Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities

Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson’s Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research, diagnostics and monitoring the effect of secondary therapeutic agents on lysosomal enzyme activity in drug development for the lysosomal storage disorders and allied diseases.溶酶体（Lysosomes）是一类呈酸性的胞质细胞器，广泛存在于所有有核哺乳动物细胞中，参与质膜修复、病原体防御、胆固醇稳态、骨重塑、代谢、细胞凋亡及细胞信号转导等多种细胞过程。溶酶体酶活性缺陷与多种神经系统疾病相关，包括帕金森病、溶酶体贮积病、阿尔茨海默病与亨廷顿病。本研究合成了可用于活细胞成像的溶酶体及其他酸性细胞器标记的荧光探针，实验证实其可用于监测溶酶体代谢活性。这些新型靶向底物由具有低pKa值的荧光染料制备而成，可在溶酶体所处的低生理pH环境下实现最佳荧光效果。随后对其进行修饰，引入靶向基团以使其富集于溶酶体，同时引入酶可切割功能，从而可通过活细胞染色格式监测特定酶活性。将该底物应用于染色异染性脑白质营养不良、克拉伯病和戈谢病患者的血液及皮肤样本来源的细胞，以及健康人成纤维细胞和白细胞对照细胞，与已知溶酶体染料LysoTracker® Red DND-99及抗LAMP1抗体染色结果相比，染色结果均显示其定位于溶酶体。当用氯喹抑制细胞代谢时，酯酶底物的染色信号减弱，证实该底物可用于测量细胞代谢。将其应用于患病细胞时，染色强度与患病细胞中的溶酶体酶水平相一致。针对戈谢病或克拉伯病患者细胞系中酶缺陷的特异性底物，其染色强度相较于非患病细胞有所降低。这些新型溶酶体靶向荧光底物可用于溶酶体贮积病及相关疾病的药物研发中的研究、诊断，以及监测辅助治疗剂对溶酶体酶活性的影响。