In vitro and in vivo characterization of erythrosin B and derivatives against Zika virus

Abstract

Zika virus (ZIKV) causes significant human diseases without specific therapy. Previously we found erythrosin B, an FDA-approved food additive, inhibited viral NS2B-NS3 interactions, leading to inhibition of ZIKV infection in cell culture. In this study, we performed pharmacokinetic and in vivo studies to demonstrate the efficacy of erythrosin B against ZIKV in 3D mini-brain organoid and mouse models. Our results showed that erythrosin B is very effective in abolishing ZIKV replication in the 3D organoid model. Although pharmacokinetics studies indicated that erythrosin B had a low absorption profile, mice challenged by a lethal dose of ZIKV showed a significantly improved survival rate upon oral administration of erythrosin B, compared to vehicle control. Limited structure-activity relationship studies indicated that most analogs of erythrosin B with modifications on the xanthene ring led to loss or reduction of inhibitory activities towards viral NS2B-NS3 interactions, protease activity and antiviral efficacy. In contrast, introducing chlorine substitutions on the isobenzofuran ring led to slightly increased activities, suggesting that the isobenzofuran ring is well tolerated for modifications. Cytotoxicity studies indicated that all derivatives are nontoxic to human cells. Overall, our studies demonstrated erythrosin B is an effective antiviral against ZIKV both in vitro and in vivo.

Keywords: AUC, area under he curve; Antiviral; DENV, dengue virus; DMSO, dimethyl sulfoxide; Dengue virus; EB, erythrosin B; Erythrosin B; FDA, US Food and Drug Administration; FRET, fluorescence resonance energy transfer; Flavivirus; NS, non-structural protein; ORF, open reading frame; PFU, plaque-forming unit; PK, pharmacokinetic; PP, polyprotein precursor; Protease inhibitor; SAR, structure−activity relationship; SLC, split luciferase complementation; UTR, untranslated region; WHO, World Health Organization; ZIKV, Zika virus; Zika virus; aa, amino acid; dpi, day post infection; ip, intraperitoneal.

Graphical abstract

Scheme 1

The general synthetic routes of erythrosin B (EB) and its derivatives.

Figure 1

(A) ZIKV organoid infected with ZIKV-Venus. The 3D organoids were infected with PBS (Mock), or ZIKV untreated (DMSO), or ZIKV treated with EB (3.0 μmol/L), or Mock treated with EB (3.0 μmol/L). Upper panel, bright field image of intact organoids. Lower panel, Venus fluorescence image (excitation 515 nm, emission 528 nm) of the intact 3D organoids. (B) Relative mean fluorescence intensity of ZIKV-Venus infected organoids treated with DMSO or EB (3.0 μmol/L). n = 3. ∗∗∗P < 0.001. (C) Immunofluorescence assay (IFA) imaging of slices of organoid infected with ZIKV PRVABC59. The 3D organoids were infected with PBS (Mock), or ZIKV untreated (DMSO), or ZIKV treated with EB. IFA using anti-E 4G2 antibody (green); blue, DAPI. (D) ZIKV production from the 3D organoids at 5 dpi. Culture supernatants were collected, and virus production was quantified by PFU assay. n = 3. Error bars in panel D represent the standard deviations at each concentration. ∗∗∗P < 0.001. Student t-test was used for data in panels B and D.

Figure 2

Pharmacokinetic study of EB. Adult female B6 mice were given a single dose of EB at (A) 50 mg/kg (ip) or (B) 100 mg/kg (oral). Plasma was obtained at various times after dosing. EB was extracted from the plasma and then analyzed by LC−MS/MS as described in the Experimental Section. n = 3.

Figure 3

In vivo antiviral activity of EB against ZIKV. (A) and (B) Survival percentage for four-week-old A129 male mice infected with ZIKV PRVABC59 (1.7 × 10⁴ PFU) and treated with EB 200 mg/kg (n = 12) (A) or EB 400 mg/kg (n = 4) (B) or vehicle (n = 9) via oral gavage. (C) Survival percentage of female mice infected with ZIKV (1.7 × 10⁵ PFU) and treated with EB 200 mg/kg (n = 15) or vehicle (n = 15). All survival curves were compared using the Log-rank test. ∗∗∗∗P < 0.0001; ∗∗P < 0.005; ∗P < 0.05.

Figure 4

Inhibition of NS2B−NS3 interaction by the SLC assay (A), NS2B−NS3 protease activity (B), anti-Zika virus activity (C) by EB and JMX0902 and cytotoxicity of EB and JMX0902 (D). (A) Dose-dependent inhibition of SLC upon binding of NLuc-NS2B₄₉₋₆₆ to GST-CLuc-NS3. n = 3. (B) Dose−response inhibitions of the His-NS2B/His-MBP-NS3 protease activity. n = 3. (C) Dose-dependent inhibition of ZIKV infectivity. n = 3. (D) Cell viability assay. A549 cells were incubated with various concentrations of compounds and then assayed for viability at 48 h post incubation. n = 3. Error bars in all panels represent the standard deviations at each concentration. The DMSO control was set as 100%.