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. 2024 Aug 25;14(9):213. doi: 10.1007/s13205-024-04054-y

Structural insights into Furin enzyme inhibition to block SARS-CoV-2 spike protein cleavage: an in-silico approach

Ramakrishnan Jaganathan 1,✉, Poomani Kumaradhas 2
PMCID: PMC11345345  PMID: 39193012

Abstract

This study investigates the binding affinity and interactions of the Furin enzyme with two inhibitors, Naphthofluorescein and decanoyl-RVKR-chloromethylketone (CMK), using molecular docking and molecular dynamics (MD) simulations. Molecular docking results showed binding affinities of − 9.18 kcal/mol for CMK and − 5.39 kcal/mol for Naphthofluorescein. To further understand the stability and conformational changes of these complexes, MD simulations were performed. Despite CMK’s favorable docking score, MD simulations revealed that its binding interactions at the Furin-active site were unstable, with significant changes observed during the simulation. In contrast, Naphthofluorescein maintained strong and stable interactions throughout the MD simulation, as confirmed by RMSD and RMSF analyses. The binding-free-energy analysis also supported the stability of Naphthofluorescein. These findings indicate that Naphthofluorescein exhibits greater stability and binding affinity as a Furin inhibitor compared to CMK. The results of this in-silico study suggest that Naphthofluorescein, along with CMK, holds the potential for repurposing as a treatment for COVID-19, subject to further validation through clinical studies.

Keywords: SARS-CoV-2, Spike protein, Furin, Repurposing drugs, MD simulation, Binding-free energy

Introduction

In late 2019 the novel human coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome-Corona Virus 2) was detected in Wuhan, China; which is an urgent public health emergency and it is creating a severe impact on universal health (Cheng et al. 2020). COVID-19 is a worldwide pandemic disease that causes high death rates. To date, there is no approved effective drug available to treat COVID patients. As per reports, this virus changes its structure and emerges as a variety of variants in different countries. This is a very difficult situation for people who suffer from low immunity power. To escape from this critical situation, new drugs to be designed and it is much needed to treat the various targets of the virus as well as host cells. The Furin-like cleavage site is present in the spike protein (682–689 amino-acid residues) of SARS-CoV-2, which plays an important role in the mechanism of the virus entering the host cell (Duan et al. 2020; Hoffmann et al. 2020). Furin protease also relates to the calcium-dependent prohormone/proprotein convertase (PCs) family, these are widely expressed in humans and its range is notably elevated in lung cystic fibrosis (Henrich et al. 2003; Ornatowski et al. 2007; Dahms et al. 2016; Vankadari 2020) and it is also known for cleaving different viral-envelope glycoproteins such as influenza and HIV by intensifying the fusion the viral protein with the membrane of the host cell (Hallenberger et al. 1992; Follis et al. 2006; Schneck et al. 2020). Reported Furin crystal structure studies states that His194, Asn295, and Ser368 are the catalytic site residues which includes the conformational changes of the protein (Dahms et al. 2016).

Both MERS-CoV-2 and SARS-CoV-2 spike proteins have a Furin-cleavage site at the S1/S2 intersection promotes viral entry into lung cells, and the cleavage site contributes to viral pathogenesis in SARS-CoV-2 animal models (Zhou et al. 2020; Johnson et al. 2021; Peacock et al. 2021; Wu and Zhao 2021). In the SARS-CoV-2 spike protein, the Furin-cleavage site mediates syncytium formation and it is suppressed by specific Furin inhibitors (Wang et al. 2023; Reuter et al. 2023). Ya-Wen Cheng et al (2020) reported that the cleavage and the syncytium are eradicated by hospitalization with decanoyl-RVKR-chloromethylketone (CMK) and Naphthofluorescein which are the Furin inhibitors (Coppola et al. 2008; Remacle et al. 2010; Imran et al. 2019). These two inhibitors show the antiviral reactions on SARS-CoV-2 infected cells by reducing the production of viruses and cytopathic effects (Cheng et al. 2020; Jiang et al. 2024). The latest work examined the inhibition assays for Furin using inhibitors including Naphthofluorescein, CMK, hexa-D-arginine amide, and SSM3 trifluoroacetate. The results of the study demonstrated that Naphthofluorescein could prevent the cleavage of the Furin protein (Yu et al. 2024). To comprehend the structural binding nature of the inhibitors with the Furin-active site, we conducted binding-free-energy calculations, molecular docking, and MD simulations in this study. This in-silico work allowed for the prediction of the binding site itself as well as an analysis of the dynamic properties of the inhibitor-Furin docked complex. The study’s findings make it possible to comprehend how these tiny molecules are inhibited by Furin, which is essential to move on with additional clinical research.

