Inhibitors of NAD biosynthesis for the treatment of dengue virus infections

Abstract

Dengue virus (DENV) is a mosquito-borne flavivirus responsible for dengue fever. Studies have shown that DENV interacts with metabolic pathways, in particular the glycolysis is increased in DENV-infected cells. However, the dependence of DENV on this pathway has not been investigated in details. The inventors identified an interaction between the non-structural protein 3 (NS3) of DENV and GCKR, a host protein that regulates the liver-specific hexokinase GCK. NS3 expression alone was found to increase glucose consumption and lactate secretion in an hepatic cell line expressing GCK. The inventors observed that GCKR interaction with GCK decreases DENV replication, supporting the role of NS3 as an inhibitor of GCKR function. By targeting NAD(H) biosynthesis with the antimetabolite 6-Amino-Nicotinamide (6-AN), the inventors decreased cellular glycolytic activity and inhibited DENV replication in hepatic cell. The present invention thus relates to the use of inhibitors of NAD biosynthesis for the treatment of dengue virus infections.

Description

INHIBITORS OF NAD BIOSYNTHESIS FOR THE TREATMENT OF DENGUE VIRUS INFECTIONSFIELD OF THE INVENTION:The present invention is in the field of medicine, in particular virology.BACKGROUND OF THE INVENTION:Viruses depend on host cell metabolism to acquire the energy and metabolites they need for their replication. They feed on cellular resources but in many cases, they have also evolved mechanisms to actively induce metabolic reprogramming to meet their needs, modulating the expression or activity of numerous metabolic enzymes[l-3]. Viruses interfere especially with central carbon metabolism (CCM;[4, 5]) including glycolysis which produces both ATP and intermediate metabolites required for the biosynthesis of complex molecules. By characterizing the metabolic pathways modulated by viruses, these studies have identified key enzymatic cascades essential for virus replication. This provides basic knowledge on metabolic pathways and virus-host interactions, but has led also to the development of innovative therapeutic approaches based on the targeting of host cell metabolism to restrict viral growth.Research carried out on different species of the Flaviviridae family, which includes major human pathogens such as hepatitis C virus (HCV), dengue virus (DENV), West-Nile virus (WNV), Japanese encephalitis virus (JEV) and Zika virus (ZIKV), illustrates these “viro- metabolic” interactions. Flaviviridae are enveloped viruses with a single-stranded RNA genome of positive polarity encoding a large polyprotein that is processed by viral and cellular proteases into mature structural and non-structural (NS) proteins. Because HCV is associated with liver steatosis in chronically infected patients, this virus has been extensively studied for its ability to interfere with carbohydrate-lipid metabolism. At cellular level, HCV infection stimulates the synthesis of neutral lipids in hepatocytes, promoting the formation of replication complexes on the surface of cytosolic lipid droplets. Furthermore, HCV uses the lipoprotein synthesis pathway to produce low-density virions enriched in triglycerides (TG), called lipo- viral particles (LVP), which are essential for its propagation^, 6-8], Several key proteins related to lipid metabolism were shown to be induced or activated by HCV, leading to increased lipogenesis and lipid accumulation in infected hepatocytes[9, 10], In addition, HCV infection has been associated with increased consumption of simple metabolites, notably glucose, to fuellipogenesis and meet the needs for replication [11, 12], Importantly, this ability to modulate lipid metabolism is not unique to HCV, but extends to mosquito-borne Flaviviridae that are responsible for acute infections such as ZIKV

[0013] , WNV

[0014] , JEV

[0015] and DENV[16, 17], In the case of DENV, several studies showed that infection increases glycolysis and lipogenesis in infected cellsf 17— 19] . An interaction was identified between the NS3 protein of DENV and fatty acid synthase (FASN) at the virus replication site

[0016] . This interaction was associated with higher fatty acid biosynthesis in DENV-infected cells, while de novo synthesized lipids preferentially co-fractionate with DENV RNA, suggesting a direct effect of the viral protein on enzymatic activity

[0016] . In addition, it has been shown that intracellular fatty acids become a source of energy via beta-oxidation for infected cells

