Abstract

Virtual screening can be a helpful approach to propose treatments for COVID-19 by developing inhibitors for blocking the attachment of the virus to human cells. This study uses molecular docking, recovery time and dynamics to analyze if potential inhibitors of main protease (M^pro) of SARS-CoV-2 can interfere in the attachment of nanobodies, specifically Nb20, in the receptor binding domain (RBD) of SARS-CoV-2. The potential inhibitors are four compounds previously identified in a fluorescence resonance energy transfer (FRET)-based enzymatic assay for the SARS-CoV-2 M^pro: Boceprevir, Calpain Inhibitor II, Calpain Inhibitor XII, and GC376. The findings reveal that Boceprevir has the higher affinity with the RBD/Nb20 complex, followed by Calpain Inhibitor XII, GC376 and Calpain Inhibitor II. The recovery time indicates that the RBD/Nb20 complex needs a relatively short time to return to what it was before the presence of the ligands. For the RMSD the Boceprevir and Calpain Inhibitor II have the shortest interaction times, while Calpain Inhibitor XII shows slightly more interaction, but with significant pose fluctuations. On the other hand, GC376 remains stably bound for a longer duration compared to the other compounds, suggesting that they can potentially interfere with the neutralization process of Nb20.

Keywords

SARS-CoV-2, Main protease M^pro, Boceprevir, Calpain Inhibitor II, Calpain Inhibitor XII, GC376, Nanobody Nb20, In Silico

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1. Introduction

Global efforts have focused on developing vaccines and antiviral drugs to combat the COVID-19 disease caused by the SARS-CoV-2 coronavirus, which reached the pandemic in March 2020 [1] - [6] . As of June 2023, the virus has infected over 767 million individuals and caused over 6.9 million deaths worldwide [7] [8] . Recent successes have resulted in the production of vaccines being administered [9] [10] [11] [12] . However, despite the initiation of vaccination programs, no effective treatment for individuals already infected with the virus has been universally agreed upon.

Nanobodies, which are small antibodies found in camelids like llamas and camels, possess unique characteristics that make them highly effective in binding to specific antigens, including proteins of the SARS-CoV-2 virus [6] [13] [14] . These nanobodies have garnered considerable attention as a promising therapeutic approach against COVID-19. They can be engineered to target various virus proteins, such as the spike protein found in the receptor binding domain (RBD), the main protease, and other viral proteins. By doing so, they inhibit the function of these proteins and hinder viral entry into host cells and replication [15] [16] .

Compared to traditional antibodies, nanobodies offer several potential advantages. They have a compact size, which facilitates large-scale production and enhances stability. Moreover, they can be precisely designed to bind to specific regions of viral proteins, reducing the risk of mutations that could undermine the treatment’s effectiveness [17] [18] .

At the same time, numerous drugs have been extensively studied and proposed to combat SARS-CoV-2, employing diverse mechanisms, whether acting on the RBD of SARS-CoV-2 or on the main protease (M^pro) that participates in the viral replication process [19] [20] [21] [22] . These drugs can potentially interfere with the function of nanobodies or work synergistically to enhance treatment outcomes. Given these features, we selected four compounds: Boceprevir, Calpain Inhibitor II, Calpain Inhibitor XII, and GC376. These compounds were previously identified in a fluorescence resonance energy transfer (FRET)-based enzymatic assay targeting the SARS-CoV-2 the M^pro [23] [24] . They have demonstrated potent activity, with IC₅₀ values ranging from single-digit to submicromolar concentrations in the enzymatic assay [23] .

In this study, we conducted molecular docking and dynamics analyses to investigate the potential interactions of the four previously mentioned compounds, which are known to interact with M^pro. We aimed to estimate whether these compounds could interfere with the neutralization process of RBD of SARS-CoV-2 by the human nanobody Nb20 or potentially act synergistically to improve the treatment against SARS-CoV-2.

