Erythrosin B is a potent and broad-spectrum orthosteric inhibitor of the flavivirus NS2B-NS3 protease

doi:10.1016/j.antiviral.2017.12.018

Antiviral Research

Volume 150, February 2018, Pages 217-225

https://doi.org/10.1016/j.antiviral.2017.12.018 Get rights and content

Highlights

•
We identified erythrosin B as a potent inhibitor for flavivirus protease.
•
Erythrosin B inhibited the DENV2 protease via a non-competitive mechanism.
•
Erythrosin B interferes the interactions between viral NS3 and its co-factor NS2B.
•
Erythrosin B is a potent inhibitor for Zika virus and Dengue virus.

Abstract

Many flaviviruses, such as Zika virus (ZIKV), Dengue virus (DENV1-4) and yellow fever virus (YFV), are significant human pathogens. Infection with ZIKV, an emerging mosquito-borne flavivirus, is associated with increased risk of microcephaly in newborns and Guillain-Barré syndrome and other complications in adults. Currently, specific therapy does not exist for any flavivirus infections. In this study, we found that erythrosin B, an FDA-approved food additive, is a potent inhibitor for flaviviruses, including ZIKV and DENV2. Erythrosin B was found to inhibit the DENV2 and ZIKV NS2B-NS3 proteases with IC₅₀ in low micromolar range, via a non-competitive mechanism. Erythrosin B can significantly reduce titers of representative flaviviruses, DENV2, ZIKV, YFV, JEV, and WNV, with micromolar potency and with excellent cytotoxicity profile. Erythrosin B can also inhibit ZIKV replication in ZIKV-relevant human placental and neural progenitor cells. As a pregnancy category B food additive, erythrosin B may represent a promising and easily developed therapy for management of infections by ZIKV and other flaviviruses.

Introduction

The genus Flavivirus contains more than 70 viruses, many of which cause serious human diseases. The four serotypes of Dengue virus (DENV1-4), yellow fever virus (YFV), West Nile virus (WNV), Zika virus (ZIKV), St. Louis encephalitis virus (SLEV), Japanese encephalitis virus (JEV), Powassan virus (POWV), and tick-borne encephalitis virus (TBEV) are categorized as global emerging pathogens. Due to global travel, ZIKV outbreaks recently occurred worldwide, and was imported to many new territories including the UK, Canada, and the US (Attar, 2016, Bogoch et al., 2016, Chen, 2016, Korhonen et al., 2016). ZIKV transmission has been further increased by transfer between humans through sexual activities and blood transfusions (Foy et al., 2011, Musso et al., 2014, Musso et al., 2015, Patino-Barbosa et al., 2015). Importantly, ZIKV infection increases the risk of microcephaly in babies born to infected mothers, and may cause Guillain-Barré syndrome and other complications in adults (Calvet et al., 2016, Martines et al., 2016, Rodriguez-Morales, 2016, Thomas et al., 2016). Published data in mice and humans have demonstrated that ZIKV infects the central nervous system during fetal development, including (but not limited to) regions like the cortex, thalamus, and hypothalamus, as well as neurogenic regions including the subventricular and subgranular zones, with fetal infection occurring through placenta cells (Cugola et al., 2016, Lancaster et al., 2013, Tabata et al., 2016, Tang et al., 2016). Infection with ZIKV results in a phenotypic and genetically measured infection in stem cell and neural progenitor cells with cortical thickness, cell apoptosis, and most critically, a phenotype similar to microcephaly (Cugola et al., 2016, Lancaster et al., 2013, Li et al., 2016, Tang et al., 2016).

