an attractive target for the design of anticoronaviral drugs (Xue et al., 2008; Anand et al., 2003) .
To present, these are still no clinically approved antibodies or drugs specific for coronaviruses, which makes it more difficult for curing 2019-nCoV caused diseases and controlling the associated pandemic. With the hope to identify candidate drugs for 2019-nCoV, we adopted a computational approach to screen for available commercial medicines which may function as inhibitors for the M pro of 2019-nCoV.
A previous attempt to predict drugs for the M pro of SARS-CoV has identified two HIV-1 protease inhibitors, namely lopinavir and ritonavir, as potential candidates, both of which bind to the same target site of M pro . Clinical application of these two drugs on 2019-nCoV patients also appears to be effective, whereas the nearby non-conserved amino acids locate far away from the binding pocket, thus would not affect its structural conservation (Figure 1b) . Virtual docking of lopinavir/ritonavir to 2019-nCoV M pro also showed high binding ability to the pocket site (Figure 1c) , similar to previous report for SARS-CoV M pro . Amino acids Thr24, Thr26, and Asn119 were predicted to be the key residues for binding the drugs (Figure 1c and Supplementary Figure 1) , forming 2 hydrogen bonds with lopinavir and 2 hydrogen bonds with ritonavir, respectively. author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the . Basing on these results, we performed virtual docking to screen for commercial medicines in the DrugBank database that could bind to the above mentioned pocket site of 2019-nCoV M pro , and identified 10 candidate clinical medicines (Table 1, Figure 1d and Supplementary Figure 2) . These drugs could form hydrogen bonds with one or more residues among Thr24-Asn28 and Asn119, theoretically, are capable to bind to author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.01.29.924100 doi: bioRxiv preprint the pocket formed by these amino acids and interfere the function of 2019-nCoV M pro .
In summary, basing on the structural information of clinical effective medicines for 2019-nCoV, we have predicted a list of commercial medicines which may function as inhibitors for 2019-nCoV by targeting its main protease M pro . Compared to lopinavir/ritonavir, most of these predicted drugs could form more hydrogen bounds with 2019-nCoV M pro , thus may have higher mutation tolerance than lopinavir/ritonavir.
The binding pockets of these drugs on M pro are conserved between SARS-CoV M pro and 2019-nCoV M pro , indicating the potential of these drugs to function as inhibitors for other coronaviruses with similar M pro binding sites and pocket structures. Table 1 . Predicted commercial medicines as potential inhibitors for 2019-nCoV M pro .
author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.01.29.924100 doi: bioRxiv preprint
The protein sequences of SARS-CoV M pro (Accession: 1UK3_A) and 2019-nCoV polyprotein orf1ab (Accession: YP_009724389.1) were downloaded from GenBank 
Molecular structures of commercial available medicines were downloaded from the DrugBank database (http://www.drugbank.ca) (Wishart et al., 2018) . The original indications of medicines were collected according to DrugBank descriptions. Virtual screening of medicines with binding potential to the pocket site of 2019-nCoV M pro was performed using the Libdock algorithm of Discovery Studio 3.5 (Accelrys Software Inc). The pocket site of 2019-nCoV M pro was identified by homology comparison to a previous published work . Spatial conformations of medicines were generated using CAESAR algorithm of Discovery Studio 3.5 using default parameters. Maximal hits of the docking process was set to 50.