[0020] , illustrating the fine tuning of anabolic vs catabolic pathways by DENV.In addition to lipids, Flaviviridae also increase the consumption of glucose which is a major source of metabolites and energy. However, the molecular mechanisms involved are still poorly characterized. Large-scale mapping of virus-host interactions provide valuable insights[21, 22], and a few studies have identified some direct modulation of cellular enzyme activity by viral proteins[12, 15, 23-25], In particular, we have demonstrated that the NS5A protein of HCV interacts directly and increases the activity of hexokinases, which are the first rate-limiting enzymes of glycolysis that catalyze glucose phosphorylation to produce glucose-6-phosphate (G6P)[12, 25], This virus-host interaction leads to increased glucose consumption and higher level of triglyceride biosynthesis. For DENV, an interaction between NS3 and glyceraldehyde- 3 -phosphate dehydrogenase (GAPDH) was reported to decrease GAPDH glycolytic activity

[0026] , Meanwhile, DENV infection increases glycolysis, which suggests the possible accumulation of glycolysis intermediates essential to the connected anabolic pathways[19, 20, 23], but underlying mechanisms are still unknown.The human genome contains five genes encoding distinct hexokinase isoenzymes, known as HK1, HK2, HK3, GCK (for glucokinase) and HKDC1, with distinct enzyme properties and tissue distributions. GCK is exclusively expressed in pancreas and liver, and is a monomeric allosteric enzyme with a higher Km compared to other hexokinases. Whereas GCK is inactive at normal blood glucose concentration, it becomes highly active when glucose availability rises, thus contributing to normalize glycemia. When necessary, to slow down cellular glycolysis, GCK activity is negatively regulated by glucokinase regulatory (GCKR) protein through direct interact! on

[0027] , In order to understand how DENV interfere with glycolysis in infected cells,we analyzed virus-host protein interactions and serendipitously found that NS3 interacts with GCKR. This observation is unsettling given that the prevalence of liver damage in dengue hemorrhagic fever is as high as 80% in some studies and hepatomegaly increases the risk of developing a severe form of dengue infection

