# Evaluation of Natural Peptides to Prevent and Reduce the Novel SARS-CoV-2 Infection

Authors: María Paredes-Ramos; Enma Conde Piñeiro; Horacio Pérez-Sánchez; José M. López-Vilariño
Journal: Journal of Food Quality
 (2022)

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## Abstract

In a preventive context, natural peptides can play a major role against SARS-CoV-2, so their character of GRAS (generally recognized as safe) means they would not need innocuity analyses to be employed. This study analyses the potential of pea peptides, LSDRFS and SDRFSY, and amaranth peptides, GGV, IGV, IVG, VGVL, and VIKP, against the SARS-CoV-2 hosts, ACE2 (angiotensin-converting enzyme 2), ACE (angiotensin-converting enzyme), and CD26 (cluster of differentiation 26), and SARS-CoV-2 enzymes, spike glycoprotein and 3CLpro (3-chymotrypsin-like protease). Also, currently used drugs were analysed to contrast drug and peptide behaviour. Employing docking, virtual screening, and molecular dynamics assays, SDRFSY, LSDRFS, and VIKP were detected as potential bioactive peptides by blocking ACE2 and CD26 or reducing the inflammation associated with COVID-19. Enzyme inhibition analyses were also performed, proving the ability of SDRFSY and LSDRFS as ACE2-blocking agents against the spike glycoprotein with inhibition capacities above 80%.


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## Body

## 1. Introduction

The high transmission (R0 1.5–6.8) [1, 2] and mortality rates (1–3%) [3] of SARS-CoV-2 had caused a global epidemic. Although several vaccines were developed in a record time, they are not yet available in many parts of the world, so another strategy is required not only to treat COVID-19 patients, but also to prevent the infection of healthy individuals and the reinfection of recovered patients or, at least, to mitigate the harmful effects of the infection.Great efforts were done by the scientific community to reposition several approved antiviral and anti-inflammatory drugs [4–7]. In this regard, it is well known that some hydrolysed peptides show very high bioactivity when interacting with different human proteins, generating functional benefits in some cases and food allergies in others [8]. Therefore, these peptides could be a good option to take a step forward against SARS-CoV-2 infection. They are directly extracted from natural sources, with no modification of their molecules, so they are not structurally optimized to exert a certain effect and they possess less efficacy than drugs. Nevertheless, the absence of toxic effects related to their consumption makes them excellent candidates to be used as a preventive treatment or even as a treatment for those patients with minor symptoms, acting similarly as cough syrups for mild colds.To fight this novel coronavirus, two different strategies can be applied: the direct treatment of the virus or the treatment of the host. Several studies were performed to identify the main receptors of both, and among them, spike glycoprotein, 3-chymotrypsin-like protease (3CLpro), RNA-dependent RNA polymerase (RdRp), and papain-like protease (PLpro) are signalled as the key proteins of the virus. The spike glycoprotein is located on the outer envelope of the virion and contributes to the cell receptor binding, and the 3CLpro enzyme is indispensable to the viral replication and infection process, thereby making them ideal targets for antiviral therapy [9]. Among the host receptors, angiotensin-converting enzyme 2 (ACE2) and dipeptidyl peptidase, known as cluster of differentiation 26 (CD26), are identified as the most important human cell proteins employed as hosts by SARS-CoV-2 [5, 10–13].Also, critical patients of COVID-19 show extremely high inflammatory parameters, including creatine-reactive protein (CRP) and proinflammatory cytokines (e.g., IL-6 and IL-8) [14, 15]. These proinflammatory cytokines are triggered by the nuclear factor kappa B (NF-kB), and thus, NF-kB can be also an appropriate target to overcome not the SARS-CoV-2 itself but one of their acutest side effects [16]. One of the cell enzymes that mediate the NF-kB pathway is angiotensin-converting enzyme (ACE), so its inhibition would reduce the production of the proinflammatory cytokines already mentioned [17–20]. Hence, ACE is also considered a promising host target.To find inhibitors for these receptors, computer-aided methods are required. Since the 1990s, huge efforts have been made to test an almost unlimited number of drug-like compounds in an automatic way [21]. Hence, computer-aided drug design (CADD) software is nowadays commonly employed for discovering the bioactive molecules, which can then be employed for the pharmaceutical industry as drugs or for the food industry as nutraceutical supplements or functional food/beverages.Accordingly, a computational approach rooted in molecular docking-based virtual screening is employed to examine the receptors, namely, 3CLpro (PDB code: 6lu7), ACE2 (PDB code: 6m18), spike glycoprotein (PDB code: 6vsb), CD26 (PDB code: 6l8q), and ACE (PDB code: 1o8a). As inhibitors, a library of pea and amaranth peptides was studied, including LSDRFS, SDRFSY, GGV, IGV, IVG, VIKP, and VGVL. Their computed binding affinities and docking poses were compared to FDA-approved drugs. Then,in vitro assays were performed to test the capacity of these peptides to inhibit the spike glycoprotein-ACE2 interaction.


## 2. Materials and Methods

### 2.1. Resources and Programs

UniProt Knowledgebase (UniProtKB), Swiss Protein (SwissProt) database, ChemSpider database, and Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) were searched.Openbabel GUI 2.4.1 [22], Acpype [23], Gromacs 2018 [24–26], Chimera UCSF 1.13.1 [27], AutoDock Tools 4.2 [28], AutoDock Vina 2.0 [29], PyMOL 2.3 (The PyMOL Molecular Graphics System, version 2.3 Schrödinger, LLC), Python 2.7.6 (Python Software Foundation), PoseView 1.1.2 (ZBH University of Hamburg, BioSolveIT GmbH), Omega 2.5.1.4 (OpenEye Scientific Software) [30], PLIP 1.3.2 [31], Maestro suite 2019.4, Schrödinger LLC, and the Shuttlemol suite of HPC scripts for virtual screening (https://bio-hpc.eu) were used.


### 2.2. Materials and Reagents

Synthetic GGV, VIKP, LSDFRS, and SDFRSY peptides were purchased from APeptide Co. Ltd. (Shanghai, China) and presented a purity degree above 95%. The Spike glycoprotein-ACE2 binding assay kit was purchased from RayBiotech® (Atlanta, United States) and the ACE2 inhibitor screening kit from AssayGenie Ltd. (London, United Kingdom).


### 2.3. Molecular Modelling

#### 2.3.1. Selection of Ligands

Several amaranth and pea peptides were identified as bioactive peptides in numerous studies. Some amaranth peptides are well known for their 3-hydroxy-3-methylglutaryl reductase (HMGCR) and angiotensin-converting enzyme (ACE) inhibition capacities, thus reducing cholesterol production and hypertension [32–37]. Several pea peptides are proved to be ACE inhibitors but also ACE2 enhancers, boosting Ang 1–7 and Ang 1–9 production and consequently acting as vasodilators [38, 39].Accordingly, GGV, IGV, IVG, VIKP, and VGVL from amaranth and LSDFRS and SDFRSY from pea are selected as ligands.


#### 2.3.2. Selection of Receptors

There are two different target groups that can be employed to overcome the COVID-19 infection: (1) receptors of the virus and (2) receptors of the host [5, 10–13].The spike glycoprotein (protein S) is mainly responsible for the SARS-CoV-2 entry at the host receptor, so it fuses the virus and the host membranes [40, 41]. Accordingly, this receptor is selected for docking studies (PDB code: 6vsb). Also, there are several virus proteins (3CLpro, RdRd, and PLpro) that are considered important factors for the infection [5, 42]. Among them, the 3CLpro is considered the key for virus replication, so it is selected as a virus receptor for docking analysis (PDB code: 6lu7).As host enzymes, angiotensin-converting enzyme 2 (ACE2) [40, 43], dipeptidyl peptidase (CD26) [13, 44, 45], and angiotensin-converting enzyme (ACE) [19] are identified as the most important human cell enzymes, so PDB codes 6m18, 6l8q, and 1o8a corresponding to these receptors were downloaded from RCSB.


#### 2.3.3. Molecular File Preparation

3CLpro (PDB code: 6lu7), ACE2 (PDB code: 6m18), spike glycoprotein (PDB code: 6vsb), CD26 (PDB code: 6l8q), and ACE (PDB code: 1o8a) were downloaded from RCSB.Ligands were removed from pdb files employing PyMOL 2.3. Then, AutoDock Tools 4.2 was used to remove water molecules, add hydrogens, assign AD4 type to all atoms, compute Gasteiger charges, and save protein files both in pdb and pdbqt formats.Ligand molecules were downloaded from ChemSpider in mol format and converted to sdf format using the Openbabel GUI 2.4.1. Then they were converted to mol2 format with the Molconvert script from ChemAxon and minimized in the Merck Molecular Force Field (mmff94). AutoDock Tools 4.2 was employed to convert files to pdbqt format.


#### 2.3.4. Blind Docking Analysis

A blind docking (BD) study was performed to detect the regions of interaction between ligands and the aforementioned receptors: ACE, ACE2, spike glycoprotein, CD16, and 3CLpro [46].Series of single docking simulations were performed in each alpha carbon of the protein, detecting the most favourable binding pockets in terms of bond energy but also the spatial conformation of the ligand and its bound residues. Affinities of ligand-protein interactions are determined with AutoDock 4.2 and individual energies of each bonded residue are calculated with PoseView 1.1.2, representing global bond energy and key residues bond energy, respectively.


#### 2.3.5. Virtual Screening Analysis

To delve deeper into the binding pockets of each ligand, virtual screening (VS) studies were performed, allowing ligands to rotate and move within a box of 30 Å side and increasing the exhaustiveness of the blind docking analysis. The coordinates employed in this step had been obtained from the previous blind docking analyses.


#### 2.3.6. Molecular Dynamics Simulation

Molecular dynamics (MD) is an approximation of the behaviour of the system against real conditions and allows to test the stability of the ligand-protein contacts, which were detected during the BD and VS analyses, during a period of time.MD simulations were carried out using the GPU version of Desmond included with Maestro suite 2019.4 (Schrödinger LLC) on a workstation with a NVIDIA QUADRO 5000. The system conformed by the ligand and protein of interest was solvated in an aqueous environment, in a cubic box with a minimal distance of 10 Å between the biomolecule and the box boundary (for periodic boundary conditions). Next, systems were neutralized and maintained in 0.15 M NaCl. The OPLS3 force field and the TIP3P-TIP4P water model were employed. Initially, the systems were simply energy-minimized for 2000 time steps. Next, systems were allowed to execute free dynamics in the NPT ensemble; pressure was controlled using the Martyna–Tobias–Klein methodology, and the Nose–Hoover thermostat was employed to maintain the system near 310K. Production-grade MD trajectories were extended to a total duration of 20 ns per system.MD trajectories were characterized in terms of the root mean square deviation (RMSD) of fluctuations of ligand and receptor, particularly in terms of the main interactions with the top interacting residues. The trajectories were also used to assess the stabilities of the protein secondary structures (in complex with potential inhibitor) by plotting RMSDs.