The flavivirus genomic RNA is single-stranded with positive polarity. The viral genome is approximately 11 kb in length, composed of a 5′ untranslated region (UTR), a single long open reading frame (ORF), and a 3’ UTR. The single ORF encodes a large precursor polyprotein (PP) that requires post-translational processing by host and viral proteases (Chambers et al., 1990). These processes lead to mature viral proteins, including three structural proteins (capsid, pre-membrane or membrane, and envelope), and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Among the viral proteins, NS3 is the second largest protein encoded by flavivirus, and has multi-enzymatic functions, including protease, RNA triphosphatase, nucleoside triphosphatase, and helicase activities (Brecher et al., 2013, Lim et al., 2013, Luo et al., 2015, Sampath and Padmanabhan, 2009). The viral protease is encoded by the N-terminal region (∼180 amino acids (aa)) of the NS3 protein. The viral NS2B protein is a membrane-associated protein, with a central hydrophobic core region (∼40 aa) as an essential co-factor for the NS3 protease function. As an essential enzyme, the flavivirus NS2B-NS3 protease is highly conserved (Brecher et al., 2013, Brecher et al., 2017, Chambers et al., 1991, Falgout et al., 1991).

In a recent study, we developed a split luciferase complementation (SLC)-based high-throughput screening assay to identify inhibitors blocking the essential interactions between the DENV2 protease NS3 and its co-factor NS2B (Li et al., 2017a). Pilot screening of the NCATS Chemical Genomic Center (NCGC) Pharmaceutical Collection compound library resulted in a few priority “hits” as potent orthosteric inhibitors capable of abolishing NS2B-NS3 interactions (Li et al., 2017a).

In this study, we explored additional hit compounds that were not characterized by previous study. Among these uncharacterized compounds, erythrosin B (EB), an FDA-approved food additive, was also found to act as a potent inhibitor for the flavivirus NS2B-NS3 protease. Erythrosin B moderately inhibited the interactions between the DENV2 NS2B and NS3 with IC_50-SLC of 15 μM, and significantly inhibited the DENV2 and ZIKV protease activities with IC_50-pro in low micromolar range via a non-competitive mechanism (IC₅₀: half maximal inhibitory concentration, defined as the compound concentration required to inhibit protease activity (IC_50-pro) or SLC (IC_50-SLC) by 50%). Results from cell-based assays indicated that EB significantly reduced titers of DENV2, ZIKV, WNV, YFV, and JEV with EC₅₀ in low micromolar potency (EC_50: half maximal effective concentration, defined as the compound concentration required to reduce virus titer by 50%). In contrast, significantly higher concentrations of EB (>150 μM) will be required to cause death of human cells, including human placental epithelial cells and neural progenitor cells relevant to ZIKV pathogenesis. As an FDA pregnancy category B food additive, EB may represent a promising and easily developed therapy for management of infections by DENV, ZIKV and other flaviviruses.

Section snippets

Compounds

Erythrosin B was procured from Sigma-Aldrich.

Cloning, expression and purification

All clones were generated as described previously (Li et al., 2017a). The MBP-NS3 mutants were generated using the Strategene QuickChange II mutagenesis strategy with primer pairs shown in Supplemental Table S1. All proteins were expressed in Escherichia coli strain Rosetta 2(DE3) (EMD Biosciences) and purified through a nickel-nitrilotriacetic acid (NTA) column (Qiagen) or Glutathione sepharose 4B (GE HealthCare), followed by a gel filtration 16/60

Erythrosin B abolishes interactions between NS2B and NS3 of DENV2

Previously, we developed an SLC-based NS2B-NS3 interaction assay by generating GST-CLuc398-NS3 and His-NLuc416-NS2B_49-66 fusion proteins in which the C-terminal fragment (CLuc398) of firefly luciferase (FLuc, aa 398–550) was fused between the GST-tag and viral NS3, and the N-terminal fragment (NLuc416) of FLuc (aa 1–416) was fused between the His-tag and viral NS2B peptide 49–66 (Li et al., 2017a). Interactions between the NS2B peptide 49–66 and NS3 result in complementation of NLuc and CLuc to

Discussion

Since the outbreak of WNV in the New York City in 1999, the flavivirus protease enzyme has been an encouraging drug target. Recent study shows that inhibitors preventing the NS2B-NS3 interaction provide a promising approach for therapeutic development (Li et al., 2017a).

