Phenotypic Screening Using High-Content Imaging to Identify Lysosomal pH Modulators in a Neuronal Cell Model.

Area of Science:

• Cell Biology
• Biochemistry
• Neuroscience

Background:

• Lysosomes are vital organelles for cellular waste breakdown, requiring an acidic internal pH for enzyme function.
• Disrupted lysosomal acidity is implicated in impaired protein homeostasis and age-related diseases, including neurodegeneration.
• Developing tools to modulate lysosomal pH is critical for understanding and treating these conditions.

Purpose of the Study:

• To screen for small molecules capable of acidifying lysosomal pH using a novel biosensor.
• To identify molecular targets and regulatory pathways governing lysosomal pH.
• To discover potential therapeutic agents for neurodegenerative diseases by targeting lysosomal function.

Main Methods:

• Development and application of a cell-based fluorescence assay utilizing the FIRE-pHLy lysosomal pH biosensor.
• High-throughput screening (HTS) of 1835 bioactive compounds for lysosomal pH modulatory effects.
• Dose-response assays and data analysis to validate hit compounds and identify top acidifiers.

Main Results:

• A total of 1835 compounds were screened, with 45 initially selected as potential lysosomal pH modulators.
• Twenty-three compounds were further evaluated through dose-response studies.
• OSI-027 and PP242 were identified as potent small molecules that significantly acidify lysosomal pH.

Conclusions:

• The study successfully identified novel small molecules, OSI-027 and PP242, that acidify lysosomal pH.
• These compounds can serve as valuable tools for investigating lysosomal pH regulation and autophagy.
• Targeting lysosomal acidification presents a potential therapeutic strategy for neurodegenerative diseases.