Development of in Vitro Screening Assay to Detect Inhibition of Mitochondrial RNA Polymerase by Anti‐viral Drugs

Many antiviral agents have been shown to have an off‐target inhibitory effect on mitochondrial DNA (mtDNA) and RNA (mtRNA) polymerases. Quantification of mitochondrial DNA relative to nuclear DNA content by qPCR has been proven as a reliable and fast approach to screen for mitochondrial DNA toxicity. Methods used to assess the function of mitochondrial RNA polymerase (POLRMT) include biochemical and cell‐based assays, and are not optimal for routine screening. Here we studied the utility of RT‐qPCR for the analysis of mitochondrial gene expression as a function of POLRMT in a standard cell culture model. HepG2 cells were treated with 5 and 10mM 2′C‐methyladenosine and 10 and 50uM balapiravir (RG1626, prodrug of 4‐azidocytosine) every 48 hours for 7 and 14 days. Both test compounds have previously been shown to be chain terminators upon mtRNA transcription. 2′ 3′‐dideoxycytidine (ddC) and fialuridine were used as positive controls and 2‐epi‐fialuridine was used as negative control for the study. Upon harvest cells were counted and extracted RNA and DNA were used for gene expression and mtDNA copy number analysis. RT‐qPCR was performed for five mitochondrially encoded (MT‐ND1, MT‐ND4L, MT‐ND5, MT‐CYB, MT‐RNR2) and two nuclear (GAPDH, ACTB) genes. Gene expression analysis in cells treated with the test compounds revealed a dose dependent decrease in the expression of mitochondrial genes when compared to untreated controls, while mtDNA copy number remained unchanged. The degree of expression of each gene had a reverse correlation with the distance between the gene positon in the mitochondrial genome and the initiation transcription site IT H2 . The mRNA levels of the housekeeping genes, GAPDH and ACTB, transcribed by RNA Pol II remained unchanged across all samples and could be used for data normalization. As a conclusion, analysis of mitochondrial gene expression using RT‐qPCR can be utilized as a simple and sensitive first pass method to screen for POLRMT inhibition in vitro and would aid in identification of mitochondrial toxicity of anti‐viral compounds prior to in vivo testing. Support or Funding Information TS, JLZ, and ML are employees of Abbvie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication