Molecular Dynamics Simulations Indicate the SARS-CoV-2 Mpro Is Not a Viable Target for Small-Molecule Inhibitors Design

Maria Bzówka; Karolina Mitusinska; Agata Raczynska; Aleksandra Samol; Jack Tuszynski; Artur Góra

The genome of coronaviruses typically contains a positive-sense, single-stranded RNA but it differs in size ranging between ~26 and ~32 kb. It also includes a variable number of open reading frames (ORFs)from 6 to 11. The first ORF is the largest, encoding nearly 70% of the entire genome and 16 non-structural proteins (nsps) [3, 8] . Of the nsps, the main protease (Mpro, also known as a chymotrypsin-like cysteine protease, 3CLpro), encoded by nsp5, has been found to play a fundamental role in viral gene expression and replication, thus it is an attractive target for anti-CoV drug design [9] . The remaining ORFs encode accessory protease: one, referred to as SARS-CoV Mpro N3 (PDB ID: 2amq), co-crystallised with the same N3 inhibitor, and the other without an inhibitor (PDB ID: 1q2w), which we refer to as SARS-CoV Mpro. The SARS-CoV-2 Mpro and SARS-CoV Mpro structures differ by only 12 amino acids located mostly on the proteins' surface ( Figure 1A , Supplementary Table S1 ). Both enzymes share the same structural composition; they comprise three domains: domains I (residues 1-101) and II (residues 102-184) consist of an antiparallel β-barrel, and the α-helical domain III (residues 201-301) is required for the enzymatic activity [27] . Both enzymes resemble the structure of cysteine proteases, although their active site is lacking the third catalytic residue [28] ; their active site comprises a catalytic dyad, namely H41 and C145, and a particularly stable water molecule forms at least three hydrogen bond interactions with surrounding residues, including the catalytic histidine, which corresponds to the position of a third catalytic member ( Figure 1B ). It should be also noted that one of the differing amino acids in SARS-CoV-2 Mpro, namely S46, is located on a C44-P52 loop, which is flanking the active site cavity. Close-up of the active site cavity and bound N3 inhibitor into SARS-CoV (black sticks) and SARS-CoV-2 (blue sticks) Mpros. The catalytic water molecule that resembles the position of the third member of the catalytic triad adopted from the cysteine proteases is shown for both SARS-CoV (black sphere) and SARS-CoV-2 (blue sphere) Mpros. The active site residues are shown as red sticks and the proteins' structures are shown in surface representation. The differing residues in position 46 located near the entrance to the active site are marked with an asterisk (*) on the (A) and as blue and black lines on the (B) panel.