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36 INNOSC Theranostics and Pharmacological Sciences, 2022, Vol. 5, No. 2 Das et al.
2.3. Structure and sequence analysis of S protein and analyzed using Discovery Studio 2017 R2
and ACE- 2 receptor Client [23-25].

The cryogenic electron microscopic structure of 2.6. Protein-protein interaction
the S protein of SARS-CoV-2 (Protein Data Bank
[PDB] ID: 6vsb) with a resolution of 3.46 Å and the Protein-protein interaction studies were carried out
X-ray diffraction structure of the ACE-2 receptor using flexible docking to investigate the interactions
(PDB ID: 1r42) with a resolution of 2.2 Å were of specific protein complexes [1,2]. First, the
retrieved from the PDB. The FASTA sequences of interactions between the S protein with mHTCC
the other reported human coronaviruses (HCoV- and NCMC were examined using molecular
229E, MERS-CoV, HCoV-NL63, and SARS-CoV) docking. Subsequently, the resulting S protein-
were obtained from the PDB database. These mHTCC and S protein-NCMC complexes were
sequences were then used for multiple sequence studied in their interaction with the ACE2 receptor.
alignment to identify the similarity of the amino To assess the binding affinity of the S protein for
acid sequences of the three chains of the spike the ACE2 receptor in the presence of mHTCC
and NCMC similarly, the interaction between the
protein.
S protein and ACE2 receptor without mHTCC
2.4. Phylogenetic analysis and NCMC was also analyzed [26,27]. For this
analysis, the clusPro 2.0 software (https://cluspro.
The FASTA sequence of the S-protein was bu.edu/publications.php) was employed, utilizing
retrieved from the PDB database and evolutionary the fast Fourier transform (FFT) docking methods.
analysis of genetic distance and diversity was The selections of the filtered conformations were
conducted using MEGA-X. Phylogenetic analysis based on the assessment of empirical free energy.
was performed using the Substitution Model Both the lowest de-solvation and electrostatic
Jones-Taylor-Thornton (JTT) [21], and standard energies were taken into account for the evaluation
error estimates were obtained through a bootstrap of free energy. Piper being a FFT-based rigid
procedure (1000 replicates). The phylogenetic tree docking tool, serves the clusPro clustering program
was then constructed using the maximum likelihood for measure the native sites by providing 1000 low
statistical method. energy outcomes [28]. The native site is assumed

2.5. Molecular docking to possess a wide range of free energies to yield
a great number of results. Initially, a sample of
The evaluation of the free binding energy of approximately 10 positions of the ligand with
9
energy-minimized chitosan subunits and their respect to the receptor was taken. From this set,
monomeric and oligomeric derivatives with the only the top 10 positions were selected for further
3
S protein and ACE2 receptor was conducted analysis, considering all relative ligand positions
using the molecular docking program AutoDock corresponding to the receptor.
Tools 1.5.6. Molecular docking studies were
also performed to assess the binding affinity 3. Results
of chitosan derivatives, namely, mHTCC and 3.1. Phylogenetic analysis
NCMC, with the RBD of the S protein. The
protein binding affinity of S protein, RBD, and The S protein of SARS-CoV-2 showed the highest
ACE2 receptor with chitosan monomeric and sequence identity (73.9090%) with SARS-CoV,
oligomeric derivatives was examined using while its lowest amino acid sequence similarity
AutoDock Vina1.1.2. Various parameters, such was observed with HCoV-229E (10.6077%). In
as binding affinity, receptors interacting atom, addition, the S protein shares 22.9037% sequence
receptor pocket atom, receptor-ligand interaction identity with MERS-CoV and 18.5559% with
site, atomic contact energy (ACE), and side amino HcoV-NL63. Phylogenetic tree analysis of
acid residues, were studied to identify the binding SARS-CoV-2 and SARS-CoV reveals their close
sites of the S protein and ACE2 receptor [22]. The relationship, as they share the same operational
results of the docking studies were visualized taxonomic unit (out) (Figure S1).

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