Full title: “Proposing the structure of a new drug as a potential treatment for COVID-19 by inhibition of the Receptor Binding Domain of the Spike (S) Protein on SARS-CoV-2”
Authors: Aditi Das and Tanya Ghai, McMaster University
This article endeavours to find a solution to the COVID 19 pandemic by proposing a structure of a potential inhibitor drug that would be able to prevent the virus from affecting the human body. Severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2, which is the causative agent if Covid 19 pandemic, uses spike (S) protein to infect the human body. During viral entry into the host cell, the S protein on the envelope of the virus binds to the host cell receptor, angiotensin converting enzyme 2 (ACE2). This results in the fusion of the viral membrane with the host cell and release of viral RNA in the host body for replication. To design a drug that would be able to inhibit the SARS-CoV-2 spike protein, Pymol and molecular docking software were used. It was hypothesized that the designed drug would be able to mimic the binding ability of the ACE2 receptor protein and in an environment where both designed drugs and the receptor protein are present, the former would be able to more efficiently bind to the S protein first, thus blocking any chance for virus to interact with the ACE2 to gain entry in human cell. To create a good inhibitor, it was found to be beneficial to add heteroatoms and hydroxyl, amine and thiol groups that would be capable of forming hydrogen bonds with the receptor binding domain (RBD) of the viral S protein. Moreover, some bulky aromatic groups were included to facilitate interaction with the aromatic residues on the binding site. When the docking simulation was run, a docking score of -4.4 was generated. The more negative the docking score, the higher the binding affinity. The docking score of ACE2 itself is -4.1, which is a less negative value compared to the designed drug. This implies that the latter has higher binding affinity than the receptor protein and is more efficient than ACE2. This drug is predicted to be able to competitively bind to viral cells and inhibit it, thus aiding in preventing the virus from spreading in the body. This research may provide an important opportunity for the accelerated development of potential treatment measures for COVID-19. Nevertheless, more clinical trials need to be done to confirm the effectiveness of the inhibitor since the docking simulation does not take all physiological factors into consideration.
The recent coronavirus pandemic has brought the entire world to a standstill and affected almost every facet of human lives. After almost a year of the initial outbreak, the virus still continues to spread across the world, taking many lives with it and leaving many lasting effects on the social, economic and personal aspects of daily life. Severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2 was identified as the causative agent of the COVID-19 outbreak. SARS-CoV-2 is a 100nm sphere virion which comes from Pisonivircetes class (Shang et al., 2020). The most important structural proteins of CoV are spike (S) protein (trimeric), membrane (M) protein, envelope (E) protein, and the nucleocapsid (N) protein ( Prajapat et al., 2020) . Protein of interest for this research is the Spike protein that interfaces with the target organisms receptor. To fulfill its function, it uses a novel metallocarboxyl peptidase, angiotensin converting enzyme 2 or ACE2 to gain entry into human cells ( Huang et al., 2020). Using the available knowledge, this research paper will endeavour to design a drug that will have a potential to inhibit the function of S protein, causing the viral cell unable to bind with the host cell. It is hypothesized that, in an environment where both ACE2 and the designed drug are present, the latter would be able to reach and interact with the receptor binding site of the spike protein first, blocking the ACE2’s access to it and thus, competitively inhibit the function of the spike protein. Through the process of Pymol and molecular docking software, it would be possible to look closely to examine the structure and to determine the binding affinity of the designed inhibitor to the RBD of the S protein.
In order to create a good inhibitor, it was found that it is beneficial to have heteroatoms, some thiol groups, hydroxyl and amine groups, and some bulky aromatic groups that would interact with the residues on the target. Docking software will determine how well the inhibitor binds to RBD. The docking score for the inhibitor designed in this study was -4.4 ( More than Molecule, 2020) . Thus, when this inhibitor gets injected into the bloodstream of the human body, it will competitively inhibit the receptor binding site on S protein and stop the spread of the virus. Through simulations and during the active process of designing the inhibitor, the inhibitor seems to work well in theory, but the physiological conditions need to be taken into account. The docking simulation cannot predict how the physiological factors will affect the inhibitor binding. Due to this reason, clinical trials need to be conducted to consider its future implications such as, to determine the effectiveness of the inhibition.
