key: cord-0885838-5w992gjo
authors: Hemmati, Seyed Ali; Tabein, Saeid
title: Insect protease inhibitors; promising inhibitory compounds against SARS-CoV-2 main protease
date: 2022-01-13
journal: Comput Biol Med
DOI: 10.1016/j.compbiomed.2022.105228
sha: 6527d14fd03864431d94ec398b380e6928325466
doc_id: 885838
cord_uid: 5w992gjo

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has adversely affected global health since its emergence in 2019. The lack of effective treatments prompted worldwide efforts to immediately develop therapeutic strategies against COVID-19. The main protease (M(pro)) of SARS-CoV-2 plays a crucial role in viral replication, and therefore it serves as an attractive target for COVID-19-specific drug development. Due to the richness and diversity of insect protease inhibitors, we docked SARS-CoV-2 M(pro) onto 25 publicly accessible insect-derived protease inhibitors using the ClusPro server, and the regions with high inhibitory potentials against M(pro) were used to design peptides. Interactions of these inhibitory peptides with M(pro) were further assessed by two directed docking programs, AutoDock and Haddock. AutoDock analysis predicted the highest binding energy (−9.39 kcal/mol) and the lowest inhibition constant (130 nM) for the peptide 1KJ0-7 derived from SGCI (Schistocerca gregaria chymotrypsin inhibitor). On the other hand, Haddock analysis resulted in the discovery of a different peptide designated 2ERW-9 from infestin, a serine protease inhibitor of Triatoma infestans, with the best docking score (−131), binding energy (−11.7 kcal/mol), and dissociation constant (2.6E-09 M) for M(pro). Furthermore, using molecular dynamic simulations, 1KJ0-7 and 2ERW-9 were demonstrated to form stable complexes with M(pro). The peptides also showed suitable drug-likeness properties compared to commercially available drugs based on Lipinski's rule. Our findings present two peptides with possible protease inhibitor activities against M(pro) and further demonstrate the potential of insect-derived peptides and computer-aided methods for drug discovery.

the binding affinities of the designed peptides towards M pro were characterized using molecular docking and molecular dynamics simulations. The in-silico approach adopted in this study enabled the discovery of novel drug candidates with potential inhibitory effects against M pro , mainly targeting the enzyme's active site. Our findings suggest that domain-specific M pro inhibitory peptides may prove to be a new generation of drugs to be used against SARS-CoV-2.

The amino acid sequence (Uniport code: P0DTD1) and the three-dimensional (3D) structure (PDB ID: 6LU7) [22] of SARS-CoV-2 M pro were retrieved from the universal protein resource (Uniprot) database (www.uniprot.org/) and the protein data bank (PDB) archive (https://www.rcsb.org/), respectively. We estimated various physicochemical properties of M pro , including protein length, molecular weight, the total number of negatively and positively charged residues, theoretical isoelectric point, instability index, aliphatic index, and grand average of hydropathicity index using ExPASy ProtParam tools (http://web.expasy.org/protparam) [23] . The secondary structures of M pro were predicted using the self-optimized prediction method with alignment (SOPMA) (http://npsa-pbil.ibcp.fr/cgibin/npsa_automat.pl?page=npsa_sopma.html) [24] . Transmembrane topology prediction was performed using the transmembrane hidden Markov model (TMHMM) 6 

The pro-region of proteases is required for the proper folding of the protease domain and can also function as a potent inhibitor of the mature enzyme [27, 28, 29] . Amino acid sequences of multiple pro-regions and other naturally occurring protease inhibitors from different insect species were obtained from the national center for biotechnology information (NCBI) 

To investigate the interaction of insect-derived inhibitors with SARS-CoV-2 M pro , we performed a blind docking between the inhibitors and the 3D structure of SARS-CoV-2 M pro using the ClusPro server (https://cluspro.org) without changing the program default settings [32] . The ClusPro server combines conformational sampling, root mean square deviation (RMSD)-based clustering of the predicted protein-protein complexes, and energy refinement to generate a list of near-native structures [32] . These top-ranked near-native structures were run through the WHAT-IF server (https://swift.cmbi.umcn.nl/servers/html/index.html) [33] to refine the inhibitory peptide design. For this purpose, the change in accessible surface area (ΔASA) of free and protein-bound ligands was calculated for the aforementioned top-ranked inhibitors in complex with residues in the active site of M pro . The regions within inhibitors that showed higher ΔASA values were considered to be actively involved in ligand-protein interaction and therefore exerted an inhibitory effect on M pro . 8 The molecular dynamic (MD) simulation was carried out to analyze the dynamic interactions of screened peptides in complex with M pro . MD simulations were performed using the GROMACS simulation package version 5.1.4 within the gromos 54a7 force field [36] .