### 2.4.In Vitro Assay

#### 2.4.1. Spike-ACE2 Binding Assay

Thein vitro enzyme-linked immunosorbent assay is a sensitive method to characterize the binding of the spike-ACE2 complex in the presence of potential inhibitors. The assay uses a 96-well plate coated with recombinantly expressed receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. The testing reagent-of-choice is then added to the wells in the presence of recombinant human ACE2 protein. Unbound ACE2 is removed by washing, and a goat anti-ACE2 antibody that binds to the spike-ACE2 complex is added.HRP-conjugated anti-goat IgG is then applied to the wells in the presence of 3,3′, 5,5′-tetramethylbenzidine (TMB) substrate. The HRP-conjugated anti-goat IgG binds to the ACE2 antibody and reacts with the TMB solution, producing a blue colour that is proportional to the amount of bound ACE. The HRP-TMB reaction is halted with the addition of the stop solution, resulting in a blue-to-yellow colour change. The intensity of the yellow colour is then measured at 450 nm. The percentage of inhibition of the formation of the spike-ACE2 complex was calculated as follows:(1)Spike−ACE2binding inhibition%=Acontrol−AsampleAcontrol·100,where Acontrol is the absorbance without inhibitor (considered as 100% Spike-ACE2 binding) and Asample is the absorbance of sample with inhibitor.


#### 2.4.2. ACE2 Inhibitor Screening Assay

The ACE2 inhibitor screening kit was used to detect potent inhibitors of ACE2 activity. It utilizes the ability of an active ACE2 to cleave a synthetic MCA-based peptide substrate to release a free fluorophore. The released MCA is easily quantified using a fluorescence microplate reader (excitation wavelength 320 nm and emission wavelength 420 nm). In the presence of an ACE2-specific inhibitor, the enzyme loses its peptidase activity which results in the decrease of fluorescence intensity. The ACE2 percentage inhibition was calculated as follows:(2)ACE2inhibition%=Fcontrol−FsampleFcontrol·100,where Fcontrol is the fluorescence with ACE2 and without inhibitor (considered as 100% enzyme activity) and Fsample is the fluorescence of sample with ACE2 and inhibitor.


## 2.1. Resources and Programs

UniProt Knowledgebase (UniProtKB), Swiss Protein (SwissProt) database, ChemSpider database, and Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) were searched.Openbabel GUI 2.4.1 [22], Acpype [23], Gromacs 2018 [24–26], Chimera UCSF 1.13.1 [27], AutoDock Tools 4.2 [28], AutoDock Vina 2.0 [29], PyMOL 2.3 (The PyMOL Molecular Graphics System, version 2.3 Schrödinger, LLC), Python 2.7.6 (Python Software Foundation), PoseView 1.1.2 (ZBH University of Hamburg, BioSolveIT GmbH), Omega 2.5.1.4 (OpenEye Scientific Software) [30], PLIP 1.3.2 [31], Maestro suite 2019.4, Schrödinger LLC, and the Shuttlemol suite of HPC scripts for virtual screening (https://bio-hpc.eu) were used.


## 2.2. Materials and Reagents

Synthetic GGV, VIKP, LSDFRS, and SDFRSY peptides were purchased from APeptide Co. Ltd. (Shanghai, China) and presented a purity degree above 95%. The Spike glycoprotein-ACE2 binding assay kit was purchased from RayBiotech® (Atlanta, United States) and the ACE2 inhibitor screening kit from AssayGenie Ltd. (London, United Kingdom).


## 2.3. Molecular Modelling

### 2.3.1. Selection of Ligands

Several amaranth and pea peptides were identified as bioactive peptides in numerous studies. Some amaranth peptides are well known for their 3-hydroxy-3-methylglutaryl reductase (HMGCR) and angiotensin-converting enzyme (ACE) inhibition capacities, thus reducing cholesterol production and hypertension [32–37]. Several pea peptides are proved to be ACE inhibitors but also ACE2 enhancers, boosting Ang 1–7 and Ang 1–9 production and consequently acting as vasodilators [38, 39].Accordingly, GGV, IGV, IVG, VIKP, and VGVL from amaranth and LSDFRS and SDFRSY from pea are selected as ligands.


### 2.3.2. Selection of Receptors

There are two different target groups that can be employed to overcome the COVID-19 infection: (1) receptors of the virus and (2) receptors of the host [5, 10–13].The spike glycoprotein (protein S) is mainly responsible for the SARS-CoV-2 entry at the host receptor, so it fuses the virus and the host membranes [40, 41]. Accordingly, this receptor is selected for docking studies (PDB code: 6vsb). Also, there are several virus proteins (3CLpro, RdRd, and PLpro) that are considered important factors for the infection [5, 42]. Among them, the 3CLpro is considered the key for virus replication, so it is selected as a virus receptor for docking analysis (PDB code: 6lu7).As host enzymes, angiotensin-converting enzyme 2 (ACE2) [40, 43], dipeptidyl peptidase (CD26) [13, 44, 45], and angiotensin-converting enzyme (ACE) [19] are identified as the most important human cell enzymes, so PDB codes 6m18, 6l8q, and 1o8a corresponding to these receptors were downloaded from RCSB.


### 2.3.3. Molecular File Preparation

3CLpro (PDB code: 6lu7), ACE2 (PDB code: 6m18), spike glycoprotein (PDB code: 6vsb), CD26 (PDB code: 6l8q), and ACE (PDB code: 1o8a) were downloaded from RCSB.Ligands were removed from pdb files employing PyMOL 2.3. Then, AutoDock Tools 4.2 was used to remove water molecules, add hydrogens, assign AD4 type to all atoms, compute Gasteiger charges, and save protein files both in pdb and pdbqt formats.Ligand molecules were downloaded from ChemSpider in mol format and converted to sdf format using the Openbabel GUI 2.4.1. Then they were converted to mol2 format with the Molconvert script from ChemAxon and minimized in the Merck Molecular Force Field (mmff94). AutoDock Tools 4.2 was employed to convert files to pdbqt format.


### 2.3.4. Blind Docking Analysis

A blind docking (BD) study was performed to detect the regions of interaction between ligands and the aforementioned receptors: ACE, ACE2, spike glycoprotein, CD16, and 3CLpro [46].Series of single docking simulations were performed in each alpha carbon of the protein, detecting the most favourable binding pockets in terms of bond energy but also the spatial conformation of the ligand and its bound residues. Affinities of ligand-protein interactions are determined with AutoDock 4.2 and individual energies of each bonded residue are calculated with PoseView 1.1.2, representing global bond energy and key residues bond energy, respectively.


### 2.3.5. Virtual Screening Analysis

To delve deeper into the binding pockets of each ligand, virtual screening (VS) studies were performed, allowing ligands to rotate and move within a box of 30 Å side and increasing the exhaustiveness of the blind docking analysis. The coordinates employed in this step had been obtained from the previous blind docking analyses.


### 2.3.6. Molecular Dynamics Simulation

Molecular dynamics (MD) is an approximation of the behaviour of the system against real conditions and allows to test the stability of the ligand-protein contacts, which were detected during the BD and VS analyses, during a period of time.MD simulations were carried out using the GPU version of Desmond included with Maestro suite 2019.4 (Schrödinger LLC) on a workstation with a NVIDIA QUADRO 5000. The system conformed by the ligand and protein of interest was solvated in an aqueous environment, in a cubic box with a minimal distance of 10 Å between the biomolecule and the box boundary (for periodic boundary conditions). Next, systems were neutralized and maintained in 0.15 M NaCl. The OPLS3 force field and the TIP3P-TIP4P water model were employed. Initially, the systems were simply energy-minimized for 2000 time steps. Next, systems were allowed to execute free dynamics in the NPT ensemble; pressure was controlled using the Martyna–Tobias–Klein methodology, and the Nose–Hoover thermostat was employed to maintain the system near 310K. Production-grade MD trajectories were extended to a total duration of 20 ns per system.MD trajectories were characterized in terms of the root mean square deviation (RMSD) of fluctuations of ligand and receptor, particularly in terms of the main interactions with the top interacting residues. The trajectories were also used to assess the stabilities of the protein secondary structures (in complex with potential inhibitor) by plotting RMSDs.


## 2.3.1. Selection of Ligands

Several amaranth and pea peptides were identified as bioactive peptides in numerous studies. Some amaranth peptides are well known for their 3-hydroxy-3-methylglutaryl reductase (HMGCR) and angiotensin-converting enzyme (ACE) inhibition capacities, thus reducing cholesterol production and hypertension [32–37]. Several pea peptides are proved to be ACE inhibitors but also ACE2 enhancers, boosting Ang 1–7 and Ang 1–9 production and consequently acting as vasodilators [38, 39].Accordingly, GGV, IGV, IVG, VIKP, and VGVL from amaranth and LSDFRS and SDFRSY from pea are selected as ligands.


## 2.3.2. Selection of Receptors

There are two different target groups that can be employed to overcome the COVID-19 infection: (1) receptors of the virus and (2) receptors of the host [5, 10–13].The spike glycoprotein (protein S) is mainly responsible for the SARS-CoV-2 entry at the host receptor, so it fuses the virus and the host membranes [40, 41]. Accordingly, this receptor is selected for docking studies (PDB code: 6vsb). Also, there are several virus proteins (3CLpro, RdRd, and PLpro) that are considered important factors for the infection [5, 42]. Among them, the 3CLpro is considered the key for virus replication, so it is selected as a virus receptor for docking analysis (PDB code: 6lu7).As host enzymes, angiotensin-converting enzyme 2 (ACE2) [40, 43], dipeptidyl peptidase (CD26) [13, 44, 45], and angiotensin-converting enzyme (ACE) [19] are identified as the most important human cell enzymes, so PDB codes 6m18, 6l8q, and 1o8a corresponding to these receptors were downloaded from RCSB.


## 2.3.3. Molecular File Preparation

3CLpro (PDB code: 6lu7), ACE2 (PDB code: 6m18), spike glycoprotein (PDB code: 6vsb), CD26 (PDB code: 6l8q), and ACE (PDB code: 1o8a) were downloaded from RCSB.Ligands were removed from pdb files employing PyMOL 2.3. Then, AutoDock Tools 4.2 was used to remove water molecules, add hydrogens, assign AD4 type to all atoms, compute Gasteiger charges, and save protein files both in pdb and pdbqt formats.Ligand molecules were downloaded from ChemSpider in mol format and converted to sdf format using the Openbabel GUI 2.4.1. Then they were converted to mol2 format with the Molconvert script from ChemAxon and minimized in the Merck Molecular Force Field (mmff94). AutoDock Tools 4.2 was employed to convert files to pdbqt format.