Materials and methods

Protein and ligands preparation

The crystal structure of the Furin protein was downloaded from the Protein Data Bank (PDB ID: 5JXG) to prepare the Furin target to perform Induced Fit Docking (IFD) simulation (Berman et al. 2002). Before IFD, the protein was prepared with a protein preparation wizard module incorporated into the Schrödinger package, further, the structure was optimized and minimized with the OPLS4 force field (Sastry et al. 2013; Lu et al. 2021). Both Furin inhibitors (decanoyl-RVKR-chloromethylketone (CMK) and Naphthofluorescein) are downloaded from the PubChem database in SDF format (Kim et al. 2021). Ligand molecules were neutralized and prepared for docking using the Epik program on the LigPrep module which is included in the Schrödinger Maestro application (Shelley et al. 2007; Schrödinger 2020).

Induced fit docking

Molecular docking simulation was performed for Furin with its inhibitors using the IFD method with extra precision (XP) mode (Sherman et al. 2006). In IFD, the receptor can alter the binding-site conformations to build the inhibitor-Furin complex. Before the docking process, to find the best binding site of the Furin, the SiteMap program was performed which allowed us to predict the best binding site from the SiteMap results (Halgren 2007). The IFD result gives different conformers of both inhibitors with the active site of Furin protein.

Molecular dynamics simulation and binding-free-energy calculation

From the IFD results, the best conformers of inhibitors were identified based on their binding energy and intermolecular interactions with the Furin enzyme. The MD simulations were performed for the selected complexes of each molecule to analyze the structural stability of the molecules within the active site of the Furin enzyme. The MD simulations were carried out using the Amber20 package for 200 ns, with the ff19SB force field (Tian et al. 2019; Case et al. 2020). The solvent box setup was prepared with a TIP3P water model with a 10 Å distance on each side with an NPT ensemble (Mark and Nilsson 2001). Temperature and pressure were maintained at 310 K and 1 atm; further, the system was neutralized by adding Na + /Cl- ions. The RMSD and RMSF plots are generated from the results obtained from the MD simulation. These findings are used to understand the conformational stability of both inhibitors in the active site of Furin; and also, the fluctuation of the amino acids of Furin enzyme during the MD simulation. From the trajectories of the MD simulation, the binding-free energies of both complexes were calculated using MM/GBSA/PBSA methods. The binding-free energy (ΔG) reveals the binding stability of ligands within the active site of Furin during the MD simulation.

Results and discussion

Induced fit-docking analysis

The inhibitors CMK and Naphthofluorescein with Furin enzymes produced distinct conformers for the IFD simulation, and the docking findings were examined (Table 1). The best conformer was selected for additional examination based on the IFD scores and the intermolecular interactions between the inhibitor and the Furin enzyme’s active site residues. Decanoyl-RVKR-chloromethylketone (CMK) and Naphthofluorescein molecules had respective IFD score values of − 9.18 and − 5.39 kcal/mol. The glide energies of CMK and Naphthofluorescein, − 85.93 and − 52 kcal/mol are respectively sums of various energy components such as van der Waals, electrostatic, and other intermolecular interactions indicate the stability of both docked complexes. The prime energies of these complexes − 21,344.3 (CMK) and − 19,878.9 (Naphthofluorescein) kcal/mol represent the protein, ligand, and solvation effects of the complex also signify the stability of the system. IFD scores which consider both docking scores and energy required for the Furin enzyme to adjust to both CMK and Naphthofluorescein also resulting stability of the complexes with scores -− 1076.94 and − 999.98 kcal/mol, respectively.