In this study, we identified erythrosin B as a candidate orthosteric inhibitor blocking interactions between flavivirus NS2B and NS3. Erythrosin B, also known as Red No. 3, erythrosine, or food additive E127, is an organoiodine

Acknowledgement

We thank J. Tang at the Wadsworth Center Tissue Culture Core facility for help with cell culture and K. McClive-Reed for helpful suggestions. We also thank other core facilities at the Wadsworth Center, including the Applied Genomic Technologies Core for DNA sequencing, the Advanced Light Microscopy Core for immunofluorescence imaging, and the Tissue Culture Core for media. M.B. was partially supported by the NIH Biodefense and Emerging Infectious Disease training grant AI055429. This work was

References (45)

A.K. Bera et al.
Functional characterization of cis and trans activity of the Flavivirus NS2B-NS3 protease
J. Biol. Chem.
(2007)
Bogoch et al.
Anticipating the international spread of Zika virus from Brazil
Lancet
(2016)
G. Calvet et al.
Detection and sequencing of Zika virus from amniotic fluid of fetuses with microcephaly in Brazil: a case study
Lancet Infect. Dis.
(2016)
H. Chen et al.
Selective inhibition of the West Nile virus methyltransferase by nucleoside analogs
Antivir. Res.
(2013)
C. Li et al.
Zika virus disrupts neural progenitor development and leads to microcephaly in mice
Cell Stem Cell
(2016)
Z. Li et al.
Flavivirus NS2B/NS3 protease: structure, function, and inhibition
S.P. Lim et al.
Ten years of dengue drug discovery: progress and prospects
Antivir. Res.
(2013)
D. Luo et al.
The flavivirus NS2B-NS3 protease-helicase as a target for antiviral drug development
Antivir. Res.
(2015)
X. Qian et al.
Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure
Cell
(2016)
A.J. Rodriguez-Morales
Zika and microcephaly in Latin America: an emerging threat for pregnant travelers?
Trav. Med. Infect. Dis.
(2016)

A. Sampath et al.

Molecular targets for flavivirus drug discovery

Antivir. Res.

(2009)

T. Tabata et al.

Zika virus targets different primary human placental cells, suggesting two routes for vertical transmission

Cell Host Microbe

(2016)

T. Tanaka

Reproductive and neurobehavioural toxicity study of erythrosine administered to mice in the diet

Food Chem. Toxicol.

(2001)

H. Tang et al.

Zika virus infects human cortical neural progenitors and attenuates their growth

Cell Stem Cell

(2016)

(ANS), E.P.o.F.A.a.N.S.a.t.F

Scientific Opinion on the re-evaluation of Erythrosine (E 127) as a food additive

EFSA J.

(2011)

N. Attar

ZIKA virus circulates in new regions

Nat. Rev. Microbiol.

(2016)

M. Brecher et al.

Novel broad spectrum inhibitors targeting the flavivirus methyltransferase

PLos One

(2015)

M. Brecher et al.

A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease

PLoS Pathog.

(2017)

M. Brecher et al.

The flavivirus protease as a target for drug discovery

Virol. Sin.

(2013)

S.M. Chambers et al.

Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling

Nat. Biotechnol.

(2009)

T.J. Chambers et al.

Processing of the yellow fever virus nonstructural polyprotein: a catalytically active NS3 proteinase domain and NS2B are required for cleavages at dibasic sites

J. Virol.

(1991)

T.J. Chambers et al.

Flavivirus genome organization, expression, and replication

Annu. Rev. Microbiol.

