In vitro activity: Ombitasvir (formerly known as ABT-267) is an N-phenylpyrrolidine-based inhibitor of hepatitis C virus (HCV) NS5A with excellent potency, metabolic stability, and pharmacokinetics. Ombitasvir was found to be a pan-genotypic HCV inhibitor with an EC50 range of 1.7-19.3 pM against GT1a, -1b, -2a, -2b, -3a, -4a, and -5a and 366 pM against GT6a. It decreased HCV RNA up to 3.10 log10 IU/mL during 3-day monotherapy in treatment-naive HCV GT1-infected subjects. Ombitasvir has been approved by the Food and Drug Administration (FDA) for use in combination with other antiviral agents (paritaprevir, ritonavir and dasabuvir) under the name of Viekira Pak.

Kinase Assay: The methods used for measurement of the effects of individual amino acid variants on the activity of an inhibitor in HCV replicon cell culture assays were described previously. Briefly, the resistance-associated variants in NS5A were each introduced into the genotype 1a-H77 or 1b-Con1 or one of the chimeric genotype 2 to 6 replicons using the Change-IT multiple-mutation site-directed mutagenesis kit (Affymetrix, Santa Clara, CA). After the presence of the variant was confirmed by sequence analysis, the plasmid was linearized and the TranscriptAid T7 high-yield transcription kit (Fermentas, Glen Burnie, MD) was used to transcribe the HCV subgenomic RNA from the plasmid. In a transient assay, the replicon RNA containing the variant was transfected via electroporation into a Huh-7 cell line. The EC50s were calculated as described in the previous sections.

Cell Assay: A genotype 1b-Con1 HCV replicon-based shuttle vector cassette with the luciferase reporter but without the Neo gene was constructed for assessing the phenotypes of NS5A genes derived from individuals infected with HCV genotypes 1 to 6. A NotI restriction site was cloned into the 1b-Con1 subgenomic replicon vector 90 nucleotides upstream of NS5A in the 3′ end of NS4B, and a ClaI site was cloned after the NS5A amino acid 413 codon. The NS5A region from genotype 1-infected patients was inserted between the NotI and ClaI restriction sites. The 1b-Con1 shuttle vector with NotI and BlpI restriction sites (described in the previous section) was used to evaluate the ability of ombitasvir to inhibit the NS5A region encompassing amino acids 1 to 214 from non-genotype 1 HCV. The NS5A gene from clinical samples was amplified and ligated into the shuttle vector. In a transient assay, the HCV subgenomic replicon RNA containing the NS5A gene from each clinical sample was transfected via electroporation into a Huh-7 derived cell line. The cells were incubated in the presence of ombitasvir for 4 days. The luciferase activity in the cells was measured, and the EC50 was calculated as described above.