Avasimibe: A novel hepatitis C virus inhibitor that targets the assembly of infectious viral particles

Longbo Hu, Jinqian Li, Hua Cai, Wenxia Yao, Jing Xiao, Yi-Ping Li, Xiu Qiu, Huimin Xia, Tao Peng

Abstract

Direct-acting antivirals (DAAs), which target hepatitis C virus (HCV) proteins, have exhibited impressive efficacy in the management of chronic hepatitis C. However, the concerns regarding high costs, drug resistance mutations and subsequent unexpected side effects still call for the development of host-targeting agents (HTAs) that target host factors involved in the viral life cycle and exhibit pan-genotypic antiviral activity. Given the close relationship between lipid metabolism and the HCV life cycle, we investigated the anti-HCV activity of a series of lipid-lowering drugs that have been approved by government administrations or proven safety in clinical trials. Our results showed that avasimibe, an inhibitor of acyl coenzyme A:cholesterol acyltransferase ACAT), exhibited marked pan-genotypic inhibitory activity and superior inhibition against HCV when combined with DAAs. Moreover, avasimibe significantly impaired the assembly of infectious HCV virions. Mechanistic studies demonstrated that avasimibe induced downregulation of microsomal triglyceride transfer protein expression, resulting in reduced apolipoprotein E and apolipoprotein B secretion. Therefore, the pan-genotypic antiviral activity and clinically proven safety endow avasimibe exceptional potential as a candidate for combination therapy with DAAs. In addition, the discovery of the antiviral properties of ACAT inhibitors also suggests that inhibiting the synthesis of cholesteryl esters might be an additional target for the therapeutic intervention of chronic HCV infection
Keywords: HCV; avasimibe; lipid-lowering drug; ACAT; antiviral activity; host-targeting agents

1. Introduction

Hepatitis C virus (HCV), an enveloped positive-sense RNA virus, belongs to the Hepacivirus genus of the Flaviviridae family (Scheel and Rice, 2013). More than 180 million individuals worldwide are infected with HCV; approximately 75%-85% of them develop chronic infection, of which, 10% to 20% develop progressive liver injury, fibrosis, cirrhosis or hepatocellular carcinoma over a period of 20 to 30 years (Chen and Morgan, 2006; Thrift et al., 2017). Currently, there is no effective prophylactic or therapeutic vaccine against HCV. In the past, the combination of ribavirin plus interferon (IFN)-α was the standard anti-HCV therapy, but its disappointing efficacy and severe side effects compelled researcher to identify other advanced antiviral agents(Carter et al., 2017). Recently, revolutionary direct-acting antivirals (DAAs) have demonstrated amazing efficacy in the management of chronic hepatitis C infection, making it a curable disease in the majority of treated patients (Li and De Clercq, 2017). However, the extremely high cost of DAAs reduces their accessibility to patients even in high-income countries. Furthermore, concerns remain regarding drug resistance mutations as well as subsequent unexpected side effects (Buti and Esteban, 2016; El Kassas et al., 2016; Esposito et al., 2016). Thus, identifying other novel anti-HCV targets is still urgently needed.
Antiviral agents can be classified into two categories: DAAs, which directly target viral proteins and host-targeting agents (HTAs), which target host factors involved in the viral life cycle (Baugh et al., 2013; Zeisel et al., 2015). Unlike DAAs, which have a high risk of inducing drug resistance mutations due to the high genetic variability of HCV, HTAs induce few resistance and exhibit pan-genotypic antiviral activity because of the low genetic variability of host factors (Pawlotsky, 2014). Therefore, combination therapy involving DAAs and HTAs could offer a promising option for HCV treatment. The search for HTAs relies heavily on the elucidation of HCV-host interaction mechanisms. Host lipid metabolism related proteins are involved in every step of the HCV life cycle (Grassi et al., 2016; Popescu et al., 2014). First, the low-density lipoproteins receptor and the high-density lipoprotein receptor scavenger receptor class B type I facilitate the binding and entry of HCV particles (Agnello et al., 1999; Scarselli et al., 2002). Then, HCV replication occurs in the membranous web and the lipid kinase phosphatidylinositol-4-kinase IIIα, the oxysterol-binding protein and the sterol regulatory element-binding protein play important roles in this process (Reiss et al., 2011; Wang et al., 2014; Waris et al., 2007). In addition, the HCV particles assembly relies primarily on lipid droplets, the neutral lipids storage organelles in cells (Suzuki, 2011). Furthermore, the secretion of HCV virions hijacks the very low-density lipoprotein secretion pathway(Suzuki, 2012). Thus, small interfering RNAs (siRNAs) or inhibitors that target host factors involved in LD biogenesis including diacylglycerol acyltransferase 1, apolipoprotein E (ApoE) and apolipoprotein B (ApoB), and,microsomal triglyceride transfer protein (MTTP) which transfers triglyceride to apolipoprotein in the liver and intestine in the initial step during the formation of very-low-density lipoproteins (VLDL) and chylomicrons (Barter and Rye, 2016), can effectively inhibit infectious HCV production(Chang et al., 2007; Herker et al., 2010; Huang et al., 2007; Nahmias et al., 2008).
Given the close association between lipid metabolism and the HCV life cycle, lipid-lowering drugs show great potential as HTAs (Villareal et al., 2015). Therefore, in this study, we tested the anti-HCV activity of candidate lipid-lowering drugs that have been approved by government administrations or proven safety in clinical trials in consideration of future clinical translation to treat HCV. Avasimibe, an inhibitor of acyl coenzyme A:cholesterol acyltransferase (ACAT), exhibited marked pan-genotypic inhibitory activity and a great potential for combination therapy with DAAs. al., 2014). The plasmids of these HCV recombinants were kindly provided by Dr. Jens Bukh (University of Copenhagen and Hvidovre Hospital, Denmark). For titration, the viral supernatant was centrifuged to remove cell debris and serially diluted to infect Huh7.5.1 cells. After 48 hours, the cells were fixed with 4% paraformaldehyde and observed under a microscope. Virus infectivity titers were calculated as focus forming units per milliliter (FFU/ml). The dose-response curves of HCV derived from cell culture (HCVcc) to avasimibe were plotted and the EC50 of supernatant HCV titers were represented as the relative titer calculated as percentages of GFP-positive rate related to the control.