Table 1.

The docking scores (kcal/mol) of the different conformers of decanoyl-RVKR-chloromethylketone (CMK) and Naphthofluorescein with Furin complexes were obtained from the IFD simulation

Conformer decanoyl-RVKR-chloromethylketone (CMK) Naphthofluorescein
Docking score Glide energy Prime energy IFD score Docking score Glide energy Prime energy IFD score
1 − 9.18 − 85.39 − 21,344.3 − 1076.94 − 5.39 − 52 − 19,878.9 − 999.98
2 − 9.06 − 88.74 − 21,362.9 − 1075.83 − 5.05 − 50.05 − 19,883.1 − 999.61
3 − 9.05 − 90.9 − 21,347 − 1075.4 − 4.76 − 46.62 − 19,886.5 − 998.82
4 − 8.78 − 92.29 − 21,350.9 − 1074.95 − 4.39 − 48.27 − 19,879.3 − 997.97
5 − 8.67 − 82.12 − 21,351.2 − 1074.89 − 4.36 − 47.1 − 19,858.8 − 997.95
6 − 8.28 − 68.87 − 21,350.3 − 1074.42 − 4.26 − 46.05 − 19,870.3 − 997.75
7 − 8.10 − 75.56 − 21,335.8 − 1074.26 − 4.25 − 45.8 − 19,874.2 − 997.64
8 − 7.58 − 77.81 − 21,339.8 − 1073.96 − 4.04 − 46.42 − 19,873.3 − 997.43
9 − 7.47 − 74.2 − 21,340 − 1073.64 − 4.04 − 46.57 − 19,874.9 − 997.29
10 − 7.38 − 74.27 − 21,333.9 − 1073.57 − 4.00 − 43.82 − 19,867.9 − 997.12
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Both docked complexes were analyzed to understand the intermolecular interactions of the binding of the inhibitors with the active-site residues of the Furin enzyme using Discovery Studio and PyMol software (DeLano 2002; BIOVIA 2016) (Fig. 1). For the factual understanding, the top conformers were chosen and analyzed their intermolecular interactions (Table 2). To predict the best complex, the docking scores and the inhibitors that form interactions with the key residues of Furin were compared with the reported structures (Dahms et al. 2016; Vankadari 2020).

Fig. 1.

Fig. 1

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Intermolecular interactions of decanoyl-RVKR-chloromethylketone (CMK) (right) and Naphthofluorescein (left) within the active site of the Furin enzyme, as obtained from Induced Fit Docking studies. The figure illustrates how each inhibitor binds to the Furin-active site, highlighting the key residues involved in the binding interactions

Table 2.

Intermolecular interactions and the contact distance (Å) between the decanoyl-RVKR-chloromethylketone (CMK) and Naphthofluorescein with the active site residues of Furin obtained from IFD docking and MD simulations