## 2.3.4. Blind Docking Analysis

A blind docking (BD) study was performed to detect the regions of interaction between ligands and the aforementioned receptors: ACE, ACE2, spike glycoprotein, CD16, and 3CLpro [46].Series of single docking simulations were performed in each alpha carbon of the protein, detecting the most favourable binding pockets in terms of bond energy but also the spatial conformation of the ligand and its bound residues. Affinities of ligand-protein interactions are determined with AutoDock 4.2 and individual energies of each bonded residue are calculated with PoseView 1.1.2, representing global bond energy and key residues bond energy, respectively.


## 2.3.5. Virtual Screening Analysis

To delve deeper into the binding pockets of each ligand, virtual screening (VS) studies were performed, allowing ligands to rotate and move within a box of 30 Å side and increasing the exhaustiveness of the blind docking analysis. The coordinates employed in this step had been obtained from the previous blind docking analyses.


## 2.3.6. Molecular Dynamics Simulation

Molecular dynamics (MD) is an approximation of the behaviour of the system against real conditions and allows to test the stability of the ligand-protein contacts, which were detected during the BD and VS analyses, during a period of time.MD simulations were carried out using the GPU version of Desmond included with Maestro suite 2019.4 (Schrödinger LLC) on a workstation with a NVIDIA QUADRO 5000. The system conformed by the ligand and protein of interest was solvated in an aqueous environment, in a cubic box with a minimal distance of 10 Å between the biomolecule and the box boundary (for periodic boundary conditions). Next, systems were neutralized and maintained in 0.15 M NaCl. The OPLS3 force field and the TIP3P-TIP4P water model were employed. Initially, the systems were simply energy-minimized for 2000 time steps. Next, systems were allowed to execute free dynamics in the NPT ensemble; pressure was controlled using the Martyna–Tobias–Klein methodology, and the Nose–Hoover thermostat was employed to maintain the system near 310K. Production-grade MD trajectories were extended to a total duration of 20 ns per system.MD trajectories were characterized in terms of the root mean square deviation (RMSD) of fluctuations of ligand and receptor, particularly in terms of the main interactions with the top interacting residues. The trajectories were also used to assess the stabilities of the protein secondary structures (in complex with potential inhibitor) by plotting RMSDs.


## 2.4.In Vitro Assay

### 2.4.1. Spike-ACE2 Binding Assay

Thein vitro enzyme-linked immunosorbent assay is a sensitive method to characterize the binding of the spike-ACE2 complex in the presence of potential inhibitors. The assay uses a 96-well plate coated with recombinantly expressed receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. The testing reagent-of-choice is then added to the wells in the presence of recombinant human ACE2 protein. Unbound ACE2 is removed by washing, and a goat anti-ACE2 antibody that binds to the spike-ACE2 complex is added.HRP-conjugated anti-goat IgG is then applied to the wells in the presence of 3,3′, 5,5′-tetramethylbenzidine (TMB) substrate. The HRP-conjugated anti-goat IgG binds to the ACE2 antibody and reacts with the TMB solution, producing a blue colour that is proportional to the amount of bound ACE. The HRP-TMB reaction is halted with the addition of the stop solution, resulting in a blue-to-yellow colour change. The intensity of the yellow colour is then measured at 450 nm. The percentage of inhibition of the formation of the spike-ACE2 complex was calculated as follows:(1)Spike−ACE2binding inhibition%=Acontrol−AsampleAcontrol·100,where Acontrol is the absorbance without inhibitor (considered as 100% Spike-ACE2 binding) and Asample is the absorbance of sample with inhibitor.


### 2.4.2. ACE2 Inhibitor Screening Assay

The ACE2 inhibitor screening kit was used to detect potent inhibitors of ACE2 activity. It utilizes the ability of an active ACE2 to cleave a synthetic MCA-based peptide substrate to release a free fluorophore. The released MCA is easily quantified using a fluorescence microplate reader (excitation wavelength 320 nm and emission wavelength 420 nm). In the presence of an ACE2-specific inhibitor, the enzyme loses its peptidase activity which results in the decrease of fluorescence intensity. The ACE2 percentage inhibition was calculated as follows:(2)ACE2inhibition%=Fcontrol−FsampleFcontrol·100,where Fcontrol is the fluorescence with ACE2 and without inhibitor (considered as 100% enzyme activity) and Fsample is the fluorescence of sample with ACE2 and inhibitor.


## 2.4.1. Spike-ACE2 Binding Assay

Thein vitro enzyme-linked immunosorbent assay is a sensitive method to characterize the binding of the spike-ACE2 complex in the presence of potential inhibitors. The assay uses a 96-well plate coated with recombinantly expressed receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. The testing reagent-of-choice is then added to the wells in the presence of recombinant human ACE2 protein. Unbound ACE2 is removed by washing, and a goat anti-ACE2 antibody that binds to the spike-ACE2 complex is added.HRP-conjugated anti-goat IgG is then applied to the wells in the presence of 3,3′, 5,5′-tetramethylbenzidine (TMB) substrate. The HRP-conjugated anti-goat IgG binds to the ACE2 antibody and reacts with the TMB solution, producing a blue colour that is proportional to the amount of bound ACE. The HRP-TMB reaction is halted with the addition of the stop solution, resulting in a blue-to-yellow colour change. The intensity of the yellow colour is then measured at 450 nm. The percentage of inhibition of the formation of the spike-ACE2 complex was calculated as follows:(1)Spike−ACE2binding inhibition%=Acontrol−AsampleAcontrol·100,where Acontrol is the absorbance without inhibitor (considered as 100% Spike-ACE2 binding) and Asample is the absorbance of sample with inhibitor.


## 2.4.2. ACE2 Inhibitor Screening Assay

The ACE2 inhibitor screening kit was used to detect potent inhibitors of ACE2 activity. It utilizes the ability of an active ACE2 to cleave a synthetic MCA-based peptide substrate to release a free fluorophore. The released MCA is easily quantified using a fluorescence microplate reader (excitation wavelength 320 nm and emission wavelength 420 nm). In the presence of an ACE2-specific inhibitor, the enzyme loses its peptidase activity which results in the decrease of fluorescence intensity. The ACE2 percentage inhibition was calculated as follows:(2)ACE2inhibition%=Fcontrol−FsampleFcontrol·100,where Fcontrol is the fluorescence with ACE2 and without inhibitor (considered as 100% enzyme activity) and Fsample is the fluorescence of sample with ACE2 and inhibitor.


## 3. Results and Discussion

### 3.1. Molecular Docking and Virtual Screening

Angiotensin-converting enzyme 2 is known to be one of the essential host receptors for SARS coronaviruses, and it is also proved to be the major cellular receptor for the novel SARS-CoV-2. ACE2 is a membrane protein expressed in the lungs, kidneys, heart, and intestine, and it is responsible for vasoconstriction and blood pressure control, being its decreased expression associated with cardiovascular diseases.COVID-19 disease causes ACE2 downregulation, affecting the tissues where it is expressed, but with major implications for lung and heart tissues, causing both pulmonary and cardiovascular complications [47, 48]. There are several studies that identify the region of interaction between the virus and this receptor, proving that the α1 and α2 helices are the most important areas of interaction [10–12]. Hence, the development of effective treatment should consider the blockage of this area to avoid SARS-CoV-2 binding, but the capacity for ACE2 expression enhancement should be also kept in mind.ACE2 infection is caused due to the fusion between this enzyme and the spike glycoprotein of SARS-CoV-2. The S1 and S2 subunits of the spike are known to be responsible for the receptor binding (S1) and for the membrane fusion among the viral subunit and ACE2 (S2) [49].The crystal structure between ACE2 and the spike glycoprotein is elucidated, and the residues which take part in the binding process are identified. To avoid virus infection, the blockage of the spike glycoprotein or the host cell receptors should be considered.Similar to ACE2, dipeptidyl peptidase is considered an important entry receptor for SARS coronaviruses, including MERS-CoV, SARS-CoV, and SARS-CoV-2 [44, 50, 51].CD26 has a key role in immune regulation as a T-cell activation molecule and in immune-mediated disorder, thus acting as a key immunoregulatory factor in viral infections. The S1 loop is considered to be the area of interaction of this receptor and SARS-CoV-2, being its key residues clearly identified, so therapies to overcome the COVID-19 can be developed by targeting this region [13].As a SARS-CoV-2 host receptor, angiotensin-converting enzyme was also identified as an important target. ACE is the precursor of angiotensin II, which is a strong inducer of vascular smooth muscle cells (VSMCs) by activating the AT1 receptor. The VSMCs are essential to maintain vascular function and homeostasis, being its upregulation responsible for the overexpression of proinflammatory cytokines and growth factors as IL-6, IL-8, and NF-kB, among others [14].Lungs are one of the most affected organs when the overexpression of the inflammatory cascade takes place. Accordingly, high inflammation levels are associated with major complications of COVID-19, so ACE inhibition may be a suitable strategy to avoid or mitigate this process [19, 20]. The ACE residue Glu384 is considered the key of the hot spot, so its regulation can be achieved by triggering this amino acid.A different strategy to fight the COVID-19 infection can be the direct treatment of the virus. The viral protease 3CLpro has been proved to be essential for coronavirus replication and is thus considered the most potent drug target among the virus receptors [5, 9]. The dyad His41-Cys145 conforms the hot spot of this receptor, so an effective SARS-CoV-2 disabling can be developed by targeting these key residues.To target these five receptors, the pea peptides, LSDRFS and SDRFSY, and the amaranth peptides, GGV, IGV, IVG, VGVL, and VIKP, are proposed. Its binding capacities are analysed in a docking study which detects, first, the regions of interaction of each peptide among the whole target receptor. This will indicate whether the peptide reaches the hot spot or remains trapped in ineffective areas of the protein.Thereafter, these effective interactions are characterized in terms of affinity energy. The different types of interaction (hydrogen bond, hydrophobic interaction,π-π interaction, and salt bridge) between ligand and receptor will determine its magnitude giving an associated energy value or score. Hence, those ligands which reach the hot spot area with sufficient energy are susceptible of being effectively bound to the receptor, enhancing or inhibiting its associated biological activity.


