icon-folder.gif   Conference Reports for NATAP  
 
  57th Annual Meeting of the American Association
for the Study of Liver Diseases
(AASLD)
October 27-31, 2006
Boston, MA
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In Vitro Resistance Studies of AG-021541, a Novel Nonnucleoside Inhibitor of the Hepatitis C Virus RNA-dependent RNA Polymerase
 
 
  Reported by Jules Levin
AASLD, Oct 2006, Boston, MA
 
Stephanie T. Shi, Koleen J. Herlihy, Javier Gonzalez, Amy K. Patick, Rohit Duggal Department of Virology, Pfizer PGRD La Jolla Laboratories,
 
ABSTRACT
A novel class of nonnucleoside HCV polymerase inhibitors characterized by a dihydropyrone core was identified by high throughput screening. Crystalographical studies of these compounds in complex with the polymerase identified an allosteric binding site close to the thumb and finger domains, approximately 30A away from the enzyme's catalytic center. AG-021541, a representative compound from this series, displayed measurable in vitro antiviral activity against the HCV subgenomic replicon with a mean EC50 value of 2.9 uM. To identify mutations conferring in vitro resistance to AG-021541, resistance selection was carried out using HCV replicon cells harboring a genotype 1b subgenomic reporter replicon either by serial passages in increasing concentrations of AG-021541 or by direct colony formation at fixed concentrations of the compound. Both populatyion and clonal sequencing were performed to identify genotypic changes in AG-021541-resistant cell lines. We identified several amino acid substitutions in the inhibitor-binding region of the polymerase, including M423T, M423V, and M494A. The most predominant mutation, M423T, conferred an 87-fold reduction in susceptibility to AG-021541 but no change in susceptibility to interferon. AG-021541-resistance replicon cell lines provide a valuable tool for specificity and mechanism of action studies of dihydropyrone polymerase inhibitors. The clinical relevance of in vitro resistance to HCV polymerase inhibitors remains to be investigated.
 
Conclusions
In vitro resistance studies of AG-021541 by either serial passages or instant colony formation using the replicon cels identified amino acid changes at the thumb-base allosteric site.
 
The most predominant mutation, M423T, conferred an 87-fold reduction in susceptibility to AG-021541, but no change in susceptibility to IFN.
 
Introduction of M423T, M423V, or V494A into the wild-type replicon resulted in reduced replication fitness and resistance to AG-021541 and structurally related compounds.
 
Resistance mutations identified in the replicon system may have future clinical implications.
 
RESULTS
 

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M423T conferred an 87-fold reduction in susceptibility to AG-021541 but no change in susceptibility to IFN.
 
The M423T mutant replicon displayed reduced replication fitness.
 
(9) Susceptibility of the M423T Mutant Enzyme and Replicon to AG-021541 and Related Compounds
 
M423T conferred resistance to AG-021541 and structurally related compounds in biochemical & replicon assays.
 
AG-021541-resistant mutants conferred resistance to AG-021541 and a structurally related compound in the transient replication assay.
 
A reduction in replication fitness was observed with the mutant replicons.
 
OBJECTIVES
-- To select for HCV replicons resistant to AG-021541, a nonnucleoside HCV polymerase inhibitor
-- To evaluate replicon fitness of AG-021541-resistant replicons
-- To confirm target/MOA of AG-021541 and obtain information on residues involved in the interaction between the inhibitor and the target
-- To evaluate cross resistance of AG-021541 to other HCV inhibitors
-- To identify resistance mutations that may have future clinical implications
 
MATERIALS and METHODS
Replicon and Cell Lines

The BB7 replicon, containing the HCV NS3-NS5B derived from the Con1 strain of genotype 1b, was licensed from Apath LLC. The replicon was modified to contain three adaptive mutations and a humanized Renilla luciferase (hRLuc) gene and electroporated into Huh7 cells to generate a reporter replicon cell line, DSR (17).
 

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In vitro Selection of AG-021541-Resistant Replicon Cells
For serial passage experiments, selection started in 0.2xEC90 (3 uM) of AG-021541, which was increased by 2 fold after every 3 passages. Cells grown in 0.8x, and 3.2xEC90 of AG-021541 were seeded for colony formation in 1.6x, 3.2x, and 6.4xEC90, respectively. For resistance selection at fixed concentrations, DSR cells were seeded for instant colony formation in 3xEC90 and 6xEC90 of AG-021541. For both procedures, resistant colonies were isolated about 2-3 weeks after the start of colony formation. Colonies were expanded to cell lines and subjected to replication fitness and resistance phenotye determinations. RNA were isolated from resistant lines for sequencing of the entire NS5B region.
 
Luciferase Reporter Assay
DSR cells were seeded in 96-well plates at 2x10-4th per well in the absence of selection antibodies. Compounds were tested at half-log serial dilutions over a range of concentrations with appropriate solvent controls. For transient replication assays, Huh7.5 cells were electroporated with in vitro replicon transcripts, plated in 96-well plates at 6x10-4th cells per well, and allowed to adhere for 4 at 37 degrees C before compound addition. Cells were incubated with compounds for 3 days at 37 degrees C. hRLuc activity was determined using Promega Renilla Luciferase Assay System in a Perkin Elmer 1450 MicroBeta Jet.
 
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