WO2019185579A1

WO2019185579A1 - Use of quercetin-3-o-glucoside for the treatment of flavivirus infections

Info

Publication number: WO2019185579A1
Application number: PCT/EP2019/057500
Authority: WO
Inventors: Chaker EL KALAMOUNI; Philippe Despres
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Universite De La Reunion Saint Denis; Institut De Recherche Pour Le Développement (Ird); Centre National De La Recherche Scientifique (Cnrs)
Priority date: 2018-03-27
Filing date: 2019-03-26
Publication date: 2019-10-03

Abstract

Flaviviruses are largely pathogenic to humans and other mammals and mostly are transmitted by mosquitoes. To combat flavivirus infections, it is urgent to propose safe and effective anti-viral compounds not only to impair the virus spread but also to mitigate the associated morbidities. Despite the development of antiviral agents from synthetic sources, a novel antiviral approach (economical, simple and environmentally friendly) is to use natural sources such as nutraceutical as preventive treatments against viral infections. The inventors demonstrated that flavonoid isoquercitrin exerts antiviral activity against African historical and Asian epidemic strains of ZIKV in human hepatoma, epithelial and neuroblastoma cell lines. They showed that isoquercitrin acts on ZIKV entry by preventing the internalization of virus particles into the host cell. Next the inventors demonstrated that Q3G exert antiviral activity against Yellow Fever Virus (YFV). These results suggest that Q3G represents a potential prophylactic agent targeting the entry of two medically relevant flavivirus and could thus be used to prevent flavivirus infection. Accordingly, the the present invention relates to use of quercetin-3 -O-glucoside for the treatment of flavivirus infections.

Description

USE OF QUERCETIN-3-O-GLUCOSIDE FOR THE TREATMENT OF FLAVIVIRUS

INFECTIONS

FIELD OF THE INVENTION:

The present invention relates to use of quercetin-3 -O-glucoside for the treatment of flavivirus infections.

BACKGROUND OF THE INVENTION:

Flaviviruses are members of the genus Flavivirus, which is classified within the family Flaviviridae. The flaviviruses are largely pathogenic to humans and other mammals. Flaviviruses that inflict disease on humans include yellow fever virus, Zika virus, Japanese encephalitis virus (JEV), dengue virus (including the four serotypes dengue- 1, dengue-2, dengue-3 and dengue-4), tick-home encephalitis virus, St. Louis encephalitis vims (SLEV), and others. Many flaviviruses including JEV are transmitted to humans and other host animals by mosquitoes. They therefore occur over widespread areas, and their transmission is not easily interrupted or prevented. For instance, JEV affects adults and children, and there is a high mortality rate among infants, children, and the elderly; in areas of tropical and subtropical Asia. Among survivors, there are serious neurological consequences, related to the symptoms of encephalitis, that persist after infection. Dengue vims disease is also mosquito-bome, occurring globally in regions with tropical and sub-tropical climates. Symptoms include fever, rash, severe headache and joint pain, but mortality from dengue is low. Epidemics of dengue vims are sufficiently frequent and widespread that the disease represents a major public health problem. Nevertheless, safe and effective vaccines to protect against dengue are not available, despite decades of effort. Yellow fever is prevalent in tropical regions of South America and sub-Saharan Africa, and is transmitted by mosquitoes. Infection leads to fever, chills, severe headache and other pains, anorexia, nausea and vomiting, with the emergence of jaundice. The Mosquito-bome Zika vims (ZIKV), which was historically identified in Africa, has recently gained global attention due to the recent epidemics in South Pacific islands and then Americas, and its newly recognised association with Guillain-Barre syndrome and dramatic congenital malformations in infants bom from infected mothers. ZIKV can be shed in different human fluids including the semen and vaginal secretions of humans, leading to sexual transmissions. ZIKV infection could also be a cause of severe damages to sexual organs. To date, there is no vaccination or anti-ZIKV therapy available. To combat flavivirus infections, it is urgent to propose safe and effective anti- viral compounds not only to impair the virus spread but also to mitigate the associated morbidities. Despite the development of antiviral agents from synthetic sources, a novel antiviral approach (economical, simple and environmentally friendly) is to use natural sources such as nutraceutical as preventive treatments against viral infections ( Estoppey , David, et al. "The natural product cavinafungin selectively interferes with Zika and dengue virus replication by inhibition of the host signal peptidase. " Cell reports 19.3 (2017): 451-460; Lani, Rafidah, et al. "Antiviral activity of selected flavonoids against Chikungunya virus. " Antiviral research 133 (2016): 50-61; Martin, Karen W., and Edzard Ernst. "Antiviral agents from plants and herbs: a systematic review. " Focus on Alternative and Complementary Therapies 8.1 (2003): 152-152 ). The family of flavonoid molecules emerged from many promising antiviral candidates ( dos Santos, Alda E., et al. "Quercetin and quercetin 3-O- glycosides from Bauhinia longifolia (Bong.) Steud. show anti-Mayaro virus activity. " Parasites & vectors 7.1 (2014): 130; Khachatoorian, Ronik, et al. "Divergent antiviral effects of bioflavonoids on the hepatitis C virus life cycle. " Virology 433.2 (2012): 346-355. Kim, Yunjeong, Sanjeev Narayanan, and Kyeong-Ok Chang. "Inhibition of influenza virus replication by plant-derived isoquercetin. "Antiviral research 88.2 (2010): 227-235; Lim, Hee- jung, et al. "Inhibitory effect of flavonoids against NS2B-NS3 protease of ZIKA virus and their structure activity relationship. "Biotechnology letters 39.3 (2017): 415-421; Sajitha Lulu, S., et al. "Naringenin and quercetin-potential anti-HCV agents for NS2 protease targets. " Natural product research 30.4 (2016): 464-468 ). Flavonoids are widely found in fruits, vegetables, nuts, seeds, flowers, tea and wine. Intrinsically, flavonoids are the most abundant polyphenols in human diet. The basic structure of flavonoids is a diphenylpropane skeleton linked by a three carbons chain that forms a closed pyran ring. Varieties of functional groups are grafted at different positions on this skeleton making the flavonoids one of the larger and diversified groups of bioactive phytochemicals. Flavonoids exhibit a multitude of biological activities including antiviral activity against viruses of medical concern ( Dayem , Ahmed Abdal, et al. "Antiviral effect of methylated flavonol isorhamnetin against influenza. " PloS one 10.3 (2015): e0121610.; Calland, Noemie, et al. "Polyphenols inhibit hepatitis C virus entry by a new mechanism of action. "Journal of virology 89.19 (2015): 10053-10063.; Cotin, Sebastien, et al. "Eight flavonoids and their potential as inhibitors of human cytomegalovirus replication. " Antiviral research 96.2 (2012): 181-186.; Frabasile, Sandra, et al. "The citrus flavanone naringenin impairs dengue virus replication in human cells. " Scientific reports 7 (2017): 41864; Grienke, Ulrike, et al. "Discovery of prenylated flavonoids with dual activity against influenza virus and Streptococcus pneumoniae. " Scientific reports 6 (2016): 27156 ). Recently, it has been reported that epigallocatechin gallate (EGCG), curcumin, and isoquercitrin (quercetin-3 -O-glucoside or Q3G) are efficient against ZIKV ( Mounce , Bryan C., et al. "Curcumin inhibits Zika and chikungunya virus infection by inhibiting cell binding. " Antiviral research 142 (2017): 148-157; Sharma, Nitin, et al. "Epigallocatechin gallate, an active green tea compound inhibits the Zika virus entry into host cells via binding the envelope protein. " International journal of biological macromolecules 104 (2017): 1046-1054; Wong, Gary, et al. "Antiviral activity of quercetin-3-f-OD-g/ucoside against Zika virus infection. " Virologica Sinica 32.6 (2017): 545-547).

SUMMARY OF THE INVENTION:

The present invention relates to use of quercetin-3 -O-glucoside for the treatment of flavivirus infections. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

The first object of the present invention relates to a method of treating a flavivirus infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of quercetin-3 -O-glucoside (Q3G).

As used herein, the term "flavivirus" has its general meaning in the art and refers collectively to members of the Flaviridae family of single stranded (-) RNA viruses. The family includes Dengue virus serotype 1 (DEN1), Dengue virus serotype 2 (DEN2), Dengue virus serotype 3 (DEN3), Dengue virus serotype 4 (DEN4), Zika virus, West Nile virus (WNV), St. Louis Encephalitis virus, Japanese Encephalitis virus, Yellow Fever virus, Kunjin virus, Kyasanur Forest Disease virus, Tick-home Encephalitis vims (TBEV), Murray Valley vims, LANGAT vims, Louping disease vims and Powassan vims and Omsk hemorrhagic fever vims. Members of the Flavivirus genus may produce a wide variety of disease states, such as fever, arthralgia, rash, hemorrhagic fever, and/or encephalitis. The outcome of infection is influenced by both the vims and host-specific factors, such as age, sex, genetic susceptibility, and/or pre exposure to the same or a related agent. Some of the various diseases associated with members of the genus Flavivims are Yellow Fever; Dengue Fever; and West Nile, Japanese, and St. Louis Encephalitides. In some embodiments, the flavivims is not a Zika vims.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

According to the present invention, Q3G is particularly suitable for inhibiting internalization of the virus (i.e. virus entry) and thus can be suitable for reducing the progression of the infection or preventing the infection.