Coronaviruses (CoVs) have a single-stranded RNA genome, covered by an enveloped structure. The shape is either pleomorphic or spherical, and it is characterized by club-shaped projections of glycoproteins of diameter 80–120 nm on its surface ( Prajapat et al., 2020) . Among all the RNA viruses, the RNA genome of CoV is one among the largest (Shang et al., 2020). By September 2020, the World Health Organization confirmed more than 25.88 million cases of COVID-19 and 859,000 associated deaths in 216 countries (Lan et al., 2020). The spike protein for SARS-CoV-2 is responsible for receptor recognition and membrane fusion. During viral entry into the host cell, the spike proteins (S) on the envelope of SARS-CoV-2 are cleaved into S1 and S2 subunit ( Huang et al., 2020) . S2 does not interact with the receptor but it harbors the functional elements required for membrane fusion of the virion. S1 contains receptor binding domain (RBD) and directly binds to the peptidase domain of ACE 2 and its proteolytic activation by human proteases allows entry into the host cell ( Huang et al., 2020) . The spike protein is 1273 amino acids long and consists of extracellular N terminus (NTD), an intracellular C terminus (CTD) and a transmembrane (TM) domain (Huang et al., 2020). This trimer forms a bulbous structure around the viral particle. A short signal peptide located at N terminus that expands from residue 1-13. It is also divided into two subunits, S1 and S2. The S1 subunit expands from residue 14 to 685 and is responsible for binding to the host cell receptor (Huang et al., 2020). It contains a N terminal domain (NTD) and a receptor binding domain (RBD) extending from residue 14 to 309 and residues 319 to 541 respectively (Huang et al., 2020). The S2 subunit extends from residue 686 to 1273 and has fusion peptide (FP) from residue 788 to 806, heptapeptide repeat sequences, HR1 and HR2, extending from residues 912-984 and 1163-1213 respectively, the TM domain found on residue 1213-1237 and the cytoplasm domain from 1237-1273 (Huang et al., 2020). This subunit is responsible for the fusion of the viral membrane with the host cell and release of viral RNA in the host body for replication.
Pymol and molecular docking software were used to examine the structure and to determine the binding affinity (More than Molecule., 2020). To obtain a good inhibitor, it was found to be beneficial to have heteroatoms that are capable of forming hydrogen bonds on the inhibitor, so it can competitively interact with the hydrogen bonding residues on RBD. On top of that, some thiol groups were necessary to be added to the inhibitor to disrup the disulfide bonds in RBD and interact with cysteine residue instead. Moreover, hydroxyl and amine groups were added to aid with formation of strong hydrogen bonds with different residues on the target and some bulky aromatic groups were included to facilitate interaction with the aromatic residues on the RBD. Use of docking software allowed the use of an algorithm, which was used to determine the location of where the substrate binds and how well it binds to the enzyme. When the docking simulation was run, a negative integer value was generated. The more negative the docking score, the higher the binding affinity. The docking score of ACE2 was -4.1. Whereas, the docking score for the inhibitor designed in this study was -4.4. Thus, it is more efficient than ACE2 in binding to the active site of a target and thus, inhibiting its activity. Hence, such research may provide an important opportunity for the accelerated development of potential treatment measures for COVID-19.
The spike (S) protein for SARS-CoV-2 is responsible for receptor recognition and membrane fusion. These proteins are found on the surface of the viral structure and identify the ACE2 receptor on the host body and bind to it. The binding of the protein with the receptor activates the S protein and initiates host invasion. The viral cell membrane fuses with the host cell body and releases it’s RNA material and replicates inside the host cell. It is possible that if the functions of the S protein is somehow inhibited, the viral cell would not be able to bind or fuse with the host cell.
To accomplish this, a inhibitor was designed that would mimic the substrate that binds to the S protein, which in this case is the ACE2 protein. The inhibitor would be able to mimic the substrate’s binding mechanism and bind to the active site through competitive inhibition. The inhibitor will inhibit the protein and block the receptor’s access to the spike protein.
The binding of the spike protein with the ACE2 receptor initiates the viral infection. Only after the spike protein attaches to the ACE2 receptor on the host cell, the cell proteases cleave the S1 and S2 unit and activate the protein (Huang et al., 2020). The receptor binding domain (RBD) situated in the S1 subunit is responsible for binding to the ACE2 protein (Huang et al., 2020). The binding efficiency is stabilized by the presence of NTD and CTD (Huang et al., 2020). TheS1 subunit as a whole ensures proper viral binding to the receptor. Due to this reason, inhibiting the RBD domain might help stop the viral binding to the receptor and thus, prevent the viral infection.