#### 3.1.1. Angiotensin-Converting Enzyme 2 (ACE2)

Angiotensin-converting enzyme 2 (ACE2) is considered one of the major host receptors ofβ-coronaviruses, including SARS-CoV, MERS-CoV, and SARS-CoV-2. Regarding the SARS-CoV-2, its interaction takes place among the α1 and α2 helices of ACE2, where residues Gln24, Asp30, His34, Tyr41, Gln42, Met82, Lys353, and Arg357 play a major role in reinforcing virus interaction [11, 12].There is some controversy about the regulation of ACE2. Some authors indicate that ACE2 expression reduction could be beneficial, so the main host receptor for SARS-CoV-2 would be diminished, involving a less acute infection [52]. Nevertheless, this would cause an increase of the ratio Ang II:Ang 1–7, exacerbating the pulmonary tissue damage initially provoked by SARS-CoV-2 [47, 48, 53]. It is true that cardiovascular tissues or cells that express ACE2 are potentially at risk for SARS-CoV-2 infection; however, ACE2 expression reduction would be also detrimental.The moderate enhancing production of ACE2 with a blocking agent, which helps to protect the area of interaction between ACE2 and SARS-CoV-2, may be a good approach to prevent COVID-19 disease while an adequate ratio Ang II:Ang 1-7 is maintained to avoid pulmonary tissue damage.Several pea peptides had been proved to be bioactive against ACE2, so ligands as LSDRFS and SDRFSY are known to interact in the hot spot of this receptor [39]. Besides, it is well known that when a ligand shows bioactivity against one receptor, it would bind its hot spot but also other nonactive areas of the target. Thus, considering the key area of interaction of SARS-CoV-2 in ACE2, a blind docking analysis was performed to detect both the region of interaction and its magnitude for the pea peptides, LSDRFS and SDRFSY, and the amaranth peptides, GGV, IGV, IVG, VGVL, and VIKP.Table1 shows the global affinity values, calculated with AutoDock 4.2, and the affinities related to the key residues for virus interaction, calculated with PoseView 1.1.2.Table 1
Angiotensin-converting enzyme 2 (ACE2) docking analysis.
Drug/peptideE bondglobal (kcal/mol)E bondkey residues (kcal/mol)Drug/peptideE bondglobal (kcal/mol)E bondkey residues (kcal/mol)Ritonavir (347980)−7,98>−2IGV−5,05−2,557−5,86−2,274−2,080−4,7>−2−5,79−2,279−2,055−2,313IVG−5,25−3,559Lopinavir (83706)−5,88−2,557−4,88−2,106−5,3−2,903VGVL−5,14>−2−4,85>−2−4,93−4,000Emtricitabine (54859)−4,94−5,184−4,28−5,587−4,83−4,453VIKP−5,18−2,049Hydroxychloroquine (3526)−5,06>−2−4,69>−2−4,71>−2LSDRFS−4,66−2,543−4,46−3,965−5,93−4,115−8,610Chloroquine (2618)−4,8>−1−5,69−5,649−2,366−2,557−4,39>−2−5,51−2,351−4,18>−2−4,69−11,390−2,191GGV−5,78−4,866SDRFSY−7,56−5,494−4,76−2,557−6,32−2,759−2,352−4,389−4,32−3,576−6,05−6,452LSDRFS and SDRFSY show the best performance against ACE2 (Table1), even compared to drugs that were studied for drug repositioning such as ritonavir, lopinavir, emtricitabine, hydroxychloroquine, or chloroquine [54–56]. Regarding the global interaction energy, SDRFSY has a better affinity value. Nevertheless, the ACE2 key residues that are fundamental for virus infection (Figure 1, yellow colour) are more strongly bound to LSDRFS than to SDRFSY. This means that these positions would be involved in the LSDRFS interaction, blocking the access of the SARS-CoV-2 to this area (Figure 1).Figure 1
Angiotensin-converting enzyme 2 blind docking analysis. (a) LSDRFS, (b) SDRFSY, (c) GGV, and (d) LSDRFS and GGV. Yellow = hot spot area/key residues. ACE2 PBD: 6m18.
(a)(b)(c)(d)Comparing the area of interaction of these two peptides, LSDRFS covers a more extensive area of the hot spot region than SDRFSY. There are four positions of interaction that are involved with key residues (Figure1(a)), and SDRFSY peptides let exposed the key residues Tyr41 and Lys353, located at the left side of α2 helix, and Gln24 and Met82, located at the right side of both helixes. Thus, due to the stronger interactions and larger blocked area, LSDRFS is expected to be more effective than SDRFSY to prevent ACE2 infection.VGVL and VIKP also show good global affinity values, but only two positions of the key binding area are effectively blocked (Supplementary Figures29 and 33), within key residue energies lower than −2.00 kcal/mol (Supplementary Figures 30 and 36), so this would probably cause poor blocking capacity against SARS-CoV-2 infection.GGV also shows a good performance, regarding the global and key residue energies, but the small size of this amaranth peptide does not allow to cover the whole area where the virus interacts (Figure1(c)). However, the combination of GGV and LSDRFS can be a promising match. Figure 1(d) shows that there are no competitive interactions between these ligands, so they interact along the whole α1 and α2 helices covering the main area of virus interaction and matching their strong binding capacities. This LSDRFS-GGV combination may be the most promising strategy to protect ACE2 from SARS-CoV-2 infection.


#### 3.1.2. Spike Glycoprotein

The spike glycoprotein or S protein of coronaviruses, including SARS-CoV-2, is one of their most important structural proteins. It recovers the external capsid of the virion, giving the appearance of a crown. Conformed by three major sections, ectodomain, single-pass transmembrane, and intracellular tail, the most important one is the ectodomain. This section is divided in two different subunits: the S1, responsible for the receptor binding to the host cells, and the S2, the membrane fusion unit [40, 49, 50].The main host receptor for SARS-CoV-2 and thus spike glycoprotein is ACE2. Several studies had identified the residues that take part in this interaction, revealing the importance of Tyr449, Tyr453, Leu455, Phe456, Phe486, Asn487, Tyr489, Gln493, Gly496, Gln498, Thr500, Asn501, Gly502, and Tyr505 [41, 51].Blind docking and virtual screening analyses were performed to detect the possible interactions between spike glycoprotein and the amaranth peptides, GGV, IGV, IVG, VGVL, and VIKP, and the pea peptides, LSDRFS and SDRFSY (Supplementary Table1, Supplementary Figures 44–64). Regarding the affinity scores (Supplementary Table 1), it seems that these ligands are not suitable to shield the crown of the SARS-CoV-2 and prevent an infection (Supplementary Figures 44–64). Nevertheless, this can be considered as a beneficial behaviour. It is well known that a certain molecule can act as a protein-protein stabilizer, enhancing the interaction between two (or more) different proteins [57]. This ineffective interaction with the protein S proves that there is no possibility for these peptides to enhance the ACE2 infection, so these results reinforce the promising behaviour of LSDRFS, SDRFSY, and the combination LSDRFS-GGV against ACE2 host receptor.


#### 3.1.3. Cluster of Differentiation 26 (CD26)

The cluster of differentiation 26 (CD26), also known as dipeptidyl peptidase, has been recognized as an important host receptor of SARS-CoV-2. Residues Lys267, Gln286, Ile287, Thr288, Ala289, Ala291, Leu294, Ile295, Arg317, Tyr322, Arg336, and Asn338 from S1 loop were identified as key residues in virus interaction [13].A blind docking analysis was performed to detect the possible interactions between this receptor and the proposed ligands LSDRFS, SDRFSY, GGV, IGV, IVG, VGVL, and VIKP. Also, the approved drugs chloroquine, hydroxychloroquine, and ritonavir, studied for drug repurposing, were tested following the same procedure. Accordingly, peptide and drug results can be compared (Supplementary Table2, Supplementary Figures 65–74), showing similar responses for SDRFSY and hydroxychloroquine, with high-affinity values against CD26.The hydroxychloroquine exerts a good performance against CD26, with great binding energies and blocking Val341, Asn338, Arg336, Gln286, and Lys287 from virus interaction (Supplementary Figure66, Supplementary Table 2). Nevertheless, the central area conformed by residues Ala291, Tyr322, Leu294, Ile295, and Arg317 is uncovered, exposing important residues from the hot spot and allowing the virus binding. SDRFSY performance can be compared with the hydroxychloroquine (Supplementary Figure 74, Supplementary Table 2), so it has also good energy values for global and key residues interaction. Due to the size of this pea peptide, a broad area of CD26 is expected to be inhibited, so Supplementary Figure 74 shows almost a total binding of the hot spot, being only Tyr322 and Arg336 unbound. This would help to protect the receptor against virus entry. Regarding the other studied drugs and peptides, low binding capacities are detected, so they are not expected to be effective virus blockers (Supplementary Table 2, Supplementary Figures 65–74).


#### 3.1.4. Angiotensin-Converting Enzyme (ACE)

Angiotensin-converting enzyme was selected as target because of its connexion to the inflammatory process mediated by NF-kB transcription factor, which is overexpressed in patients with major COVID-19 complications.Several pea and amaranth peptides are proved to have ACE inhibitory capacity, so LSDRFS, SDRFSY, GGV, IGV, IVG, VGVL, and VIKP ligands are studied against ACE by means of blind docking and virtual screening analyses. Also, the approved drug captopril was tested following the same procedure to contrast drug and peptide results (Supplementary Table3, Supplementary Figures 75–90).Glu384 is considered by UniProtKB database as the key residue to inhibit the hypertension effect caused by ACE. Supplementary Table3 summarizes the global and key residue binding energies of the proposed drugs and peptides, showing that LSDRFS and VIKP are expected to behave as effective hypertension reduction agents, thus also helping to reduce the inflammation levels of the organism. IGV and VGVL show also great inhibitory potentials, being only GGV results below captopril ones (Supplementary Figures 75–90). This is an indication of the angiotensin reduction capacity of these peptides, which may be suitable to inhibit the NF-kB transcription factor overexpression while helping to block the area of interaction of ACE2 and SARS-CoV-2. Hence, peptides as LSDRFS, with favourable binding capacities against ACE2 and ACE, are expected to exert a double action against COVID-19.


#### 3.1.5. Chymotrypsin-Like Protease (3CLpro)

The chymotrypsin-like protease enzyme (3CLpro) is indispensable to the viral replication and infection process, so it is considered nowadays the most important target to develop an antiviral therapy [9]. Several approved drugs are being studied for drug repositioning by targeting this receptor, such as demeclocycline, doxycycline, oxytetracycline, lymecycline, nicardipine, telmisartan, and conivaptan [5].It is unlikely that the proposed peptides would bind and inhibit 3CLpro, but due to the favourable binding of some of these ligands with ACE and ACE2, it is important to test their possible interferences with the virus itself. The hot spot of the 3CLpro receptor is the catalytic dyad His41-Cys145 [5, 9]. Thus, blind docking and virtual screening analyses were performed for 3CLpro and peptides LSDRFS, SDRFSY, GGV, IGV, IVG, VGVL, and VIKP regarding this binding site. Also, demeclocycline, doxycycline, oxytetracycline, lymecycline, nicardipine, telmisartan, and conivaptan are studied following the same procedure to contrast drug and peptide results (Supplementary Table 4, Supplementary Figures 91–103).The poor bond energies of this analysis suggest, as it was expected, an ineffective interaction between these ligands and the 3CLpro virus receptor results (Supplementary Table4, Supplementary Figures 91–103). However, this noninteraction between peptides and the main virus receptor can be favourable because it proves that there is no possibility of these peptides to act as a protein–protein stabilizer, enhancing the interaction between two (or more) different proteins [57]. Hence, these peptides are well-suited to inhibit the regions of interaction of ACE2 (LSDRFS and SDRFSY peptides) and mitigate the inflammation process by triggering ACE (LSDRFS and VIKP peptides) without exerting an attraction effect against the 3CLpro nor the spike protein SARS-CoV-2 receptors.