The method of the invention may be carried out with any subject. The subject is preferably a mammal, more preferably a primate and more preferably still, a human. Subjects may be male or female and may be of any age, including prenatal (i.e., in utero), neonatal, infant, juvenile, adolescent, adult, and geriatric subjects. Thus, in some cases the subjects may be pregnant female subjects.

As used herein, the term“quercetin-3 -O-glucoside” or“Q3G” has its general meaning in the art and refers to 4-[5,7-dihydroxy-4-oxo-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)oxan-2-yl]oxychromen-2-yl]-2-hydroxyphenolate. The term is also known as 4H-l-benzopyran-4-one, 2-(3,4-dihydroxyphenyl)-3-(beta-D-glucofuranosyloxy)-5,7- dihydroxy flavone, 3,3',4',5,7-pentahydroxy-, 3-beta-D-glucofuranoside, isoquercitin, isoquercitrin, isoquercitroside, isotrifoliin, quercetin 3-(beta-D-glucofuranoside), quercetin 3- O-beta-D-glucofuranoside, quercetin-3 -glucoside and quercetin-3-O-beta-glucoside. The compound is commercially available.

Typically Q3G is administered to the subject with a therapeutically effective amount. By a "therapeutically effective amount" of Q3G as above described is meant a sufficient amount of Q3G to treat the viral infection at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the specific agonist employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

Q3G may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Galenic adaptations may be done for specific delivery in the small intestine or colon. Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising Q3Gs of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. Q3G can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifusoluble agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intrap eritoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Q3G may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered. In addition to Q3Gs of the invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Q3G prevents infection of A549 cells by epidemic strain of ZIKV. A549 cells were infected 24 h with PF-25013-18 at a multiplicity of infection (MOI) of 2 in presence of increasing concentrations of Q3G or vehicle. In (a), immunofluorescence quantification of viral protein E expression in ZIKV-infected A549 cells. In (b), ZIKV progeny production was quantified by plaque-forming assay. Data represent the means ± SD of four independent experiments performed in triplicate. In (c), the amount of viral genomic RNA in ZIKV-infected A549 cells was determined by RT-qPCR. Results are expressed as fold change of viral RNA transcripts in ZIKV-infected and Q3G-treated cells relative to those in vehicle-treated cells. Data represent the means ± SD of four independent experiments performed in triplicate. In (d), detection of intracellular E protein in ZIKV-infected A549 cells by immunoblot assay using anti-E mAh. b-tubulin served as loading control. Bands were quantified by densitometry using ImageJ software and the ZIKV.E:P-tubulin ratio values are indicated in the table.

Figure 2. Q3G inhibits ZIKV growth across three human cell lines. Viral clone ZIKV MR766MC was used to infect A549 and Huh-7 cells at MOI of 1 and SH-SY5Y cells at MOI of 10. Increasing concentrations of Q3G or vehicle were added concurrently to virus input. Virus progeny production was determined at 48 hours post-infection. Data represent the means ± SD of four independent experiments performed in triplicate.

Figure 3. Effect of Q3G on ZIKV infectivity. In (a), RNase A protection assay on ZIKV. MR766MC was incubated with 100 mM Q3G or mock-treated in presence of RNase A for 1 h at 37°C. Viral RNA was extracted and amplified by RT-PCR with ZIKV E primers. Viral RNA extracted from ZIKV and treated with RNase A or vehicle served as controls. Results from a representative experiment (n = 3 repeats) are shown. In (b), Viral inactivation assay. ZIKV_GFP was incubated with 100 mM Q3G or mock-treated at 37°C for 1 h and the mixture was assessed for viral infectivity on A549 cells at MOI of 0.5 (2 mM Q3G final concentration). At 24 h p.i., the percentage of GFP -positive cells was determined by FACS analysis. Data represent the means ± SD of four independent experiments performed in triplicate.

