Cyclosporin derivatives inhibit hepatitis B virus entry without interfering with NTCP transporter activity

Abstract

Background & aims: The sodium taurocholate co-transporting polypeptide (NTCP) is the main target of most hepatitis B virus (HBV) specific entry inhibitors. Unfortunately, these agents also block NTCP transport of bile acids into hepatocytes, and thus have the potential to cause adverse effects. We aimed to identify small molecules that inhibit HBV entry while maintaining NTCP transporter function.

Methods: We characterized a series of cyclosporine (CsA) derivatives for their anti-HBV activity and NTCP binding specificity using HepG2 cells overexpressing NTCP and primary human hepatocytes. The four most potent derivatives were tested for their capacity to prevent HBV entry, but maintain NTCP transporter function. Their antiviral activity against different HBV genotypes was analysed.

Results: We identified several CsA derivatives that inhibited HBV infection with a sub-micromolar IC₅₀. Among them, SCY446 and SCY450 showed low activity against calcineurin (CN) and cyclophilins (CyPs), two major CsA cellular targets. This suggested that instead, these compounds interacted directly with NTCP to inhibit viral attachment to host cells, and have no immunosuppressive function. Importantly, we found that SCY450 and SCY995 did not impair the NTCP-dependent uptake of bile acids, and inhibited multiple HBV genotypes including a clinically relevant nucleoside analog-resistant HBV isolate.

Conclusions: This is the first example of small molecule selective inhibition of HBV entry with no decrease in NTCP transporter activity. It suggests that the anti-HBV activity can be functionally separated from bile acid transport. These broadly active anti-HBV molecules are potential candidates for developing new drugs with fewer adverse effects.

Lay summary: In this study, we identified new compounds that selectively inhibited hepatitis B virus (HBV) entry, and did not impair bile acid uptake. Our evidence offers a new strategy for developing anti-HBV drugs with fewer side effects.

Keywords: Antiviral; Bile acids and salts; Cyclophilins; Cyclosporine; HBV; Hepatitis B virus; Hepatitis D virus; Infection; Membrane transport proteins; NTCP; PreS1; Replication.

Graphical abstract

Fig. 1

Anti-HBV activity of cyclosporin A (CsA) derivatives. HepG2-hNTCP-C4 cells were exposed to HBV in the presence of compounds [DMSO 0.16%, preS1 peptide 100 nM, CsA and its derivatives (SCYs) 8 μM] for 16 h. The cells were then washed out free HBV inoculum and compounds, and were cultured in the absence of compounds for an additional 12 days. HBs antigen secreted into the culture supernatant (A) and HBc protein in the cells (C, D) at day 13 post infection were detected by ELISA and immunofluorescence, respectively. Cell viability was also measured by MTT assay (B). In (D), red and blue signals indicate HBc protein and nucleus respectively. Fluorescent signals for HBc protein were quantified using Lumina Vision image analysis software and are shown in (C) as a graph. (C) shows the quantified HBc levels for all the samples, but (D) shows the results for only the compounds that significantly reduced the HBc levels. The data show the means of three independent experiments. Error bars represent SD. Statistical significance was determined using Student’s t test (∗p <0.05, ∗∗p <0.01).

Fig. 2

CsA derivatives, SCY806, SCY446, SCY450, and SCY995, were highly active to inhibit HBV infection. (A) Chemical structures of CsA, SCY806, SCY446, SCY450, and SCY995. (B, C) Primary human hepatocytes were exposed to HBV with or without compounds [DMSO 0.16%, preS1 peptide 100 nM, CsA and its derivatives (SCYs) 8 μM] for 16 h. HBV DNA (B) and cccDNA (C) in the cells were detected by real-time PCR analysis at day 16 post infection. (D) Dose-response curves for anti-HBV activity. Primary human hepatocytes were infected with HBV with or without various concentrations of indicated compounds (0.25, 0.5, 1, 2, 4, and 8 μM). HBe antigens secreted into the culture supernatant were quantified at day 13 post infection.