Simulations were run using an Intel Core i7 Processor Extreme Edition on CentOS Linux 6.8

with graphics processing unit acceleration by NVIDIA GeForce GTX 970. We applied three similar MD simulations to refine the structure of M pro in apo and inhibitor-bound states. In each simulation, the initial structure was placed in the center of a cubic box and solvated by the random distribution of water molecules in an extended single-point charge (SPC/E) model followed by adding counter ions to reach a neutral system. The system was first subjected to energy minimization using the steepest descent energy minimization for 50,000 steps. The energy minimization step was followed by a pre-equilibration simulation for 500 ps in the NVT ensemble with a time constant of 0.1 ps. Next, the NVT equilibrium simulation was performed with the Berendsen thermostat for temperature control (300 K) [37] . Then, each system underwent a 500-ps run in an NPT ensemble, which used the Parrinello-Rahman barostat at 1 bar with the coupling constant set at 0.2 ps [38] . The linear constraint solver (LINCS) algorithm was utilized to constrain bonds during simulation [39] . The periodic boundary condition (PBC) was applied in x, y, and z directions to minimize the 'edge effects'. The Lennard-Jones (LJ) potential with a cut-off radius of 1.4 nm was used for the short-range van der Waals interactions. The particle-mesh Ewald (PME) algorithm was used to calculate long-range electrostatic interactions of Coulomb potential energies with the real space contribution to the Columbic interactions truncated at 0.9 nm applied to the system [40] . The initial velocity of particles was assigned according to Maxwell distributions. Finally, 10 ns MD simulation was produced to remove the structural clashes of the lone protein besides the 100 ns MD simulation for the peptides/M pro complexes to examine the binding phenomena. All MD simulations were carried out when RMSD values reached a plateau.

Lipinski's rule of five was used to evaluate drug-likeness of the designed peptides. Moreover, pharmacokinetic properties of peptides, including absorption, distribution, metabolism, excretion and toxicity (ADMET) profiling of peptides, were determined using the admetSAR [41] and ProTox web tools [42] . Ritonavir and Lopinavir, as two FDA-approved protease inhibitors, were used as reference compounds.

M pro plays an essential role in SARS-CoV-2 replication [13] and is, therefore, an attractive target for drug development against COVID-19. In February 2020, the crystal structure of

Methyl}But-2-Enyl)-L-Leucinamide) was made publicly available by Jin et al. [22] , which was retrieved to perform primary and secondary structure analysis for M pro using ExPASy ProtParam, SOPMA, and TMHMM. The results of sequence analyses and secondary structure prediction are summarized in 1 ).

Results of primary docking of 25 insect-derived protease inhibitors to SARS-CoV-2 M pro using ClusPro web server are presented in Table 2 . ClusPro docking of protease inhibitors with M pro resulted in various clusters, and most of the protease inhibitors were demonstrated to interact with M pro through multiple regions. Inhibitor/M pro complexes were ranked based on the lowest binding energy and the cluster size (the number of members in clusters). The top-ranked cluster (cluster "0") was selected for further analysis. Within cluster 0, seven insect protease inhibitors, including 1CCV, 1KMA, 2OZQ, 2XXT, 3SSB, 2M5X, and 2ERW were predicted to have the lowest binding energy, which suggests the great potential of these peptides to inhibit SARS-CoV-2 M pro . Furthermore, selected regions within these 25 insect-derived protease inhibitors were predicted to be involved in binding to the active site of M pro based on changes in accessible surface areas (∆ASA) of residues, which resulted in the generation of 60 peptide inhibitors with potential inhibitory effect against SARS-CoV-2 M pro . Finally, structural models of these 60 designed peptides were constructed using PEP-FOLD3 for peptide-protein docking.

The molecular docking results of the 60 designed peptides with inhibitory properties against M pro are listed in Table 3 . Peptide molecules were ranked based on the binding energy and 

To further elucidate the inhibitory effect of the designed peptides against M pro , docking studies were also carried out with the HADDOCK web server. The 60 designed peptides were docked into the active site of M pro ( 

SARS-CoV-2 M pro is a cysteine protease whose active site has an unusual catalytic dyad formed by C145 and H41 [41] . A catalytic water molecule forms three hydrogen bond interactions with H41, H164, and D187 in the active site of M pro . A salt bridge interaction between D187 and R40 is important to maintain the architecture of the catalytic cavity. It has been reported that L141, N142, S46, Q189, E166, P168, A191, and T190 in the solventexposed region of the M pro substrate-binding site are involved in trapping of the substrate [44] .

The docking procedure was validated using the coordination information of the 6LU7 PDBID of M pro by manually removing and redocking the peptide-like N3 inhibitor following the same docking procedure used to run HADDOCK and AutoDock. The re-docked complex was then superimposed onto the reference co-crystallized complex using AutoDock tools 1.5.7, and the RMSD value was calculated. ΔRMSD values (the differences between the predicted dock structure to the reference ligand N3 position in the PDB ID 6LU7 coordinate) were calculated to be 0.05 nm by AutoDock, and 0.08 nm by HADDOCK ( Supplementary Fig. 2 

The drug-likeness of a novel compound is investigated using Lipinski's rule of five in the drug discovery process [48] . The rule determines essential pharmacokinetic properties of drug molecules, including the absorption, distribution, metabolism, and excretion (ADMET) [41, 48] . were similar to our findings for the binding site of the designed peptides. Insects are known to produce a wide range of protease inhibitors [20] . However, in comparison with other natural sources, insects have been relatively neglected for drug development [21] .