2. Materials and Methods

2.1. Protein Preparation

Figure 1 presents the critical proteins involved in the RBD/Nb20 interaction obtained from Protein Data Bank, ID 7JVB [17] . In Figure 1(a), the Nb20 protein is blue, while the Sars-CoV-2 RBD is red. We highlighted the inhibition region between these proteins with a black square in Figure 1(b). The GLN493, ARG31, TYR104, GLU484, and ARG97 are the residues that interact via hydrogen bonds within the RBD/Nb20 interface. Table 1 shows the residues participating in the Nb20/SARS-Cov-2 interactions and respective distances for the hydrogen bonds. Figure 1(c) illustrates the binding site surface using the following color code: grey, red, blue, and white for carbon, oxygen, nitrogen, and hydrogen atoms, respectively. The protein resolution is 2.45 Å, and no pKa prediction was performed. We have considered all the crucial residues in the RDB/Nb20 interface and only metal ions in the docking study.

2.2. Ligand Preparation

The Boceprevir, Calpain Inhibitor II, Calpain Inhibitor XII, and GC376 3D structures were extracted from PubChem [25] . The chemical structures of these compounds are presented in Figure 2, while relevant information such as PubChem ID, molecular weight, and molecular formula is shown in Table 2.

2.3. Molecular Docking Simulations

We used molecular docking simulations to analyze the non-covalent binding between the RBD/Nb20 protein complex (see Figure 1) and four small molecules presented in Figure 2. The docking simulations were conducted using the Webina-autodock 1.0.2 server [26] , which employs the Chemistry at HARvard Macromolecular Mechanics (CHARMM) force field and a blind docking strategy [27] .

The Protein-Ligand Interaction Profiler (PLIP) server [28] was used to characterize the interaction between each compound and the RBD/Nb20 complex, with a focus on the interface between the two proteins, which is crucial for blocking coronavirus entry and replication, see Figure 1(b). The simulation box used for ligand screening was also limited to this interface, with dimensions of 28 Å × 0 Å × 10 Å, and a center point of (32, −36, 2) Å, which corresponds to the region of primary interest, consisting of the main interaction site between the RBD of SARS-CoV-2 and Nb20 protein. The ligand positions and binding affinities were estimated with an accuracy of ±2 Å, and ±0.01 Kcal/mol, respectively.

2.4. Recovering Time Calculations

The recovery time ( $\tau$ ) [29] [30] of a substance after it has adsorbed on a solid surface can be calculated using the following equation from transition state theory:

$\tau ={\nu }_0^{-1}{\text{e}}^{E_{ad}/k_{B}T},$ (1)

were, ${\nu }_0$ is the attempt frequency, which is approximately 10¹² s⁻¹ [31] , T is the temperature (set to 300 K), $k_B$ is Boltzmann’s constant, and $E_{ad}$ is the desorption energy. Desorption refers to releasing a substance from the interface between a solid surface and a solution. According to the equation above, a more negative value of $E_{ad}$ leads to a longer recovery time. In other words, the activation energy ( $E_a$ ) required to overcome the desorption process is directly proportional to $E_{ad}$ . Thus, the recovery time can be used to estimate the time required for a complex to return to what it was before the presence of the ligand.

2.5. Molecular Dynamics Simulations

Additionally, we performed a molecular dynamics (MD) simulation with a time on the order of the smallest value obtained for the recovery time, using the NAMD computational package [32] and CHARMM36 [33] force field, to estimate if the ligands can quickly escape the chosen binding site and, thus, not interfere with the RBD/Nb20 interactions. If the ligands are not stable in the region of RBD/Nb20 interaction, they are expected to not compete with Nb20. The number of molecules, pressure, and temperature were kept constant using Langevin dynamics in the NPT ensemble at 300 K and 1 atm, respectively. We computed the system’s temporal evolution to calculate the Root Mean Square Deviation (RMSD). The MD snapshots, trajectories, and RMSD calculations were obtained using the visualization and analysis software (VMD) [34] .

3. Results and Discussion

We begin our discussion by presenting the molecular docking results. The ligand poses with the lowest binding energy affinity (ΔG free energy) were selected based on their docking scores, corresponding to the most likely and stable binding modes [35] .

The results of the docking simulations are presented in Table 3, and they indicate that all four ligands have significant binding affinities for the target proteins. Among them, Boceprevir shows the highest binding affinity for the RBD/Nb20 proteins, with a value of approximately −7.3 Kcal/mol, while Calpain02 exhibited the lowest binding affinity of about −5.4 Kcal/mol. We observed that the ligands with a higher molecular weight tended to have more stable docking results, evident when contrasting information in Table 2 and Table 3.

