Synergistic inhibition of hepatitis C virus infection by a novel microtubule inhibitor in combination with daclatasvir

Abstract

Even though substantial progress has been made in the treatment of hepatitis C virus (HCV) infection, viral resistance and relapse still occur in some patients and additional therapeutic approaches may ultimately be needed should viral resistance become more prevalent. Microtubules play important roles in several HCV life cycle events, including cell attachment, entry, cellular transportation, morphogenesis and progeny secretion steps. Therefore, it was hypothesized that microtubular inhibition might be a novel approach for the treatment of HCV infection. Here, the inhibitory effects of our recently developed microtubule inhibitors were studied in the HCV replicon luciferase reporter system and the infectious system. In addition, the combination responses of microtubule inhibitors with daclatasvir, which is a clinically used HCV NS5A inhibitor, were also evaluated. Our results indicated that microtubule targeting had activity against HCV replication and showed synergistic effect with a current clinical drug.

Keywords: Combinational therapy; Hepatitis C virus; Microtubule inhibitor; Viral resistance.

Fig. 1

(A) Compounds structure. Compounds 27a, 27b, 9f, 19a, 31b and 31d are our recently developed microtubule inhibitors which target the colchicine binding site. Daclatasvir and ledipasvir are clinically used drugs which target HCV NS5A. (B) Immunofluorescence images of FFU assays to determine infectivity titer. Huh7.5.1 cells were infected by various dilutions of cell culture medium (a, mock infected), JFH-1 D183 virus stock (b) and newly produced viral supernatant (c) separately. After 72 h infection, Huh7.5.1 cells were fixed and stained for NS5A (red) and nuclei (blue). Clusters of 2–50 NS5A positive cells were considered as foci. (C) Quantitative real time PCR (qRT-PCR) results (left panel) and toxicity assay results (right panel) of daclatasvir and compound 9f at 24 h and 48 h. HCV RNA was relatively quantified by normalization to untreated control. GAPDH was used as housekeeping gene to guarantee the same base level. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

(A)(B) Combenefit software plotted the combinational responses of daclatasvir with compound 9f using Loewe model. Upper panel, 24 h. Lower panel, 48h. (A) D-r shift map when added with a range of concentrations of different compounds. (B) Synergy surface map (left panel) and Matrix score table (right panel), N means the number of replicates, the number under the score is standard deviation, stars indicate the level of significance (*P < 0.05, **P < 0.001, ***P < 0.0001). (C)(D) Compusyn software plotted the combinational responses of daclatasvir with compound 9f using median effect equation. Upper panel, 24 h. Lower panel, 48 h. (C) Fa-CI plot (left panel), synergism (CI < 1), additivity (CI = 1), antagonism (CI > 1). Classic isobologram (right panel), synergism (middle combination data point below the line), additivity (middle combination data point on the line), antagonism (middle combination data point above the line). (D) CI data for compounds combination group at different Fa levels.

Fig. 3

Immunofluorescence images of BM4-5 Feo cells after incubation with daclatsvir (125 pM) and compound 9f (25 nM) and the combination group respectively after (A)24 h (B) 48h. Cells were fixed and immunostained for nuclei (blue), microtubule (green) and HCV NS5A (red). Photos were captured by confocal SP8 microscope (630 magnification). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)