Mitochondrial biogenesis assay

Detect mitochondrial toxicity of novel therapeutics using Cyprotex’s mitochondrial biogenesis assay.

Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

Mitochondrial biogenesis assay using HCS

Mitochondrial proteins are encoded by both mitochondrial and nuclear genomes. Mitochondrial biogenesis is defined as the growth and division of pre-existing mitochondria.¹ Certain drugs are able to affect mitochondrial biogenesis through inhibition of mtDNA replication or mitochondrial encoded protein synthesis.
There is certain similarity between mitochondrial biogenesis and bacterial and viral replication. As a consequence, many antobacterial and antiviral agents cause mitochondrial toxicity through effects on human mitochondrial biogenesis. In fact, the FDA suggest that all antiviral drugs should be tested for effects on mitochondrial function.²
Cyprotex use high content screening and fluorescently labelled antibodies to evaluate ratios of an mtDNA-encoded protein (COX-1, a subunit of Complex IV) and an nDNA-encoded protein (SDH-A, a subunit of Complex II).
The mitochondrial biogenesis assay can be used to identify drugs cause toxicity through inhibition of mitochondrial biogenesis.

Mitochondria are the only organelles outside of the nucleus that contain their own genome and replicate itself in an independent manner from the nuclear genome

³Moreira AC et al, (2011), In: Biosensors for Health, Environment and Biosecurity, Serra PD (Ed.), 411-444

Protocol

Mitochondrial biogenesis assay protocol

Method	High content screening using fluorescently labelled antibodies for COX-1 and SDH-A
Cell Types	HepG2 or HepaRG (others available on request)
Exposure Time	5-8 days depending on cell line and customer needs
Number of Concentrations	10 concentrations (n=3 wells per concentration)
Positive Control	Chloramphenicol
Negative Control	Lincomycin
Data Delivery	COX-1 protein expression SDH-A protein expression COX-1/SDH-A protein expression ratio Cell loss

Data

Data from Cyprotex's mitochondrial biogenesis assay

Cyprotex’s mitochondrial biogenesis assay uses high content imaging to detect COX-1 (mtDNA-encoded) and SDH-A (nDNA-encoded) protein expression. Using the ratio of COX-1/SDH-A protein expression, specific effects on mitochondrial biogenesis can be identified. The positive control for the assay, chloramphenicol (antibiotic), inhibits mitochondrial biogenesis in a dose-dependent manner (figure 1).

Figure 1
Dose curve for choramphenicol in the mitochondrial biogenesis assay

Figure 2A
Dose curve for R1479 demonstrating the effect of the antiviral, R1479 (4’-azidocytidine) on mitochondrial biogenesis as illustrated by the impact on COX-1/SDH-A protein expression.

Figure 2B
High content images illustrating the effect of increasing concentrations of R1479 (4'-azidocytidine) on COX-1/SDH-1 protein expression

References

¹ Jornayvaz FR and Shulman GI (2010) Regulation of mitochondrial biogenesis. Essays Biochem 47; 69-84
² Food and Drug Administration. Guidance for Industry: Antiviral product development - Conducting and submitting virology studies to the agency (https://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/
UCM070953.pdf)
³ Moreira AC et al, (2011) Mitochondria as a biosensor for drug-induced toxicity - Is it really relevant? Biosensors for Health, Environment and Biosecurity, Serra PA (Ed.); 411-444