Abstract

Anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer is driven predominantly by EML4-ALK fusion, and various generations of ALK-tyrosine kinase inhibitors (TKIs), including first-generation (crizotinib), second-generation (alectinib, brigatinib, ceritinib), and third-generation (lorlatinib), have been used to target this oncogenic driver. Lorlatinib is used to treat patients who have developed resistance to earlier TKIs. However, sequential treatment with first-generation (1G), second-generation (2G), and then third-generation (3G) TKIs often leads to the emergence of compound mutations within the ALK kinase domain, conferring high-level resistance to even the most potent inhibitor, lorlatinib, as well as to other TKIs. These compound mutations arise due to the mechanism of acquired or on-target resistance to ALK inhibitors. In this study, we investigated two clinically reported triple mutations, G1202R+L1204V+G1269A (TM1) and G1202R+S1206F+G1269A (TM2), identified in EML4-ALK variant 3 patients conferring resistance to lorlatinib. The G1202R, L1204V, and S1206F mutations were identified as deleterious, while the G1202R, L1204V, and G1269A mutations had a destabilizing effect on ALK, as predicted by various in silico tools. We propose two potential lead compounds, CID 118003002 and CID 145224731, which demonstrated favorable ADMET properties and were prioritized for further computational analysis. Molecular docking showed strong binding affinities, with CID 118003002 against G1202R+L1204V+G1269A (–8.02 kcal/mol), and CID 145224731 against G1202R+S1206F+G1269A (–8.49 kcal/mol), respectively. Molecular dynamics simulations further confirmed that both complexes, TM1 with CID 118003002 and TM2 with CID 145224731, maintained significant structural stability, compactness, and consistent hydrogen bonding throughout the 100 ns simulation period. To the best of our knowledge, this is the first comprehensive in silico study aimed at identifying potential lead compounds targeting lorlatinib-resistant ALK triple mutations – G1202R+L1204V+G1269A and G1202R+S1206F+G1269A.

Key words: ALK-positive NSCLC, EML4-ALK variant 3, lorlatinib resistance, compound mutations, molecular docking, molecular dynamics simulation