Anti-SARS and anti-HCV drugs repurposing against the Papain-like protease of the newly emerged coronavirus (2019-nCoV)

A new mysterious coronavirus outbreak started last month in China. The World Health Organization (WHO) termed the new virus strain 2019-nCoV to be the seventh reported human (HCoV). A seafood market in Wuhan city, central China was the starting point of the emergence with unknown animal causes the first animal to human infection. Until today 904 confirmed deaths and more than 40000 cases confirmed in China and 28 countries. There is a massive fear of the human to human transmission of 2019-nCoV that reported last week by the Chinese government. The most famous two strains of HCoV are the Severe Acute Respiratory Syndrome coronavirus (SARS CoV) and the Middle East Respiratory Syndrome coronavirus (MERS CoV). The former had emerged in China in 2002 while the latter emerged in the Middle East region in 2012 and south Korea in 2015. In this study, the newly emerged 2019-nCoV papain-like protease (PL pro ) is targeted by anti-SARS PL pro drugs and the anti-Hepatitis C Virus (HCV) Non-structural protein 3 (NS3) serine protease drugs. Sequence analysis, modeling, and docking are used to get a valid model for 2019-nCoV PL pro . The results suggest the effectiveness of the anti-SARS drugs (GRL-0667, GRL-0617, and Mycophenolic acid) and the anti-HCV drugs (Grazoprevir, Telaprevir, and Boceprevir) as potent inhibitors against the newly emerged coronavirus.

Until 20 days ago, 41 cases are confirmed to be 2019-nCoV positives leaving one dead and seven in critical care. This number is grossly increasing every day and the number of confirmed cases at the date of writing this manuscript is more than 1400 and 80 deaths are confirmed in China (50 infections confirmed outside China) 7 . The National Health Commission of China confirmed the human-to-human transmission of the Wuhan outbreak (2019-nCoV) five days ago 7 . The symptoms include fever, malaise, dry cough, shortness of breath, and respiratory distress 1 .
2019-nCoV is a member of Betacoronaviruses family such as the Severe Acute Respiratory Syndrome Human coronavirus (SARS HCoV (8000 infections from which 774 dead)) and the Middle-East Respiratory Syndrome Human coronavirus (MERS HCoV (2500 infections from which 858 dead)) 8,9 . All the human coronaviruses (HCoVs) are zoonotic viruses that transmit from animals to humans through direct contact. Until today, seven different strains of Human coronaviruses (HCoVs) have been reported, including the newly emerged 2019-nCoV 1,10 . 229E and NL63 strains of HCoVs belong to Alphacoronaviruses while OC43, HKU1, SARS, MERS, and 2019-nCoV HCoVs belong to Betacoronaviruses 1,8 . SARS and MERS HCoV are the most aggressive strains of coronaviruses, leaving about 800 deaths each. SARS HCoV has a 10% mortality rate, while MERS HCoV has a 36% mortality rate, according to the WHO 8, [10][11][12][13] . For the newly emerged coronavirus, the mortality rate is far lower (3%) than that of SARS and MERS but the fear is from its high transmission rate (human to human).
HCoVs generally are positive-sense single-stranded RNA (30kb) viruses. Two groups of protein characterize HCoVs; structural, such as Spike (S), Nucleocapsid (N) Matrix (M) and Envelope (E), and non-structural proteins such as RNA dependent RNA polymerase (RdRp) (nsp12) and the Papain-like protease PL pro 8 . PL pro is an essential enzyme in the life cycle of RNA viruses, including coronaviruses. PL pro is a multifunctional cysteine protease that processes the viral polyprotein and host cell proteins by hydrolysing the peptide and isopeptide bonds in viral and cellular substrates leading to the virus replication (Baez-Santos et al. 2014). PL pro is targeted in different coronaviruses viruses, including SARS, MERS, and HCV 14-18 .
In this study, the 2019-nCoV PL pro model is generated using homology modeling after sequence comparison to the solved structures in the protein data bank 19 . Molecular docking is then performed to test some drugs (anti-SARS PL pro and anti-HCV NS3) against 2019-nCoV PL pro . The results suggest possible inhibition for the currently available therapeutics against the newly emerged coronavirus in silico.