In previous studies, similar binding affinity results for other ligand species were reported for the interaction of RBD with the human angiotensin-converting enzyme 2 (ACE-2) protein, with docking scores ranging from −3.2 to −9.8 kcal/mol [36] [37] . In Table 3, the docking affinities of the ligands are comparable to the ones for (S,S)-2-1-carboxy-2-[3-(3,5-dichlorobenzyl)-3H-imidazol4-yl]-ethylamino-4-methylpentanoic acid (MLN-4760). This potent inhibitor alters the conformation of ACE-2 and prevents the binding of SARS-CoV-2 and ACE-2 [38] . The docking score of MLN-4760 was −7.28 kcal/mol, similar to Boceprevir’s docking score in this study.

In Figure 3, the binding site surfaces (BSS) are presented for the ligand conformations with the RBD/Nb20 proteins, showing the best results in molecular docking simulations. Only the region of interest displayed in Figure 1(b) is depicted. The color code used to represent the different atoms is grey, red, blue, and white, indicating carbon, oxygen, nitrogen, and hydrogen, respectively. The ball-and-stick representation uses cyan, red, and white to show carbon, oxygen, and hydrogen atoms. Overall, the ligands fit nicely inside the cavity, which is the core pocket region of the RBD/Nb20 proteins, shown in Figure 1(a). Boceprevir, Calpain Inhibitor II, Calpain Inhibitor XII, and GC376 exhibited adsorption regions in the interaction with the RBD/Nb20 complex and tended to interact with oxygen and nitrogen atoms.

The interactions between amino acid residues of RBD/Nb20 proteins and ligands are depicted in Figure 4, generated using PLIP. The ligands are represented as sticks, with carbon and oxygen atoms in orange and red, respectively. The color code highlights hydrophobic interactions in red, hydrogen bonds in green, π-stacking in blue, and salt bridges in purple. Figure 4 illustrates that Boceprevir (Figure 4(a)), Calpain Inhibitor II (Figure 4(b)), Calpain Inhibitor XII (Figure 4(c)), and GC376 (Figure 4(d)) interact with RBD/Nb20 proteins mainly through 2, 4, 1, and 3 hydrogen bonds and 6, 3, 5, and 3 hydrophobic interactions with distinct amino acid residues in both RBD and Nb20 proteins. Additionally, Figure 4(c) shows an π-stacking interaction between RBD/Nb20 and Calpain Inhibitor XII, while a salt bridge interaction with GC376 is visible in Figure 4(d).

Table 4 presents all the interactions between amino acid residues of RBD/Nb20 proteins and the ligands, along with their respective distances. Thirteen amino acid residues of the RBD/Nb20 proteins were identified as interacting with the ligands. Specifically, the RBD amino acid residues involved were ILE294 (5), ALA224 (1), GLU295 (2), SER162 (1), and HIS225 (1), while the

Nb20 amino acid residues were TYR117 (1), TYR173 (5), TYR163 (2), TYR121 (2), ARG71 (4), PHE165 (2), GLY164 (3), and ASN169 (4). Based on their higher occurrence, the results suggest that the target amino acid residues for this set of ligands are ARG71 and ASN169 from Nb20 and ILE294 from RBD.

Notably, Calpain Inhibitor II and Calpain Inhibitor XII exhibit higher hydrophobic interactions, while Boceprevir shows higher hydrogen bonds, and GC376 has quantities of hydrophobic interactions and hydrogen bonds equal. Additionally, the number of times the amino acid with the highest interaction (TYR) interacts with the ligands can be estimated, corresponding to ten interactions (eight hydrogen bonds and two hydrophobic interactions). This finding suggests that TYR is a significant amino acid in promoting attachment to RBD for this set of ligands (see Figure 5).

Based on the docking results, we calculate the recovery time (Equation (1)) of the proteins/ligand complexes to estimate if the ligands (Boceprevir, Calpain Inhibitor II, Calpain Inhibitor XII, and GC376) interacted with the complex RBD/Nb20 for a short or long time. The recovery time represents the time necessary for the proteins to return to their original conformation. Table 5 presents the complex RBD/Nb20 recovery times with each inhibitor, with Boceprevir

demonstrating the highest time in order of 10⁻⁷ s. The recovery time range in order from 10⁻⁹ s to 10⁻⁷ s. This order of value indicates that the complex RBD/Nb20 needs a relatively short time to return to what it was before the presence of the inhibitors.

