key: cord-0908486-xfo0b1uu
authors: Ramakrishnan, Jaganathan; Kandasamy, Saravanan; Iruthayaraj, Ancy; Magudeeswaran, Sivanandam; Chinnasamy, Kalaiarasi; Poomani, Kumaradhas
title: Strong Binding of Leupeptin with TMPRSS2 Protease May Be an Alternative to Camostat and Nafamostat for SARS-CoV-2 Repurposed Drug: Evaluation from Molecular Docking and Molecular Dynamics Simulations
date: 2021-01-29
journal: Appl Biochem Biotechnol
DOI: 10.1007/s12010-020-03475-8
sha: c9e01ad7fe5fb433b36377f5ac99e8b641480f0d
doc_id: 908486
cord_uid: xfo0b1uu

The unprecedented coronavirus SARS-CoV-2 outbreak at Wuhan, China, caused acute respiratory infection to humans. There is no precise vaccine/therapeutic agents available to combat the COVID-19 disease. Some repurposed drugs are saving the life of diseased, but the complete cure is relatively less. Several drug targets have been reported to inhibit the SARS-CoV-2 virus infection, in that TMPRSS2 (transmembrane protease serine 2) is one of the potential targets; inhibiting this protease stops the virus entry into the host human cell. Camostat mesylate, nafamostat, and leupeptin are the drugs, in which the first two drugs are being used for COVID-19 and leupeptin also tested. To consider these drugs as the repurposed drug for COVID-19, it is essential to understand their binding affinity and stability with TMPRSS2. In the present study, we performed the molecular docking and molecular dynamics (MD) simulation of these molecules with the TMPRSS2. The docking study reveals that leupeptin molecule strongly binds with TMPRSS2 protein than the other two drug molecules. The RMSD and RMSF values of MD simulation confirm that leupeptin and the amino acids of TMPRSS2 are very stable than the other two molecules. Furthermore, leupeptin forms interactions with the key amino acids of TMPRSS2 and the same have been maintained during the MD simulations. This structural and dynamical information is useful to evaluate these drugs to be used as repurposed drugs, however, the strong binding profile of leupeptin with TMPRSS2, suggests, it may be considered as a repurposed drug for COVID-19 disease after clinical trial. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12010-020-03475-8.

The crystal structure of TMPRSS2 protein is not available; hence, the homology model structure was generated from the amino acid sequence of TMPRSS2 with catalytic domain retrieved from the UniprotKB (Uniprot Accession O15393) using Schrödinger homology modeling interface [24] . In this homology modeling, the Hepsin structure (PDB: 5CE1) was used as a template, which was chosen based on the sequence similarity (BLAST search) [25] . The ligand molecule bound to the template structure was removed. The inbuilt secondary structure prediction (SSP) algorithm of Prime was used to predict the secondary structure of TMPRSS2. The ClustralW method incorporated to Prime was employed for aligning the sequence of TMPRSS2 with the template sequence. The unnecessary gaps found in the secondary structure were removed by manual adjustments. After the sequence alignment, the homology model of TMPRSS2 was generated. Finally, the modeled structure was optimized and refined by protein preparation wizard [26] . The structure was validated with the Ramachandran plot ( Figure 1S ) [27] .

To understand the binding affinity and the intermolecular interaction of the chosen three drug molecules camostat, nafamostat, and leupeptin [16, [18] [19] [20] (https://clinicaltrials.gov/ct2/show/ NCT04353284) with the active site amino acids of TMPRSS2, the molecular docking simulation was performed. In prior to the docking simulation, the abovesaid three drug molecules were prepared by the Ligprep module of Schrödinger software [28] . Further, Camostat mesylate Nafamostat Leupeptin Fig. 1 Chemical structure of the drug molecules Applied Biochemistry and Biotechnology these molecules were docked with the TMPRSS2 by extra precision mode (XP) incorporated in the induced fit docking (IFD) method. Epik was employed to generate the ionization states of the ligands at pH 7.0 +/− 2.0. Thus, prepared ligand inputs were minimized using OPLS3e force field [29] .

The induced fit docking (IFD) resulted in different conformations for each ligand-TMPRSS2 complex. Among these, the complex exhibits top docking score, lowest energy conformation, and the ligand forming expected interactions with the catalytic site residues of TMPRSS2 was selected for further studies. PyMOL software [30] was used to analyze the intermolecular interactions between the ligand and TMPRSS2 complexes.

To understand the stability, intermolecular interactions, and the binding energy of the three ligand-TMPRSS2 complexes, the molecular dynamics (MD) simulation has been performed using the OPLS3e force field [29] implemented in Desmond v5 package [31] . Further, the system was built with the pre-defined TIP4P water model and orthorhombic periodic boundary conditions at the distances 10 Å. Then, the counterions were used to neutralize the charge of these complexes with the balancing Na + /Cl − ions. Further, the constructed system for each ligand-TMPRSS2 complex was energy minimized by heating and equilibrium processes before the MD simulation. For energy minimization, the minimization and heating protocol was fixed based on the steepest descent method, annealing temperature at 0-300 K, and 2000 steps with the time steps of 0.001 ps. Further, the system was normalized in an equilibrium state at 1000 steps with a time step of 0.001 ps. Finally, the production step of the systems was continued up to 100 ns with the time steps of 0.001 ps, 300 K, and 1 atm pressure was applied using the Nose-Hoover method with NPT ensemble [32] . Intermolecular interactions and conformation of each ligand-protein complex were analyzed from the final results of MD simulation. Among the 1000 fractions, the 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 ns fractions were used to determine the binding-free energy (MM/GBSA) of the ligand-TMPRSS2 complexes by Prime application incorporated to Schrödinger software package [33] .

TMPRSS2 is the serine protease, which mediates the S-protein of SARS-CoV-2 for the virus entry through the ACE2 enzyme. Reportedly, by inhibiting the TMPRSS2 protease, the SARS-CoV-2 fusion can be stopped [13] [14] [15] [16] [17] . In TMPRSS2, the ligand-binding active site has catalytic triad consists of Ser186, His41, and Asp180 amino acids; to inhibit the function of this enzyme, ligands necessarily bind with these key amino acids. To understand the binding mechanism, initially, the molecular docking simulations for the drug molecules camostat, nafamostat, and leupeptin with TMPRSS2 have been performed using induced fit IFD method. (Table 1) . On comparing the abovesaid values reveal that the leupeptin consistently exhibits high score. The magnitude of difference in docking scores and glide energy reflects the nature of their intermolecular interactions with the neighboring amino acids present in the active site of TMPRSS2 protease (Fig. 2 ). In the catalytic site, the camostat and leupeptin are forming strong interactions with Asp180, Ser186, and His41 than the nafamostat (Table 2) ; however, the interactions of leupeptin with the catalytic amino acids Asp180, Ser186, and His41are found stronger and the interaction distances are 1.9, 2.1, 2.0, and 3.5 Å, respectively. Figure 3 shows the superimposed form of the three drug molecules in the active site of TMPRSS2 revealing the orientation of the molecules and differences. From this, we observed that these three molecules have a tendency to bind with the serine 2 protease TMPRSS2. However, the nafamostat is lacking the Ser186 and His41 interactions with TMPRSS2 ( Table 2 ). The intermolecular interactions of camostat, nafamostat, and leupeptin with the catalytic triad of TMPRSS2 were compared with the reported complexes [34, 35] [ Table 3 ]. In the present study, the camostat molecule forms intermolecular interactions with the expected three catalytic triad residues (His41, Asp180, Ser186), whereas the reported complex [34] exhibits only two interactions with the active site catalytic triad residues (His296 and Ser441) of TMPRSS2. And the nafamostat molecule forms only one interaction with the catalytic triad residue (Asp180), which is unlike the reported complex [34] (His296). Interestingly, leupeptin molecule forms interaction with all the residues (His41, Asp180, Ser186) of catalytic triad, whereas the reported structure [35] shows leupeptin forms two interactions with the catalytic triad residues (His57 and Ser214). From this comparison, it is clear that how the three molecules of the present study forms interactions with the active site catalytic triad of TMPRSS2 and also how these interactions differ from the reported structures. Further, this study also probed the sustainability of these interactions and the binding affinity of the three drug molecules with the TMPRSS2 during the molecular dynamics simulations.

Molecular dynamics simulation is the computational technique, which allows to understand the biological function of proteins. Here, we performed 100 ns MD simulation for the three ligand-TMPRSS2 complexes; the results explore the stability and the binding affinity of the Fig. 4(a-c) , whereas the RMSD of protein in the camostat-TMPRSS2 and nafamostat-TMPRSS2 complexes are found high comparing with the leupeptin bound TMPRSS2 complex (2.4 Å). Figure 5 (a-c) displays the variations in the RMSF of TMPRSS2 of three complexes; among these, a strong fluctuation is found in the region of residue number 60 of camostat-TMPRSS2 complex and no such incidence is noticed in the RMSF map of other two complexes. The superimposed form of the three ligand-TMPRSS2 complexes (Fig. 6 ) obtained from the docking and MD simulations is showing the conformational difference of ligands and TMPRSS2 protein in the respective complex; specifically, this allows to visualize that how the conformation and the orientation of the ligands and proteins are altered during the MD simulation. Overall, the fluctuation of the leupeptin molecule is found to be very less and it is normal. Furthermore, surprisingly, the superimposed form of docked and MD complexes of leupeptin (Fig. 6) are showing not much deviation, whereas the other two complexes deviated considerably, this confirms that leupeptin is very stable while it binds with the TMPRSS2 protein.

