Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -
cancer agents. However, their clinical applications have been limited by off target effects. Development of
antiviral ribonucleosides for treatment of hepatitis C virus (HCV)
infection has been hampered by appearance of toxicity during clinical trials that evaded detection during preclinical studies. It is well established that the human
mitochondrial DNA polymerase is an off target for deoxyribonucleoside
reverse transcriptase inhibitors. Here we test the hypothesis that triphosphorylated metabolites of therapeutic ribonucleoside analogues are substrates for cellular
RNA polymerases. We have used ribonucleoside analogues with activity against HCV as model compounds for therapeutic
ribonucleosides. We have included ribonucleoside analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents that are non-obligate chain terminators of the HCV
RNA polymerase. We show that all of the anti-HCV ribonucleoside analogues are substrates for human
mitochondrial RNA polymerase (POLRMT) and eukaryotic core
RNA polymerase II (Pol II) in vitro. Unexpectedly, analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents were inhibitors of POLRMT and Pol II. Importantly, the proofreading activity of
TFIIS was capable of excising these analogues from Pol II transcripts. Evaluation of transcription in cells confirmed sensitivity of POLRMT to
antiviral ribonucleosides, while Pol II remained predominantly refractory. We introduce a parameter termed the mitovir (
mitochondrial dysfunction caused by
antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by
nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of
ribonucleosides with a lower potential for toxicity.