Molecular interactions and inhibition of the SARS‐CoV‐2 main protease by a thiadiazolidinone derivative

Abstract We report molecular interactions and inhibition of the main protease (MPro) of SARS‐CoV‐2, a key enzyme involved in the viral life cycle. By using a thiadiazolidinone (TDZD) derivative as a chemical probe, we explore the conformational dynamics of MPro via docking protocols and molecular dynamics simulations in all‐atom detail. We reveal the local and global dynamics of MPro in the presence of this inhibitor and confirm the inhibition of the enzyme with an IC50 value of 1.39 ± 0.22 μM, which is comparable to other known inhibitors of this enzyme.

Pose 1 is characterized by a hydrogen bond from the residues C145 and G143 to the first oxygen atom of CCG-50014 and a hydrogen bond from the residue Q189 to the second oxygen atom of CCG-50014. Notably, the ligand is not exposed to the solvent and is enclosed inside the surface of the protein. In the second pose, we observed three hydrogen bonds to the first oxygen atom of CCG-50014, one each from G143, S144, and C145, and we again report a hydrogen bond from the residue Q189 to the second oxygen atom of the ligand. Poses 1 and 2 are similar in orientation and interactions formed in the active site, but in pose 2 CCG-50014 formed an additional hydrogen bond with S144, and we also observed solvent exposure of the fluorine atom on the aromatic side-chain. Notably, pose 1 had a more favorable MM-GBSA value (-71.51 kcal/mol) than pose 2 (-53.30 kcal/mol) suggesting that the hydrogen bond formed with S144 in pose 2 does not increase the binding affinity of the ligand.
In Pose 3, we once again observed hydrogen bonds between the residues G143 and C145, and the first oxygen atom of CCG-50014, we also report a third hydrogen bond between the residue E166 and the second oxygen atom of CCG-50014. Unique to the third pose, pi-cation and pi-pi stacking interactions were observed between H41 and and toluene side-chain of CCG-50014. In the fourth pose, we observed a hydrogen bond between the first oxygen atom of CCG-50014 and the residue E166, and a second hydrogen bond between the second oxygen atom of CCG-50014 and the residue Q189. Unique to this pose, we report pi-cation interactions between the residue H41 and both the central aromatic ring and the toluene side-chain of CCG-50014. In the fifth pose we report the fewest number of initial interactions, with only two hydrogen bonds, one between the residue C145 and the first oxygen atom of the ligand and a second between the residue N142 and the second oxygen atom of CCG-50014. Notably, in all non-covalent poses we observed CCG-50014 interacting with several of the key active site residues that we observed the ligand forming hydrogen bonds and stacking interactions with in the covalently-bound form of M Pro .
In the resulting non-covalent MD simulations, we observed that the dissociation time of CCG-50014 in each protein/ligand conformation was impacted by the initial docking pose. Pose 2, which had a relatively high binding affinity of -53.30 kcal/mol, dissociated from the active site in 13.06 ns and was the only conformation in which we initially observed the solvent exposure of the fluorine atom of CCG-50014. Fluorine is a polar atom, which has strong affinity to the solvent, and pulls the ligand out of the binding pocket. The most stable pose was pose 1, which had a binding affinity of -71.51 kcal/mol, and remained in the active site for 101.94 ns.