(1990)

Cited by (58)

Synthesis and evaluation of anti-yellow fever virus activity of new 6-aryl-3-R-amino-1,2,4-triazin-5(4H)-ones
2023, European Journal of Medicinal Chemistry
Yellow fever disease is one of public health concerns in the tropics. Despite its significant medicinal and economic impact among large groups of the population, there is a lack of effective treatment against yellow fever. In this regard, here we describe the synthesis of a series of new 6-aryl-3-R-amino-1,2,4-triazin-5(4H)-ones and evaluation of their in vitro inhibitory activity against yellow fever virus. Among all tested compounds 4 derivatives possessing strong inhibitory activity at μM concentrations were identified. All the active compounds revealed a good toxicity profile. These facts make the compounds interesting candidates for further evaluation of their efﬁcacy in the treatment of yellow fever virus infection in vivo.
Therapeutics for flaviviral infections
2023, Antiviral Research
Flaviviruses are vector-borne pathogens capable of causing devastating human diseases. The re-emergence of Zika in 2016 notoriously led to a widescale epidemic in the Americas. New daunting evidence suggests that a single mutation in Zika virus genome may increase transmission and pathogenesis, further highlighting the need to be prepared for flavivirus outbreaks. Dengue, in particular infects about 400 million people each year, leading to reoccurring local outbreaks. Public health efforts to mitigate flavivirus transmission is largely dependent on vector control strategies, as only a limited number of flavivirus vaccines have been developed thus far. There are currently no commercially available antivirals for flaviviruses, leaving supportive care as the primary treatment option. In this review, we will briefly paint a broad picture of the flavivirus landscape in terms of therapeutics, with particular focus on viral targets, promising novel compounds entering the drug discovery pipeline, as well as model systems for evaluating drug efficacy.
Allosteric quinoxaline-based inhibitors of the flavivirus NS2B/NS3 protease
2023, Bioorganic Chemistry
Citation Excerpt :
Structural and binding mode characterizations have proven challenging for non-competitive inhibitors of flaviviral proteases. The non-competitive inhibitors previously reported were suggested to bind in the allosteric site or the NS2B binding site [24,25,40], but reliable cocrystal structures or direct binding affinity measurements have been elusive. In addition to the relatively modest potencies of those inhibitors [24–27], experimental characterization is challenged by the extremely dynamic nature of the protease and especially the NS2B cofactor.
Viruses from the Flavivirus genus infect millions of people worldwide and cause severe diseases, including recent epidemics of dengue virus (DENV), and Zika virus (ZIKV). There is currently no antiviral treatment against flavivirus infections, despite considerable efforts to develop inhibitors against essential viral enzymes including NS2B/NS3 protease. Targeting the flavivirus NS2B/NS3 protease proved to be challenging because of the conformational dynamics, topology, and electrostatic properties of the active site. Here, we report the identification of quinoxaline-based allosteric inhibitors by fragment-based drug discovery approach as a promising new drug-like scaffold to target the NS2B/NS3 protease. Enzymatic assays and mutational analysis of the allosteric site in ZIKV NS2B/NS3 protease support noncompetitive inhibition mechanism as well as engineered DENV protease construct indicating the compounds likely compete with the NS2B cofactor for binding to the protease domain. Furthermore, antiviral activity confirmed the therapeutic potential of this new inhibitor scaffold.
Structure-based design of a novel inhibitor of the ZIKA virus NS2B/NS3 protease
2022, Bioorganic Chemistry
Zika virus (ZIKV) has been a serious public health problem, and there is no vaccine or drug approved for the prevention or treatment of ZIKV yet. The ZIKV NS2B/NS3 protease plays an important role in processing the virus precursor polyprotein and is thus a promising target for antiviral drugs development. In order to discover novel inhibitors of this protease, we carried out a fragment-based hit screening and characterized protein-inhibitor interactions using the X-ray crystallography together with isothermal titration calorimetry. We reported two high-resolution crystal structures of the protease (bZiPro^C143S) in complex with an active fragment as well as a tetrapeptide, revealing that there is domain swapping in the protein structures and two ligands only occupy the substrate-binding pocket of one copy in a symmetric unit. Based on the detailed binding modes of two ligands revealed by crystal structures, we designed a novel inhibitor which inhibits the NS2B/NS3 protease with a higher potency than the fragment and possesses a higher ligand-binding efficiency and a comparable IC₅₀ compared to the tetrapeptide. These results thus provide a structural basis and valuable hint for development of more potent inhibitors of the ZIKV NS2B/NS3 protease.
Editorial of Special Column on Antiviral Drug Discovery and Pharmacology
2022, Acta Pharmaceutica Sinica B
Enhancement of E. coli inactivation by photosensitized erythrosine-based solar disinfection under weakly acidic conditions
2022, Water Research
Cost-effective disinfection technology is urgently needed in poor rural areas. Erythrosine (ERY)-based solar disinfection (SODIS) provides a promising solution because of its effective inactivation of viruses and gram-positive bacteria at low cost. However, the poor gram-negative bacteria (G⁻, e.g., Escherichia coli) inactivation of photosensitized ERY inhibits its application. Herein, for the first time, the protonation of ERY was found to greatly enhance its G⁻ inactivation, and 99.99999% (7.0 log) of E. coli were completely inactivated within only 30 s using 2.5 mg/L ERY under 200 mW/cm² visible light irradiation. The inactivation rate constant (k) reached 17.5 min⁻¹ at pH 4.0, which was 4730 times higher than that at pH 7.0. At a lower pH, more severe cell wall and genomic DNA damage was observed. A linear correlation between k and monoanionic ERY (HE⁻) content was obtained, indicating that HE⁻ rather than dianionic ERY (E²⁻) participated in the inactivation at pH 5.0–7.0, which was further explained by the higher production of reactive oxygen species and bacterial adsorption of HE⁻ than E²⁻. Both ¹O₂ and $O_{2}^{-}$ • dominated bacterial inactivation, contributing 56.8% and 43.2%, respectively. $O_{2}^{-}$ • but not ¹O₂ caused ERY photobleaching. $OH$ • was not involved in either inactivation or photobleaching. Humic acid and salts (NaCl, Na₂SO₄, CaCl₂, and MgCl₂) slightly inhibited inactivation, while NaHCO₃ accelerated inactivation. Complete inactivation (99.9999%) of E. coli was achieved within ∼30 min at pH 5.0 in ERY-based SODIS with good adaptation to various water matrices and weather (sunny or partly cloudy). This work will help to promote the application of ERY-based disinfection especially for SODIS in poor rural areas.