[0028] . Thus, although dengue infects many cell types, liver infection correlates with the severity of the disease. Hence, DENV-NS3 interaction with GCKR suggests a specific interference between DENV infection and regulation of GCK in the liver.SUMMARY OF THE INVENTION:The present invention is defined by the claims. In particular, the present invention relates to the use of inhibitors of NAD biosynthesis for the treatment of dengue virus infections.DETAILED DESCRIPTION OF THE INVENTION:Viruses have developed sophisticated strategies for controlling the metabolic activity of the cells they infect in order to supply their replication machinery with energy and metabolites. Dengue virus (DENV), a mosquito-borne flavivirus responsible for dengue fever, is no exception. Previous reports have documented DENV interactions with metabolic pathways and shown in particular that glycolysis is increased in DENV-infected cells. However, the underlying molecular mechanisms are still poorly characterized and the dependence of DENV on this pathway has not been investigated in details. Here, the inventors identified an interaction between the non-structural protein 3 (NS3) of DENV and GCKR, a host protein that regulates the liver-specific hexokinase GCK. NS3 expression alone was found to increase glucose consumption and lactate secretion in an hepatic cell line expressing GCK. Interestingly, the inventors observed that GCKR interaction with GCK decreases DENV replication, supporting the role of NS3 as an inhibitor of GCKR function. Accordingly, the inventors observed in the same cells that DENV replication both induced and depended on glycolysis. Indeed, by targeting NAD(H) biosynthesis with the antimetabolite 6-Amino-Nicotinamide (6-AN), the inventors decreased cellular glycolytic activity and inhibited DENV replication in hepatic cell. Infection of primary organotypic liver cultures (OLiC) from hamsters was also inhibited by 6- AN. Altogether, the results show that DENV has evolved to hijack glycolytic enzymes in the liver, which could account for hepatic dysfunctions associated to DENV infection. Besides, the findings suggest that lowering intracellular availability of NAD(H) could be a valuable therapeutic strategy to control glycolysis and inhibit DENV replication in the liver.Main definitions:As used herein, the term “dengue virus” or “DENV” as is general meaning in the art and denotes an RNA virus of the family Flaviviridae genus Flavivirus. Other members of the same genus include yellow fever virus, West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, tick-borne encephalitis virus, Kyasanur forest disease virus, and Omsk hemorrhagic fever virus. Most are transmitted by arthropods (mosquitoes or ticks), and are therefore also referred to as arboviruses (arthropod-borne viruses). According to the present invention, the dengue virus may be of any serotype, i.e. serotype 1, 2, 3 or 4.As used herein, the term “dengue virus disease” means any disease caused, directly or indirectly, by one of the four serotypes of a dengue virus, which is a flavivirus. Dengue is an acute febrile disease characterized by sudden onset, with headache, fever, prostration, joint and muscle pain, lymphadenopathy, and a rash that appears simultaneously with a temperature rise. A second phase of temperature rise may appear following an afebrile period. Dengue hemorrhagic fever / dengue shock syndrome is an acute disease occurring primarily in children characterized by an abrupt febrile onset followed by hemorrhagic manifestations and circulatory collapse. Four grades of severity are recognized: grade I: fever and constitutional symptoms, grade II: grade I plus spontaneous bleeding (of skin, gums or gastrointestinal tract), grade III: grade II plus agitation and circulatory failure and grade IV: profound shock. The disease is transmitted by a mosquito of the genus Aedes (generally A. aegyptil, but frequently, A. albopictus). Also called Aden, bouquet, breakbone, dandy, date, dengue (hemorrhagic) or polka, solar fever, stiffneck fever, scarlatina rheumatica or exanthesis arthorosia. “Hemorrhagic dengue” is a more pathogenic epidemic form of dengue which has erupted in a number of epidemic outbreaks in the Pacific region in recent years.As used herein, the term "subject" or "patient" and "subject in need thereof" or "patient in need thereof", is intended for a human or non-human mammal infected or likely to be infected with a dengue virus.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, andincludes suppression of clinical relapse. The treatment may be administered to a patient 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 patient 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 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.]).As used herein, the term “NAD” has its general meaning in the art and refers to nicotinamide adenine dinucleotide that is a coenzyme central to metabolism. Found in all living cells, NAD is called a dinucleotide because it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an adenine nucleobase and the other, nicotinamide. NAD exists in two forms: an oxidized and reduced form, abbreviated as NAD+ and NADH (H for hydrogen), respectively.The term "NAD biosynthesis" refers to the biosynthetic pathway that leads to the synthesis of NAD. NAD+ can be synthesized de novo by the conversion of the amino acid tryptophan through multiple enzymatic steps to nicotinic acid mononucleotide (NaMN). NaMN is converted to nicotinic acid dinucleotide (NaAD+) by NMN / NaMN adenylyltransferases (NMNATs) and then amidated to NAD+ by NAD+ synthetase. In mammals, a