Figure 4. Q3G targets early stages of ZIKV infection. In (a), schematic representation of synchronised ZIKV_GFP infection and Q3G treatment assays in A549 cells. Q3G (100 mM) or vehicle were used for treatment of A549 cells throughout the infection (Throughout), concurrently to virus input (Co -treatment), pretreatment of naive cells (Pre- treatment), after virus exposure (Post-infection) with specific washing steps and incubation periods. For viral attachment assay (Binding), the test samples were used to treat A549 cells concurrently with ZIKV_GFP at 4°C before washing and shifting the temperature to 37°C. Black arrows indicate the presence of Q3G during the infection. In (b), the percentages of GFP- positive cells were determined by flow cytometry assay. The data represent the means ± SD of four independent experiments performed in triplicate, and are expressed as relative values compared to vehicle-treated cells.

Figure 5. Q3G inhibits ZIKV entry in A549 cells. A549 cells were incubated 1 h with ZIKVGFP at 4°C and temperature was shifted to 37°C in absence (vehicle) or presence of 100 mM Q3G. The percentage of GFP -positive cells was determined at 24 h p.i. In (a), attached and non- internalized virus particles were removed with citrate buffer at different time points post-adsorption. In (b), Q3G was added at different time points post temperature shift. The left axis represents the inhibition of ZIKV entry calculated from the percentage of GFP- positive cells. The right axis represents the virus titers. Data represent the means ± SD of four independent experiments performed in triplicate.

Figure 6. Q3G inhibits YFV entry in A549 cells. Yellow Fever Virus (YFV) vaccine 17D was used to infect A549 cells at MOI of 2. Increasing concentrations of Q3G or vehicle were added concurrently to virus input. Virus progeny production was determined at 48 hours post-infection. Data represent the means ± SD of two independent experiments performed in triplicate.

EXAMPLE 1: THE FLAVONOID ISOQUERCITRIN PRECLUDES INITIATION OF ZIKA VIRUS INFECTION IN HUMAN CELLS

Methods:

Cells, virus and reagents

Human lung epithelial A549 cells (ATCC, CCL-185), Vero cells (ATCC, CCL-81), human-derived Huh-7 hepatoma cells (ATCC, PTA-8561) and human neuroblastoma SH- SY5Y cells (ATCC, CRL2266) were grown in minimum essential medium (MEM: Gibco/Invitrogen, Carlsbad, CA) supplemented with non-essential amino acids and 10% heat- inactivated fetal bovine serum (Invitrogen), under a 5% C0₂ atmosphere at 37°C. ZIKV strains PF-25013-18, MR766MC and the mutant ZIKVGFP have been previously described [38, 42] The ZIKV progeny production is determined by measuring the quantity of infectious virus particles into the supernatant of infected cells by plaque-forming assay on Vero cells as previously described [38] Q3G, hyperoside, kaempferol and quercetin were purchased from Sigma- Aldrich (France) and stock solutions were prepared in sterile dimethyl sulfoxide (Sigma- Aldrich, France). Growth culture medium supplemented with 0.2% of DMSO was used as a vehicle control. The mouse anti-pan flavivirus envelope E protein mAB 4G2 was purchased from RD Biotech (France).

Cytotoxicity assay

The cytotoxicity was evaluated by spectrophotometric MTT (3-[4,5-dimethylthiazol-2- yl]-2,5- diphenyltetrazolium bromide) assay as described previously [37] The concentration that inhibited viability in 50% of cells (CC50) was obtained by performing nonlinear regression following the construction of a sigmoidal concentration-response curve (variable slope; Graphpad Prism; La Jolla, CA, USA).

Immunofluorescence and flow cytometry assays

For immunofluorescence assay, cells grown on glass coverslips were fixed with 3.7% formaldehyde at room temperature for 10 min. Fixed cells were permeabilized with Triton X- 100 (0.15%) in PBS for 4 min and stained using the mouse anti-pan flavivirus envelope E protein mAh 4G2 (1 : 1,000 dilution). Nucleus was stained with DAPI. The coverslips were mounted with Vectashield (Vector Labs), and fluorescence was observed using a Nikon Eclipse E2000-U microscope (Nikon, France). Images were captured and treated using a Hamamatsu ORCA-ER camera and the imaging software NIS-Element AR (Nikon, France). For flow cytometry assay, cells were fixed with 3.7% paraformaldehyde in PBS for 20 min, washed twice with PBS, and then submitted to a flow cytometric analysis using FACScan flow cytometer (BD Bioscience, USA). Results were analysed using FlowJo software.