The intermolecular interactions between the ligand and the active site amino acids of TMPRSS2 of three complexes obtained from the MD simulations are shown in Fig. 7(a-c) . On comparing these interactions with the interactions of ligand-TMPRSS2 complexes (Fig. 2) from docking simulation reveals that during the MD simulation, some interactions became strong and in addition that few new interactions also formed (Fig. 7) . Further, when we examine the interactions present in the camostat-TMPRSS2 complex, the terminal NH 2 group of camostat molecule forms interactions with one of the key amino acids Asp180 at the distances 1.6 and 1.7 Å, and these interactions are also very stable during the MD simulation, whereas in the nafamostat-TMPRSS2 complex, we observed a similar trend, in which the Table 3 Comparison of intermolecular interactions of leupeptin, camostat, and nafamostat with the catalytic triad residues of TMPRSS2 and reported complexes

Camostat-TMPRSS2

Camostat-TMPRSS2 [34] Nafamostat-TMPRSS2 [34] Leupeptin-Trypsin [35] His296

His296 leupeptin-TMPRSS2 complex, as found in the docked complex, the MD simulation also displays the same hydrogen bonding interactions between leupeptin and the key amino acids Asp180, His41, and Ser186 at the distances, 1.8, 1.8, and 1.7 Å. These important interactions were highly stable throughout the 100-ns MD simulations and this can be visualized from Figs. 8(a-c), 9(a-c) and Figure 2S . The characterization of interactions between the drug molecules and TMPRSS2 reveals that the leupeptin molecule is highly stable and forms the expected interactions with the key amino acids Asp180, His41, and Ser186 of catalytic site and found 

To address the urgent need of drugs to treat the COVID-19 pandemic disease, repurposing drugs is one of the best solutions comparing with the other techniques like vaccine development and new plasma transfusion. In the line of drug searching process, we have undertaken camostat, nafamostat, and leupeptin drug molecules to inhibit the TMPRSS2 serine 2 protease, which is the activator of SARS-CoV-2 for the fusion of the virus enter into the host human cell. The molecular docking study reveals that these three drug molecules form interactions and has binding affinity towards TMPRSS2. However, the leupeptin forms strong interactions with the key amino acids Ser186, His41, and Asp180 of catalytic triad present in the active site of TMPRSS2 as the other two molecules lacking Ser186 and His41 interactions, whereas in the molecular dynamics simulations of these three complexes, leupeptin is highly stable (RMSD & RMSF) and this molecule also forms stable interactions with the key amino acids Ser186, His41, and Asp186 of TMPRSS2 during the 100-ns MD simulations. On comparing the binding-free energy of all the three molecules, notably, leupeptin has high binding affinity towards TMPRSS2. From the above static and dynamic studies, it is confirmed that leupeptin is very stable and it could strongly inhibit the TRMPRSS2 serine 2 protease and this leads to stop the fusion of SARS-CoV-2 virus onto the host human cell, hence it is considered as a repurposed drug after clinical studies.

Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1007/s12010-020-03475-8.

The authors thank the Computer Centre, Periyar University, Salem, for performing the computational work in the High Performance Cluster (HPC) Computer. 

Coronavirus genomics and bioinformatics analysis. Viruses

Discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus

Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome

Epidemiology and cause of severe acute respiratory syndrome (SARS) in Guangdong, People's Republic of China

Middle East respiratory syndrome

Outbreak of Middle East respiratory syndrome coronavirus in Saudi Arabia: a retrospective study

A novel coronavirus outbreak of global health concern

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. The Lancet

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. The Lancet

Supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron cryomicroscopy

Cryo-electron tomography of mouse hepatitis virus: insights into the structure of the coronavirion

The novel coronavirus 2019 (COVID-19) uses the SARS-1 coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells

Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus Spike protein for membrane fusion and reduces viral control by the humoral immune response

TMPRSS2 contributes to virus spread and immunopathology in the airways of murine models after coronavirus infection

Simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitorsprevents severe acute respiratory syndrome coronavirus entry

Protease inhibitors targeting coronavirus and filovirus entry

TMPRSS2-inhibitors play a role in cell entry mechanism of COVID-19: an insight into camostat and nafamostat

Nafamostat mesylate blocks activation of SARS-CoV-2: new treatment option for COVID-19

Homology modeling and docking studies of TMPRSS2 with experimentally known inhibitors camostatmesylate, nafamostat and bromhexinehydrochloride to control SARS-Coronavirus-2

Inhibition of the growth of human coronavirus 229E by leupeptin

Identification of potential molecules against COVID-19 main protease through structure-guided virtual screening approach

Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry

Basic local alignment search tool

Protein Preparation Wizard; Epik version 2.3, Schrödinger, LLC

Stereochemistry of polypeptide chain configurations

Schrödinger Release 2020-1: LigPrep, Schrödinger, LLC

OPLS3: a force field providing broad coverage of drug-like small molecules and proteins

Molecular Graphics System. Version 2.0 Schrödinger, LLC

Maestro-Desmond Interoperability Tools

The Nose-Hoover thermostat

The VSGB 2.0 model: a next generation energy model for high resolution protein structure modelling

Computer-aided screening for potential TMPRSS2 inhibitors: a combination of pharmacophore modeling, molecular docking and molecular dynamics simulation approaches

Two crystal structures of the leupeptin-trypsin complex

Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.