To investigate the impact the non-covalently bound ligand had on the dynamics of the seven key active site residues (Fig. 1B), we calculated the ligand-active site distances, RMSF of all protein residues while CCG-50014 was interacting with active site residues, and the RMSF of all protein residues once CCG-50014 had dissociated from the active site, which we report in Fig. S8. From the MD simulation of the first non-covalently bound pose we report the following ∆RMSF values: -0.858Å (M49), -0.16Å (G143), -0.139Å (H163), -0.174Å (H164), -0.159Å (E166), -0.096Å (P168), and -0.461Å (Q189), indicating that all of the key active site residues were stabilized in this MD simulation. We report correlation in the ∆RMSF values between the covalent simulation and this non-covalent simulation in the residues M49, H164, E166, P168, and Q189. In the MD simulation based on the initial coordinates of the fifth pose, which had the second longest dissociation time of 83.84 ns, we observed correlation between the non-covalent and covalent simulations in the ∆RMSF values of the residues M49, G143, and H163. Further, in the MD simulation based on the initial coordinates of the fourth pose, with the third longest dissociation time of 43.94 ns, we observed correlated ∆RMSF values between the non-covalent and covalent simulations in the residues M49, G143, and H163.
In these three MD simulations, based on the first, fifth, and fourth poses, we observed the longest dissociation times, and only the residue M49 showed consistent behavior between the three non-covalent simulations and the covalent simulations. Further, we observed across the five non-covalent simulations that the residue M49 was consistently stabilized, with maximum and minimum ∆RMSF values of -3.1896Å and 0.471Å, respectively. In the covalently-bound form of M Pro the ∆RMSF of the residue M49 was -1.78Å, so in both the covalent and non-covalently protein/ligand complex the drifting of the residue M49 is limited, a dynamic correlated with the "flipped out" φ state of the side-chain of the residue H41. Interestingly, in the simulation based on the first pose, which had the longest dissociation time, we observed similar fluctuations in all the key active site residues, exclud-ing G143 and H163, between the non-covalent and covalent MD simulations, indicating that the stabilization of the key active site residues M49, H164, E166, P168, and Q189 is important for a stable binding pose. In the MD simulations based on poses 2 and 3, we observed the least correlation with the covalent MD simulations, and these poses also had the shortest dissociation times, showing that the covalently-bound pose impacts the active site similarly to the most stable non-covalent poses.
Across the five non-covalent simulations, CCG-50014 had an average dissociation time of 56.66 ns, with a maximum dissociation time of 101.94 ns, and a minimum dissociation time of 13.06 ns. In all MD simulations of the non-covalently bound poses the ligand dissociated quickly, but the poses that affected the key active site residues similarly to the covalently-bound pose were the most stable in MD simulations.  Table S3. Community partitioning details of the apo form of M Pro . We provide the total number of residues in each community, the specific residues assigned to each community, and the critical nodes in communities. The critical nodes are defined as nodes that form an edge with a neighboring node in a different community. The community data is generated using the Girvan-Newman algorithm (see supplemental methods for further details.) total residue# critical nodes C1 43 9-17; 29; 95; 97; 99; 100-102; 104; 111-116; 122; 16; 112; 159; 112; 149 124-128; 140; 148-160 C2 1 191 C3 23 131; 137; 139; 197-199; 201; 226-236; 238-242; 265 131; 197; 198 ; 200; 202-207; 209; 220; 222; 224; 243-259; 261; 286; 288-299 200; 209; 292 C9 43 14; 17; 28; 112-127; 136; 138-149; 161; 163; 166-173 28; 112; 172; C10 11 133; 135; 137; 190-197 135; 190         representations where the narrow side shows the orientation of the side-chain of the residue and the wide side shows the orientation of the backbone of the residue, within 5Å of CCG-50014. Also shown are van der Waals edges of the negatively charged residues (red), hydrophobic residues (green), and polar residues (blue). 11 Figure S8. Data from MD simulations of non-covalently docked poses (see Fig. S7). (left) We show the measured distance from the center of mass of the ligand to the center of mass of the seven key active site residues. We set a cutoff distance of 15Å to consider the ligand as dissociated from the active site. The dissociation times are reported in Table S5. (center) We show the RMSF values of all residues in M Pro , from each non-covalent simulation, while the ligand was still in the active site. We show domain I (orange), domain II (purple), domain III (blue), and the loop (gray) in the background. (right) We show the RMSF of all residues in M Pro after CCG-50014 dissociated from the active site.