Multiparameter phenotypic screening for endogenous TFEB and TFE3 translocation identifies novel chemical series...

Area of Science:

• Neurobiology
• Cell Biology
• Pharmacology

Background:

• Defects in the autophagy-lysosome pathway are linked to toxic protein aggregate accumulation in neurodegenerative diseases.
• Modulating transcription factor EB (TFEB), the master regulator of lysosome function, can ameliorate disease phenotypes.

Purpose of the Study:

• To identify novel kinase regulators of TFEB and TFE3 using a high-throughput screen.
• To discover compounds that promote TFEB/TFE3 nuclear translocation and enhance lysosomal function for potential neurodegenerative disease therapies.

Main Methods:

• A multi-parameter high-throughput screen of the Published Kinase Inhibitor Set 2 (PKIS2) library.
• Assays for cellular toxicity and lysosomal function to ensure positive effects on lysosome activity.
• KINOMESCAN and cell-based KiNativ kinase profiling, CRISPRi knockdown, and orthogonal PRKD inhibitors for target validation.

Main Results:

• Identified quinazoline-derivative compounds that induce TFEB and TFE3 translocation, with novel analogs showing higher potency.
• Compounds demonstrated high binding for the PRKD kinase family but activity was independent of PRKD inhibition.
• Potent analogs upregulated the CLEAR gene network and cleared pathological HTT protein in a cellular model of proteinopathy.

Conclusions:

• Novel compounds effectively promote TFEB/TFE3 translocation and enhance lysosomal function.
• These compounds demonstrate potential for alleviating neurodegeneration-relevant phenotypes by clearing toxic protein aggregates.
• The findings support the development of new therapeutic strategies targeting the autophagy-lysosome pathway.