In silico studies on milk derived peptides as potential inhibitors against SARS CoV-2 spike protein receptor binding domain

COVID-19 (Corona Virus Infected Diseases-19) is caused by a strain of coronavirus called SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2). There’s no permanent diagnosis available till date to combat the disease. The viral infection in humans is initiated by binding of RBD (receptor binding domain) of spike protein to human angiotensin-converting enzyme 2 (hACE2) receptor protein. In this computational study, milk-derived peptides are screened against Receptor Binding Domain (RBD) of spike protein of the virus. Milk is considered as one of the most nutrient-rich liquid foods having several antibacterial and antiviral activities. Milk derived peptides including Casein and Whey derived peptides are known to have profound anti-viral and immunomodulatory activities. After extensive literature search, some peptides having anti-viral activities against different viruses, are shortlisted for this study and their three-dimensional structures are modelled for protein-peptide docking against SARS-CoV-2 spike protein RBD. After performing protein-peptide docking and protein-protein docking using different servers such as HPEPDOCK, FIREDOCK, HADDOCK, HDOCK, it has been observed that in presence of the peptides, the interaction between spike RBD and hACE2 has been reasonably decreased, which implies that milk-derived peptides can be potential peptide-inhibitors against the RBD of the virus along with other medications. Further studies on milk-derived peptides should be performed to develop peptide drugs based on milk-derived peptides.


Introduction
A new strain of coronavirus was rst detected in December 2019 at Wuhan, China. This virus is designated as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and the disease caused by the virus is known as COVID-19 [1]. As per the latest update on March 17, 2021, a total of 121 M people has been affected by the disease and it causes the demises of 2.67 M people worldwide [2]. There's no permanent medication available to combat the virus till date! Keeping in mind the urgent need of a potential anti-viral against SARS-CoV-2, drug repurposing can be a very useful route to ght against the viral infection [3].
SARS-CoV-2 is a type of beta-coronavirus with positive-sense single-stranded RNA [4]. Like other coronaviruses, SARS-CoV-2 also has four structural and various nonstructural proteins. The structural proteins are: spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid (N) protein. Spike, envelope and membrane proteins form the viral envelope together [5]. The spike (S) protein allows the virus to be attached into the host surface by interacting with human angiotensin-converting enzyme-2 (hACE2) receptors which are expressed in many organs including the lung, small intestine, testis, and kidney [6] [7].
The S protein comprises S1 and S2 domains. The S1 domain is responsible for binding to ACE2 receptors via its receptor-binding domain (RBD), whereas the S2 domain performs the fusion, enabling viral genome entry [8]. The viral infection in humans is initiated by binding of RBD (receptor binding domain) of spike protein to human angiotensin-converting enzyme 2 (hACE2) receptor protein [9], which suggests that if a therapeutic agent can disrupt the protein-protein interaction between RBD and hACE-2, the viral infection might be terminated or reduced at a very early stage of infection. Small molecules or peptide inhibitors can be designed to disrupt the PPI but small molecules are not suitable for targeting large protein-protein interactions; on the other hand, peptides, due to their large surface area can target the protein binding interface more e ciently than that of small molecules [10]. Recently, several works have been done by researchers around the world in this context. A group has proposed human ACE-2 alphahelix based peptide inhibitors by computational studies [11]. Another work has been done on natural food preservative peptide nisin to interact with the SARS-CoV-2 spike protein receptor human ACE2 [12].
Some experimental studies suggest that milk proteins can e ciently interfere with viral infections [13] [14]. Lactoferrin is one of the most studied milk-protein for its antiviral potential [15]. In this in silico study, some milk-derived peptides having immunomodulatory effects and antiviral activities against other viruses have been selected for experimentation [16]. At rst, all those peptides, human ACE2 and human ACE2 alpha-helix were computationally docked against SARS-CoV-2 spike protein RBD, then RBD-peptide complexes were again docked against ACE2 to check whether the binding a nity between the RBD and hACE-2 were decreased in the presence of the peptides or not. Peptides with a reasonable binding a nity with the spike protein RBD have been reported as potential peptide inhibitor against SARS-CoV-2 viral infection. Various physicochemical properties of the peptides such as hydrophobicity, IC50, toxicity, B. Bioinformatic analyses of antiviral potential of the peptides: Using iAMPPred web-server (http://cabgrid.res.in:8080/amppred/index.html) antiviral potential of the peptides have been estimated. iAMPPred server uses Support Vector Machine (SVM) algorithm to predict the antimicrobial, antiviral and antifungal peptides [17].
C. de novo structure determination and validation: De novo structure predicting PEP-FOLD3 web server (https://bioserv.rpbs.univ-parisdiderot.fr/services/PEP-FOLD3/)was used to model selected peptides. It's based on structural alphabet (SA) letters to describe the conformations of four consecutive residues, couples the predicted series of SA letters to a greedy algorithm and a coarse-grained force eld [18]. The structures have been further validated by Ramachandran plot with the help of Ramachandran plot server by zlab (https://zlab.umassmed.edu/bu/rama/) [19] [20].