2. Materials and methods

Drug Combination Analysis

Huh7.5.1 cells were infected with HCV-GFP in the presence of avasimibe alone or in combination with each DAA at the different concentration ratios for 72 h. The infection efficiency of HCV-GFP was detected by flow cytometry and the inhibition efficiency of alone drug and drug combination using GraphPad Prism software. Student’s t-test was used to evaluate the significance between control and treatment groups. P<0.05 was considered to be statistically significant.

3. Results

3.1. Avasimibe significantly inhibits HCV infection.

To evaluate the ability of lipid-lowering drugs to inhibit HCV infection, Huh7.5.1 cells were infected with HCV-GFP in the presence of candidate drugs, and then the infection efficiency and the cellular toxicity of the compounds were measured using flow cytometer and AlamarBlue assay, respectively. The antiviral activity was evaluated by the reduction of infected cells. As shown in Fig. 1, under non-cellular-toxic concentration, the tested statins (mevastatin, lovastatin, fluvastatin, simvastatin, or atorvastatin) showed minor anti-HCV activity (Fig. 1A and 1B). For the fibrates, bezafibrate and fenofibrate displayed slight anti-HCV activity, while gemfibrozil and ciprofibrate showed no obvious anti-HCV activity (Fig. 1C and 1D). However, two inhibitors of ACAT, CI-976 and avasimibe, displayed differential anti-HCV activity. Compared with CI-976 that inhibited HCV infection starting from 5 µg/ml (12.5 nM) (Fig. 1E and 1F), avasimibe (CI-1011) effectively inhibited HCV infection starting from a rather lower concentration at 0.31 µg/ml (0.6 nM), and reached approximately 90% inhibition at 5µg/ml (Fig. 1G and 1H). To further evaluate the antiviral activities of avasimibe and CI-976, the dose-response curves were constructed to determine the cytotoxic concentration 50% (CC50) and inhibitory concentration (IC50). As shown in Fig. S1, CI-976 showed slight anti-HCV activity beyond toxicity as the IC50 and CC50 of CI-976 are 6.25 µg/ml and 14.31 µg/ml. However, avasimibe displayed superior anti-HCV activity as the IC50 and CC50 of avasimibe are 0.67 µg/ml and 11.57 µg/ml.
To confirm the inhibition of HCV infection by avasimibe, we quantified the viral protein expression, the number of intracellular and supernatant HCV RNA copies and the titer of the supernatant virus of the HCV-infected Huh7.5.1 cells at various concentrations of avasimibe. Consistent with the above results, avasimibe dramatically reduced the expression of viral proteins (NS3 and core, Fig. 2A), and the number of both intracellular (Fig. 2B) and supernatant HCV RNA copies (Fig. 2C) in a dose-dependent manner. In addition, the infectivity titers in the viral infection. telaprevir and boceprevir to inhibit HCV infection in combination with avasimibe.