### 3.2. Molecular Dynamics Simulation

Molecular dynamics simulation study was performed to test the stability of the interactions detected during the molecular blind docking and virtual screening analyses. The LSDRFS, SDRFSY, and VIKP–ACE2 systems were studied following this protocol. Also, hydroxychloroquine was submitted to the same process to contrast peptide and drug performances [54]. Each ligand interaction with ACE2 was analysed, reporting the main protein-interacting residues during the simulation period and its correspondent RMSD plots (Figures 2–5, Supplementary Figures 104 and 105).Figure 2
Molecular dynamics simulation LSDRFS-ACE2. Analysis of positions 1 and 2: interactions and RMSD fluctuation.Figure 3
Molecular dynamics simulation LSDRFS–ACE2. Analysis of positions 3 and 4: interactions and RMSD fluctuation.Figure 4
Molecular dynamics simulation SDRFSY-ACE2. Analysis of positions 1, 2, and 3: interactions and RMSD fluctuation.Figure 5
Molecular dynamics simulation VIKP–ACE2. Analysis of positions 1, 2, and 3: interactions and RMSD fluctuation.The first docking position of LSDRFS (Figure2) shows a persistent interaction with Gln24 during the first 10 ns, but it becomes unstable after that, and a more intermittent but stable interaction with Met82. There are no persistent contacts during the whole simulation, so this is translated to the RMSD values, which show a high value around 18 Å. The second docking position (Figure 2) interacts persistently with Asn322 and Gln552, and more intermittently but stably with Phe555. This position is maintained during the whole simulation, causing lower and stable RMSD values (around 7 Å). Furthermore, the peptide head and tail are involved in these interactions, fixing the whole position of this peptide and thus blocking a wide area which otherwise would be available for spike glycoprotein reception.A quite similar behaviour is present at the third docking position (Figure3), which has stable interactions with Gln305, Trp328, and Ser331 and a persistent interaction with Glu329. Accordingly, the RMSD values are moderate and stable around 8 Å. The fourth position analysis (Figure 3) reveals persistent interactions with Asn90, Thr92, and Glu564 and high stable interactions with Leu91, Gln388, Arg559, and Ser563. This is translated to the RMSD value, which is stable and low (around 6 Å). The whole structure of this peptide (head, centre, and tail) is well fixed to the protein, causing a high stable interaction and allowing to block a big area where the spike glycoprotein interacts.LSDRFS shows stable interactions with ACE2, specially the second and fourth positions, followed by the third one (Figures2 and 3). This is expected to efficiently block the virus entry by covering an important area where the spike glycoprotein causes the ACE2 infection (Figure 1(a)).The first docking position of SDRFSY shows a persistent interaction with Asp30 and high stable interactions both with Glu37 and Ala387. Also, it has more intermittent but stable interactions with Val93 and Gln96 (Figure4). The peptide is fixed to the protein in its central zone, leaving the head and tail amino acids free to rotate. This makes the RMSD values slightly high (around 8.5 Å), but still covers a large area of the protein in a stable way. The second position (Figure 4) shows persistent interactions with Phe40, Ala348, Trp349, Asp350, Asp382, Arg393, Asn394, and Gly395 and also stable but slightly more intermittent interactions with Phe390, His401, and Arg514. This high number of contacts makes the peptide interaction very stable, with RMSD values around 4 Å. In addition, the whole peptide structure is bound to the protein, covering a large area of the protein in a very stable way, so a high capacity to prevent virus binding is expected. The third position (Figure 4) has persistent interactions with Trp48, Asn49, Asn53, Asn59, and Arg357. It also interacts slightly more intermittently with Asn61 and Asn330. The head amino acids are specially well fixed to the protein (Figure 4), leaving the tail free to rotate, which causes mid-low RMSD values around 7 Å.SDRFSY shows three interactions along the area of interaction of ACE2 and spike glycoprotein (Figure1(b)). These three docking positions have a high number of persistent contacts with ACE2 (Figure 4), making SDRFSY interactions very stable and thus preventing virus binding by covering its main area of interaction.VIKP shows intermittent and unstable interactions for the first and second docking positions (Figure5), with high RMSD values around 12 Å. Only the third position is considered stable, so it has persistent interactions with Trp48, Asn53, and Asn58 and low RMSD values around 4 Å (Figure 5).Blind docking and virtual screening analyses reveal VIKP as a weaker candidate for ACE2 binding. This was also proved by molecular dynamics analysis (Figure5), which show intermittent and unstable interactions specially for the first and second docking positions. Despite the stable interaction of the third position, the small size of this peptide does not allow to cover a large area of ACE2, so it is not expected to act as spike glycoprotein blocker.The high number of persistent contacts and low RMSD values of SDRFSY show the high stability of SDRFSY–ACE2 interactions, so this peptide is expected to behave as a good inhibitor for spike glycoprotein binding, followed by LSDRFS, with slightly higher instability but also strong interactions with several ACE2 key residues.These molecular dynamics results were compared with hydroxychloroquine, showing a better performance to prevent the virus infection than this drug which was used for COVID-19 treatment (Supplementary Figures104 and 105), so SDRFSY and LSDRFS peptides are potential candidates to act preventively against mid SARS-CoV-2 infection.


### 3.3.In Vitro Assays

Thein vitro analyses were carried out following the fluorimetric procedure explained before. The percentages of inhibition of peptides LSDRFS, SDRFSY, VIKP, and GGV for spike glycoprotein-ACE2 interaction and ACE2 activity are shown in Table 2.Table 2
Inhibitory effects of peptides on the interaction between SARS-CoV-2 spike glycoprotein and the human ACE2 receptor and on the ACE2 activity. a = 15 g/L, b = 7.5 g/L, c = 15 g/L (1 : 1), and d = 15 g/L (2 : 1), n.d. = not detected.
PeptidesSpike-ACE2ACE2PI (%)aPI (%)cPI (%)dPI (%)aPI (%)bLSDRFS79.56——95.5052.17SDRFSY84.65——95.8171.33VIKP62.03——94.3716.4GGV34.99——6.07n.dLSDRFS + GGV—4.0838.29——SDRFSY and LSDRFS show a higher inhibitory potential at spike–ACE2 and also ACE2 analysis. This is an indicative of the strong interaction of these peptides within the ACE2 receptor, which would prevent virus (spike glycoprotein) and host (ACE2) from forming an effective interaction. This is in accordance with the results obtained by the computational method, which predicted a good performance for SDRFSY and LSDRFS (Table1, Figures 1–4).VIKP shows lower inhibition values, as it was also reported by the docking and molecular dynamics analyses (Table1, Figure 5). However, results for the combination of LSDRFS and GGV widely differ from the molecular modelling ones, which foresaw a synergetic effect for the combination of these peptides (Figure 1(d)). This is probably due to the incorrect positioning of the peptides in the interaction zone of ACE2 and spike glycoprotein. Although after the BD analysis these ligands were located at compatible poses and positions, avoiding binding competition between them, regarding the obtained results, GGV probably presents a higher instability that interferes with LSDRFS interaction positions, causing poor inhibition capacities.


## 3.1. Molecular Docking and Virtual Screening

Angiotensin-converting enzyme 2 is known to be one of the essential host receptors for SARS coronaviruses, and it is also proved to be the major cellular receptor for the novel SARS-CoV-2. ACE2 is a membrane protein expressed in the lungs, kidneys, heart, and intestine, and it is responsible for vasoconstriction and blood pressure control, being its decreased expression associated with cardiovascular diseases.COVID-19 disease causes ACE2 downregulation, affecting the tissues where it is expressed, but with major implications for lung and heart tissues, causing both pulmonary and cardiovascular complications [47, 48]. There are several studies that identify the region of interaction between the virus and this receptor, proving that the α1 and α2 helices are the most important areas of interaction [10–12]. Hence, the development of effective treatment should consider the blockage of this area to avoid SARS-CoV-2 binding, but the capacity for ACE2 expression enhancement should be also kept in mind.ACE2 infection is caused due to the fusion between this enzyme and the spike glycoprotein of SARS-CoV-2. The S1 and S2 subunits of the spike are known to be responsible for the receptor binding (S1) and for the membrane fusion among the viral subunit and ACE2 (S2) [49].The crystal structure between ACE2 and the spike glycoprotein is elucidated, and the residues which take part in the binding process are identified. To avoid virus infection, the blockage of the spike glycoprotein or the host cell receptors should be considered.Similar to ACE2, dipeptidyl peptidase is considered an important entry receptor for SARS coronaviruses, including MERS-CoV, SARS-CoV, and SARS-CoV-2 [44, 50, 51].CD26 has a key role in immune regulation as a T-cell activation molecule and in immune-mediated disorder, thus acting as a key immunoregulatory factor in viral infections. The S1 loop is considered to be the area of interaction of this receptor and SARS-CoV-2, being its key residues clearly identified, so therapies to overcome the COVID-19 can be developed by targeting this region [13].As a SARS-CoV-2 host receptor, angiotensin-converting enzyme was also identified as an important target. ACE is the precursor of angiotensin II, which is a strong inducer of vascular smooth muscle cells (VSMCs) by activating the AT1 receptor. The VSMCs are essential to maintain vascular function and homeostasis, being its upregulation responsible for the overexpression of proinflammatory cytokines and growth factors as IL-6, IL-8, and NF-kB, among others [14].Lungs are one of the most affected organs when the overexpression of the inflammatory cascade takes place. Accordingly, high inflammation levels are associated with major complications of COVID-19, so ACE inhibition may be a suitable strategy to avoid or mitigate this process [19, 20]. The ACE residue Glu384 is considered the key of the hot spot, so its regulation can be achieved by triggering this amino acid.A different strategy to fight the COVID-19 infection can be the direct treatment of the virus. The viral protease 3CLpro has been proved to be essential for coronavirus replication and is thus considered the most potent drug target among the virus receptors [5, 9]. The dyad His41-Cys145 conforms the hot spot of this receptor, so an effective SARS-CoV-2 disabling can be developed by targeting these key residues.To target these five receptors, the pea peptides, LSDRFS and SDRFSY, and the amaranth peptides, GGV, IGV, IVG, VGVL, and VIKP, are proposed. Its binding capacities are analysed in a docking study which detects, first, the regions of interaction of each peptide among the whole target receptor. This will indicate whether the peptide reaches the hot spot or remains trapped in ineffective areas of the protein.Thereafter, these effective interactions are characterized in terms of affinity energy. The different types of interaction (hydrogen bond, hydrophobic interaction,π-π interaction, and salt bridge) between ligand and receptor will determine its magnitude giving an associated energy value or score. Hence, those ligands which reach the hot spot area with sufficient energy are susceptible of being effectively bound to the receptor, enhancing or inhibiting its associated biological activity.