Virus binding and internalization assays

For binding assay, A549 cell monolayers were pre-chilled at 4°C for 30 min and subsequently infected with ZIKV in presence or absence of Q3G for 60 min at 4°C. After infection, cells were washed twice with ice-cold PBS to remove unbound virus. The cells were further incubated with fresh medium at 37°C for 24 h before being subjected to cytometry assay. For internalization assay, A549 cells monolayers were pre-chilled at 4°C for 30 min and subsequently infected with ZIKV for 1 h at 4°C. Cells were washed with PBS then treated with citrate buffer (pH 3, citric acid 40 mM, potassium chloride 10 mM, sodium chloride 135 m) for 1 min to remove attached but non-intemalized virus. Cells were then washed with PBS and further incubated with fresh medium for 24 h at 37°C before being subjected to cytometry and virus titration assays.

RNase protection assay RNase protection assay was performed as previously described with some minor modifications [43] Briefly cell- free virus particles were incubated with Q3G and 15 pg.mF-l RNase A (USB-Affymetrix, Sigma- Aldrich, France) for 1 h at 37°C, and viral RNA was extracted using a QIAamp Viral RNA Mini Kit (Qiagen, Germany). The viral RNA samples were subjected to RT-PCR and gel electrophoresis. The RNA was reverse transcribed using 50 pmol of random hexamers (Eurofins, Germany) and MMLV Reverse Transcriptase (Promega, France). PCR amplification was performed using GoTaq polymerase (Promega, France) with ZIKV E primers (forward 5’-gtcttggaacatggagg-3’ and reverse 5’-ttcaccttgtgttgggc-3’ ), which were designed to match both MR766-NIID and PF-25013-18 sequences.

Viral inactivation assay

Viral inactivation assay was performed as previously described with minor modifications [44] Briefly, ZIKV_GFP particles (6.7 log PFU) was mixed with 100 mM Q3G and then incubated at 37°C for 1 h. As a control, a same dose of ZIKV_GFP was mixed with Q3G and then directly tested without an incubation period. Prior addition of the sample on A549 cells grown on a 6-well plate, the virus-Q3G mix was diluted to 50-fold in growth cell medium in order to reduce Q3G concentration below its effective dose against ZIKV and to get a virus input about 1 PFU/cell. After 2 h of adsorption at 37°C, the samples were discarded and the cells were washed twice with PBS. The cells were further incubated with fresh medium at 37°C for 24 h before being subjected to cytometry as above described.

RT-qPCR

Total RNA including genomic viral RNA was extracted from cells with RNeasy kit (Qiagen) and reverse transcribed using 500 ng of total RNA, as above described. Quantitative PCR was performed on a ABI7500 Real-Time PCR System (Applied Biosystems, Fife Technologies, France). Briefly, 10 ng of cDNA were amplified using 0.2 mM of each primer and IX GoTaq Master Mix (Promega, France). Data were normalised to the internal standard GAPDH. For each single-well amplification reaction, a threshold cycle (Ct) was calculated using the ABI7500 program (Applied Biosystems, Fife Technologies, France) in the exponential phase of amplification. Relative changes in gene expression were determined using the AACt method and reported relative to the control. The couple of primer for ZIKV E gene has been described elsewhere [37]

Western blot analysis

Cells were lysed in RIPA buffer and cell lysates were analysed by immunoblot assay as previously described [45] Primary antibodies were prepared at 1 : 1,000 dilutions. Secondary antibodies: anti-rabbit immunoglobulin- horseradish peroxidase and anti-mouse immunoglobulin-horseradish peroxidase conjugates were prepared at 1 :2,000 dilutions. Blots were revealed with ECL detection reagents.

Data analysis

Statistical analysis consisted of one-way A OVA followed by Dunnett’s test for multiple comparisons with a significance of p < 0.05. All statistical tests were performed using Prism software (Graphpad version 7.0; La Jola, CA, USA). Degrees of significance are indicated as follow: *p<0.05; **p<0.0l; ***p<0.00l, ns = not significant.

Results:

Q3G precludes ZIKV infection in human cells of various tissue origin

We reported that ZIKV PF-25013-18 clinical isolate from the French Polynesia during the epidemic in 2013 replicates efficiently in human lung epithelial A549 cells [1] First, we determined the Q3G cytotoxicity in A549 cells using a MTT assay (data not shown). The highest non-cytotoxic concentration of Q3G was 200 mM. To assess whether Q3G inhibited contemporary epidemic ZIKV strain, A549 cells were infected 24 h with PF-25013-18 at the MOI of 2. ZIKV-infected A549 cells were incubated with increasing concentrations of Q3G up to 100 mM from the beginning of infectious virus cycle. By IF analysis using anti- flavi virus E mAb 4G2, we showed that Q3G treatment resulted in a severe viral growth restriction in A549 cells (Figure 1A). At the non-cytotoxic concentration of 100 mM, Q3G reduced the viral progeny production by at least 4 logio (Figure IB) and the amount of intracellular viral RNA by 90% (Figure 1C). Also, viral protein production was severely restricted in a concentration- dependent manner (Figure ID). We concluded that isoquercitrin is a potent inhibitor of ZIKV growth in human cells.

