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Published on: March 10, 2017
Multiparameter phenotypic screening for endogenous TFEB and TFE3 translocation identifies novel chemical series
Phillippa J Carling1,2, Brent J Ryan1, William McGuinness1
1Department of Physiology, Anatomy and Genetics, Oxford Parkinson's Disease Centre, University of Oxford, Oxford, UK.
Abstract:
The accumulation of toxic protein aggregates in multiple neurodegenerative diseases is associated with defects in the macroautophagy/autophagy-lysosome pathway. The amelioration of disease phenotypes across multiple models of neurodegeneration can be achieved through modulating the master regulator of lysosome function, TFEB (transcription factor EB). Using a novel multi-parameter high-throughput screen for cytoplasmic:nuclear translocation of endogenous TFEB and the related transcription factor TFE3, we screened the Published Kinase Inhibitor Set 2 (PKIS2) library as proof of principle and to identify kinase regulators of TFEB and TFE3. Given that TFEB and TFE3 are responsive to cellular stress we have established assays for cellular toxicity and lysosomal function, critical to ensuring the identification of hit compounds with only positive effects on lysosome activity. In addition to AKT inhibitors which regulate TFEB localization, we identified a series of quinazoline-derivative compounds that induced TFEB and TFE3 translocation. A novel series of structurally-related analogs was developed, and several compounds induced TFEB and TFE3 translocation at higher potency than previously screened compounds. KINOMEscan and cell-based KiNativ kinase profiling revealed high binding for the PRKD (protein kinase D) family of kinases, suggesting good selectivity for these compounds. We describe and utilize a cellular target-validation platform using CRISPRi knockdown and orthogonal PRKD inhibitors to demonstrate that the activity of these compounds is independent of PRKD inhibition. The more potent analogs induced subsequent upregulation of the CLEAR gene network and cleared pathological HTT protein in a cellular model of proteinopathy, demonstrating their potential to alleviate neurodegeneration-relevant phenotypes. Abbreviations: AD: Alzheimer disease; AK: adenylate kinase; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; HD: Huntington disease; PD: Parkinson disease; PKIS2: Published Kinase Inhibitor Set 2; PRKD: protein kinase D; TFEB: transcription factor EB.
Insights
Researchers identified novel compounds that enhance TFEB and TFE3 activity, promoting lysosomal function and clearing toxic proteins. These findings offer potential therapeutic strategies for neurodegenerative diseases like Alzheimer
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.