3.2. Avasimibe exhibits pan-genotypic activity against HCV.

HCV strains are classified into six epidemically important genotypes (Thrift et al., 2017). Both IFN-based treatment and DAAs display differential efficacy against different HCV genotypes (De Re, 2010; Li and De Clercq, 2017).To investigate the efficacy of avasimibe against different genotypes of HCV, we tested its antiviral activity using infectious HCV cell culture systems, 4A, compared with IFN-α and telaprevir, whose antiviral activity was dramatically impaired when applied more than 24 hours post infection, the antiviral activity of avasimibe reduced significantly when it was added at 48 hours post infection. Pretreatment with avasimibe (6 hours before infection) did not increase its antiviral activity, similar to IFN-α and telaprevir. These results suggested that avasimibe targeted the late stage of the HCV life cycle. To further elucidate which stage of the HCV life cycle was disrupted by avasimibe, we evaluated the effect of avasimibe on each stage of the HCV life cycle. As shown in Fig. 4B, 4C and S5, avasimibe treatment did not influence HCV pseudoparticles (HCVpp) infection, viral RNA level, the NS3 protein expression in Huh7.5.1 cells harboring HCV subgenomic replicon (SGR) and mean fluorescence intensity of GFP within HCV-GFP infected cells, suggesting that avasimibe did not affect the virus entry, the RNA replication or protein translation stages of HCV. However, when we measured the extracellular/intracellular titer ratio to evaluate the HCV virion release stage, as well as the specific infectivity to evaluate the HCV virion assembly process (Cai et al., 2016; Hu et al., 2014), we found that avasimibe only strongly impaired the specific infectivity in the supernatant in a dose-dependent manner (Fig. 4E), while it had no effect on the extracellular/intracellular titer ratio (Fig. 4D). These results suggested that avasimibe specifically inhibited the assembly process of HCV virions but might not be involved in HCV particle release.

3.4 Avasimibe inhibits HCV infection partly by downregulating MTTP.

Apolipoproteins are essential to the formation of infectious HCV particles during HCV virion assembly (Falcon et al., 2017). Highly infectious HCV particles are usually associated with more ipoproteins and hence have a low buoyant density. Since avasimibe inhibits ACAT, we wondered whether avasimibe impaired HCV assembly by modulating the associated lipoprotein. Avasimibe treatment resulted in a shift of the buoyant density of HCV particles towards a higher density (Fig. 4F and S6), suggesting that avasimibe reduced the production of highly infectious HCV particles by modulating the associated lipoprotein components of the virions.
ApoB and ApoE have been suggested to associate with HCV virions (Nielsen et al., 2006). Thus, we quantified the supernatant and intracellular ApoB and ApoE proteins upon avasimibe treatment to investigate whether avasimibe impaired virion assembly by modulating ApoB and ApoE levels. The protein levels of ApoB and ApoE in supernatant decreased upon avasimibe treatment, while the intracellular ApoB and ApoE proteins levels increased (Fig. 5A and 5B). We next examined how the intracellular ApoB and ApoE levels were modulated by avasimibe by detecting the expression of apolipoprotein- and lipid metabolism-related genes. Avasimibe did not alter the mRNA level of ApoB, ApoC1, ApoE, PLA2G12B, or HNF4α, while it reduced MTTP at both the mRNA and protein levels in a dose-dependent manner (Fig. 5C and 5D). The downregulation of the MTTP expression seemed to be specific to avasimibe, as the other ACAT inhibitors, CI-976 and TMP-153, had no influence on the expression of MTTP (Fig. 5E and data not shown). Moreover, the knockdown of MTTP mimicked the reduction in ApoB and ApoE secretion as well as the inhibition of HCV infection caused by avasimibe (Fig. 5F and 5G). Furthermore, MTTP overexpression partially impaired avasimibe-induced inhibition of HCV infection (Fig. 5H). These results suggested that avasimibe modulated the secretion of ApoB and ApoE by downregulating MTTP expression, resulting in the production of HCV virions with low infectivity. In addition, since other ACAT inhibitors did not affect MTTP expression and displayed inferior anti-HCV activity to avasimibe, we speculate that the modulation of MTTP expression might explain the superior anti-HCV activity of avasimibe compared with other ACAT inhibitors.

