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The ongoing coronavirus-19 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has claimed more than 2.8 million lives worldwide. Authorities are struggling to prevent the spread of the disease due to the highly infectious nature of SARS-CoV-2. Therefore, reducing the ability of the virus to infect an individual’s respiratory epithelial cells could prove to be an effective measure to prevent disease transmission.
The SARS-CoV-2 Spike (S) protein is primarily responsible for facilitating entry into host cells. During this process, the S protein is primed by the transmembrane serine protease 2 (TMPRSS2), the S1 domain of the protein interacting with the angiotensin converting enzyme 2 (ACE2) of the host cell. After the interaction, ACE2, which is present on the lipid rafts of the cell membranes of the hosts, induces endocytosis and, therefore, infection occurs.
Many researchers have focused on preventing viral infections by blocking the ability of S protein to interact with ACE2. This process requires new inhibitors with a strong affinity for the S protein. Another approach is to develop ACE2 inhibitors using its native substrate, namely the peptide angiotensin II (Ang II), as a starting structure. The main physiological function of ACE2 is to cleave a single amino acid from the C-terminus of the Ang II octapeptide, which helps control blood pressure.
ACE2 can be categorized as a gluzincin due to the presence of a zinc-coordinating amino acid sequence at the Ang II binding site. Previous studies have revealed that this class of metalloproteases undergoes catalysis via the nucleophilic addition of a water molecule, coordinated to zinc in ACE2. Even though Ang II has a strong affinity for ACE2, it does not share an active binding site with S protein. Therefore, Ang II cannot inhibit only S protein binding. However, the researchers perceived that the incorporation of a peptide-nanoparticle conjugate to the Ang II peptide would allow the blocking of the receptor site, which would inhibit the interaction between the S protein and the ACE2.
The success of this strategy strongly depends on the affinity of Ang II for ACE2. The researchers indicated that the affinity of Ang II for ACE2 can potentially be improved by altering the primary structure of Ang II.
A new study, published on the bioRxiv* pre-print server, seeks to determine Ang II mutants from previous studies and to identify new sequences of Ang II mutants that have high binding affinity with ACE2, using computational methods. In this study, the native peptide and each Ang II mutant were anchored to zinc in all probable coordination sites found in the peptide by replacing the existing water molecule at the coordination site. The potential impact of peptide binding to ACE2 on the structure of the S protein binding region was also evaluated.
Many scientists have adopted computer methods to discover molecules essential to the design of new drugs. In this method, possible inhibitors are anchored to the receptor, after which molecular dynamics (MD) simulations have been performed, and free binding energy is calculated. This technique makes it possible to precisely determine new potential inhibitory molecules for protein S and ACE2. This molecule is then validated by various laboratory experiments. The success of the experiments is highly dependent on assigning appropriate parameters to all atoms and the interactions obtained in MD simulations, and then calculating the free binding energy.
Current research has selected mutants based on the results of the previous study which evaluated the DRVYVYPF and DRVYIYPF peptides. Both mutants exhibited potent ACE2 inhibition over native Ang II on a fluorescent-quenched substrate assay. These mutants also showed higher stability to enzymatic degradation. The previous study revealed that the 6th residue of Ang II could play an essential role in binding to ACE2. In the current study, DRVYIEPF was also taken into account. The researchers incorporated cysteine and glutamate as the sixth residue because they can bind to zinc ions. The 4th amino acid residue was also studied. This study revealed that native Ang II and DRVYVYPF form a complex at ACE2 with significantly strong binding free energy. The 4th amino acid residue of the mutant sequence was also observed to bind to the coordinates of zinc.
(A) The structure of Ang II (red) linked to ACE2 (teal). The slit in ACE2 where Ang II resides is visible in both the surface rendered image (left) and the ribbon diagram (right). Residues on ACE2 that interact with the spike protein are shown in black. (BE) Enlarged views of the Ang II-ACE2 binding, illustrating the coordination of Ang II to zinc (orange) at four possible residues of D, Y, H and the C-terminus, shown in B , C, D and E, respectively. The corresponding coordination residues on ACE2 are shown in purple.
A limitation of this study is the lack of entropy calculations of the MM / PBSA protocol, but since the error tends to be marginal, it would not contradict the projected result. This study also did not include the impacts of kinetics because it requires more computationally expensive techniques.
Thus, this research revealed that among the sequences studied, only one native Ang II sequence exhibited strong binding energy and could bind to the ACE2 slit. The strong binding energy was linked to the tyrosine residue at the fourth position of the peptide sequence coordinating with the zinc atom in the slit. Another interesting finding from this study is that the S1 binding region and mutant Ang II peptides alone cannot block the interaction between ACE2 and S protein. binding of viruses to cells.
*Important Notice
bioRxiv publishes preliminary scientific reports which are not peer reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behaviors, or treated as established information.
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