### 3.1.1. Angiotensin-Converting Enzyme 2 (ACE2)

Angiotensin-converting enzyme 2 (ACE2) is considered one of the major host receptors ofβ-coronaviruses, including SARS-CoV, MERS-CoV, and SARS-CoV-2. Regarding the SARS-CoV-2, its interaction takes place among the α1 and α2 helices of ACE2, where residues Gln24, Asp30, His34, Tyr41, Gln42, Met82, Lys353, and Arg357 play a major role in reinforcing virus interaction [11, 12].There is some controversy about the regulation of ACE2. Some authors indicate that ACE2 expression reduction could be beneficial, so the main host receptor for SARS-CoV-2 would be diminished, involving a less acute infection [52]. Nevertheless, this would cause an increase of the ratio Ang II:Ang 1–7, exacerbating the pulmonary tissue damage initially provoked by SARS-CoV-2 [47, 48, 53]. It is true that cardiovascular tissues or cells that express ACE2 are potentially at risk for SARS-CoV-2 infection; however, ACE2 expression reduction would be also detrimental.The moderate enhancing production of ACE2 with a blocking agent, which helps to protect the area of interaction between ACE2 and SARS-CoV-2, may be a good approach to prevent COVID-19 disease while an adequate ratio Ang II:Ang 1-7 is maintained to avoid pulmonary tissue damage.Several pea peptides had been proved to be bioactive against ACE2, so ligands as LSDRFS and SDRFSY are known to interact in the hot spot of this receptor [39]. Besides, it is well known that when a ligand shows bioactivity against one receptor, it would bind its hot spot but also other nonactive areas of the target. Thus, considering the key area of interaction of SARS-CoV-2 in ACE2, a blind docking analysis was performed to detect both the region of interaction and its magnitude for the pea peptides, LSDRFS and SDRFSY, and the amaranth peptides, GGV, IGV, IVG, VGVL, and VIKP.Table1 shows the global affinity values, calculated with AutoDock 4.2, and the affinities related to the key residues for virus interaction, calculated with PoseView 1.1.2.Table 1
Angiotensin-converting enzyme 2 (ACE2) docking analysis.
Drug/peptideE bondglobal (kcal/mol)E bondkey residues (kcal/mol)Drug/peptideE bondglobal (kcal/mol)E bondkey residues (kcal/mol)Ritonavir (347980)−7,98>−2IGV−5,05−2,557−5,86−2,274−2,080−4,7>−2−5,79−2,279−2,055−2,313IVG−5,25−3,559Lopinavir (83706)−5,88−2,557−4,88−2,106−5,3−2,903VGVL−5,14>−2−4,85>−2−4,93−4,000Emtricitabine (54859)−4,94−5,184−4,28−5,587−4,83−4,453VIKP−5,18−2,049Hydroxychloroquine (3526)−5,06>−2−4,69>−2−4,71>−2LSDRFS−4,66−2,543−4,46−3,965−5,93−4,115−8,610Chloroquine (2618)−4,8>−1−5,69−5,649−2,366−2,557−4,39>−2−5,51−2,351−4,18>−2−4,69−11,390−2,191GGV−5,78−4,866SDRFSY−7,56−5,494−4,76−2,557−6,32−2,759−2,352−4,389−4,32−3,576−6,05−6,452LSDRFS and SDRFSY show the best performance against ACE2 (Table1), even compared to drugs that were studied for drug repositioning such as ritonavir, lopinavir, emtricitabine, hydroxychloroquine, or chloroquine [54–56]. Regarding the global interaction energy, SDRFSY has a better affinity value. Nevertheless, the ACE2 key residues that are fundamental for virus infection (Figure 1, yellow colour) are more strongly bound to LSDRFS than to SDRFSY. This means that these positions would be involved in the LSDRFS interaction, blocking the access of the SARS-CoV-2 to this area (Figure 1).Figure 1
Angiotensin-converting enzyme 2 blind docking analysis. (a) LSDRFS, (b) SDRFSY, (c) GGV, and (d) LSDRFS and GGV. Yellow = hot spot area/key residues. ACE2 PBD: 6m18.
(a)(b)(c)(d)Comparing the area of interaction of these two peptides, LSDRFS covers a more extensive area of the hot spot region than SDRFSY. There are four positions of interaction that are involved with key residues (Figure1(a)), and SDRFSY peptides let exposed the key residues Tyr41 and Lys353, located at the left side of α2 helix, and Gln24 and Met82, located at the right side of both helixes. Thus, due to the stronger interactions and larger blocked area, LSDRFS is expected to be more effective than SDRFSY to prevent ACE2 infection.VGVL and VIKP also show good global affinity values, but only two positions of the key binding area are effectively blocked (Supplementary Figures29 and 33), within key residue energies lower than −2.00 kcal/mol (Supplementary Figures 30 and 36), so this would probably cause poor blocking capacity against SARS-CoV-2 infection.GGV also shows a good performance, regarding the global and key residue energies, but the small size of this amaranth peptide does not allow to cover the whole area where the virus interacts (Figure1(c)). However, the combination of GGV and LSDRFS can be a promising match. Figure 1(d) shows that there are no competitive interactions between these ligands, so they interact along the whole α1 and α2 helices covering the main area of virus interaction and matching their strong binding capacities. This LSDRFS-GGV combination may be the most promising strategy to protect ACE2 from SARS-CoV-2 infection.


### 3.1.2. Spike Glycoprotein

The spike glycoprotein or S protein of coronaviruses, including SARS-CoV-2, is one of their most important structural proteins. It recovers the external capsid of the virion, giving the appearance of a crown. Conformed by three major sections, ectodomain, single-pass transmembrane, and intracellular tail, the most important one is the ectodomain. This section is divided in two different subunits: the S1, responsible for the receptor binding to the host cells, and the S2, the membrane fusion unit [40, 49, 50].The main host receptor for SARS-CoV-2 and thus spike glycoprotein is ACE2. Several studies had identified the residues that take part in this interaction, revealing the importance of Tyr449, Tyr453, Leu455, Phe456, Phe486, Asn487, Tyr489, Gln493, Gly496, Gln498, Thr500, Asn501, Gly502, and Tyr505 [41, 51].Blind docking and virtual screening analyses were performed to detect the possible interactions between spike glycoprotein and the amaranth peptides, GGV, IGV, IVG, VGVL, and VIKP, and the pea peptides, LSDRFS and SDRFSY (Supplementary Table1, Supplementary Figures 44–64). Regarding the affinity scores (Supplementary Table 1), it seems that these ligands are not suitable to shield the crown of the SARS-CoV-2 and prevent an infection (Supplementary Figures 44–64). Nevertheless, this can be considered as a beneficial behaviour. It is well known that a certain molecule can act as a protein-protein stabilizer, enhancing the interaction between two (or more) different proteins [57]. This ineffective interaction with the protein S proves that there is no possibility for these peptides to enhance the ACE2 infection, so these results reinforce the promising behaviour of LSDRFS, SDRFSY, and the combination LSDRFS-GGV against ACE2 host receptor.


### 3.1.3. Cluster of Differentiation 26 (CD26)

The cluster of differentiation 26 (CD26), also known as dipeptidyl peptidase, has been recognized as an important host receptor of SARS-CoV-2. Residues Lys267, Gln286, Ile287, Thr288, Ala289, Ala291, Leu294, Ile295, Arg317, Tyr322, Arg336, and Asn338 from S1 loop were identified as key residues in virus interaction [13].A blind docking analysis was performed to detect the possible interactions between this receptor and the proposed ligands LSDRFS, SDRFSY, GGV, IGV, IVG, VGVL, and VIKP. Also, the approved drugs chloroquine, hydroxychloroquine, and ritonavir, studied for drug repurposing, were tested following the same procedure. Accordingly, peptide and drug results can be compared (Supplementary Table2, Supplementary Figures 65–74), showing similar responses for SDRFSY and hydroxychloroquine, with high-affinity values against CD26.The hydroxychloroquine exerts a good performance against CD26, with great binding energies and blocking Val341, Asn338, Arg336, Gln286, and Lys287 from virus interaction (Supplementary Figure66, Supplementary Table 2). Nevertheless, the central area conformed by residues Ala291, Tyr322, Leu294, Ile295, and Arg317 is uncovered, exposing important residues from the hot spot and allowing the virus binding. SDRFSY performance can be compared with the hydroxychloroquine (Supplementary Figure 74, Supplementary Table 2), so it has also good energy values for global and key residues interaction. Due to the size of this pea peptide, a broad area of CD26 is expected to be inhibited, so Supplementary Figure 74 shows almost a total binding of the hot spot, being only Tyr322 and Arg336 unbound. This would help to protect the receptor against virus entry. Regarding the other studied drugs and peptides, low binding capacities are detected, so they are not expected to be effective virus blockers (Supplementary Table 2, Supplementary Figures 65–74).


### 3.1.4. Angiotensin-Converting Enzyme (ACE)

Angiotensin-converting enzyme was selected as target because of its connexion to the inflammatory process mediated by NF-kB transcription factor, which is overexpressed in patients with major COVID-19 complications.Several pea and amaranth peptides are proved to have ACE inhibitory capacity, so LSDRFS, SDRFSY, GGV, IGV, IVG, VGVL, and VIKP ligands are studied against ACE by means of blind docking and virtual screening analyses. Also, the approved drug captopril was tested following the same procedure to contrast drug and peptide results (Supplementary Table3, Supplementary Figures 75–90).Glu384 is considered by UniProtKB database as the key residue to inhibit the hypertension effect caused by ACE. Supplementary Table3 summarizes the global and key residue binding energies of the proposed drugs and peptides, showing that LSDRFS and VIKP are expected to behave as effective hypertension reduction agents, thus also helping to reduce the inflammation levels of the organism. IGV and VGVL show also great inhibitory potentials, being only GGV results below captopril ones (Supplementary Figures 75–90). This is an indication of the angiotensin reduction capacity of these peptides, which may be suitable to inhibit the NF-kB transcription factor overexpression while helping to block the area of interaction of ACE2 and SARS-CoV-2. Hence, peptides as LSDRFS, with favourable binding capacities against ACE2 and ACE, are expected to exert a double action against COVID-19.