View all citing articles on Scopus

View full text

Erythrosin B is a potent and broad-spectrum orthosteric inhibitor of the flavivirus NS2B-NS3 protease

Highlights

Abstract

Introduction

Section snippets

Compounds

Cloning, expression and purification

Erythrosin B abolishes interactions between NS2B and NS3 of DENV2

Discussion

Acknowledgement

J. Biol. Chem.

Lancet

Lancet Infect. Dis.

Antivir. Res.

Cell Stem Cell

Antivir. Res.

Antivir. Res.

Cell

Trav. Med. Infect. Dis.

Antivir. Res.

Cell Host Microbe

Food Chem. Toxicol.

Cell Stem Cell

Scientific Opinion on the re-evaluation of Erythrosine (E 127) as a food additive

EFSA J.

ZIKA virus circulates in new regions

Nat. Rev. Microbiol.

Novel broad spectrum inhibitors targeting the flavivirus methyltransferase

PLos One

A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease

PLoS Pathog.

The flavivirus protease as a target for drug discovery

Virol. Sin.

Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling

Nat. Biotechnol.

Processing of the yellow fever virus nonstructural polyprotein: a catalytically active NS3 proteinase domain and NS2B are required for cleavages at dibasic sites

J. Virol.

Flavivirus genome organization, expression, and replication

Annu. Rev. Microbiol.