The receptor binding domain has a twisted five stranded antiparallel beta sheet formed by beta strands β1, β2, β3, β4 and β7, which are connected by helices and loops (Lan et al. 2020). Between the β4 and β7, the receptor binding motif (RBM) is found which is composed of short beta strands, β5 and β6, and α4 and α5 helices, which are connected by loops (Lan et al. 2020) . The RBM is the site that contains most residues that directly interact with receptor protein. The outer surface of the RBM interacts with the N terminal helix, on the ACE2, which is the binding site on the receptor protein. The RBM has a slightly concave surface with a ridge that binds to the claw-like outer surface of the ACE2 (Shang et al., 2020). There are in total 13 hydrogen bonds at the binding interface (Lan et al, 2020). These are formed by the interactions of the viral residues Tyr449, Tyr489, Tyr505 and Arg408 with polar hydroxyl groups on ACE2 (Lan et al, 2020). Keeping this in mind, it might be beneficial to have some heteroatoms atoms that are capable of forming hydrogen bonds on the inhibitor, so that it can competitively interact with the hydrogen bonding residues on the RBD.
The ACE2 binding ridge in the RBD is very compact and contains cystines that form disulfide bonds (Shang et al, 2020). There are 9 cysteine residues in the receptor binding domain (Lan et al, 2020). Among them, Cys336- Cys361, Cys379- Cys432, Cys391- Cys525 and Cys480-Cys488 form disulfide bridges, stabilizing the active site (Lan et al., 2020) . Based on this information, some thiol groups were added to the design inhibitor so that they can disrupt the disulfide bridges within the RBD and instead, interact with the cysteine residues, and thus destabilize the beta structure.
The binding ridge also contains a motif containing two bulky glutamate, two flexible glycines, one valine and one threonine (Shang et al, 2020). This creates the compact structure and allows the Ala475 in RBD to move closer to ACE2 and form hydrogen bond with N terminal residue Ser19 (Shang et al, 2020). To mimic this interaction, some amine groups, some hydrophobic groups and bulky aromatic rings were added to the inhibitor. Taking all the previously mentioned factors into consideration, the following inhibitor structure was proposed:
The thiol groups in the drug would interact with the cysteine in the target, hydroxyl and amine groups that would facilitate formation of strong hydrogen bonds and the bulky aromatic groups would interact with the aromatic residues on the RBD.
In order to determine the affinity of the designed inhibitor to the RBD of the S protein, a docking simulation called 1-Click Docking was used. The structure was put into the docking website and it’s the binding orientation within the target was determined. The simulation also determined the affinity of the target to the inhibitor and calculated the docking score based on that.
To determine the affinity of the target to the ACE2 in comparison to the designed inhibitor, the docking score was also determined for the ACE2 protein. The docking scores of the ACE2 and the designed inhibitor were compared to check if the inhibitor would successfully compete with the substrate and bind to the target’s active site.
After running the predicted inhibitor on the docking stimulation, a docking score of -4.4 was obtained (More than molecule, 2020) . This value is more negative than the docking score of ACE2, which is the main substrate for the binding site on the S protein. The docking score of ACE2 is -4.1 (More than molecule, 2020) . The more negative the docking score of a compound, the better it’s ability to bind to the active site of an enzyme (More Than Molecule, 2020). Based on this, it can be said that the designed inhibitor will efficiently interact with the RBD of the S protein and inhibit its activity. Since it has higher affinity to the target than ACE2, in an environment where both ACE2 and the inhibitor is present at the same time, the inhibitor will be more likely to attach to the RBD of the target protein and prevent it from reacting with the ACE2 receptor by blocking the receptor’s access to the binding site.
Figure 2 demonstrates that the inhibitor interacts strongly with the receptor binding domain on the S protein of SARS-COV-2. The hydroxyl and amine groups present in the inhibitor from hydrogen bonds with the residues Tyr449, Tyr489, Tyr505 and Arg408 (Y449, Y489, Y505 and R408) of the receptor binding motif (RBM). The thiol groups in the molecule interact with the cysteine residues on the RBD and contribute to stabilizing the binding of the inhibitor with the S protein.
