Center of Cancer Research, Comprehensive Cancer Center, Medical University of Vienna, Borschkegasse 8a, 1090, Vienna, Austria.
Institute of Inorganic Chemistry, Faculty of Chemistry, University of Vienna, Währinger Straße 42, 1090, Vienna, Austria; Doctoral School in Chemistry (DoSChem), University of Vienna, Währinger Straße 40-42, 1090, Vienna, Austria.
Chem Biol Interact. 2024 Nov 1;403:111243. doi: 10.1016/j.cbi.2024.111243. Epub 2024 Sep 14.
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 lysosome-specific 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 non-protonatable NIN derivatives, exhibited lysosome-associated birefringence signals co-localizing with the NIN-derived fluorescence emission. Sensitivity of both parameters towards vATPase inhibitors confirmed pH-dependent, 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.
尼达尼布（NIN）是一种多靶点酪氨酸激酶抑制剂，临床上已被批准用于特发性肺纤维化和肺癌，其特点是质子依赖性溶酶体趋向性和出现溶酶体特异性荧光发射特性。在这里，我们研究了在酸性细胞区室中是否存在一种迄今为止未知的 NIN 物质，这种物质是否是导致这些意外发光特性的原因，并研究了其对溶酶体功能的影响。用 NIN 处理的细胞的溶酶体，但不是不可质子化的 NIN 衍生物，表现出与 NIN 衍生的荧光发射共定位的溶酶体相关双折射信号。这两个参数对 vATPase 抑制剂的敏感性证实了溶酶体中新型结晶 NIN 结构的自发形成是 pH 依赖性的。因此，从缓冲溶液中结晶 NIN 导致形成具有 pH 依赖性荧光特性的多种晶体多晶型物。在溶酶体样 pH 条件下生长的无细胞晶体在荧光模式、光漂白动力学和拉曼光谱方面与用 NIN 处理的细胞溶酶体相似。然而，在双折射强度和 FAIM 确定的各向异性以及与（内）溶酶体膜结构的主要关联方面的差异表明，在酸性溶酶体中形成了一种半固态 NIN 结晶物质。尽管具有相似的靶激酶抑制作用，但 NIN 而不是其不可质子化的衍生物，会损害溶酶体功能，导致大量细胞空泡化、增强自噬、脂质代谢失调，并诱导非典型磷脂沉积病。此外，NIN 还表现出独特的光毒性，这种毒性严格依赖于溶酶体微结晶事件。口服 NIN 处理的小鼠肠道黏膜中也观察到 NIN 结晶结构的自发形成。总之，这里描述的 NIN 对溶酶体功能的激酶抑制作用无关的影响介导了其几种细胞生物学活性，并可能导致 NIN 的不良反应。
