title: Antiviral Nucleotide Incorporation by Recombinant Human Mitochondrial RNA Polymerase Is Predictive of Increased In Vivo Mitochondrial Toxicity Risk.
abstract: Nucleoside or nucleotide inhibitors are a highly successful class of antivirals due to selectivity, potency, broad coverage, and high barrier to resistance. Nucleosides are the backbone of combination treatments for HIV, hepatitis B virus, and, since the FDA approval of sofosbuvir in 2013, also for hepatitis C virus (HCV). However, many promising nucleotide inhibitors have advanced to clinical trials only to be terminated due to unexpected toxicity. Here we describe the in vitro pharmacology of compound 1, a monophosphate prodrug of a 2'-ethynyluridine developed for the treatment of HCV. Compound 1 inhibits multiple HCV genotypes in vitro (50% effective concentration [EC50], 0.05 to 0.1 μM) with a selectivity index of >300 (50% cytotoxic concentration [CC50], 30 μM in MT-4 cells). The active triphosphate metabolite of compound 1, compound 2, does not inhibit human α, β, or γ DNA polymerases but was a substrate for incorporation by the human mitochondrial RNA polymerase (POLRMT). In dog, the oral administration of compound 1 resulted in elevated serum liver enzymes and microscopic changes in the liver. Transmission electron microscopy showed significant mitochondrial swelling and lipid accumulation in hepatocytes. Gene expression analysis revealed dose-proportional gene signature changes linked to loss of hepatic function and increased mitochondrial dysfunction. The potential of in vivo toxicity through mitochondrial polymerase incorporation by nucleoside analogs has been previously shown. This study shows that even moderate levels of nucleotide analog incorporation by POLRMT increase the risk of in vivo mitochondrial dysfunction. Based on these results, further development of compound 1 as an anti-HCV compound was terminated.
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, California, USA martijn.fenaux@novartis.com.
authors.forename: Martijn
authors.initials: M
authors.lastname: Fenaux
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, California, USA.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Lin
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore, Singapore.
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Yokokawa
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, California, USA.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Sweeney
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, California, USA.
authors.forename: Oliver
authors.initials: O
authors.lastname: Saunders
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, California, USA.
authors.forename: Lili
authors.initials: L
authors.lastname: Xie
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore, Singapore.
authors.forename: Siew Pheng
authors.initials: SP
authors.lastname: Lim
authors.affiliation: Novartis Pharmaceuticals, Basel, Switzerland.
authors.forename: Marianne
authors.initials: M
authors.lastname: Uteng
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, California, USA.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Uehara
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, California, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Warne
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore, Singapore.
authors.forename: Wang
authors.initials: W
authors.lastname: Gang
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, California, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Jones
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, California, USA.
authors.forename: Satya
authors.initials: S
authors.lastname: Yendluri
authors.affiliation: Novartis Pharmaceuticals, East Hanover, New Jersey, USA.
authors.forename: Helen
authors.initials: H
authors.lastname: Gu
authors.affiliation: Novartis Institute for BioMedical Research, Cambridge, Massachusetts, USA.
authors.forename: Keith
authors.initials: K
authors.lastname: Mansfield
authors.affiliation: Novartis Pharmaceuticals, Basel, Switzerland.
authors.forename: Julie
authors.initials: J
authors.lastname: Boisclair
authors.affiliation: Novartis Pharmaceuticals, East Hanover, New Jersey, USA.
authors.forename: Tycho
authors.initials: T
authors.lastname: Heimbach
authors.affiliation: Novartis Pharmaceuticals, East Hanover, New Jersey, USA.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Catoire
authors.affiliation: Novartis Institute for BioMedical Research, Cambridge, Massachusetts, USA.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Bracken
authors.affiliation: Novartis Institute for BioMedical Research, Cambridge, Massachusetts, USA.
authors.forename: Margaret
authors.initials: M
authors.lastname: Weaver
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, California, USA.
authors.forename: Heinz
authors.initials: H
authors.lastname: Moser
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, California, USA.
authors.forename: Weidong
authors.initials: W
authors.lastname: Zhong
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000008:administration & dosage / Q000493:pharmacokinetics* / Q000633:toxicity*; D002460:Cell Line; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000473:pathology; D008297:Male; D009705:Nucleosides / Q000493:pharmacokinetics*; D011122:Polyphosphates / Q000378:metabolism; D011355:Prodrugs / Q000493:pharmacokinetics / Q000633:toxicity; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018675:Toxicity Tests / Q000379:methods; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D012321:DNA-Directed RNA Polymerases; D009705:Nucleosides; D011122:Polyphosphates; D011355:Prodrugs; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C519567:POLRMT protein, human; C005692:triphosphoric acid