While in theory, the inhibitor seems to work well, the physiological conditions need to be taken into account. The docking simulation cannot predict how the physiological factors will affect the inhibitor binding. Due to this reason, clinical trials need to be conducted to determine the effectiveness of the inhibition and its nature of administration.
The main focus on this research was the spike protein on SARS-COV-2 and the inhibitor was designed specifically keeping that in mind. However, the receptor binding domain is not highly conserved within the SARS-COV family. While there are 9 ACE2 contacting residues that are fully conserved and 4 that are partially conserved, some antibodies in SARS-CoV do not work within SARS-CoV-2, which indicates differences in antigenicity (Huang et al., 2020). This region is also highly mutable. A drug designed to target the RBD of SARS-COV-2 might not be applicable to SARS-CoV. To ensure the potential prevention of infection by the virus, regardless of the types and mutations, it is important to target a more conserved site.
In this case, S2 subunit might be a better target. The FP domain is conserved and consists of 15-20 predominantly hydrophobic amino acids like glycine and alanine (Huang et al., 2020). It helps to anchor the viral body to the host membrane. The HR1 and HR2 is made of repetitive heptapeptide, HPPHCPC, where H is hydrophobic and bulky residue, P is polar and hydrophilic and C is charged (Huang et al, 2020). This forms a six helical bundle that helps in the viral fusion and subsequent entry to the host cell (Huang et al, 2020). The highly conserved nature of this subunit makes it a good target for inhibitors. However, more research is needed to confirm that. The inhibitor designed in this research has a good potential to be effective in preventing SARS-COV-2 infection and since, this specific type of SARS-COV is primarily responsible for most of the severe COVID-19 cases found so far and is significantly more infectious than the other types (Shang et al, 2020) , the designed inhibitor might potentially be a good solution. It is suggested that more research be done to find a way to modify the designed drug to increase its effectiveness on the other types of virus within the SARS-COV family.
The proposed drug has higher affinity to the RBD of the S protein than the ACE2 receptor. This indicates that if the drug is injected into the bloodstream, then it would potentially be able to competitively inhibit the receptor binding site on the S protein, thus, preventing the virus from spreading in the body and providing a possible treatment solution for millions who might be suffering from such deadly virus. Nevertheless, more clinical trials need to be done to confirm the effectiveness of the inhibitor since the docking simulation does not take all physiological factors into consideration. Moreover, this inhibitor was only designed for SARS-CoV-2, so it might not work for a different type of pathogen, such as SARS-CoV. Such limitations must be taken into consideration for next steps in this research. This research has been solely focused on the SARS-CoV-2.
- Huang, Y., Yang, C., Xu, X., Xu, W., & Liu, S. (2020, August 03). Structural and functional properties of SARS-CoV-2 spike protein: Potential antiviral drug development for COVID-19. Retrieved November 21, 2020, from https://www.nature.com/articles/s41401-020-0485-4
- Lan, J., Ge, J., Yu, J., Shan, S., Zhou, H., Fan, S., . . . Wang, X. (2020, March 30). Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Retrieved November 21, 2020, from https://www.nature.com/articles/s41586-020-2180-5
- Shang J;Ye G;Shi K;Wan Y;Luo C;Aihara H;Geng Q;Auerbach A;Li F;. (n.d.). Structural basis of receptor recognition by SARS-CoV-2. Retrieved November 21, 2020, from https://pubmed.ncbi.nlm.nih.gov/32225175/
- More than Molecule. (2020). 1-Click Docking. Retrieved on November 19, 2020 from https://mcule.com/apps/1-click-docking/?utm_source=ccl%26utm_medium=maillist%26utm_ca mpaign=1-click-docking
- Prajapat, M., Sarma, P., Shekhar, N., Avti, P., Sinha, S., Kaur, H., . . . Medhi, B. (2020). Drug targets for coronavirus: A systematic review. Retrieved November 21, 2020, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7074424/
- UniProt ConsortiumEuropean Bioinformatics InstituteProtein Information ResourceSIB Swiss Institute of Bioinformatics. (2020, October 07). Spike glycoprotein. Retrieved November 21, 2020, from https://www.uniprot.org/uniprot/P0DTC2