Discovery of a potent ROR1 inhibitor using μs-scale MD simulations, wt-metadynamics, and absolute binding free energy calculations.

contextReceptor tyrosine kinase-like orphan receptor 1 (ROR1) is a cancer-associated pseudokinase with low expression in normal adult tissues but elevated levels in various malignancies, making it a promising therapeutic target. Among ~ 4 million compounds, CHEMBL3926946 emerged as the most promising candidate, demonstrating a persistent binding pose and a well-defined free energy basin. Well-tempered metadynamics (wt-MetaD) revealed a deep minimum of 26.00 ± 2.44 kcal/mol, indicating a highly stable interaction. CHEMBL3926946 exhibited a favourable Absolute Binding Free Energy Perturbation (ABFEP) of - 16.52 ± 0.37 kcal/mol, significantly outperforming the inhibitor Ponatinib (- 8.67 ± 0.94 kcal/mol), supported by persistent interactions with GLU523 and LEU479. This study highlights CHEMBL3926946 as a robust lead for ROR1-targeted cancer therapy and emphasizes the utility of combining wt-MetaD and ABFEP for reliable hit prioritization.

methodsWe employed a multilayered in silico pipeline integrating high-throughput virtual screening, long-timescale molecular dynamics (MD), wt-MetaD, and ABFEP. Ligands and protein were prepared using the OPLS2005 force field, and all stages up to wt-MetaD were conducted in Maestro (v12.8.117) using the same force field. A library of ~ 4 million compounds yielded 137 candidates, Further shortlisted via MD. 7 high-confidence molecules underwent 5 independent MD replicates with randomized seeds to ensure statistical robustness. The top 3 compounds were validated by 1 μs (1000 ns) simulations to assess long-term conformational stability and wt-MetaD to reveal deep minimum. ABFEP calculations were performed using the CGenFF force field in NAMD 3.0. We benchmarked ABFEP protocol against experimentally validated ligands, successfully reproducing experimental binding free energies (ΔG), confirming the protocol's predictive reliability.