E. Computation of various physical and chemical parameters:
Protparam (https://web.expasy.org/protparam/) is a tool for computing various physical and chemical parameters for a given protein or peptide. The computed parameters include the molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coe cient, estimated half-life, instability index, aliphatic index and grand average of hydropathicity (GRAVY). Amino acid sequence of the peptides has been given as an input to determine the physicochemical properties of the peptides [31]. Half maximal inhibitory concentration (IC 50) has been calculated using the AVP-IC50 Pred web-server (http://crdd.osdd.net/servers/ic50avp/) [32]. Hemolytic or hemotoxic or RBC lysing potential of the peptides have been estimated by HemoPI: Hemolytic Peptide Identi cation Server (https://webs.iiitd.edu.in/raghava/hemopi/) [33]. Toxicity of the peptides was determined by ToxinPred server (http://crdd.osdd.net/raghava/toxinpred/) [34].

Results And Discussions
A. Peptide sequence retrieval: From a book chapter named "Milk Derived Peptides with Immune Stimulating Antiviral Properties" By Haiyan Sun and Håvard Jenssen from a book "Milk Protein" edited by Walter Hurley, University of Illinois (https://www.intechopen.com/books/milk-protein/milk-derived-peptides-with-immune-stimulatingantiviral-properties?fbclid=IwAR0WLVwxgv-vfmw4mk9RZtEWCd-wJrVFw0DTne3XdxODgqgOqganNcvyjl8), six (6) milk-derived peptides having antiviral and/ or immunomodulatory effects have been identi ed and selected for docking against SARS CoV-2 viral protein [16]. A detailed description of the proteins given below:  According to iAMPPred web-server, a peptide having > = 0.5 antiviral activity can be referred to as antiviral.

C. Computation of Physicochemical Properties:
All the physicochemical properties of the six peptides have been summed up in Table V. Among the six peptides, Peptide 02 and Peptide 03 have greater half-life (estimated) than the others. A protein having instability index < 40 can be treated as stable, whereas if instability index > = 40, the corresponding protein will be unstable, from that point of view, none of the peptides are stable. As the peptides are unstable, the stability should be enhanced using various biochemical techniques such as cyclization of the peptide, replacement of natural peptide bonds by pseudo-peptide bonds, selective chemical modi cation etc. before experimental validation against spike protein [42]. Otherwise, peptidomimetic drugs can be produced based on the functionality of the peptides, which are basically small organic molecules mimicking the characteristics of the peptides, but a way more stable than the peptides [43]. In brief, some processing techniques, such as chemical modi cation or incorporation of synthetic amino acids, can be applied to increase peptide stability and, consequently, lower susceptibility to hydrolysis by proteases.
The aliphatic index of a protein is the relative volume occupied by aliphatic side chains (alanine, valine, isoleucine, and leucine). The GRAVY value for a peptide or protein is calculated as the sum of hydropathy values of all the amino acids, divided by the number The heat stability of peptide was indicated by its aliphatic index. The higher aliphatic index means higher heat stability. The hydrophilicity and hydrophobicity of peptide were predicted by GRAVY. The peptide was hydrophobic when the GRAVY value was plus; otherwise, it was hydrophilic. Interestingly, in the case of Peptide 05, aliphatic index is zero (0), signi es the absence of aliphatic side chains (Ala, Val, Ile, Leu). According to the ToxinPred web server, none of the peptides are toxic. For toxicity prediction, SVM (Swiss-Prot) + Motif based method was used and other options were set as default. HemoPI server was used to identify the hemolytic potency of the peptides and hybrid method (SVM + Motif based) was used for prediction. Hemolytic potency of a peptide is decided by PROB score, which is the normalized SVM score and ranges between 0 and 1, where 1 means the peptide is very likely to be hemolytic and 0 means it is likely to be non-hemolytic. For the aforementioned peptides, the PROB score ranges from 0.46 (peptide 06) to 1.00 (Peptide 01 and 05), which signi es that there is a high probability for them of being

Conclusions
Peptide therapeutics have various advantages like low toxicity, ease of synthesis, high target speci city etc. In this present computational study, according to the docking score and free energy calculations, Peptide 03 shows comparatively better binding a nity with the spike protein RBD. Presence of Peptide 03 also reduces the interaction between human ACE2 and spike protein RBD. Being a low molecular weight peptide and readily bioavailable, its binding to RBD is expected to interfere the bonding interaction between RBD of spike of SARS-CoV-2 and human ACE2 and could reduce the infection. Molecular dynamics simulation is planned to be conducted to further verify the interactions of these protein-peptide complexes. Further in vitro and in vivo validation should be done to determine its doses and e cacies against SARS-CoV-2 spike proteins.

Declarations
Con ict of Interest: Protein-peptide interactions for spike protein RBD and peptide 01 Protein-peptide interactions for spike protein RBD and peptide 03

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. SupportingInformation1.pdf