Interacting residues of Furin enzyme···Ligand atoms Type of interactions Distance (Å)
Docking MD
100 ns 200 ns
Decanoyl-RVKR-chloromethylketone (CMK)
ASN133/HD22⋅⋅⋅O2 Hydrogen bond – 2.52 –
ALA139/O⋅⋅⋅H(41) Hydrogen bond – 2.58 –
ASP154/OD2⋅⋅⋅H(39) Hydrogen bond 2.95 – –
ASP191/O⋅⋅⋅H(39) Hydrogen bond 2.57 – –
ARG193/HE⋅⋅⋅O(5) Hydrogen bond 2.25 – –
LEU227/O⋅⋅⋅H(6) Hydrogen bond 1.93 – –
ASP228/OD1⋅⋅⋅H(50) Hydrogen bond 2.58 – –
SER253/O⋅⋅⋅H(56) Hydrogen bond 2.43 – -
TRP254⋅⋅⋅C(4) Pi-Alkyl 5.41 – –
PRO256/O⋅⋅⋅H(2) Hydrogen bond 1.97 – –
GLU257/OE1⋅⋅⋅H(38) Hydrogen bond 2.19 – –
GLY294/HA3⋅⋅⋅N(4) Hydrogen bond 3.03 – –
ASN295/HD21⋅⋅⋅O(3) Hydrogen bond 2.64 – –
ARG298/HH22⋅⋅⋅O(3) Hydrogen bond 2.25 – –
ASP306/OD2⋅⋅⋅H(7) Hydrogen bond 1.98 – –
TRP328⋅⋅⋅C(6) Pi-Alkyl 4.40 – –
TYR329⋅⋅⋅C(6) Pi-Alkyl 4.08 – –
HIS364/ND1⋅⋅⋅H(15) Hydrogen bond 2.14 – –
THR365/OG1⋅⋅⋅H(13) Hydrogen bond 1.88 – –
GLN399/OE1⋅⋅⋅H(7) Hydrogen bond – – 2.04
GLY432/HA2⋅⋅⋅O(2) Hydrogen bond – 2.51 –
ALA436⋅⋅⋅C(15) Alkyl – 3.73 –
ASN440/O⋅⋅⋅H(59) Hydrogen bond – – 1.96
THR442/HN⋅⋅⋅N9 Hydrogen bond – – 2.60
LEU578⋅⋅⋅C(15) Alkyl – – 4.78
Naphthofluorescein
ASP153/OD2⋅⋅⋅H(4) Hydrogen bond 3.70 7.50 6.90
ASP154/OD2⋅⋅⋅H(16) Hydrogen bond 1.97 – 1.78
ARG193/NH2⋅⋅⋅Benzene Pi-Cation 5.30 – –
HIS194/ND1⋅⋅⋅Benzene Pi-Cation 3.30 5.70 6.00
LEU227⋅⋅⋅Benzene Pi-Alkyl 4.22 4.18 4.06
GLU230/OE2⋅⋅⋅H(16) Hydrogen bond – 1.70 5.70
VAL231⋅⋅⋅Benzene Pi-Alkyl 3.93 2.37 3.01
GLU236/OE1⋅⋅⋅Benzene Pi-Anion 4.30 4.32 4.25
SER253/HG⋅⋅⋅O(4) Hydrogen bond 3.40 7.60 -
TRP254⋅⋅⋅Benzene Pi-Pi Stacked 5.41 4.70 4.57
PRO256/HA⋅⋅⋅O(3) Hydrogen bond 3.70 2.47 2.29
PRO256⋅⋅⋅Benzene Pi-Alkyl 2.20 3.40 3.10
GLU257/H⋅⋅⋅O(3) Hydrogen bond 5.10 2.41 3.60
ASP258/OD2Z⋅⋅⋅H(15) Hydrogen bond 1.86 – –
ASN295/HD21⋅⋅⋅O(4) Hydrogen bond 2.09 – –
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CMK forms the hydrogen bonding interactions with the Furin residues Asp154, Asp191, Arg193, Leu227, Asp228, Ser253, Pro256, Glu257, Gly294, Asn295, Arg298, Asp306, His364, and Thr365 with the distance 2.95, 2.57, 2.25, 1.93, 2.58, 2.43, 1.97, 2.19, 3.03, 2.64, 2.25, 1.98, 2.14 and 1.88 Å, respectively. Whereas, the Naphthofluorescein forms the hydrogen bonding interactions with Furin residues Asp153, Asp154, Ser253, Asp258, and Asn295 with the distances 3.70, 1.97, 3.40, 1.86, and 2.09 Å, respectively. Furthermore, both inhibitors form interactions with the catalytic site residues of Furin. From these analyses, it is found that both molecules exhibit interactions with the Furin-active-site residues and are highly stable in the active site.

MD simulations

The best conformer of CMK and Naphthofluorescein in the active site of Furin obtained from the IFD simulation was chosen for the MD simulation study of the CMK-Furin and Naphthofluorescein-Furin complexes. Thus, the selected best conformer exhibits lowest energy and form interactions with the key amino acids of active site of the Furin enzyme and their binding affinity. The MD simulations for both complexes were performed up to 200 ns to understand their dynamic binding nature and the stability of the molecules in the Furin binding-site residues.