### 3.1.5. Chymotrypsin-Like Protease (3CLpro)

The chymotrypsin-like protease enzyme (3CLpro) is indispensable to the viral replication and infection process, so it is considered nowadays the most important target to develop an antiviral therapy [9]. Several approved drugs are being studied for drug repositioning by targeting this receptor, such as demeclocycline, doxycycline, oxytetracycline, lymecycline, nicardipine, telmisartan, and conivaptan [5].It is unlikely that the proposed peptides would bind and inhibit 3CLpro, but due to the favourable binding of some of these ligands with ACE and ACE2, it is important to test their possible interferences with the virus itself. The hot spot of the 3CLpro receptor is the catalytic dyad His41-Cys145 [5, 9]. Thus, blind docking and virtual screening analyses were performed for 3CLpro and peptides LSDRFS, SDRFSY, GGV, IGV, IVG, VGVL, and VIKP regarding this binding site. Also, demeclocycline, doxycycline, oxytetracycline, lymecycline, nicardipine, telmisartan, and conivaptan are studied following the same procedure to contrast drug and peptide results (Supplementary Table 4, Supplementary Figures 91–103).The poor bond energies of this analysis suggest, as it was expected, an ineffective interaction between these ligands and the 3CLpro virus receptor results (Supplementary Table4, Supplementary Figures 91–103). However, this noninteraction between peptides and the main virus receptor can be favourable because it proves that there is no possibility of these peptides to act as a protein–protein stabilizer, enhancing the interaction between two (or more) different proteins [57]. Hence, these peptides are well-suited to inhibit the regions of interaction of ACE2 (LSDRFS and SDRFSY peptides) and mitigate the inflammation process by triggering ACE (LSDRFS and VIKP peptides) without exerting an attraction effect against the 3CLpro nor the spike protein SARS-CoV-2 receptors.


## 3.1.1. Angiotensin-Converting Enzyme 2 (ACE2)

Angiotensin-converting enzyme 2 (ACE2) is considered one of the major host receptors ofβ-coronaviruses, including SARS-CoV, MERS-CoV, and SARS-CoV-2. Regarding the SARS-CoV-2, its interaction takes place among the α1 and α2 helices of ACE2, where residues Gln24, Asp30, His34, Tyr41, Gln42, Met82, Lys353, and Arg357 play a major role in reinforcing virus interaction [11, 12].There is some controversy about the regulation of ACE2. Some authors indicate that ACE2 expression reduction could be beneficial, so the main host receptor for SARS-CoV-2 would be diminished, involving a less acute infection [52]. Nevertheless, this would cause an increase of the ratio Ang II:Ang 1–7, exacerbating the pulmonary tissue damage initially provoked by SARS-CoV-2 [47, 48, 53]. It is true that cardiovascular tissues or cells that express ACE2 are potentially at risk for SARS-CoV-2 infection; however, ACE2 expression reduction would be also detrimental.The moderate enhancing production of ACE2 with a blocking agent, which helps to protect the area of interaction between ACE2 and SARS-CoV-2, may be a good approach to prevent COVID-19 disease while an adequate ratio Ang II:Ang 1-7 is maintained to avoid pulmonary tissue damage.Several pea peptides had been proved to be bioactive against ACE2, so ligands as LSDRFS and SDRFSY are known to interact in the hot spot of this receptor [39]. Besides, it is well known that when a ligand shows bioactivity against one receptor, it would bind its hot spot but also other nonactive areas of the target. Thus, considering the key area of interaction of SARS-CoV-2 in ACE2, a blind docking analysis was performed to detect both the region of interaction and its magnitude for the pea peptides, LSDRFS and SDRFSY, and the amaranth peptides, GGV, IGV, IVG, VGVL, and VIKP.Table1 shows the global affinity values, calculated with AutoDock 4.2, and the affinities related to the key residues for virus interaction, calculated with PoseView 1.1.2.Table 1
Angiotensin-converting enzyme 2 (ACE2) docking analysis.
Drug/peptideE bondglobal (kcal/mol)E bondkey residues (kcal/mol)Drug/peptideE bondglobal (kcal/mol)E bondkey residues (kcal/mol)Ritonavir (347980)−7,98>−2IGV−5,05−2,557−5,86−2,274−2,080−4,7>−2−5,79−2,279−2,055−2,313IVG−5,25−3,559Lopinavir (83706)−5,88−2,557−4,88−2,106−5,3−2,903VGVL−5,14>−2−4,85>−2−4,93−4,000Emtricitabine (54859)−4,94−5,184−4,28−5,587−4,83−4,453VIKP−5,18−2,049Hydroxychloroquine (3526)−5,06>−2−4,69>−2−4,71>−2LSDRFS−4,66−2,543−4,46−3,965−5,93−4,115−8,610Chloroquine (2618)−4,8>−1−5,69−5,649−2,366−2,557−4,39>−2−5,51−2,351−4,18>−2−4,69−11,390−2,191GGV−5,78−4,866SDRFSY−7,56−5,494−4,76−2,557−6,32−2,759−2,352−4,389−4,32−3,576−6,05−6,452LSDRFS and SDRFSY show the best performance against ACE2 (Table1), even compared to drugs that were studied for drug repositioning such as ritonavir, lopinavir, emtricitabine, hydroxychloroquine, or chloroquine [54–56]. Regarding the global interaction energy, SDRFSY has a better affinity value. Nevertheless, the ACE2 key residues that are fundamental for virus infection (Figure 1, yellow colour) are more strongly bound to LSDRFS than to SDRFSY. This means that these positions would be involved in the LSDRFS interaction, blocking the access of the SARS-CoV-2 to this area (Figure 1).Figure 1
Angiotensin-converting enzyme 2 blind docking analysis. (a) LSDRFS, (b) SDRFSY, (c) GGV, and (d) LSDRFS and GGV. Yellow = hot spot area/key residues. ACE2 PBD: 6m18.
(a)(b)(c)(d)Comparing the area of interaction of these two peptides, LSDRFS covers a more extensive area of the hot spot region than SDRFSY. There are four positions of interaction that are involved with key residues (Figure1(a)), and SDRFSY peptides let exposed the key residues Tyr41 and Lys353, located at the left side of α2 helix, and Gln24 and Met82, located at the right side of both helixes. Thus, due to the stronger interactions and larger blocked area, LSDRFS is expected to be more effective than SDRFSY to prevent ACE2 infection.VGVL and VIKP also show good global affinity values, but only two positions of the key binding area are effectively blocked (Supplementary Figures29 and 33), within key residue energies lower than −2.00 kcal/mol (Supplementary Figures 30 and 36), so this would probably cause poor blocking capacity against SARS-CoV-2 infection.GGV also shows a good performance, regarding the global and key residue energies, but the small size of this amaranth peptide does not allow to cover the whole area where the virus interacts (Figure1(c)). However, the combination of GGV and LSDRFS can be a promising match. Figure 1(d) shows that there are no competitive interactions between these ligands, so they interact along the whole α1 and α2 helices covering the main area of virus interaction and matching their strong binding capacities. This LSDRFS-GGV combination may be the most promising strategy to protect ACE2 from SARS-CoV-2 infection.


## 3.1.2. Spike Glycoprotein

The spike glycoprotein or S protein of coronaviruses, including SARS-CoV-2, is one of their most important structural proteins. It recovers the external capsid of the virion, giving the appearance of a crown. Conformed by three major sections, ectodomain, single-pass transmembrane, and intracellular tail, the most important one is the ectodomain. This section is divided in two different subunits: the S1, responsible for the receptor binding to the host cells, and the S2, the membrane fusion unit [40, 49, 50].The main host receptor for SARS-CoV-2 and thus spike glycoprotein is ACE2. Several studies had identified the residues that take part in this interaction, revealing the importance of Tyr449, Tyr453, Leu455, Phe456, Phe486, Asn487, Tyr489, Gln493, Gly496, Gln498, Thr500, Asn501, Gly502, and Tyr505 [41, 51].Blind docking and virtual screening analyses were performed to detect the possible interactions between spike glycoprotein and the amaranth peptides, GGV, IGV, IVG, VGVL, and VIKP, and the pea peptides, LSDRFS and SDRFSY (Supplementary Table1, Supplementary Figures 44–64). Regarding the affinity scores (Supplementary Table 1), it seems that these ligands are not suitable to shield the crown of the SARS-CoV-2 and prevent an infection (Supplementary Figures 44–64). Nevertheless, this can be considered as a beneficial behaviour. It is well known that a certain molecule can act as a protein-protein stabilizer, enhancing the interaction between two (or more) different proteins [57]. This ineffective interaction with the protein S proves that there is no possibility for these peptides to enhance the ACE2 infection, so these results reinforce the promising behaviour of LSDRFS, SDRFSY, and the combination LSDRFS-GGV against ACE2 host receptor.


## 3.1.3. Cluster of Differentiation 26 (CD26)

The cluster of differentiation 26 (CD26), also known as dipeptidyl peptidase, has been recognized as an important host receptor of SARS-CoV-2. Residues Lys267, Gln286, Ile287, Thr288, Ala289, Ala291, Leu294, Ile295, Arg317, Tyr322, Arg336, and Asn338 from S1 loop were identified as key residues in virus interaction [13].A blind docking analysis was performed to detect the possible interactions between this receptor and the proposed ligands LSDRFS, SDRFSY, GGV, IGV, IVG, VGVL, and VIKP. Also, the approved drugs chloroquine, hydroxychloroquine, and ritonavir, studied for drug repurposing, were tested following the same procedure. Accordingly, peptide and drug results can be compared (Supplementary Table2, Supplementary Figures 65–74), showing similar responses for SDRFSY and hydroxychloroquine, with high-affinity values against CD26.The hydroxychloroquine exerts a good performance against CD26, with great binding energies and blocking Val341, Asn338, Arg336, Gln286, and Lys287 from virus interaction (Supplementary Figure66, Supplementary Table 2). Nevertheless, the central area conformed by residues Ala291, Tyr322, Leu294, Ile295, and Arg317 is uncovered, exposing important residues from the hot spot and allowing the virus binding. SDRFSY performance can be compared with the hydroxychloroquine (Supplementary Figure 74, Supplementary Table 2), so it has also good energy values for global and key residues interaction. Due to the size of this pea peptide, a broad area of CD26 is expected to be inhibited, so Supplementary Figure 74 shows almost a total binding of the hot spot, being only Tyr322 and Arg336 unbound. This would help to protect the receptor against virus entry. Regarding the other studied drugs and peptides, low binding capacities are detected, so they are not expected to be effective virus blockers (Supplementary Table 2, Supplementary Figures 65–74).


## 3.1.4. Angiotensin-Converting Enzyme (ACE)

Angiotensin-converting enzyme was selected as target because of its connexion to the inflammatory process mediated by NF-kB transcription factor, which is overexpressed in patients with major COVID-19 complications.Several pea and amaranth peptides are proved to have ACE inhibitory capacity, so LSDRFS, SDRFSY, GGV, IGV, IVG, VGVL, and VIKP ligands are studied against ACE by means of blind docking and virtual screening analyses. Also, the approved drug captopril was tested following the same procedure to contrast drug and peptide results (Supplementary Table3, Supplementary Figures 75–90).Glu384 is considered by UniProtKB database as the key residue to inhibit the hypertension effect caused by ACE. Supplementary Table3 summarizes the global and key residue binding energies of the proposed drugs and peptides, showing that LSDRFS and VIKP are expected to behave as effective hypertension reduction agents, thus also helping to reduce the inflammation levels of the organism. IGV and VGVL show also great inhibitory potentials, being only GGV results below captopril ones (Supplementary Figures 75–90). This is an indication of the angiotensin reduction capacity of these peptides, which may be suitable to inhibit the NF-kB transcription factor overexpression while helping to block the area of interaction of ACE2 and SARS-CoV-2. Hence, peptides as LSDRFS, with favourable binding capacities against ACE2 and ACE, are expected to exert a double action against COVID-19.


