High throughput fluorescence assays for the measurement of mitochondrial activity in intact human neuroblastoma cells
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1Department of Biochemistry, Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Harlow, Essex, UK.
Abstract:
The mitochondrial permeability transition event is implicated in the activation phase of apoptosis and necrosis, and is therefore postulated to play a role in many disease states. Mitochondrial permeability transition is therefore of increasing pharmaceutical interest. Drug discovery requires the rapid screening of compound libraries to identify functionally active ligands. We report the development of two fluorescence-based approaches for screening compound libraries for effects on mitochondrial function. These assays use the fluorometric imaging plate reader in 96-well format, and two commercially available dyes: JC-1 and calcein-AM. We show here that a JC-1 assay proved highly amenable to HTS implementation. By combining this with a calcein-based assay, these approaches gave complementary information: JC-1 facilitates the discovery of modulators of mitochondrial polarization from a library of approximately 100,000 compounds screened at 8 microM, and the calcein assay identifies permeability transition pore-specific inhibitors.
Insights
We developed two fluorescence assays to screen for compounds affecting mitochondrial function, crucial for understanding diseases. These assays enable rapid drug discovery by identifying modulators of mitochondrial polarization and permeability transition pore inhibitors.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Mitochondrial permeability transition is involved in apoptosis and necrosis, making it a target for pharmaceutical research.
- Efficient screening of compound libraries is essential for identifying drugs that modulate mitochondrial function.
Purpose of the Study:
- To develop and validate fluorescence-based assays for high-throughput screening (HTS) of compounds targeting mitochondrial permeability transition.
- To identify modulators of mitochondrial polarization and specific inhibitors of the permeability transition pore.
Main Methods:
- Utilized a fluorometric imaging plate reader in a 96-well format.
- Employed two commercially available fluorescent dyes: JC-1 for mitochondrial polarization and calcein-AM for cell viability/membrane integrity.
- Developed a JC-1 assay suitable for HTS and combined it with a calcein-based assay for complementary information.
Main Results:
- The JC-1 assay demonstrated high amenability for HTS implementation.
- Screened approximately 100,000 compounds at 8 microM using the JC-1 assay to identify modulators of mitochondrial polarization.
- The combined approach successfully identified both modulators of mitochondrial polarization and permeability transition pore-specific inhibitors.
Conclusions:
- Developed robust, complementary fluorescence-based assays for screening compound libraries for effects on mitochondrial function.
- These assays are valuable tools for drug discovery targeting diseases involving mitochondrial dysfunction.
- The JC-1 and calcein-AM assays provide efficient and reliable methods for identifying novel therapeutic agents.
