title: Sensitivity of mitochondrial transcription and resistance of RNA polymerase II dependent nuclear transcription to antiviral ribonucleosides.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America. jja5@psu.edu
authors.forename: Jamie J
authors.initials: JJ
authors.lastname: Arnold
authors.forename: Suresh D
authors.initials: SD
authors.lastname: Sharma
authors.forename: Joy Y
authors.initials: JY
authors.lastname: Feng
authors.forename: Adrian S
authors.initials: AS
authors.lastname: Ray
authors.forename: Eric D
authors.initials: ED
authors.lastname: Smidansky
authors.forename: Maria L
authors.initials: ML
authors.lastname: Kireeva
authors.forename: Aesop
authors.initials: A
authors.lastname: Cho
authors.forename: Jason
authors.initials: J
authors.lastname: Perry
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Vela
authors.forename: Yeojin
authors.initials: Y
authors.lastname: Park
authors.forename: Yili
authors.initials: Y
authors.lastname: Xu
authors.forename: Yang
authors.initials: Y
authors.lastname: Tian
authors.forename: Darius
authors.initials: D
authors.lastname: Babusis
authors.forename: Ona
authors.initials: O
authors.lastname: Barauskus
authors.forename: Blake R
authors.initials: BR
authors.lastname: Peterson
authors.forename: Averell
authors.initials: A
authors.lastname: Gnatt
authors.forename: Mikhail
authors.initials: M
authors.lastname: Kashlev
authors.forename: Weidong
authors.initials: W
authors.lastname: Zhong
authors.forename: Craig E
authors.initials: CE
authors.lastname: Cameron
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000009:adverse effects / Q000494:pharmacology*; D002417:Cattle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D016174:Hepacivirus / Q000378:metabolism*; D006526:Hepatitis C / Q000188:drug therapy / Q000201:enzymology; D008928:Mitochondria / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism*; D012367:RNA, Viral / Q000096:biosynthesis; D012263:Ribonucleosides / Q000009:adverse effects / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D012367:RNA, Viral; D012263:Ribonucleosides; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases; C519567:POLRMT protein, human