The tyrosine kinase inhibitor Nintedanib induces lysosomal dysfunctionality: Role of protonation-dependent crystallization processes

Mosca, Elena and Federa, Anja and Pirker, Christine and Schosserer, Markus and Liendl, Lisa and Eckhard, Margret and Sombke, Andy and Dömötör, Orsolya and Kirchhofer, Dominik and Timelthaler, Gerald and Baier, Dina and Gurschka, Patrizia and Gabler, Lisa and Reithofer, Michael and Chin, Jia Min and Elsayad, Kareem and Englinger, Bernhard and Tahir, Ammar and Kowol, Christian R. and Berger, Walter (2024) The tyrosine kinase inhibitor Nintedanib induces lysosomal dysfunctionality: Role of protonation-dependent crystallization processes. CHEMICO-BIOLOGICAL INTERACTIONS, 403. ISSN 0009-2797

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Abstract

Nintedanib (NIN), a multi-tyrosine kinase inhibitor clinically approved for idiopathic pulmonary fibrosis and lung cancer, is characterized by protonation-dependent lysosomotropic behavior and appearance of lysosomespecific fluorescence emission properties. Here we investigate whether spontaneous formation of a so far unknown NIN matter within the acidic cell compartment is underlying these unexpected emissive properties and investigate the consequences on lysosome functionality. Lysosomes of cells treated with NIN, but not nonprotonatable NIN derivatives, exhibited lysosome-associated birefringence signals co-localizing with the NINderived fluorescence emission. Sensitivity of both parameters towards vATPase inhibitors confirmed pHdependent, spontaneous adoption of novel crystalline NIN structures in lysosomes. Accordingly, NIN crystallization from buffer solutions resulted in formation of multiple crystal polymorphs with pH-dependent fluorescence properties. Cell-free crystals grown at lysosomal-like pH conditions resembled NIN-treated cell lysosomes concerning fluorescence pattern, photobleaching dynamics, and Raman spectra. However, differences in birefringence intensity and FAIM-determined anisotropy, as well as predominant association with (intra)lysosomal membrane structures, suggested formation of a semi-solid NIN crystalline matter in acidic lysosomes. Despite comparable target kinase inhibition, NIN, but not its non-protonatable derivatives, impaired lysosomal functionality, mediated massive cell vacuolization, enhanced autophagy, deregulated lipid metabolism, and induced atypical phospholipidosis. Moreover, NIN exerted distinct phototoxicity, strictly dependent on lysosomal microcrystallization events. The spontaneous formation of NIN crystalline structures was also observable in the gut mucosa of orally NIN-treated mice. Summarizing, the here-described kinase inhibition-independent impact of NIN on lysosomal functionality mediates several of its cell biological activities and might contribute to NIN adverse effects.

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