Intermolecular interactions between decanoyl-RVKR-chloromethylketone (CMK) and Naphthofluorescein and Furin-active-site residues

The intermolecular interactions of both molecules were analyzed from the final frame of the MD trajectories which are converted as the PDB format. This final frame file represents the MD simulation form of the complex, this allows to compare and analyze the intermolecular interactions with the docked complex; this facilitates understanding the conformational modifications and binding nature of the inhibitors (Fig. 2) during the MD simulations. Unlike as found in the docked complex, the MD simulation predicts the stability of intermolecular interactions of CMK inhibitor; notably, it forms new interactions with the binding site amino-acid residues Gln399, Asn440, Thr442, and Leu578 at the distance of 2.04, 1.96, 2.60, and 4.78 Å, respectively. These interactions confirm that the binding site of Furin for CMK was totally changing during the MD simulation (Fig. 3). Hence, the intermolecular interactions of the CMK with Furin-active-site residues are missed and so far there is no experimental studies have been reported to understand the enzymatic activities of this new binding site.

Fig. 2.

Fig. 2

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Intermolecular interactions between Furin and a Decanoyl-RVKR-chloromethylketone (CMK) or b Naphthofluorescein during the MD simulation, highlighting the key interactions between the inhibitors and Furin residues

Fig. 3.

Fig. 3

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Superimposed structures of a CMK-Furin and b Naphthofluorescein-Furin complexes, obtained from molecular docking and MD simulations, illustrating the conformational differences between the two complexes

Whereas, in the MD simulation of the Naphthofluorescein-Furin complex, the Naphthofluorescein molecule also forms a similar type of intermolecular interactions with the Furin as found in the docked complex. It forms hydrogen bonding interactions with the amino-acid residues Asp153, Asp154, Pro256, and Glu257 at the distance of 6.90, 1.78, 2.29, and 3.60 Å, respectively. This molecule also forms pi-type interactions with the residues His194, Trp254, and Pro256 at distances of 7.00, 4.57, and 3.10 Å, respectively. From this, it is confirmed that the intermolecular interactions found in the molecular docking are well maintained during the MD simulations and are not altered, indicating the Naphthofluorescein is well stable. Notably, during the MD simulation, the CMK molecule completely moved away from the Furin binding site as predicted by the molecular docking. Among these findings, unequivocally it is confirmed that the Naphthofluorescein exhibits high stability in the active site of Furin.

RMSD analysis

The RMSD variation of both complexes are plotted (Fig. 4) with the reference frame (docked complex) and calculated based on the atomic selection of MD simulation trajectories. For the CMK-Furin complex up to 100 ns simulation, the deviations are slightly uneven with the range of 1 to 2.3 Å. Further, from 100 to 200 ns simulation, the RMSD variation is found as evenly deviated with the range of 1.7 to 2.2 Å. Whereas in the Naphthofluorescein-Furin complex, the RMSD is slightly raised (1.6 to 2.7 Å) in between 60–80 ns and 175–185 ns MD simulation. And remaining trajectories show even deviation with the range of 1.5 to 1.9 Å. However, the variation of RMSD for both complexes is relatively small, this confirms that the conformational modifications of protein are not abnormal and are at an acceptable level.

Fig. 4.

Fig. 4

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RMSD variation for CMK-Furin and Naphthofluorescein-Furin complexes during the MD simulations, depicting the stability and structural deviations of the complexes over time

The RMSD of CMK shows high deviations during the MD simulation, this confirms that the CMK molecule is unstable in the active site of Furin. Whereas in the Naphthofluorescein-Furin complex, the values of RMSD of the Naphthofluorescein molecule shows that the deviation is not significant during the MD simulation; it confirms that the molecule is stable in the active site and the intermolecular interactions also not much varied during the simulation.

RMSF analysis

To understand the fluctuations of the amino-acid residues of Furin in the ligand-Furin complex, the root means square fluctuation (RMSF) values are plotted (Fig. 5). For both CMK-Furin and Naphthofluorescein-Furin complexes, the fluctuations are found in the range of 0.5 to 2.0 Å. In both complexes, the loop-region residues show high fluctuation during the simulation, in which notably, the alpha helices and beta strands are rigid than the loops. During the simulation, the N and C terminals also display high fluctuation, which is found to be higher than the other amino-acid residues of the Furin. However overall, the amino acids of both complexes do not show any significant fluctuations and are normal during the simulation; this indicates that the Furin in both complexes are found stable.