We next investigated whether Q3G also protects human hepatoma Huh-7 and neuroblastoma SH-SY5Y cells from ZIKV infection. We recently reported that MR766^MC viral clone derived from MR766-NIID historical African strain [2] replicates efficiently in A549 and SH-SY5Y cells [3] As shown in Figure 2b, the growth of MR766^MC was also efficient in Huh- 7 cells. First, we determined the sensitivity of Huh-7 and SH-SY5Y cells to increasing concentrations of Q3G using a MTT-based cell viability assay (Data not shown). Dose- dependent experiments showed that cell viability was reduced by Q3G at concentrations higher than 200 mM with a CC₅₀ up to 600 mM (Table 1).

To evaluate the anti-ZIKV activity of Q3G in different human cell lines, A549, Huh-7 and SH-SY5Y were infected two days with MR766^MC at MOI of 1 or 10 in the presence of increasing concentrations of Q3G (Figure 2). At the highest non-cytotoxic concentration of Q3G, there was no viral progeny production regardless the human cell lines tested. At 100 mM, Q3G reduced by 3 logio virus progeny production in A549 and Huh-7 cells (Figure 2a, b) whereas viral growth was still undetectable in SH-SY5Y cells (Figure 2c). The concentration that inhibited 90% of virus growth (IC90) was obtained using nonlinear regression, following the construction of a sigmoidal concentration-response curve. The IC90 was 15 mM (SH-SY5Y cells), 50 mM (Huh-7 cells), and 32 mM (A549 cells). Based on the determined cytotoxicity and antiviral efficacy, we calculated Q3G Selectivity Index (SI = CC50/IC50) which ranges from 23 to 60 (Table 1). These results confirmed that Q3G is a potent inhibitor of ZIKV infection across human cells.

Table 1. Cytotoxicity and anti-ZIKV activity of Q3G. Cytotoxic concentration (CC) and inhibitory concentration (IC) were obtained by performing nonlinear regression followed by the construction of a sigmoidal concentration-response curves Figure 2. ^a: concentration that inhibited cell viability by 50%; ^b’ ^c: concentrations that inhibited MR766^MC progeny production by 50 or 90%; ^d: Selectivity Index (CC50/IC50).

Human cell IC90 (mM)^e

CCso (mM)^: ICso (mM)·’ SI^d

lines

A549 551.2 ± 43.2 15.5 ± 2.3 32.0 ± 3.4 35.6

Huh-7 326.8 ± 45.7 14.0 ± 3.8 50.0 ± 4.7 23.3

SH-SY5Y 582.2 ± 41.4 9.7 ± 1.2 15.0 ± 2.3 60.0

Q3G targets early stages of ZIKV infection

To further investigate the mechanisms of Q3G-mediated inhibition of ZIKV, A549 cells were infected 24 h by the GFP-expressing mutant ZIKV_GFP derived from MR766^MC at the MOI of 0.5. We reported that ZIKVGFP is a reliable tool to monitor viral growth [2] At 50 mM of

Q3G, the progeny production of ZIKV_GFP was reduced by 2 logio and this was correlated with a reduction of GFP -positive A549 cells by 75% as determined by FACS analysis (data not shown). RNase protection assay was first performed to determine whether Q3G induced the release of genomic RNA from extracellular virus particles. Viral RNA was insensitive to RNase suggesting that Q3G-mediated inhibition of ZIKV was not associated with a loss of viral particle integrity (data not shown). Furthermore, results from virus inactivation assays showed no reduction in virus infectivity when ZIKVGFP was incubated with Q3G at 37°C for 2 h (Figure 3). These results indicated that Q3G does not cause disassembly of ZIKV particles nor affect their infectivity.

