Identification of Piperazinylbenzenesulfonamides as New Inhibitors of Claudin-1 Trafficking and Hepatitis C Virus Entry

Abstract

Hepatitis C virus (HCV) infection causes 500,000 deaths annually, in association with end-stage liver diseases. Investigations of the HCV life cycle have widened the knowledge of virology, and here we discovered that two piperazinylbenzenesulfonamides inhibit HCV entry into liver cells. The entry of HCV into host cells is a complex process that is not fully understood but is characterized by multiple spatially and temporally regulated steps involving several known host factors. Through a high-content virus infection screening analysis with a library of 1,120 biologically active chemical compounds, we identified SB258585, an antagonist of serotonin receptor 6 (5-HT6), as a new inhibitor of HCV entry in liver-derived cell lines as well as primary hepatocytes. A functional characterization suggested a role for this compound and the compound SB399885, which share similar structures, as inhibitors of a late HCV entry step, modulating the localization of the coreceptor tight junction protein claudin-1 (CLDN1) in a 5-HT6-independent manner. Both chemical compounds induced an intracellular accumulation of CLDN1, reflecting export impairment. This regulation correlated with the modulation of protein kinase A (PKA) activity. The PKA inhibitor H89 fully reproduced these phenotypes. Furthermore, PKA activation resulted in increased CLDN1 accumulation at the cell surface. Interestingly, an increase of CLDN1 recycling did not correlate with an increased interaction with CD81 or HCV entry. These findings reinforce the hypothesis of a common pathway, shared by several viruses, which involves G-protein-coupled receptor-dependent signaling in late steps of viral entry. The HCV entry process is highly complex, and important details of this structured event are poorly understood. By screening a library of biologically active chemical compounds, we identified two piperazinylbenzenesulfonamides as inhibitors of HCV entry. The mechanism of inhibition was not through the previously described activity of these inhibitors as antagonists of serotonin receptor 6 but instead through modulation of PKA activity in a 5-HT6-independent manner, as proven by the lack of 5-HT6 in the liver. We thus highlighted the involvement of the PKA pathway in modulating HCV entry at a postbinding step and in the recycling of the tight junction protein claudin-1 (CLDN1) toward the cell surface. Our work underscores once more the complexity of HCV entry steps and suggests a role for the PKA pathway as a regulator of CLDN1 recycling, with impacts on both cell biology and virology.

Hepatitis C Virus Entry: An Intriguingly Complex and Highly Regulated Process

“…A recent study showed that serotonin 2A receptor (5-HT 2A R) controls CLDN1 localization through protein kinase A (PKA)-mediated phosphorylation [122]. Serotonin receptor 6 (5-HT6) antagonists were similarly shown to mediate CLDN1 localization in a PKA-dependent (yet 5-HT6-independent) manner [123]. These findings are consistent with a pioneering study that demonstrated the importance of PKA for cell surface localization of CLDN1 [124].…”

Hypervariable Region 1 in Envelope Protein 2 of Hepatitis C Virus: A Linchpin in Neutralizing Antibody Evasion and Viral Entry

“…In addition, HCV relies on additional co-receptors, such as low-density lipoprotein receptor (LDLr) ( 46 – 48 ) and the late-stage entry receptors claudin-I and occludin ( 49 , 50 ). Most recently, cellular factors that modulate HCV co-receptor localization and possibly prime the cell for infection have also been described ( 51 – 55 ). While it has been reported that LDLr may facilitate non-infectious uptake of HCV ( 48 ), it seems clear that the receptor must play an important role in infectious uptake, as recently confirmed for a number of HCV co-receptors, including LDLr ( 56 ).…”

Serotonergic Drugs Inhibit Chikungunya Virus Infection at Different Stages of the Cell Entry Pathway

“…Many chemical compound library screen studies have revealed that agonist and antagonist serotonergic drugs can interfere with viral infections (22,23,47,48). For many of these compounds, the mechanism of action remains unclear, but many seem to act on cell entry of viruses.…”

“…For example, in hepatitis C virus infection, 5-HT 2 receptor antagonists inhibit cell entry at a late endocytic stage. This has been linked to alterations in the protein kinase A (PKA) pathway that interfere with claudin 1, an important receptor for postbinding steps of hepatitis C virus cell entry (17,47). For JC polyomavirus, 5-HT 2 receptor antagonists inhibit infection due to interference with binding of ␤-arrestin to the 5-HT 2A receptors, which is required for internalization of the virus via clathrin-coated vesicles (20,49,50).…”