title: In vitro resistance study of AG-021541, a novel nonnucleoside inhibitor of the hepatitis C virus RNA-dependent RNA polymerase.
abstract: A novel class of nonnucleoside hepatitis C virus (HCV) polymerase inhibitors characterized by a dihydropyrone core was identified by high-throughput screening. Crystallographic studies of these compounds in complex with the polymerase identified an allosteric binding site close to the junction of the thumb and finger domains, approximately 30 A away from the catalytic center. AG-021541, a representative compound from this series, displayed measurable in vitro antiviral activity against the HCV genotype 1b subgenomic replicon with a mean 50% effective concentration of 2.9 muM. To identify mutations conferring in vitro resistance to AG-021541, resistance selection was carried out using HCV replicon cells either by serial passages in increasing concentrations of AG-021541 or by direct colony formation at fixed concentrations of the compound. We identified several amino acid substitutions in the AG-021541-binding region of the polymerase, including M423(T/V/I), M426T, I482(S/T), and V494A, with M423T as the predominant change observed. These mutants conferred various levels of resistance to AG-021541 and structurally related compounds but remained sensitive to interferon and HCV polymerase inhibitors known to interact with the active site or other allosteric sites of the protein. In addition, dihydropyrone polymerase inhibitors retained activity against replicons that contain signature resistance changes to other polymerase inhibitors, including S282T, C316N, M414T, and P495(S/L), indicating their potential to be used in combination therapies with these polymerase inhibitors. AG-021541-resistant replicon cell lines provide a valuable tool for mechanism-of-action studies of dihydropyrone polymerase inhibitors. The clinical relevance of in vitro resistance to HCV polymerase inhibitors remains to be investigated.
authors: Department of Virology, Pfizer Global Research and Development, La Jolla Laboratories, 10777 Science Center Drive, San Diego, CA 92121, USA. stephanie.shi@pfizer.com
authors: Stephanie T
authors: ST
authors: Shi
authors: Koleen J
authors: KJ
authors: Herlihy
authors: Joanne P
authors: JP
authors: Graham
authors: Shella A
authors: SA
authors: Fuhrman
authors: Chau
authors: C
authors: Doan
authors: Hans
authors: H
authors: Parge
authors: Michael
authors: M
authors: Hickey
authors: Jingjin
authors: J
authors: Gao
authors: Xiu
authors: X
authors: Yu
authors: Fannie
authors: F
authors: Chau
authors: Javier
authors: J
authors: Gonzalez
authors: Hui
authors: H
authors: Li
authors: Cristina
authors: C
authors: Lewis
authors: Amy K
authors: AK
authors: Patick
authors: Rohit
authors: R
authors: Duggal
mesh_terms: D000495:Allosteric Site; D001665:Binding Sites; D045744:Cell Line, Tumor; D024882:Drug Resistance, Viral / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011753:Pyrones / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D012093:Replicon; D014779:Virus Replication
chemical_list: D004791:Enzyme Inhibitors; D011753:Pyrones; D012324:RNA-Dependent RNA Polymerase