Based on the results of the recovery time, we conducted an MD simulation with a time on the order of the smallest value obtained for the recovery time to investigate whether the ligands can remain unstable or stable at the binding site of the RBD/Nb20 complex within this time frame. This is done using RMSD, the results are illustrated in Figure 6. The results show that Boceprevir interacts with the RBD/Nb20 for at least 600 ps, unlinking from the interaction site after this time, as can be seen by the high fluctuations of the RMSD after 600 ps. Calpain Inhibitor II rapidly moves out of the RBD/Nb20 interaction region, which justifies it is having the lowest recovery time value. This can be visualized in the RMSD, which presented high fluctuation in less than 100 ps. The Calpain Inhibitor XII has more time of interaction, with high fluctuation of the RMSD just in about 300 ps, the moment that the ligand moves out for the region of interaction. In about 390 ps, these ligands re-interact with the proteins in one different location, but with high changes of poses along the time, indicating that is not a

strong interaction. The GC376 presented the more stable interaction, and the RMSD indicates some fluctuations (in about 100 - 200 ps and 360 - 430 ps), but this ligand follows the change in RBD/Nb20 proteins. This trend can be evidenced in the RMSD, which shows little fluctuations over time. The final pose, at 1000 ps, of Calpain Inhibitor XII and GC376 is presented in Figure 7.

4. Conclusions

In this study, we used molecular docking and dynamics simulations to examine how Boceprevir, Calpain Inhibitor II, Calpain Inhibitor XII, and GC376 interact with the RBD/Nb20 proteins. Nanobodies, such as Nb20, have shown promise in targeting the RBD of SARS-CoV-2 based on experimental studies. Our analysis of the binding site surface revealed pocket-like regions on the protein complex that primarily engage in hydrogen bond interactions with the compounds’ amino acid residues.

Our findings indicate that the affinity of the interactions ranges from −6.0 Kcal/mol to −7.2 Kcal/mol, with Boceprevir presenting the higher affinity and the Calpain Inhibitor II the minor. Additionally, the recovery time results indicate that the complex RBD/Nb20 can return to what it was before the presence of the inhibitors in a relatively short time, in the order of 10⁻⁷ s to 10⁻⁹ s. The MD simulations indicate that Boceprevir exhibits the highest binding affinity but does not fully occupy the binding site, similar to Calpain Inhibitor II. On the other hand, Calpain Inhibitor XII and GC376 interact more extensively with the RBD/Nb20 interface. However, Calpain Inhibitor XII displays high fluctuations and changes in binding poses, whereas GC376 remains stably bound for at least 1000 ps.

Therefore, our results suggest that GC376, in particular, may interfere with the neutralization process of the RBD of SARS-CoV-2 by the human nanobody Nb20. In contrast, the Boceprevir, Calpain Inhibitor II, and Calpain Inhibitor XII can be used simultaneously acting synergistically with the nanobody Nb20 in the process of treatment against SARS-CoV-2.

Acknowledgements

The authors gratefully acknowledge the financial support from Brazilian research agencies CNPq, CAPES, and FAP-DF. L.A.R.J acknowledges the financial support from Brazilian Research Council FAP-DF grants 00193.00001808/2022-71, 00193-00001247/2021-20, 00193-00000857/2021-14, 00193-00001247/2021-20, and 00193-00000811/2021-97 and CNPq grants 302236/2018-0 and 350176/2022-1. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES)-Finance Code 88887.691997/2022-00. R.G. and L.A.R.J acknowledge CENAPAD-SP for providing computational facilities. L.A.R.J. gratefully acknowledges the support from ABIN grant 08/2019. L.A.R.J. acknowledges Núcleo de Computação de Alto Desempenho (NACAD) for providing the computational facilities through the Lobo Carneiro supercomputer. L.A.R.J. acknowledges the National Laboratory for Scientific Computing (LNCC/MCTI, Brazil) for providing HPC resources of the SDumont supercomputer (URL: http://sdumont.lncc.br), which have contributed to the research results reported within this paper. RTDS is supported by CNPq—Brazilian National Research Council (Grant 310941/2022-9 PQ-1D), FAPDF—Brazilian Federal District Research Support Foundation (Grant 625/2022 SISTεR City), and the University of Brasilia (Grant 7129 UnB COPEI).

Conflicts of Interest

The authors confirm that there is no conflict of interest.

References