Page 228 - EJMO-9-3

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

integration of statistical planning from exploratory hydrogen bond occupancy – were performed to evaluate
to confirmatory phases aligns with best practices in complex stability and dynamic binding features, offering
translational cancer research and ensures rigorous evidence comprehensive insights into the affinity and interaction
generation in subsequent stages of drug development. profiles of the selected lead compounds. 65
3.13. Scope and generalizability of findings 4.1. Selection of lead molecules for MD simulations

The present study employed three molecularly distinct The selection of lansoprazole, capivasertib, and oxaliplatin
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+
breast cancer cell lines – MCF-7 (luminal A, ER /PR ), for MD simulations was based on three primary criteria.
T-47D (luminal B, ER /PR /HER2 ), and BT-474 (HER2 First, all three compounds demonstrated strong FN3K
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−
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subtype) – to explore the role of FN3K inhibition across inhibition, with reductions ranging from 99.6% to 99.99%
hormone-driven and HER2-amplified breast cancer across multiple breast cancer cell lines (MCF-7, T-47D,
models. This experimental design was intended to capture BT-474). Second, these compounds exhibited minimal off-
a broad segment of the clinical breast cancer spectrum, target effects in non-malignant Vero cells, reinforcing their
particularly those subtypes where redox adaptation tumor-selective properties. Finally, their favorable ADMET
contributes to treatment resistance. profiles further supported their candidacy: Capivasertib and
However, we acknowledge that these models do not lansoprazole showed high gastrointestinal absorption, while
encompass the full heterogeneity of breast cancer, especially oxaliplatin demonstrated high solubility, making all three
TNBC, which exhibits distinct redox vulnerabilities and suitable for further evaluation through MD simulations.
metabolic plasticity. In addition, cancer types outside the 4.2. Objective of MD simulations
breast tissue may exhibit different dependencies on FN3K
or Nrf2 signaling, potentially limiting the generalizability To investigate the molecular interactions and dynamic
of our present findings. stability of the lead candidates – lansoprazole, capivasertib,
and oxaliplatin – in complex with FN3K, MD simulations
This study was conceptualized as a focused proof-of- were conducted for 200 ns using Desmond. Several key
concept investigation into the feasibility of repurposing parameters were evaluated to characterize the behavior
FN3K-targeted agents in selected breast cancer subtypes. and binding profiles of the complexes. RMSD was
Moving forward, we aim to expand our experimental scope by employed to monitor the overall structural stability of the
incorporating TNBC cell lines and non-breast cancer models protein-ligand complex throughout the simulation, while
with known oxidative stress signatures, such as lung, liver, RMSF assessed the flexibility and mobility of amino acid
and pancreatic cancers. These comparative studies will enable
systematic mapping of FN3K expression, redox pathway residues, particularly within the active site, in response to
activation, and drug responsiveness across cancer types. ligand binding. Hydrogen bond analysis was performed
to examine the consistency and strength of hydrogen
4. Molecular dynamics (MD) simulations of bonding interactions between the ligand and FN3K over
lead molecules the simulation trajectory. In addition, binding free energy
was calculated using MM-GBSA or MM-PBSA methods
Following in vitro confirmation of FN3K inhibition by to estimate the thermodynamic favorability and strength of
lansoprazole, capivasertib, and oxaliplatin, MD simulations binding for each compound. Finally, total energy calculations
were employed to gain deeper insight into their binding were conducted within Desmond to analyze the system’s
stability, conformational behavior, and key molecular potential and interaction energies, offering insights into
interactions within the FN3K active site. Simulations were conformational stability and favorable binding orientations.
conducted using Desmond version 13.7 (Schrödinger, LLC,
USA) with the OPLS5 force field, placing the complexes in Findings from the MD simulations offer valuable
an orthorhombic simulation box solvated with TIP3P water mechanistic insights into FN3K inhibition, supporting the
molecules. The system was initially energy-minimized and therapeutic relevance of the identified lead compounds.
subsequently equilibrated under NPT ensemble conditions The full simulation trajectories and detailed parameter-
at 300 K and 1 atm pressure. A production simulation was specific results – such as RMSD, RMSF, rGyr, hydrogen
carried out for 200 ns with a 2-femtosecond timestep, bonding, and MM-GBSA binding energy analyses – are
applying the Particle Mesh Ewald (PME) method for presented in Supplementary Figures S7-S40.
long-range electrostatics and a 9 Å cutoff for van der 5. Future perspectives
Waals interactions. Post-simulation trajectory analyses –
including root mean square deviation (RMSD), root mean The findings of this study provide a strong foundation for
square fluctuation (RMSF), radius of gyration (rGyr), and exploring FN3K inhibition as a novel therapeutic strategy

Volume 9 Issue 3 (2025) 220 doi: 10.36922/EJMO025150114