Page 209 - EJMO-9-3

Eurasian Journal of
Medicine and Oncology FN3K–Nrf2 axis inhibition in breast cancer

modeling, a suitable structural template for FN3K was molecular docking tool that predicts the binding affinity
generated using SWISS-MODEL, an automated web-based and orientation of ligands within a receptor binding site. 36
tool for protein structure prediction based on sequence
24
homology. The preliminary FN3K model was further 2.1.2.7. Post-docking analysis
refined using the GalaxyWEB server, a platform providing The docking results generated by Glide were analyzed to
specialized tools for protein structure optimization. To identify hit molecules based on binding affinity (Glide
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ensure reliability and precision, the refined FN3K model score) and key interactions with the FN3K active site.
underwent rigorous validation through various analytical Binding conformations were evaluated for key molecular
techniques. Ramachandran plot analysis was performed to interactions, including hydrogen bonding, hydrophobic
evaluate the φ (phi) and ψ (psi) dihedral angles of amino contacts, and π-π stacking. 37
acid residues, confirming their placement within sterically 2.1.2.8. Absorption, distribution, metabolism, excretion, and
favorable regions of the protein structure. PROCHECK, toxicity (ADMET) screening
26
accessed through the SAVES server at UCLA (https://saves.
mbi.ucla.edu/), was used to assess the stereochemical quality The shortlisted hit molecules from the post-docking
and geometric parameters of the model. In addition, the analysis underwent the ADMET screening to assess their
27
Global Model Quality Estimation (GMQE) score provided drug-like properties and safety profiles. This filtering step
by SWISS-MODEL was considered to gauge the predicted ensured the identification of compounds with both high
reliability of the generated model. Finally, Qualitative binding affinity and desirable pharmacokinetic properties
28
Model Energy Analysis (QMEAN) values were examined for further consideration. 38
to estimate overall model quality based on multiple scoring 2.2. In vitro screening studies
functions. 29,30
2.2.1. In vitro cytotoxicity evaluation
2.1.2.2. Protein preparation
2.2.1.1. Cell lines and culture conditions
To prepare the protein structure constructed through
homology modeling, the generated model was imported The cytotoxicity of the selected compounds was assessed
into Maestro. Using the Protein Preparation Wizard within using human breast cancer cell lines – MCF-7, T47D, and
Maestro, several essential steps were undertaken to refine BT-474 – as well as non-malignant Vero cells. All cell lines
the homology-modeled protein structures. 31 were cultured in Dulbecco’s Modified Eagle’s Medium
(DMEM) supplemented with 10% fetal calf serum
2.1.2.3. Preparation of ligand libraries (FCS), 100 U/mL penicillin, 100 µg/mL streptomycin,
In preparing the ligand libraries, ligand datasets were and 50 µg/mL kanamycin. Cells were maintained at
curated from databases, such as DrugBank, PubChem, and 37°C in a humidified incubator containing 5% CO until
2
the FDA. 12,32,33 The selected ligand structures were then approximately 70% confluency was achieved. 39
imported into Maestro, where the LigPrep tool was used to 2.2.1.2. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
generate 3D conformations from the 2D structures. 34 bromide (MTT) assay

2.1.2.4. Identification of potential binding sites The cytotoxic effects of 1-deoxy-morpholino-D-fructose
Potential ligand-binding regions on the modeled FN3K (1-DMF), ritonavir, amiloride, lansoprazole, capivasertib,
structure were predicted using the SiteMap module integrated and oxaliplatin were investigated using the MTT assay,
within the Maestro software suite. SiteMap is an advanced following the ISO 10993-5:2009 guidelines for in vitro
40
computational tool designed to detect and characterize cytotoxicity assessment. Cells were dispensed into 96-well
4
binding pockets and cavities within a protein structure. 35 plates at a density of 1 × 10 cells per well and incubated
for 24 h to allow adherence under standard conditions.
2.1.2.5. Preparation of the receptor grid Thereafter, cells were treated in triplicates with a range of
Following the identification of potential binding sites using compound concentrations (12.5 – 200 µg/mL), tailored to
SiteMap, the next step involved the preparation of the individual solubility and bioactivity profiles, and incubated
41
receptor grid. The receptor grid generation tool in Maestro for an additional 24-h period.
was used for this purpose. 34 Post-treatment, 20 µL of MTT solution at 5 mg/mL,
prepared in phosphate-buffered saline (PBS), was added to
2.1.2.6. Docking
each well, followed by a 4-h incubation at 37°C to allow for
The molecular docking setup was initiated by selecting formazan crystal formation. The resulting crystals were
40
the standard-precision (SP) mode within Glide. Glide is a solubilized using 100 µL of isopropanol containing 0.04N

Volume 9 Issue 3 (2025) 201 doi: 10.36922/EJMO025150114