Fig. 5.

Fig. 5

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RMSF plots of the amino-acid residues of Furin in the CMK-Furin (blue) and Naphthofluorescein-Furin (red) complexes during the MD simulation, highlighting the fluctuations in residue mobility over the simulation period

Radius of gyration

The radius of gyration (Rg) is one of the most significant calculations, widely used to predict the structural activity of macromolecules (Falsafi-Zadeh et al. 2012). To understand the compactness of the ligand–protein, the Radius of gyration (Rg) was analyzed for both CMK and Naphthofluorescein with Furin complexes during the MD simulation (Fig. 6). For the CMK-Furin complex, the Rg values under the range from 22 to 22.4 Å up to 100 ns; further Rg values slightly increased by 0.1 Å up to 200 ns. In the Naphthofluorescein-Furin complex, the Rg values have been observed in the average range of 22.1 to 22.5 Å. However, the observed values show that both complexes are not much altered and the high folded nature is also indicated over the entire MD simulation.

Fig. 6.

Fig. 6

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Radius of gyration (Rg) plots for CMK-Furin (violet) and Naphthofluorescein-Furin (green) complexes during the MD simulation, illustrating the changes in the compactness and stability of each complex over time

Hydrogen bonding

The number of hydrogen bonding formations has been plotted for both complexes throughout the MD simulation (Fig. 7). Hydrogen bonds significantly contribute to the strong complex formation during the MD simulation. For the CMK-Furin complex, during the MD simulation, there is a slight decrease in the number of hydrogen bonding interactions noticed during the 100–150 ns time scale; a remaining number of hydrogen bonds are found in the range of 235–275. In the Naphthofluorescein-Furin complex, the number of hydrogen bonds formed in the range of 230–280, this exists throughout the MD simulation. Among these results, the Naphthofluorescein-Furin complex maintains more number of hydrogen bonds throughout the MD simulation.

Fig. 7.

Fig. 7

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Plots showing the number of hydrogen bonds formed in a CMK-Furin complex and b Naphthofluorescein-Furin complex during the MD simulation, depicting fluctuations in hydrogen-bond counts over time and illustrating interaction stability between each inhibitor and the Furin enzyme

DSSP (secondary structure analysis)

DSSP (Define Secondary Structure of Proteins) is the standard hydrogen-bond estimation algorithm for validating the secondary structures of the protein amino acids (Kabsch and Sander 1983). In this work, DSSP was analyzed for both complexes (Fig. 8) during the MD simulation using the CPPTRAJ program which availed with AmberTools20 (Roe and Cheatham 2013). Secondary structural modifications have been analyzed by comparing the protein structures of docking, 100 ns, and 200 ns (Table 3). In the CMK-Furin complex, HIS160-GLY165, ASP168-SER172, and GLY296-HIS300 have loop-helices modifications; and ILE351-GLU362 has some large sheet-loop modifications. In the Naphthofluorescein-Furin complex, LYS117-GLY126, LEU163-TYR167, and SER302-ASP306 have loop-helices modifications; and LEU337-SER342 has some large sheet-loop modifications.

Fig. 8.

Fig. 8

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DSSP plots showing the secondary structure changes of Furin complexes during the MD simulation. a CMK-Furin complex, and b Naphthofluorescein-Furin complex. The plots illustrate the evolution of secondary structural elements over time, highlighting variations in secondary structure content for each complex

Table 3.