major pathwayof NAD+ biosynthesis is the salvage pathway from nicotinamide. Nicotinamide is converted to nicotinamide mononucleotide (NMN), a key NAD+ intermediate, by nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in this pathway. NMNATs then convert NMN into NAD+.As used herein, the term “NAMPT” refers to the nicotinamide phosphoribosyltransferase that catalyzes the first reversible step in NAD biosynthesis and nicotinamide (NAM) salvage. The enzyme is designed for efficient capture of nicotinamide by coupling of ATP hydrolysis to assist in extraordinary NAM binding affinity and formation of NMN. NAMPT provides the mechanism to replenish the NAD pool in human metabolism. The structure of NAMPT is described further in, for example, Kim, et al. J Mol Biol.; 362:66-77 (2006).As used herein, the term “inhibitor” refers to a compound that decreases the magnitude of at least one activity, signaling or expression of a molecule compared to the magnitude of the activity, signaling or expression observed in the absence of the inhibitor. In some instances, an inhibitor will substantially decrease the magnitude of at least one activity, signaling or expression of a molecule compared to the magnitude of the activity or expression observed in the absence of the inhibitor. In some instances, an inhibitor will completely diminish the magnitude of at least one activity, signaling or expression of a molecule compared to the magnitude of the activity, signaling or expression observed in the absence of the inhibitor. Certain exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibodies, aptamers, antisense oligonucleotides, interfering RNA, carbohydrates or small organic molecules.As used herein, the expression "therapeutically effective amount" is meant a sufficient amount of the Inhibitor of the present invention to treat a dengue virus 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; drugsused in combination with the specific agonist employed; and like factors well known in the medical arts. For example, it is well known 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.Methods of the present invention:The first object of the present invention relates to a method of treating a dengue virus infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of NAD biosynthesis.The method of the present invention is thus particularly suitable for treating dengue virus disease.The efficacy of the treatment may be monitored using standard protocols. Indeed, treatment may be followed by determinations of DENV levels in serum (viral load) and measurement of serum alanine aminotransferase (ALT) levels. For example, the patients may be assessed for the presence of DENV RNA in their serum. DENV RNA (lU / mL) can be measured at regular intervals during the treatment, e.g., at Day 1 (pre-dose and 4, 8, and 12 hours post-dose) and pre-dose at Day 2, Day 3, Day 8, Day 15, Day 29, and at Week 12, Week 24, Week 36, Week 48, Week 72 (when applicable), and at follow up. Accordingly, the efficacy of treatment can be monitored using internationally accepted parameters: a) Serum DENV RNA levels are monitored using sensitive quantitative RT-PCR-based assays to assess the effect on viral replication b) Serum levels of ALT and / or aspartate aminotransferase (AST) are monitored to assess impact on liver inflammation and liver cell death.In some embodiments, the inhibitor of NAD biosynthesis is 6-Aminonicotinamide (6-AN) (IUPAC name: 6-aminopyridine-3-carboxamide).In some embodiments, the inhibitor of NAD biosynthesis is a NAMPT inhibitor. Non-limiting examples of small molecule NAMPT inhibitors which can be used in methods according to the invention are described, for example, in published PCT patent applications Nos. WO 2011006988, WO 2011109441, W02012031196, W02012031197, and W02012031199. As used herein, the term “small molecule” generally includes a molecule of less than 10,000 Da in molecular weight. Preferred small molecule inhibitors of NAMPT have excellent dosedependent enzyme inhibitory properties. Exemplary small molecule inhibitors of NAMPT include:- FK866 ((E)-N-[4-(l-benzoyl-4-yl)-butyl]-3-(pyridin-3-yl) acrylamide),CHS-828 (N-[6-(4-chlorophenoxy)hexyl]-Nz-cyano-Nzz-4-pyridinyl-guanidine,GNE-617 (N-(4-((3,5-difluorophenyl)sulfonyl)benzyl)imidazo[l,2-a]pyridine-6- carboxamide),GNE-618 (N-[[4-[[3-(Trifluoromethyl)phenyl]sulfonyl]phenyl]methyl]-lH- pyrazolo[3,4-b]pyridine-5-carboxamide),STF118804 (4-[5-Methyl-4-[[(4-methylphenyl)sulfonyl]methyl]-2-oxazolyl]-N-(3- pyridinylmethyl)benzamide),KPT-9274 ((E)-3-(6-aminopyridin-3-yl)-N-[[5-[4-(4,4-difluoropiperidine-l- carbonyl)phenyl]-7-(4-fluorophenyl)-l-benzofuran-2-yl]methyl]prop-2-enamide), and / orLSN3154567 (2-hydroxy-2-methyl-N-[l,2,3,4-tetrahydro-2-[2-(3-pyridinyl oxy)acetyl]-6-isoquinolinyl]-l-propane-sulfonamide. However, there are numerous additional NAMPT inhibitors that can be used within the combination therapies disclosed herein. For example, additional examples of NAMPT inhibitors are described in U.S. Pat. No. 9,555,039. Additional examples of NAMPT inhibitors include the pyridyloxyacetyl tetrahydroisoquinoline compounds as described in US20160229835. Additional examples of NAMPT inhibitors include the l,3-dihydro-2H- isoindole compounds as described in U.S. Pat. No. 9,302,989. Additional examples of NAMPT inhibitors include the 4,5-dihydroisoxazole derivative compounds as described in WO2014111871. Additional examples of NAMPT inhibitors include the 1,4-disubstituted triazoles with substitution compounds as described in US20160075682. Additional examples of NAMPT inhibitors include the quinoxaline, quinazoline