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Innovative Medicines & Omics SARS-CoV-2 inhibition by quinolines

by Calixto et al. The compound was obtained as a white effective concentration (EC ) values of the atazanavir
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50
solid with a yield of 99%. The product was confirmed by were used as an experimental control for virus inhibition
melting point value and H NMR spectrum. and cell viability in Vero E6 and Calu-3 assays. 33,34 All
1
virus manipulations were executed at biosafety level 3
2.4. Cell viability assay (BSL3) multiuser facility, according to the World Health
Vero E6 (African green monkey kidney ATCC CRL-1586) Organization guidelines. 35
and Calu-3 cells (submucosal gland cell line, generated
from a bronchial adenocarcinoma, a kind donation by 2.6. Statistical analysis
Farmanguinhos platform RPT11M) were cultivated in GraphPad Prism 10.0 software was used to create the
96-well plates (1.5 × 10 cell/well) in the presence or graphs for this work. The EC values were determined by
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absence of 4-aminoquinoline derivatives (Q1a, Q2a, non-linear regression of log (inhibitor) versus normalized
Q3a, Q4a, Q1b, Q2b, Q3b, Q4b, and Q1bS) at different response of best curve generated (R values ≥0.9). All
2
concentrations (5, 10, 20, 40, 80, and 160 µM) for 72 h to experiments were performed in at least three technical
evaluate their toxic effect by methylene blue assay. Briefly, replicates.
treated cells were washed with saline solution and stained
with methylene blue solution (HBSS, 1.25% glutaraldehyde, 2.7. Protein structure prediction and molecular
and 0.6% methylene blue) for 1 h. After this period, the dynamics (MD)
cells were washed with distilled water and dried, and the The M is a protein consisting of two monomers, each
pro
eluent solution (50% ethanol, 49% phosphate-buffered consisting of three domains. Domain I covers residues
saline [PBS], and 1% acetic acid) was added for 15 min 8 – 101, whereas domain II contains residues 102 – 184.
at room temperature. Then, the supernatant was read in Together, these form an antiparallel β-barrel structure.
a spectrophotometer (LMR-96i-4, Loccus, São Paulo, SP, The connection with the C-terminal region up to domain
Brazil) at 660 nm. III is formed by a long loop between residues 185 and 200.
The compounds used in the experiments were Domain III is made up of five α-helices and comprises
resuspended in 100% dimethyl sulfoxide (DMSO) and residues 201 – 303. The active site of the enzyme is
diluted in Dulbecco’s Modified Eagle Medium (DMEM- positioned in the space between domains I and II, forming
Gibco). The final DMSO concentrations did not exceed 1% a deep cleft characterized by a highly hydrophobic region.
(v/v), which did not affect the growth of the cells. 32 Dimerization occurs as the domains II and III of one
monomer engage with the N-terminal region of the other
2.5. Anti-SARS-CoV-2 analysis monomer. Furthermore, the catalytic domain is composed

To evaluate the capacity of compounds to inhibit the of five subpockets, designated S1, S1’, S2, S3, and S4, which
SARS-CoV-2 replication, Calu-3 and Vero E6 cells, at include residues T25, H41, M49, F140, N142, G143, C145,
the concentration of 1.5 × 10 cells/well, were infected H163, E166, M165, H172, and Q189. 36
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with either the wild-type (WT) SARS-CoV-2 (SisGen SARS-CoV-2 M plays a key role in polyprotein
pro
AC58AE2, B.1 lineage isolate, GenBank MT710714) or processing and is active in a dimeric form. Dimerization
the Omicron variant (GISAID EPI_ISL_17819005) at is critical for the catalytic activity of the enzyme as the
the multiplicity of infection (MOI) 0.01 for 1 h at 37°C, N-terminal region of each monomer interacts with the
under a 5% CO condition Afterward, the cells were GLU166 residue of the other, facilitating orientation of
.
2
treated with 4-aminoquinoline-derived compounds using the S1 binding subsite for the substrate. In addition,
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a concentration curve (0.6, 1.25, 2.5, 5 e 10 µM) for 24 h most M structures in the Protein Data Bank (PDB)
pro
(Calu-3 and Vero E6) or 48 h (Calu-3). The virus growth database are presented primarily as monomers, justifying
was determined by plaque-forming units (PFU) assay, in the necessity for comparative modeling of the dimer. To
which Vero E6 cells (1.5 × 10 cells/well) were exposed obtain a more comprehensive structure and to avoid
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to different dilutions (1:20 – 1:31200) of supernatant for incomplete sequences, we developed a dimeric model to
1 h at 37°C and 5% CO Then, carboxymethylcellulose
2.
medium (DMEM-HG 10×, 2.4% carboxymethylcellulose, be used in MD and molecular docking. Figure 1 shows the
and 2% fetal bovine serum), at the same volume of the comparative alignment between the modeled dimer and
well, was added and culture plates were cultivated for the template used (PDB 6Y2G).
pro
72 h. After this time, the cells were fixed with formalin To obtain the structure of the M of SARS-CoV-2,
4% for 3 h, stained with crystal violet 0.04% for 1 h, and we used the PDB6Y2E and PDB6Y2G templates obtained
used for virus titers determination by PFU quantification. from the PDB. We used the Modeller v9.23 program to
The cytotoxic concentration 50% (CC ) and half maximal complete the C-terminal portion of the polypeptide chains
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Volume 1 Issue 1 (2024) 90 doi: 10.36922/imo.3442