Fig. 3

CsA derivatives inhibited the process of HBV attachment to host cells. (A) HBV replication assay. HepG2.2.15.7 cells treated with compounds (DMSO 0.16%, ETV 1 μM as a positive control, CsA and its derivatives 8 μM) for six days were recovered to quantify HBV DNA released in the culture supernatant by real-time PCR. (B, C) PreS1 binding assay showing the effect of compounds on preS1-mediated HBV attachment to host cells. TAMRA-labeled preS1 peptide was exposed to HepG2-hNTCP-C4 cells in the presence or absence of indicated compounds at 40 μM. Red and blue signals indicate the preS1 attached to the host cell and nucleus, respectively. The levels for red fluorescence shown in (C) were quantified using Dynamic Cell Count (KEYENCE) and are shown in (B).

Fig. 4

Interaction of CsA derivatives with recombinant NTCP protein by surface plasmon resonance (SPR) binding analysis. CsA derivatives were injected over a sensor chip with immobilized recombinant NTCP (left) or BSA (right), and real-time binding profiles for increasing concentrations of compounds (red: 500 μM, pink: 100 μM, light blue: 50 μM, green: 10 μM, dark blue: 5 μM, purple: 1 μM) are analyzed as shown in the materials and methods. Compound-free buffer was injected after 120 s.

Fig. 5

Anti-HBV CsA derivatives, SCY450 and SCY995, had no significant effect on NTCP transporter activity. (A) Taurocholic acid (TCA) uptake activity of HepG2-hNTCP-C4 cells was measured with or without indicated compounds (DMSO 0.2%, preS1 peptide 100 nM, CsA and SCY compounds 10 μM) either in a sodium-free (light blue bars) or containing buffer (dark blue bars). (B) Sodium-dependent TCA uptake in primary human hepatocytes was assessed with or without the indicated compounds in a sodium-containing buffer. (C) Dose-response curves for CsA and its derivatives on NTCP transporter activity. NTCP transporter activity of HepG2-hNTCP-C4 cells was quantified as shown in (B) at various concentrations of compounds (0, 0.625, 1.25, 2.5, 5 and 10 μM). IC₅₀ values calculated for each compound are also shown above the graphs.

Fig. 6

Two functions of NTCP, transporting bile acids and supporting HBV infection, are separable. (A, B) An NTCP-targeting agent, sulfobromophthalein inhibited NTCP transporter activity but had no effect on HBV infection. (A) NTCP transporter activity of HepG2-hNTCP-C4 cells was measured as shown in Fig. 5 with or without compounds (preS1 peptide 100 nM, sulfobromophthalein 40 μM (light blue) and 100 μM (dark blue)]. (B) HBV infection assay was performed as shown in Fig. 1 with or without compounds. (C) NTCP transporter activity depends on sodium concentration. TCA uptake of HepG2-hNTCP-C4 cells was detected with buffers containing sodium (0, 2, 4, 8, 16, 32, 64, and 145 mM). (D) HBV infection assay was performed with primary human hepatocytes in a buffer containing various sodium concentrations (0, 4, 16, 64, and 145 mM). HBV infection was monitored with HBs antigens secreted into the culture supernatant at day 13 post infection.

Fig. 7

CsA derivatives showed pan-genotypic anti-HBV effects and were active to a nucleoside analog-resistant HBV isolate. (A) HDV infection assay. HepG2-hNTCP-C4 cells were treated with HDV in the presence or absence of the indicated compounds for 16 h. After washing out virus and compounds, the cells were further cultured for 6 days and intracellular HDV RNA was quantified by real-time RT-PCR analysis. (B–D) Primary human hepatocytes were infected with HBV genotype (B) A, (C) B, (D) C, or (E) A carrying mutations L180M/S202I/M204V with or without compounds (DMSO 0.1%, preS1 peptide 100 nM, SCY compounds 10 μM). HBs antigen in the culture supernatant was quantified at day 13 post infection.