4. Discussion

Given the high degree of HCV genetic variability, the emergence of viral resistance during DAA therapy remains a challenge. Considering their complementary mechanisms of action, HTAs and DAAs can work in a synergistic manner to prevent viral resistance and reduce viral loads. Thus, identifying effective HTAs is imperative, and several candidate HTAs, including miravirsen and treat HCV. Thus, combinatorial administration of avasimibe and DAAs would not only benefit HCV patients but also provide an optimal treatment for HCV patients who simultaneously develop cardiovascular diseases, neurodegenerative diseases or cancer.
Although avasimibe exhibited pan-genotypic activity in this study, it showed different inhibitory activity against diverse genotypes of HCV, displaying the best inhibitory activity against HCV genotype 1a infection (Fig.3A). It displayed similar activity against genotypes 1b, 2a, 2b, 4a, 5a, decreased the secretion of ApoE and ApoB with no effect on their mRNA levels in HepG2 cells or macrophages (Cignarella et al., 2005; Wilcox et al., 1999). Further investigation suggested that avasimibe downregulated MTTP (Fig. 5C), which is essential for the assembly and secretion of ApoB–containing lipoproteins (Hooper et al., 2015). The decreased ApoB and ApoE secretion in MTTP-silenced cells as well as the downregulation of MTTP upon avasimibe treatment suggested that avasimibe might impair the secretion of ApoB and ApoE by modulating MTTP (Fig. 5F). Previous studies have demonstrated that MTTP inhibitors markedly inhibit HCV particle assembly (Gastaminza et al., 2008; Huang et al., 2007). Similar suppression of HCV infection was also observed when we knocked down MTTP using siRNA, further confirming the importance of MTTP modulation to the anti-HCV activity of avasimibe (Fig. 5G). MTTP is crucial for the secretion of ApoB and ApoE, because ApoB lipidation catalyzed by MTTP is the initial step of maturation and secretion of VLDL containing ApoB, ApoE and triglycerides (Shelness and Sellers, 2001). The first step in HCV particle assembly is the translocation of an HCV nucleocapsid into the ER lumen, accompanied by the lipidation of a nascent apolipoprotein by MTTP (Boyer et al., 2014). These two components are pulled together by the association of the HCV glycoproteins E1E2 and ApoB and ApoE into a protein complex (Boyer et al., 2014). Therefore, we speculate that the downregulation of MTTP by avasimibe impaired the lipidation of ApoB and ApoE, resulting in the impaired mature and release of apolipoproteins as well as inhibited interactions between apolipoproteins (ApoE and ApoB) and HCV glycoproteins E1E2. This speculation could be supported by the decreased secretion of ApoE and ApoB as well as the shift toward higher buoyant density of HCV particles (Fig. 4F and 5).
The IC50 of CI-976 and avasimibe on ACAT are 73 nM (Krause et al., 1993) and 24 µM (Ohshiro et al., 2011), respectively. In consideration of the superior anti-ACAT activity and inferior anti-HCV activity of CI-976, we speculate that ACAT inhibition makes a minor contribution for superior anti-HCV activity of avasimibe. We believe that the modulation of MTTP might be primarily responsible for the superior inhibitory activity of avasimibe because CI-976 failed to influence MTTP expression and displayed inferior anti-HCV activity compared to avasimibe (Fig. 1F & Fig. 5D). In addition, previous studies have demonstrated that avasimibe regulates the 5. Conclusion

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