Residue level secondary structural modifications of Furin enzyme while the inhibitors (a) CMK and (b) Naphthofluorescein bound with the binding site of Furin found in docking and MD simulations

Residues Docking MD
100 ns 200 ns
CMK-Furin
 HIS160-GLY165 graphic file with name 13205_2024_4054_Figa_HTML.gif graphic file with name 13205_2024_4054_Figb_HTML.gif graphic file with name 13205_2024_4054_Figc_HTML.gif
 ASP168-SER172 graphic file with name 13205_2024_4054_Figd_HTML.gif graphic file with name 13205_2024_4054_Fige_HTML.gif graphic file with name 13205_2024_4054_Figf_HTML.gif
 GLY296-HIS300 graphic file with name 13205_2024_4054_Figg_HTML.gif graphic file with name 13205_2024_4054_Figh_HTML.gif graphic file with name 13205_2024_4054_Figi_HTML.gif
 ILE351-GLU362 graphic file with name 13205_2024_4054_Figj_HTML.gif graphic file with name 13205_2024_4054_Figk_HTML.gif graphic file with name 13205_2024_4054_Figl_HTML.gif
Naphthofluorescein-Furin
 LYS117-GLY126 graphic file with name 13205_2024_4054_Figm_HTML.gif graphic file with name 13205_2024_4054_Fign_HTML.gif graphic file with name 13205_2024_4054_Figo_HTML.gif
 LEU163-TYR167 graphic file with name 13205_2024_4054_Figp_HTML.gif graphic file with name 13205_2024_4054_Figq_HTML.gif graphic file with name 13205_2024_4054_Figr_HTML.gif
 SER302-ASP306 graphic file with name 13205_2024_4054_Figs_HTML.gif graphic file with name 13205_2024_4054_Figt_HTML.gif graphic file with name 13205_2024_4054_Figu_HTML.gif
 LEU337-SER342 graphic file with name 13205_2024_4054_Figv_HTML.gif graphic file with name 13205_2024_4054_Figw_HTML.gif graphic file with name 13205_2024_4054_Figx_HTML.gif
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Notably, there is no modifications are observed with the residues which form the intermolecular interactions with both molecules during the MD simulation. The above-mentioned structural modifications may happen in the protein structures due to their dynamic nature with a set of ensembles with bounded inhibitors.

Binding-free energy

To understand the binding-free energy of two inhibitors with Furin enzyme, the binding-free energy has been calculated using the MM/GBSA method from the MD simulation (Table 3). The binding-free energy of the CMK and Naphthofluorescein with Furin enzyme complexes are calculated from the MD trajectories using the MM/GBSA/PBSA method. However the MM/GBSA is a popular method, this is due to its commendatory computational speed and accuracy on compare with the MM/PBSA method (Hou et al. 2011; Wang et al. 2019). The MM/GBSA results show the different components and major energy contribution of binding-free energy of inhibitors, proteins, and complexes (Table 3). The MM/GBSA binding-free energy value (ΔG) of the CMK-Furin complex is − 20.06 ± 3.46 kcal/mol, whereas for Naphthofluorescein-Furin this value is − 20.67 ± 3.14 kcal/mol. The ΔG of both inhibitors indicates that they exhibit high binding affinity toward Furin, in which the Naphthofluorescein shows a slight difference compared with CMK. The MM/PBSA energy values show the high binding-free-energy value for the CMK-Furin complex (− 21.61 ± 4.19 kcal/mol) while compared with the Naphthofluorescein-Furin complex (− 18.03 ± 4.76 kcal/mol) (Table 4).

Table 4.

Binding-free energies (kcal/mol) of decanoyl-RVKR-chloromethylketone (CMK) and Naphthofluorescein with Furin complexes calculated using prime MM/GBSA

Binding-free-energy components CMK-Furin Naphthofluorescein-Furin
100 ns 200 ns 100 ns 200 ns
MM
 ∆Evdw − 36.95 ± 3.87 − 32.38 ± 3.34 − 29.64 ± 2.85 − 31.92 ± 2.89
 ∆Eele − 32.08 ± 8.06 − 12.84 ± 8.08 − 17.55 ± 12.34 − 7.96 ± 9.11
GBSA
 ∆GGB 43.89 ± 6.28 29.69 ± 7.44 29.71 ± 9.06 23.29 ± 7.76
 ∆Gsurf − 5.54 ± 0.60 − 4.53 ± 0.49 − 3.95 ± 0.29 − 4.01 ± 0.28
 ∆Ggas − 69.04 ± 9.52 − 45.22 ± 9.23 − 47.17 ± 11.53 − 39.86 ± 9.69
 ∆Gsolv 38.68 ± 5.85 25.16 ± 7.12 25.76 ± 8.94 19.20 ± 7.65
 ∆Gtotal − 30.68 ± 4.51 − 20.06 ± 3.46 − 21.41 ± 3.36 − 20.67 ± 3.14
PBSA
 ∆GPB 43.31 ± 6.62 27.32 ± 8.08 32.46 ± 8.91 25.39 ± 8.38
 ∆Gnpolar − 4.43 ± 0.27 − 3.71 ± 0.33 − 3.41 ± 0.18 − 3.56 ± 0.18
 ∆Ggas − 69.03 ± 9.52 − 45.22 ± 9.23 − 47.17 ± 11.52 − 39.86 ± 9.69
 ∆Gsolv 38.88 ± 6.45 23.61 ± 7.86 29.05 ± 8.85 21.83 ± 8.31
 ∆Gtotal − 30.16 ± 4.66 − 21.61 ± 4.19 − 18.12 ± 5.11 − 18.03 ± 4.76
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The energy contributions given in the table,

