Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an attractive target in breast cancer research due to its overexpression. This study aims to identify potential ROR1 inhibitors for investigation in triple-negative breast cancers through high-throughput virtual screening, molecular docking, molecular dynamics (MD) simulations, and cell-based in vitro validation. The binding affinity and stability of a potential inhibitor, Olverembatinib, were compared with Ponatinib, a known ROR1 inhibitor.

Virtual screening was performed using the Glide SP docking module in Schrödinger 2022.2, screening over 9,000 compounds from DrugBank against ROR1 (PDB ID: 6TU9). The best-scoring compounds were further analyzed for their binding interactions, including hydrogen bonding, hydrophobic contacts, and π-π stacking. MD simulations were conducted using Gromacs v2023.3, with input files prepared via the CHARMM-GUI server. Protein-ligand complexes were solvated in a TIP3P water box, and the system was equilibrated under NVT and NPT conditions at 303.15 K and 1 bar. A 300 ns MD simulation was performed for both Olverembatinib and Ponatinib, and key analyses such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), and interaction stability were conducted. Additionally, Olverembatinib and Ponatinib were tested on ROR1+ MDA-MB-231 and ROR1- MCF-7 breast cancer cells using the WST-1 assay in a time- and dose-dependent manner.

Olverembatinib demonstrated the best docking score (-14.511 kcal/mol) among all screened compounds, including Ponatinib (-14.023 kcal/mol), indicating its strong binding affinity to ROR1. Olverembatinib formed dual hydrogen bonds with Ile555, in addition to π-π stacking with Phe552 and a π-cation interaction with Hie613, which were absent in Ponatinib. Hydrophobic interactions with Leu487, Leu530, and Val536 further contributed to its stability. MD simulations confirmed these findings, with Olverembatinib maintaining key hydrogen bonds with Ile555 and Asp633. RMSD analysis showed lower fluctuations, while RMSF indicated minimal flexibility in critical binding site residues. Cell viability assays demonstrated that Olverembatinib had higher selectivity and inhibitory activity in ROR1+ cells than in ROR1- cells. It appeared to be more selective than Ponatinib against ROR1+ cells while demonstrating comparable effectiveness. The long-lasting effects of Olverembatinib across decreasing doses were especially remarkable.

This study highlights Olverembatinib as a promising ROR1 inhibitor with strong and persistent interactions, making it a viable lead compound for further preclinical validation and development of targeted therapies against ROR1-driven triple-negative breast cancers.