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High-throughput assays for assessing mitochondrial dysfunction caused by compounds that impair mtDNA-encoded protein
Sashi Nadanaciva1, James Murray, Casey Wilson
1Compound Safety Prediction, Worldwide Medicinal Chemistry, Pfizer Inc., Groton, Connecticut, USA.
Abstract:
Compounds that impair the synthesis of either mitochondrial DNA (mtNDA) or mtDNA-encoded proteins reduce the levels of 13 proteins essential for oxidative phosphorylation, leading to a decrease in mitochondrial ATP production. Toxicity caused by these compounds is seldom identified in 24 to 72 hr cytotoxicity assays due to the low turnover rates of both mtDNA and mtDNA-encoded proteins. Here, we describe three high-throughput screening assays that detect compounds that affect mtDNA-encoded protein levels. All three assays measure the levels of two proteins, one a mtDNA-encoded protein synthesized on mitochondrial ribosomes and the other, a nuclear DNA-encoded protein synthesized on cytosolic ribosomes. The first assay measures the levels of these two proteins by quantitative image analysis and requires a high-content imaging system. The second assay is an in-cell immunoassay that utilizes infrared dyes for detection of the two proteins and, thus, requires a LI-COR Odyssey system. The third assay is an in-cell immunoassay that utilizes colorimetric detection of the two proteins and requires an absorbance microplate reader.
Insights
New assays detect compounds that harm mitochondrial DNA (mtDNA)-encoded proteins. These methods identify toxicity missed by standard tests, aiding drug discovery and safety assessments.
Area of Science:
- Biochemistry
- Toxicology
- Molecular Biology
Background:
- Mitochondrial DNA (mtDNA) and its encoded proteins are crucial for oxidative phosphorylation and ATP production.
- Standard cytotoxicity assays often fail to detect toxicity from compounds affecting mtDNA synthesis due to slow protein turnover rates.
- Developing sensitive assays for mtDNA-related toxicity is essential for drug safety and development.
Purpose of the Study:
- To develop and validate novel high-throughput screening assays for detecting compounds that impair mitochondrial DNA-encoded protein levels.
- To address the limitations of conventional cytotoxicity assays in identifying mitochondrial toxicants.
- To provide tools for assessing the safety of chemical compounds and potential drug candidates.
Main Methods:
- Three distinct high-throughput screening assays were developed to measure levels of both mtDNA-encoded and nuclear DNA-encoded proteins.
- Assay 1: Quantitative image analysis using a high-content imaging system.
- Assays 2 & 3: In-cell immunoassays utilizing infrared (LI-COR Odyssey) or colorimetric detection (microplate reader).
Main Results:
- All three assays successfully measured the differential levels of mtDNA-encoded and nuclear DNA-encoded proteins.
- The assays provide a means to detect compounds that specifically affect the synthesis or stability of mtDNA-encoded proteins.
- These methods offer improved sensitivity for identifying mitochondrial toxicants compared to traditional assays.
Conclusions:
- The developed high-throughput assays are effective in identifying compounds that negatively impact mitochondrial protein synthesis.
- These assays offer a valuable tool for early-stage toxicity screening in drug discovery and chemical safety assessment.
- Improved detection of mitochondrial toxicants can enhance the safety profile of new therapeutic agents and chemicals.