∆Evdw- Van der Waals contribution from MM

∆Eele- Electrostatic energy calculated by MM force field

∆GGB- Solvation-free energy calculated by Generalized Born

∆GPB- Solvation-free energy calculated by Poisson Boltzmann

∆Gsurf- Surface area energy

∆Ggas- Gas-phase energy

∆Gsolv- Total solvation-free energy

∆Gnpolar- Non-polar contribution to solvation-free energy

∆Gtotal- Total binding-free energy (kcal/mol)

From the results of MM/GBSA, it can be concluded that the Naphthofluorescein-Furin complex has high stability than the CMK-Furin during the MD simulation. This may be due to the greater number of interactions in the Naphthofluorescein-Furin complex sustained as found in docking simulation. On the other hand, in CMK-Furin complex found low energy values after the MD simulation, this is unlike the molecular docking results; this may be due to the change of interactions in the Furin-binding site (Table 2). Among these results with the reports from the previous articles, MM/GBSA calculation has been considered as further confirmation. Based on the above results, the Naphthofluorescein molecule exhibits a high binding affinity toward Furin when compared with the CMK-Furin complex.

Summary and conclusion

One key strategy for reducing the severe impacts of cancer phenotypes and inhibiting the entry and replication of the SARS-CoV-2 viral protein is to inhibit Furin. Beta-coronaviruses and the other generation of coronaviruses both consist of Furin-cleavage sites. In addition, the highly transmissible and ineffective functionalities of SARS-CoV-2 are largely due to the Furin-like cleavage site. Using molecular docking and MD simulations, the current study aims to comprehend the binding mechanism of the experimentally reported Furin inhibitors CMK and Naphthofluorescein. According to the molecular docking simulation, both inhibitors have binding affinities of − 5.39 kcal/mol and − 9.18 kcal/mol, respectively, and they generate important interactions with Furin. In addition, the MD simulation demonstrates the high stability of the Naphthofluorescein-Furin complex and its ability to form intermolecular interactions with Furin; however, during the simulation, the CMK molecule was moved away from Furin-s active site and bound to another binding site, which has not been reported in any experimental studies. Both compounds exhibit strong binding affinities (20.06 ± 3.46 and − 20.67 ± 3.14 kcal/mol) for CMK and Naphthofluorescein with furin, according to the MM/GBSA. Despite having good binding-energy values during the MD simulation, CMK's intermolecular interactions have changed at the binding site. However, the MD simulation demonstrates that the Naphthofluorescein molecule has a strong binding affinity, which is supported by the findings of the binding-free energy analysis. Throughout the MD simulation, the Naphthofluorescein molecule preserved the intermolecular connections within the Furin-active site. Overall, after clinical testing, the Naphthofluorescein molecule may be regarded as a repurposed medicine for COVID-19 since it may be a lead molecule to inhibit the Furin enzyme.

Acknowledgements

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

Author’s contributions

All authors have contributed equally.

Data availability

The data supporting this study's findings are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

The authors declared that they have no conflict of interest.

Human and animal rights and informed consent

This article does not contain any studies with human or animal subjects performed by any of the authors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data supporting this study's findings are available from the corresponding author upon reasonable request.

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