Next, we used the time-of-drug addition approach to determine which stages of ZIKV infection were targeted by Q3G. As illustrated in Figure 4a, Q3G was added to A549 cells concurrently to virus input, prior to viral infection, or post-infection. As a positive control, Q3G was added throughout the infectious life cycle. We found that Q3G treatment concurrently with virus input severely reduced the percentage of GFP -positive A549 cells (Figure 4b, co treatment), whereas little or no antiviral effect was observed when Q3G was added prior to infection or after virus exposure (Figure 4b, pre-treatment and post-infection). Such results suggested that Q3G essentially targets the initial stages of infectious life cycle rather than viral replication or viral assembly and release of virus particles. To determine whether Q3G precluded the attachment of virus particles to cell surface, pre-chilled ZIKVGFP was mixed with Q3G and allowed to bind onto a A549 cell monolayer at 4°C for 1 h followed by a temperature shift to 37°C (Figure 4a, binding). The percentage of GFP-positive cells was determined 24 h after the temperature shift. There was no difference in GFP expression suggesting that the incapacity of ZIKV to initiate productive infection in presence of Q3G was not related to a defect in cell-attachment (Figure 4b, binding).

Q3G inhibits ZIKV internalization in A549 cells

Time-of-drug addition assays showed that Q3G-mediated inhibition of ZIKV relates to a post-adsorption step of the infectious virus cycle. To investigate whether Q3G inhibits internalization of virus particles, A549 cells were incubated with ZIKVGFP at 4°C for 1 h to allow virus adsorption and temperature was then shifted to 37°C to start virus penetration, in presence or not of Q3G (Figure 5a). At different times point post-adsorption, attached but not internalized virus particles were removed using citrate buffer. FACS analysis showed that, in the absence of Q3G, the percentage of GFP-positive A549 cells linearly increased to reach 50% at 45 min and a maximum at 120 min post temperature shift (Figure 5a). At 100 mM of Q3G, ZIKV_GFP-infected A549 cells remained negative for GFP expression. These results suggest that Q3G is a fast and potent inhibitor of virus internalization acting on virus particles trafficking early after the initial binding to plasma membrane.

To evaluate how early Q3G is able to inhibit endocytosis of virus particles after plasma membrane binding, ZIKVGFP was allowed to bind to the cell surface at 4°C followed by a temperature shift to 37°C. At different time points after temperature shift, cells were extensively washed and then incubated with Q3G. ZIKV_GFP-infected A549 cells were examined for virus progeny production and GFP-expression at 24 h post-infection (Figure 5b). Until 15 min after temperature shift, Q3G treatment resulted in a complete absence of viral progeny, which was correlated with a reduction of GFP-positive cells by 90%. The inhibition of ZIKV infection was much less pronounced if Q3G was added 30 min after the temperature shift. Thus, the Q3G- mediated inhibition of ZIKV growth occurs early after virus binding to the plasma membrane and could be explained by the incapacity of plasma membrane-associated virus particles to be internalized into the host cell. Conclusions:

In the present study, our data demonstrated that flavonol glucoside isoquercitrin is a potent inhibitor of ZIKV across different cell types of human origin with a remarkable SI up to 60. Importantly, isoquercitrin is effective against ZIKV strains of African and Asian lineages. It is worth thinking that isoquercitrin could be a very attractive antiviral compound as its toxicity and pharmacokinetics are fairly well studied and its administration is well tolerated in humans [4] As described for EGCG and curcumin, isoquercitrin precludes the initiation of ZIKV infection in the host cell. It has recently been reported that clathrin-mediated endocytosis pathway involving Axl/Gas6 as entry factors may play a key role in ZIKV entry into the host cell [5] Whether Q3G-mediated inhibition of ZIKV entry involves Axl/Gas6 is also an open question that remains to be urgently answered.

EXAMPLE 2: THE FLAVONOID ISOQUERCITRIN PRECLUDES INITIATION OF YELLOW FEVER VIRUS (YFV) VACCINE 17D INFECTION IN HUMAN CELLS

Methods:

Cells, virus and reagents

Human lung epithelial A549 cells (ATCC, CCL-185) and Vero cells (ATCC, CCL-81) were grown in minimum essential medium (MEM: Gibco/Invitrogen, Carlsbad, CA) supplemented with non-essential amino acids and 10% heat-inactivated fetal bovine serum (Invitrogen), under a 5% C0₂ atmosphere at 37°C. Vaccine 17D YFV (Stamaril vaccine; Sanofi Pasteur, Lyon, France) was provided by the Pasteur Institute Medical Center [6] YF17D stocks were amplified and titrated on Vero cells by plaque assay as previously described [1]

Results:

Q3G precludes Yellow Fever Virus (YFV) infection in human cells

We next wondered if Q3G exert antiviral activity against Yellow Fever Virus (YFV), another medically relevant flavivirus. The potential anti- YFV activity of Q3G was evaluated using the live attenuated YFV vaccine 17D. A549 cells were infected 48 h with YFV at the MOI of 2. YFV-infected A549 cells were incubated with increasing concentrations of Q3G up to 200 mM concurrently to virus input. Q3G showed a potent antiviral effect against YFV (Figure 6). At the non-cytotoxic concentration of 100 mM, Q3G reduced the YFV progeny production by at least 3 logio.

