Application of a homogenous membrane potential assay to assess mitochondrial function

Area of Science:

• Biochemistry
• Cell Biology
• Toxicology

Background:

• Mitochondrial membrane potential (MMP) is crucial for cellular energy production and function.
• Decreased MMP is an indicator of mitochondrial dysfunction and potential cell death.
• Accurate measurement of MMP is vital for assessing chemical toxicity and drug effects.

Purpose of the Study:

• To identify an optimal fluorescent dye for quantifying MMP decreases in a high-throughput screening (HTS) format.
• To develop and validate a homogenous cell-based assay using the optimal dye for HTS.
• To screen a library of pharmacologically active compounds for their ability to disrupt MMP.

Main Methods:

• Comparison of four fluorescent dyes (Mito-MPS, JC-1, Rhodamine 123, TM-Rhodamine) for MMP measurement in HepG2 cells.
• Optimization of a homogenous cell-based assay in a 1,536-well plate format.
• Quantitative HTS of 1,280 compounds using the developed Mito-MPS assay.

Main Results:

• Mito-MPS demonstrated superior performance for quantifying MMP decreases in HepG2 cells under HTS conditions.
• The optimized homogenous cell-based Mito-MPS assay was successfully implemented.
• Fourteen compounds were identified that disrupt MMP, with potencies ranging from 0.15 to 18 μM.
• Structure-activity relationships were observed for tyrphostin and 3'-substituted indolone analogs.

Conclusions:

• Mito-MPS is the optimal dye for HTS-based MMP assessment.
• The developed homogenous cell-based Mito-MPS assay is effective for large-scale chemical screening.
• This assay facilitates the identification of compounds that impair mitochondrial function.