Virtual screening of inhibitors against spike glycoprotein of 2019 novel corona virus: a drug repurposing approach

The novel coronavirus (2019-nCoV) is a human and animal pathogen recently emerged in the city of Wuhan in Hubei province of China, causing a spectrum of severe respiratory illnesses. Corona viruses makes entry in to human cells through its spike (S) protein that binds to cell surface receptors. Wide spread of 2019-nCoV has been attributed to relatively high affinity of S protein to its receptor. Although S protein is a highly importantdrug target, unavailability of a high-resolution crystal structure and solvent accessible binding surface has made it a tedious target for current rapid virtual screening. A homology model of the receptor binding domain (RBD) of 2019 -n CoV S protein that is reasonably acceptable for drug screening was prepared using a high resolution crystal structure of SARS corona virus (SARS CoV)S protein. Data obtained from RBDreceptor docking experiments and published molecular dynamics experiments were used to map a RBD-receptor interaction hotspot that can be used for designing small molecule inhibitors. The hot spot was then used for virtual screening of more than 3000 drugs approved by U.S Food and Drug Administration (FDA) and other authorities for human use. Two anthracycline class drugs (zorubicin and aclarubicin) and a food dye (E 155) were predicted to be potent inhibitors of RBD – receptor interaction. Results of present study provide evidence for the potential of these compounds asprophylactic medications or for use to reduce disease severity of COVID -19.


Introduction
The outbreak of coronavirus disease  which was first reported from Wuhan, China has been reported to spread around the world having potential to become a pandemic (Chan et al, 2020). According to the World Health Organization (WHO) on February 27 th 2020, there had been more than 82,000 cases globally (WHO, 2020). The novel coronavirus has been recognized as a new member of betacoronavirus genus which is closely Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 3 March 2020 doi:10.20944/preprints202003.0042.v1 related to bat coronavirus and severe acute respiratory syndrome coronavirus (SARS CoV) (Lu et al, 2020). WHO has declared the 2019-nCoV as a Public Health Emergency ofInternational Concern (PHEIC) on the 30 th of January 2020 by considering its rapid transmission from human to human and spread into many of the continents (Sun et al,2020).
A group of Chinese scientists has recently sequenced the complete genome of 2019-nCoV and disclosed that the 2019-nCoV is a new human-infecting beta-coronavirus (Lu et al, 2020). It has been reported that the 2019-nCoV is adequately divergent from SARS-CoV.
Further, research groups have published evidence that the 2019-nCoV binds to the human angiotensin converting enzyme 2 (ACE-2) receptor through densely glycosylated spike (S) protein as the initiation step of the entry mechanism to human cells. (Lu et al, 2020). The S protein binds ACE-2 through its receptor binding domains (RBD) and the RBDup conformation of the S protein is a prerequisite for the formation of RBD-ACE-2 complex (Wrapp et al,2020) Wide spread of 2019-nCoV has been attributed to relatively high affinity of S protein to its receptor. Although highly important, unavailability of a high-resolution crystal structure and solvent accessible binding surface has made it a tedious target for current rapid virtual screening research groups.(Gruber and Steinkellner2019) Cryo EM structure of 2019-nCoV S protein (pre fusion down conformation) has been published at 3.5 A resolution (Wrapp et al,2020).Availability of several high resolution X-ray crystal structures of SARS CoV S protein (UP conformation) in complexed with ACE-2, structural rigidity and high degree of sequence similarity of RBDs warrants us to generate reasonably accurate homology models for drug screening.In the current study more than 3000 compounds approved by various regulatory authorities including U.S. Food and Drug Administration (FDA) were screened against an optimized homology model of the 2019 -nCoV S protein RBD.

Molecular modeling
Homology model of the RBD was constructed using modeler 9.23 Blundell, 1993, Webb andSali, 2016) and using the crystal structure (2.9 A o ) of BRD of SARS CoV (RBD domain complexed with ACE2 (PDB: 2AJF_E) as the template (76.47% sequence identity.).
As previous studies have shown to preserve key interactions between RBD and ACE-2 of SARS CoV and 2019-nCoV, RBDs of 2019-nCoV (homology model) and SARS CoV (2AJF_E) were superimposed on RBD of SARS CoV using match maker function of chimera (Peng et al,2020). Resulted BRD -ACE-2 complex was then energy minimized using chimera (400 steps of steepest descents, 50 steps of conjugated descents) while keeping the backbone of the ACE-2 fixed (Pettersen et al, 2004, Yang et al, 2012 using general Amber force field (James et al,2015) to obtain the final model (BRDACE2). Instead of using conformational ensembles, the single model generated by this approach was selected for virtual screening as it provides adequate evidence for a reliable model for virtual screening (see section 3.1).

Selection of ligand binding site
Spike protein was selected for virtual screening instead of selecting its druggable ACE -2 receptor since compounds that block ACE -2 receptor is known to have modulatory effect on blood pressureand several Analysis of interaction surface of BRDACE2 in chimera showed strong H bonding at TYR 505 which is well exposed indicating an initial contact point with ACE-2. Further H bonding was observed at THR 500, GLY 502, TYR 449, GLN 493 and GLN 498. This approach gives interactions in consistence with pervious protein -protein docking data (Xu et al, 2020) and data arising from molecular dynamics (MD) experiments (Peng et al, 2020). Further surface analysis of the structure reveled Arg 439 > ASN 439 mutation on one side of the spike-like protrude (SpkP). This mutation seems to significantly weaken the H bond formed at ASN 439. There for the key interactions were restricted to one side of SpkP. (Figure 1b). We

Binding interaction analysis
Two anthracycline compounds zorubicin, aclarubicin and an approved food dye E 115 were identified as potent inhibitors of BRD-ACE-2 interaction (Table 1) were not involved in forming interactions with RBD. This provides insights to further ligand optimization to improve binding and/or to reduce toxicity (Edwardso, 2015). Thus we propose strong evidence of zorubicin as a potent candidate for the inhibition RBD -ACE-2 interaction. (Table 1)      According to our previous experience with protein-protein interaction inhibitors, a two hit hypothesis (targeting at least two critical proteinproten interactions) was employed to select potent inhibitors of S protein and ACE-2. This approach when coupled with high energy binding (vina energy over -7 kcal/mol) provides a better tool for inhibiting relatively strong protein interactions that span through large surface area.
In conclusion, Anthracycline class drug zorubicin and aclarubicin and food dye E 115 were predicted to be potent inhibitors of RBD-Receptor interaction. Results of present study suggest the potential of these compounds as a asprophylactic medication or use in preventive measures.

Compliance and ethics
The authors declare that they have no conflict of interest.

5.Acknowledgement
Mr. KanchanaSenanayake. Assistant network manager of the Institute of Biochemistry, Molecular Biology and Biotechnology for providing computing facility