Our work suggests that Q3G represents a potential prophylactic agent targeting the entry of two medically relevant flavivirus and could thus be used to treat patients.

REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

1. Frumence, E.; Roche, M.; Krejbich-Trotot, P.; El-Kalamouni, C; Nativel, B.; Rondeau, P.; Misse, D.; Gadea, G.; Viranaicken, W.; Despres, P. The South Pacific epidemic strain of Zika virus replicates efficiently in human epithelial A549 cells leading to IFN-beta production and apoptosis induction. Virology 2016, 493, 217-226.10. l0l6/j.virol.20l6.03.006.

2. Gadea, G.; Bos, S.; Krejbich-Trotot, P.; Clain, E.; Viranaicken, W.; El- Kalamouni, C.; Mavingui, P.; Despres, P. A robust method for the rapid generation of recombinant Zika virus expressing the GFP reporter gene. Virology 2016, 497, 157- 162.10. l0l6/j.viro 1.2016.07.015.

3. Bos, S.; Viranaicken, W.; Turpin, J.; El-Kalamouni, C.; Roche, M.; Krejbich- Trotot, P.; Despres, P.; Gadea, G. The structural proteins of epidemic and historical strains of Zika virus differ in their ability to initiate viral infection in human host cells. Virology 2018, 516, 265-273.

4. Valentova, K.; Vrba, J.; Bancirova, M.; Ulrichova, J.; Kren, V. Isoquercitrin: pharmacology, toxicology, and metabolism. Food Chem Toxicol 2014, 68, 267- 282.l0.l0l6/j.fct.20l4.03.0l8.

5. Meertens, L.; Labeau, A.; Dejamac, O.; Cipriani, S.; Sinigaglia, L.; Bonnet- Madin, L.; Le Charpentier, T.; Hafirassou, M. L.; Zamborlini, A.; Cao-Lormeau, V. M.;

Coulpier, M.; Misse, D.; Jouvenet, N.; Tabibiazar, R.; Gressens, P.; Schwartz, O.; Amara, A. Axl Mediates ZIKA Virus Entry in Human Glial Cells and Modulates Innate Immune Responses. Cell Rep 2017 , 18, 324-333.10. l0l6/j.celrep.2016.12.045.

6. Femandez-Garcia, M. D.; Meertens, L.; Chazal, M.; Hafirassou, M. L.; Dejamac, O.; Zamborlini, A.; Despres, P.; Sauvonnet, N.; Arenzana-Seisdedos, F.; Jouvenet,

N.; Amara, A. Vaccine and Wild-Type Strains of Yellow Fever Vims Engage Distinct Entry Mechanisms and Differentially Stimulate Antiviral Immune Responses. MBio 2016, 7, e0l956- 01915.10.1 !28/mBio.0l956-l5.

Claims

CLAIMS:

1. A method of treating a flavivirus infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of quercetin-3 -O- glucoside (Q3G).

2. The method of claim 1 wherein the flavivirus is selected from the group consisting of

Dengue virus serotype 1 (DEN1), Dengue virus serotype 2 (DEN2), Dengue virus serotype 3 (DEN3), Dengue virus serotype 4 (DEN4), Zika Virus, West Nile virus (WNV), St. Louis Encephalitis virus, Japanese Encephalitis virus, Yellow Fever virus, Kunjin virus, Kyasanur Forest Disease virus, Tick-home Encephalitis vims (TBEV), Murray Valley vims, LANGAT vims, Louping disease vims and Powassan vims and

Omsk hemorrhagic fever vims.

3. The method of claim 1 wherein the flavivirus is not a Zika vims.

4. The method of claim 1 wherein Q3G inhibits internalization of the vims.

5. The method of claim 1 wherein Q3G reduces the progression of the infection.

6. The method of claim 1 wherein Q3G prevents the infection.

7. The method of claim 1 wherein the subject is preferably a mammal, more preferably a primate and more preferably still, a human.

8. The method of claim 1 wherein the subject is male or female.

9. The method of claim 1 wherein the subject is of any age, including prenatal, neonatal, infant, juvenile, adolescent, adult, and geriatric subjects.

10. The method of claim 1 wherein the subject is a pregnant female.