In-depth theoretical modeling to explore the mechanism of TPX-0131 overcoming lorlatinib resistance to ALK mutation.

A number of anaplastic lymphoma kinase (ALK) inhibitors have been clinically approved, with lorlatinib, particularly as a third-generation drug, demonstrating efficacy against various drug-resistant ALK single mutations. However, continued clinical use of lorlatinib has led to the emergence of ALK double mutations conferring resistance to lorlatinib, notably ALK. TPX-0131 is a potential fourth-generation ALK inhibitor currently under development. TPX-0131 demonstrates a broader spectrum of activity against ALK-resistant mutations, efficiently inhibiting 26 single-point mutations and various double/triple mutations, including solvent front mutations and gatekeeper mutations. In this study, for the first time, a comprehensive elucidation of the molecular mechanisms by which TPX-0131 overcomes lorlatinib resistance to ALK through modeling, MD simulations, free energy calculations, and US simulations. The results indicate that the interactions between lorlatinib and key residues at the hinge region are disturbed by L1196M/G1202R double mutation, leading to the disruption of important hydrogen bonding between Glu1197 and lorlatinib. For TPX-0131, the L1196M/G1202R mutation enhances electrostatic and van der Waals interactions, causing significant conformational changes primarily in the hinge region, G-loop, and β-strands. The tight binding of TPX-0131 to residues Arg1202, Met1199 and Arg1120 contribute significantly to overcoming lorlatinib resistance in ALK mutant. These research results are expected to offer insights into the mechanism of TPX-0131 in treating ALK-induced NSCLC resistance and optimizing of ALK inhibitors.