Establishment of a robust hepatitis C virus replicon cell line over-expressing P-glycoprotein that facilitates analysis of P-gp drug transporter effects on inhibitor antiviral activity.
P-glycoprotein (P-gp) is an active efflux pump affecting the pharmacokinetic (PK) profiles of drugs that are P-gp substrates. The Caco-2 bi-directional assay is widely used to identify drug-P-gp interactions in vitro. For molecules exhibiting non-classical drug properties however, ambiguous results limit its use in lead optimization. The goal of this study was to develop a robust cell-based assay system to directly measure the role of P-gp-driven efflux in reducing the potency of hepatitis C virus (HCV) replication inhibitors. Vinblastine (Vin) was employed to select for a Vin-resistant HCV replicon (313-11) from the parental cell line (377-2). The 313-11 cell line was >50-fold resistant to Vin and over-expressed P-gp, as determined by Western immunoblots. Increased expression of P-gp was mediated by up-regulation of the MDR1 transcript. The reduced potency of different classes of HCV replication inhibitors in the 313-11 P-gp cell line was restored in the presence of known P-gp inhibitors. Addition of the P-gp inhibitor, tariquidar, increased the uptake of a radiolabeled HCV replication inhibitor by 14-fold in the 313-11 replicon cell line. Finally, a positive correlation was demonstrated between potency in the 313-11 replicon and the bi-directional Caco-2 efflux ratio for a panel of HCV protease inhibitors. In conclusion, a robust P-gp HCV replicon cell-based assay has been developed to measure the effect of the P-gp efflux pump on the potency of different classes of HCV replication inhibitors. This system establishes a direct correlation between antiviral activity and the effect of P-gp efflux in a single cell line.
Bristol-Myers Squibb, 5 Research Parkway, Wallingford, CT 06492, USA. dennis.hernandez@bms.com
Dennis
D
Hernandez
Paul
P
Falk
Fei
F
Yu
Guangzhi
G
Zhai
Yong
Y
Quan
Teresa
T
Faria
Kai
K
Cao
Paul
P
Scola
Fiona
F
McPhee
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology*; D001692:Biological Transport; D018938:Caco-2 Cells; D045744:Cell Line, Tumor / Q000187:drug effects* / Q000378:metabolism / Q000821:virology; D031541:Fluorescence Resonance Energy Transfer; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D006801:Humans; D010539:Permeability; D011480:Protease Inhibitors / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000187:drug effects; D012093:Replicon; D014747:Vinblastine / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors; D014779:Virus Replication / Q000187:drug effects; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D000998:Antiviral Agents; D011480:Protease Inhibitors; D012333:RNA, Messenger; D012367:RNA, Viral; D014747:Vinblastine; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases