Drug repurposing against MERS CoV and SARS-COV-2 PL pro in silico

Aim: The Middle East Respiratory Syndrome coronavirus (MERS-CoV) and COVID-19 cause severe acute, deadly, pneumonia. Papain-like protease (PL pro ), is HCoV cysteine protease encoded within the Non-Structural protein 3. Materials and Methods: Molecular docking is performed to test the binding performance of six protease inhibitors against MERS CoV and SARS-CoV-2 PL pro . Results: The compound, GRL-0667, shows the highest binding affinity to MERS CoV PL pro , while Grazoprevir shows the highest binding affinity against HCV NS3. Moreover, the interaction pattern in the case of HCV NS3 is the same as in the case of coronaviruses. Conclusion: The present study shows the ability of some anti-SARS CoV and anti-HCV NS3 drugs to inhibit MERS CoV PL pro , interestingly, including the newly emerged SARS-COV-2 PL pro .

On the other hand, SARS-CoV and MERS-CoV only express one PL pro . The MERS-CoV encodes many types of proteins such as the Mpro, which includes the chymotrypsin-like protease (3CL protease), and the PL pro [18]. The first three positions between non-structural proteins are cleaved by PL pro , while the other eleven positions are cleaved by 3CL pro [18]. PL pro is a multifunctional cysteine protease that processes the viral polyprotein and hosts cell proteins by hydrolyzing the peptide and isopeptide bonds in viral and cellular substrates leading to viral replication [18]. This is required for the cleavage of the polyprotein into its 16 viral proteins that involved in the formation of a membrane-associated cytoplasmic enzyme complex (the replicase complex), which is responsible for directing the replication and transcription of the viral genome [4,18]. PL pro is recognized to be involved in deubiquitination, de-ISGylation, and viral evasion of the innate immune response [18]. Despite the presence of several solved structures for SARS and MERS PL pro , no solved structure is available (at the time of writing this manuscript) for the SARS-COV-2 PL pro .
Most of the drugs that inhibit SARS and MERS-CoV targets are encoded in the 50-terminal two-thirds of the genome, within the two open reading frames (ORF1a and ORF1ab) that encode for the NSPs.
Telaprevir and Boceprevir have approved inhibitors against Hepatitis C Virus (HCV) in the year 2011 by the US Food and Drugs Administration (FDA), while Grazoprevir was approved by FDA in 2016, against HCV as well [19][20][21][22]. Grazoprevir is a direct-acting antiviral drug used as part of combination therapy to HCV. It is an inhibitor of NS3/4A, a serine protease, encoded by HCV genotypes 1 and 4 [19]. Additionally, Telaprevir and Boceprevir belong to the class of organic compounds known as hybrid peptides. [19,[23][24][25]. On the other hand, mycophenolic acid is an antibiotic compound derived from Penicillium stoloniferum. It blocks the de novo biosynthesis of purine nucleotides as it inhibits the enzyme inosine monophosphate dehydrogenase [26]. Mycophenolic acid, in addition to the two dioxolane derivatives (GRL-0667 and GRL-0617), inhibits the SARS PL pro [27].

Materials And Methods
MERS CoV PL pro sequences (FASTA) and coordinates (PDB) are downloaded from the Protein Data Bank database [28]. The PDB IDs of the downloaded MERS CoV PL pro are (4RNA, 5KO3, 4RF1, 5V69,   4PT5, 4REZ, 4RF0, 4R3D, 6BI8 and 4WUR). The resolution of the structures ranges from 1.79Å to 3.16Å, and the structures are solved during the last five years. Multiple Sequence Alignment (MSA) is carried out for the downloaded sequences using the T-Coffee web server [29]. The MSA is represented by the ESpript 3.0 web server, which provides enhanced graphical information for the aligned sequences [30]. By the aid of PyMOL software [31], PL pro structures are prepared for the docking step.
This includes removing water molecules, ions, and ligands. Additionally, polar Hydrogens are added, and the processed structures are saved in the PDB format for the docking study. Zn +2 ions are retained in the structures due to its crucial role in protein function [32][33][34].
AutoDock vina [35] is used in this study to perform molecular docking of N-(1,3-benzodioxol-5- Protein-Ligand Interaction Profiler (PLIP) web server is utilized to analyze the docking complexes [37].   Table 1 shows the amino acids involved in the interactions with GRL-0667 for MERS PL pro , SARS PL pro , SARS-COV-2 PL pro, and HCV NS3. Each interacting residue is listed with the number of formed interactions. The interactions involve Hydrophobic, H-bonding, salt bridges and π-π stacking. Table 1 The interactions formed between GRL-0667 and MERS PL pro , SARS PL pro , SARS-CoV-2 PL pro , and HCV NS3 upon docking. The Star (*) represents salt bridges while the douple stars (**) represent π-π contact. are reported to be the active site triad residues in MERS CoV PL pro [1,2]. As shown in figure 1A, the active site residues are water accessible for all solved structures of MERS CoV PL pro (cyan colored boxes under the residues H278, and D293). Additionally, the active site residues C112, H273, and D287 in SARS CoV PL pro (PDB ID: 5Y3Q (see figure 1B)) and H1057, and D1081 in HCV NS3/4A protease (PDB ID: 6DIQ (see figure 1C)) are water accessible (either blue or cyan boxes under the mentioned residues, where the relative accessibility is < 0.1). Exceptions are the C111 in MERS CoV PL pro and S1139 in HCV NS3/4A protease where the relative accessibility is less than 0.1 (white boxes below the pairwise alignment of figures 1A, and 1C).
In an attempt to test the drugs against the newly emerged Wuhan coronavirus (SARS-COV-2), we built a model for SARS-COV-2 PL pro then tested the compounds. The best (based on the binding energy) compound is found to be GRL-0667 with a binding energy of -7 kcal/mol. This value is in the same range of that for MERS HCoV PL pro (-7.62 ± 0.65 kcal/mol) and slightly less than that of SARS HCoV PL pro (-9.3 kcal/mol). Table 1 shows the detailed interaction patterns of GRL-0667 with all PL pro of the coronaviruses under the study and HCV NS3. For MERS PL pro , as indicated in Table 1, some residues form H-bonds, salt bridges, and hydrophobic contacts with GRL-0667 in most docking trials (in bold) including; D293 (9 out of 10 trials), V338 (5 out of 10 trials), F397 (8 out of 10 trials), and V404 (5 out of 10 trials). Additionally, P291, R296, T377, and Y407 also contribute (at least 3 out of 10 trials) in the interaction formed between GRL-0677 and MERS PL pro . The active site residue D293 is involved in the interactions between GRL-0667 and SARS PL pro . As we can see from table 1, the main driving force for binding in case of MERS and SARS PL pro is the H-bonds formed between GRL-0667 and D293 and at least three hydrophobic contacts around the active residue D287 (P291, R296, V338, T377, F397, V404, and Y407 in MERS PL pro , and Y239, L289, Y391, and Y400 in SARS PL pro ). For SARS-COV-2, the main residues that take part in the interaction with GRL-0667 are the residues around the active site C111 which are surface exposed like K105, W106, and A107. Figure

Competing Interest
All authors declare that there is no competing interest in this work.

Data Availability
The docking structures are available upon request from the corresponding author interaction profiler. Nucleic acids research 43(W1), W443-W447 (2015