Therefore, this study aimed to adopt a bioinformatics approach to screen for insect-derived J o u r n a l P r e -p r o o f compounds with potential inhibitory properties against M pro and to further predict interactions of these inhibitors with the enzyme in silico.

The crystal structure of SARS-CoV-2 M pro in complex with the inhibitor N3 was determined by Jin and colleagues in 2020 [22] and was made publicly accessible in the protein data bank (PDB-ID: 6LU7). It is worth noting that amino acid sequences of M pro encoded by SARS-CoV-2 and SARS-CoV (PDB-ID: 2GTB) have been previously shown to be 96% identical [53] .

Further primary and secondary structure analysis, performed in our study, revealed a high similarity between the two proteins (6LU7 and 2GTB). For instance, random coils were predicted to be predominant secondary structures followed by α-helices in both proteins. 6LU7

and 2GTB were predicted to be stable, acidic, and hydrophilic proteins (Supplementary Table   1 ). Taken together, M pro seems to be highly conserved among coronaviruses, as was also demonstrated through the superposition of 12 crystal structures of M pro by Jin et al. [22] . Being highly conserved among coronaviruses, M pro is believed to be a promising target for developing wide-spectrum inhibitors [54] .

In the present study, the publicly available 3D structure of SARS-CoV-2 M pro was run through molecular docking experiments. 60 inhibitor peptides were designed by blind docking of the protomer A of SARS-CoV-2 M pro (6LU7) to various insect protease inhibitors using the ClusPro server followed by structural model prediction of inhibitor/M pro complexes by PEP-FOLD3. Upon blind docking, directed docking of these 60 peptides was performed by two independent docking programs: AutoDock and HADDOCK. The use of two different docking programs enabled us to evaluate our adopted methodology by two programs that function on the basis of two different search algorithms, scoring functions, and pose selection schemes [55] . Furthermore, the binding affinity of the designed peptides to the active site of the enzyme, as calculated by the two docking programs, can be compared to one another. Predicting the binding affinity of inhibitors to the active site is particularly important, as enzyme inhibitors J o u r n a l P r e -p r o o f modify the catalytic properties of the target enzyme through binding to and blocking the active site [56] .

As expected, the two docking programs generated different outcomes. AutoDock analysis introduced 1KJ0-7 and 3OZQ-6 with binding energies of −9.39 and −9.26 kcal/mol, whereas HADDOCK analysis resulted in the discovery of 2ERW-9 with the binding energy of −11.70 kcal/mol. 1KJ0-7 (RKGCPPH) was predicted to have the highest binding affinity to the target enzyme with desirable binding energy, inhibition constant, and intermolecular energy using AutoDock (Table 3) . However, 2ERW-9 (NPCACFRNY) was ranked as the best M pro inhibitor by HADDOCK calculations based on several criteria including cluster size, HADDOCK score, RMSD, binding energy and dissociation constant (Table 4 ). 2ERW-9 showed the highest buried surface area value in comparison with other peptide inhibitors (Table 4) 

The Trial  for  COVID-19 Treatments. Table 1 Summary of primary structure analysis and secondary structure prediction for SARS-CoV-2 M pro (6LU7). Table 2 Blind docking of insect-derived protease inhibitors to M pro (6LU7) using ClusPro server. Table 5 Lipinski properties of the two screened peptides and two FDA-approved protease inhibitors including Ritonavir and Lopinavir as control compounds.

Lipinski properties 2ERW-9 Table 7 Pharmacokinetic properties of the two screened peptides and two FDA-approved protease inhibitors including Ritonavir and Lopinavir as control compounds. 

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Topological Polar Surface Area: 570.9 220.14 Number of rotatable bonds

NC(CS)C(=O)N1CCCC1C(=O)N2CCCC2C(=O)NC(CC3=C[NH]C=N3)

Topological Polar Surface Area: 385.92 169.15 Number of rotatable bonds

COC3=C(C)C=CC=C3C)CC4=CC=CC=C4

Topological Polar Surface Area: 120 Number of rotatable bonds

CC=CC=C2)NC(=O)OCC3=CN=CS3)

Topological Polar Surface Area: 202.26 Number of rotatable bonds

The initial structure 1KJ0-7/M pro complex in MD simulation, (B) the final state of the 1KJ0-7/M pro complex in MD simulation, (C) RMSF analysis for free M pro (Red) and the 1KJ0-7/M pro complex (Blue), (D) 3D representation of the residues involved in the binding of the 1KJ0-7 peptide (blue licorice stick) and M pro

Pi-Alkyl interactions (light purple dashed lines)

Pi-Anion, and Pi-Cation

The initial structure 2ERW-9/M pro complex in MD simulation, (B) the final state of the 2ERW-9/M pro complex in MD simulation, (C) RMSF analysis for free M pro (Red) and the 2ERW-9/M pro complex (Green), (D).3D representation of the residues involved in the binding of the 2ERW-9 7 peptide (blue licorice stick) and M pro

Pi-Alkyl interactions (light purple dashed lines)

Pi-Anion, and Pi-Cation