Sequence alignment and modeling
The deposited gene for the newly emerged 2019-nCoV NC_045512.2 (last updated 17 January 2020) is retrieved from the National Center for Biotechnology Information (NCBI) nucleotide database then translated using ExPASy translate tool 20 21 . Swiss Model is used to build a model for 2019-nCoV PL pro 22 . Using Basic Local Alignment Search Tool (BLAST) against the 2019-nCoV PL pro we found eight different solved structures for SARS PL pro (PDB IDs: 5TL6, 2FE8, 5E6J, 3MJ5, 5Y3E, 4M0W, 3E9S, and 4OVZ) that have at least 82.17% sequence identity to 2019-nCoV PL pro 23 . We choose the 5Y3E, chain A, because it is the best resolution (1.6 Å) structure among the eight SARS PL pro . Therefore, 5Y3E is  Figure 1A shows the pairwise sequence alignment of the PL pro of SARS and 2019-nCoV strains of coronavirus. SARS HCoV PL pro secondary structure is presented at the top of the alignment (PDB ID: 5Y3E chain: A) while its water accessibility is presented at the bottom with blue indicating highly accessible residues, cyan partially accessible while white for the buried residues. Three black-dashed rectangles mark the active site residues (C112, H273, and D287) of both SARS HCoV and 2019-nCoV PL pro . Figure 1B shows the pairwise sequence alignment of 2019-nCoV PL pro versus HCV NS3 (PDB ID: 3SU6). Orange-dashed rectangles surround the active site residues of HCV NS3 (H78, D102, and S159). As implied from the alignment, SARS PL pro versus 2019-nCoV PL pro shows high conservation (highlighted in red), called sequelogous. Despite the pairwise percent identity of 2019-nCoV PL pro against SARS HCoV PL pro is 82.8%, and only 11.85% for HCV NS3, the similarity to 2019-nCoV PL pro is 93.81% and 59.68% for SARS PL pro and HCV NS3, respectively. The active site triad C112, H273, and D287 of both SARS HCoV and 2019-nCoV PL pro (figure 1A) are partially surface accessible in order for PL pro to be able to attach its substrates for cleavage 41     As reflected from the docking scores, the six compounds can bind to 2019-nCoV PL pro , SARS HCoV PL pro , and HCV NS3 with good binding energy (-5.7 up to -11.0 kcal/mol). There is a reduction in the binding energies for the drugs to 2019-nCoV PL pro compared to other proteases. For GRL-0667, GRL-0617, and Mycophenolic acid, the reduction in the binding energies for 2019-nCoV PL pro is 25%, 21%, and 30%, respectively compared to SARS PL pro . While, in the case of HCV, the reduction in the 2019-nCoV PL pro binding energies is 18%, 11%, and 38% for Telaprevir, Boceprevir, and Grazoprevir, respectively. Despite this reduction, the PL pro of 2019-nCoV still able to bind the drugs with good binding energies (-5.7 up to -7 kcal/mol), which is enough to maintain the viral protein dysfunctionally.

2019-nCoV PL pro modeling
To further analyze the binding patterns, we examined the interaction complexes formed upon docking by the aid of the PLIP web server. Figures 4A and 4B   suggesting the possibility of these drugs (anti-SARS and anti-HCV drugs) to bind to and inhibit the function of the crucial viral enzyme PL pro . Table 1 The interactions formed between anti-SARS compounds (GRL-0667, GRL-0617, and Mycophenolic acid) and 2019-nCoV PL pro upon docking. The Star (*) represents salt bridges while the douple stars (**) represents π-π contact.  Table 2 The interactions formed between anti-HCV compounds (Telaprevir, Boceprevir, and Grazoprevir) and 2019-nCoV PLpro upon docking. The Star (*) represents salt bridges while the douple stars (**) represents π-π contact.

Conclusion
Wuhan's novel coronavirus has a significant health concern since the last outbreak of these types of    The interaction pattern for 2019-nCoV PLpro against the anti-SARS PLpro (A) and anti-HCV NS3 (B). PyMOL software is used to represent the docking poses using colored sticks.
Orange sticks represent the drugs while blue stick for the 2019-nCoV PLpro residues involved in the binding (labeled with its three-letter codes).