## 3.1.5. Chymotrypsin-Like Protease (3CLpro)

The chymotrypsin-like protease enzyme (3CLpro) is indispensable to the viral replication and infection process, so it is considered nowadays the most important target to develop an antiviral therapy [9]. Several approved drugs are being studied for drug repositioning by targeting this receptor, such as demeclocycline, doxycycline, oxytetracycline, lymecycline, nicardipine, telmisartan, and conivaptan [5].It is unlikely that the proposed peptides would bind and inhibit 3CLpro, but due to the favourable binding of some of these ligands with ACE and ACE2, it is important to test their possible interferences with the virus itself. The hot spot of the 3CLpro receptor is the catalytic dyad His41-Cys145 [5, 9]. Thus, blind docking and virtual screening analyses were performed for 3CLpro and peptides LSDRFS, SDRFSY, GGV, IGV, IVG, VGVL, and VIKP regarding this binding site. Also, demeclocycline, doxycycline, oxytetracycline, lymecycline, nicardipine, telmisartan, and conivaptan are studied following the same procedure to contrast drug and peptide results (Supplementary Table 4, Supplementary Figures 91–103).The poor bond energies of this analysis suggest, as it was expected, an ineffective interaction between these ligands and the 3CLpro virus receptor results (Supplementary Table4, Supplementary Figures 91–103). However, this noninteraction between peptides and the main virus receptor can be favourable because it proves that there is no possibility of these peptides to act as a protein–protein stabilizer, enhancing the interaction between two (or more) different proteins [57]. Hence, these peptides are well-suited to inhibit the regions of interaction of ACE2 (LSDRFS and SDRFSY peptides) and mitigate the inflammation process by triggering ACE (LSDRFS and VIKP peptides) without exerting an attraction effect against the 3CLpro nor the spike protein SARS-CoV-2 receptors.


## 3.2. Molecular Dynamics Simulation

Molecular dynamics simulation study was performed to test the stability of the interactions detected during the molecular blind docking and virtual screening analyses. The LSDRFS, SDRFSY, and VIKP–ACE2 systems were studied following this protocol. Also, hydroxychloroquine was submitted to the same process to contrast peptide and drug performances [54]. Each ligand interaction with ACE2 was analysed, reporting the main protein-interacting residues during the simulation period and its correspondent RMSD plots (Figures 2–5, Supplementary Figures 104 and 105).Figure 2
Molecular dynamics simulation LSDRFS-ACE2. Analysis of positions 1 and 2: interactions and RMSD fluctuation.Figure 3
Molecular dynamics simulation LSDRFS–ACE2. Analysis of positions 3 and 4: interactions and RMSD fluctuation.Figure 4
Molecular dynamics simulation SDRFSY-ACE2. Analysis of positions 1, 2, and 3: interactions and RMSD fluctuation.Figure 5
Molecular dynamics simulation VIKP–ACE2. Analysis of positions 1, 2, and 3: interactions and RMSD fluctuation.The first docking position of LSDRFS (Figure2) shows a persistent interaction with Gln24 during the first 10 ns, but it becomes unstable after that, and a more intermittent but stable interaction with Met82. There are no persistent contacts during the whole simulation, so this is translated to the RMSD values, which show a high value around 18 Å. The second docking position (Figure 2) interacts persistently with Asn322 and Gln552, and more intermittently but stably with Phe555. This position is maintained during the whole simulation, causing lower and stable RMSD values (around 7 Å). Furthermore, the peptide head and tail are involved in these interactions, fixing the whole position of this peptide and thus blocking a wide area which otherwise would be available for spike glycoprotein reception.A quite similar behaviour is present at the third docking position (Figure3), which has stable interactions with Gln305, Trp328, and Ser331 and a persistent interaction with Glu329. Accordingly, the RMSD values are moderate and stable around 8 Å. The fourth position analysis (Figure 3) reveals persistent interactions with Asn90, Thr92, and Glu564 and high stable interactions with Leu91, Gln388, Arg559, and Ser563. This is translated to the RMSD value, which is stable and low (around 6 Å). The whole structure of this peptide (head, centre, and tail) is well fixed to the protein, causing a high stable interaction and allowing to block a big area where the spike glycoprotein interacts.LSDRFS shows stable interactions with ACE2, specially the second and fourth positions, followed by the third one (Figures2 and 3). This is expected to efficiently block the virus entry by covering an important area where the spike glycoprotein causes the ACE2 infection (Figure 1(a)).The first docking position of SDRFSY shows a persistent interaction with Asp30 and high stable interactions both with Glu37 and Ala387. Also, it has more intermittent but stable interactions with Val93 and Gln96 (Figure4). The peptide is fixed to the protein in its central zone, leaving the head and tail amino acids free to rotate. This makes the RMSD values slightly high (around 8.5 Å), but still covers a large area of the protein in a stable way. The second position (Figure 4) shows persistent interactions with Phe40, Ala348, Trp349, Asp350, Asp382, Arg393, Asn394, and Gly395 and also stable but slightly more intermittent interactions with Phe390, His401, and Arg514. This high number of contacts makes the peptide interaction very stable, with RMSD values around 4 Å. In addition, the whole peptide structure is bound to the protein, covering a large area of the protein in a very stable way, so a high capacity to prevent virus binding is expected. The third position (Figure 4) has persistent interactions with Trp48, Asn49, Asn53, Asn59, and Arg357. It also interacts slightly more intermittently with Asn61 and Asn330. The head amino acids are specially well fixed to the protein (Figure 4), leaving the tail free to rotate, which causes mid-low RMSD values around 7 Å.SDRFSY shows three interactions along the area of interaction of ACE2 and spike glycoprotein (Figure1(b)). These three docking positions have a high number of persistent contacts with ACE2 (Figure 4), making SDRFSY interactions very stable and thus preventing virus binding by covering its main area of interaction.VIKP shows intermittent and unstable interactions for the first and second docking positions (Figure5), with high RMSD values around 12 Å. Only the third position is considered stable, so it has persistent interactions with Trp48, Asn53, and Asn58 and low RMSD values around 4 Å (Figure 5).Blind docking and virtual screening analyses reveal VIKP as a weaker candidate for ACE2 binding. This was also proved by molecular dynamics analysis (Figure5), which show intermittent and unstable interactions specially for the first and second docking positions. Despite the stable interaction of the third position, the small size of this peptide does not allow to cover a large area of ACE2, so it is not expected to act as spike glycoprotein blocker.The high number of persistent contacts and low RMSD values of SDRFSY show the high stability of SDRFSY–ACE2 interactions, so this peptide is expected to behave as a good inhibitor for spike glycoprotein binding, followed by LSDRFS, with slightly higher instability but also strong interactions with several ACE2 key residues.These molecular dynamics results were compared with hydroxychloroquine, showing a better performance to prevent the virus infection than this drug which was used for COVID-19 treatment (Supplementary Figures104 and 105), so SDRFSY and LSDRFS peptides are potential candidates to act preventively against mid SARS-CoV-2 infection.


## 3.3.In Vitro Assays

Thein vitro analyses were carried out following the fluorimetric procedure explained before. The percentages of inhibition of peptides LSDRFS, SDRFSY, VIKP, and GGV for spike glycoprotein-ACE2 interaction and ACE2 activity are shown in Table 2.Table 2
Inhibitory effects of peptides on the interaction between SARS-CoV-2 spike glycoprotein and the human ACE2 receptor and on the ACE2 activity. a = 15 g/L, b = 7.5 g/L, c = 15 g/L (1 : 1), and d = 15 g/L (2 : 1), n.d. = not detected.
PeptidesSpike-ACE2ACE2PI (%)aPI (%)cPI (%)dPI (%)aPI (%)bLSDRFS79.56——95.5052.17SDRFSY84.65——95.8171.33VIKP62.03——94.3716.4GGV34.99——6.07n.dLSDRFS + GGV—4.0838.29——SDRFSY and LSDRFS show a higher inhibitory potential at spike–ACE2 and also ACE2 analysis. This is an indicative of the strong interaction of these peptides within the ACE2 receptor, which would prevent virus (spike glycoprotein) and host (ACE2) from forming an effective interaction. This is in accordance with the results obtained by the computational method, which predicted a good performance for SDRFSY and LSDRFS (Table1, Figures 1–4).VIKP shows lower inhibition values, as it was also reported by the docking and molecular dynamics analyses (Table1, Figure 5). However, results for the combination of LSDRFS and GGV widely differ from the molecular modelling ones, which foresaw a synergetic effect for the combination of these peptides (Figure 1(d)). This is probably due to the incorrect positioning of the peptides in the interaction zone of ACE2 and spike glycoprotein. Although after the BD analysis these ligands were located at compatible poses and positions, avoiding binding competition between them, regarding the obtained results, GGV probably presents a higher instability that interferes with LSDRFS interaction positions, causing poor inhibition capacities.


## 4. Conclusions

β-Coronaviruses have crossed the animal–human barrier, causing several outbreaks during recent years, but none of them with the dimensions of SARS-CoV-2, which has been spread for over the world [58], so both treatment and preventive approaches are urgently required. In this scenario, natural peptides play a major role offering a preventive strategy or a treatment for non-acute patients due to its GRAS (generally recognised as safe) property.In this study, several pea and amaranth peptides were analysed against the major SARS-CoV-2 receptors—both host and virus. Molecular docking and dynamics simulation results show that SDRFSY and LSDRFS have strong and stable interactions with ACE2, so these peptides can be suitable to shield the area of ACE2 where the spike glycoprotein of the virus exerts its interaction (Table1, Figures 1(a) and 1(b)). It was also proved that none of the studied peptides interacts with the spike glycoprotein, so their presence does not cause an increase in the interactions between ACE2 and the spike glycoprotein, which verifies that these peptides do not have the potential to act as protein–protein interaction (PPI) stabilizers (Supplementary Table 2). These theoretical results were confirmed by in vitro analyses, proving that SDRFSY and LSDRFS prevent the spike glycoprotein–ACE2 interaction with inhibition percentages above 80% at 15 g/L (Table 2).Also, computational results show that CD26 can be similarly protected with SDRFSY and VIKP (Supplementary Table2), and ACE, which is inextricably linked to the cytokine cascade that causes major complications in acute COVID-19 patients, can be inhibited by LSDRFS and VIKP (Supplementary Table 3).Hence, it can be assessed that the pea peptides, SDRFSY and LSDRFS, and the amaranth peptide, VIKP, are susceptible of being useful not to overcome but to mitigate and prevent the effects of the SARS-CoV-2 in an effective and quick manner.



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*Source: 2102937-2022-03-10.xml*