and quinoline compounds asdescribed in US20180009784 and U.S. Ser. No. 10 / 144,742. Additional examples of NAMPT inhibitors include the 4-{[(pyridin-3yl-methyl) aminocarbonyl] amino} benzene-sulfone derivative compounds as described in W02012031196. Additional examples of NAMPT inhibitors include AU-4869 (Aurigene Discovery Technologies); OT-82 (OncoTartis Inc) and the cluster boron moieties compounds as described in U.S. Pat. No. 9,382,267.Typically the inhibitor of the present invention is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. As used herein, the term "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 comprisingthe compound 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. The compound of the invention 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, thesolution 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 intraperitoneal 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. The compound of the invention 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 compound 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.According to an aspect of the invention, the inhibitor of the present invention may be administered to the patient in combination with at least one other therapeutic agent, preferably in combination with at least one other antiviral compound, more preferably in combination with at least one other antiviral compound. The antiviral compound may include, but is not limited to, neuraminidase inhibitors, viral fusion inhibitors, protease inhibitors, DNA polymerase inhibitors, signal transduction inhibitors, reverse transcriptase inhibitors, interferons, nucleoside analogs, integrase inhibitors, thymidine kinase inhibitors, viral sugar or glycoprotein synthesis inhibitors, viral structural protein synthesis inhibitors, viral attachment and adsorption inhibitors, viral entry inhibitors and their functional analogs. In particular, neuraminidase inhibitors may include oseltamivir, zanamivir and peramivir. Viral fusion inhibitors may include cyclosporine, maraviroc, enfuviritide and docosanol. In particular, protease inhibitors may include saquinavir, indinarvir, amprenavir, nelfinavir, ritonavir, tipranavir, atazanavir, darunavir, zanamivir and oseltamivir. In particular, DNA polymerase inhibitors may include idoxuridine, vidarabine, phosphonoacetic acid, trifluridine, acyclovir, forscarnet, ganciclovir, penciclovir, cidoclovir, famciclovir, valaciclovir and valganciclovir. Signal transduction inhibitors include resveratrol and ribavirin. Nucleoside reverse transcriptase inhibitors (NRTIs) may include zidovudine (ZDV, AZT), lamivudine (3TC), stavudine (d4T), zalcitabine (ddC), didanosine (2',3'-dideoxyinosine, ddl), abacavir (ABC), emirivine (FTC), tenofovir (TDF),delaviradine (DLV), fuzeon (T-20), indinavir (IDV), lopinavir (LPV), atazanavir, combivir (ZDV / 3TC), kaletra (RTV / LPV), adefovir dipivoxil and trizivir (ZDV / 3TC / ABC). Nonnucleoside reverse transcriptase inhibitors (NNRTIs) may include nevirapine, delavirdine, UC- 781 (thiocarboxanilide), pyridinones, TIBO, calanolide A, capravirine and efavirenz. In particular, viral entry inhibitors may include Fuzeon (T-20), NB-2, NB-64, T-649, T-1249, SCH-C, SCH-D, PRO 140, TAK 779, TAK-220, RANTES analogs, AK602, UK-427, 857, monoclonal antibodies against relevant receptors, cyanovirin-N, clyclodextrins, carregeenans, sulfated or sulfonated polymers, mandelic acid condensation polymers, AMD-3100, and functional analogs thereof. In some embodiments, the antiviral compound is a pan-serotype dengue virus (DENV) inhibitor that blocks the NS3-NS4B interaction within the viral replication complex. Typically, said antiviral compound is Mosnodenvir (JNJ-1802).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: Inhibition of NAD metabolism inhibits DENV replication. A) Schematic representation of glycolysis, connections with pentose-phosphate pathway (PPP) and NAD+ biosynthesis salvage pathway and impact of drugs used in the present study (framed). B) Huh7- GCK+ / HK2~ cells were electroporated with sgDENV-R2A replicon and incubated in absence or presence of 0.5mM 6-AN and / or 25mM ribose and / or 0.5mM NAM Riboside as indicated on graph, for 72 h. Renilla luminescence was analyzed and normalized to the 4 h value that reflects transfection efficiency. Replication was expressed as % of the replication in absence of treatment. Presented data correspond to means ± SEM (n=3, ANOVA, ** / ?<0.01). C) Huh7- GCK+ / HK2~ cells were electroporated with sgDENV-R2A replicon and incubated in absence or presence of 1 pM Brequinar. Renilla luminescence was analyzed at 24, 48 and 72 h postelectroporation and normalized to the 4 h value. Replication was expressed as % of the maximal replication in control condition (Ctl.). Are presented means ± SEM of 3 experiments. D) Huh7- GCK+ / HK2~ cells were electroporated with sgDENV-R2A replicon and incubated in absence or presence of FK-866 ± 0.5mM NAM riboside for 72h. Renilla luminescence was analyzed and normalized to the 4 h value that reflects transfection efficiency. Replication was expressed as % of control (Ctl.). Are presented means ± SEM (n=3, ANOVA, ** / ?<0.01).Figure 2: Effect of 6-AN on cell metabolism. A) B) and C) Huh7 -GGK / HK2~ cells treated or not with 0.5mM 6-AN ± 0.5mM NAM riboside for 72h. NAD+ was quantified by enzymatic assay in cell homogenates (A), glucose consumption was determined in culture supernatant (B) and intracellular ATP determined using luminescent assay. Are presented means ± SEM (n=3, paired t-test * <0.05). D) and E) Huh7 -GGK / HK2~ cells were treated or not with 6-AN or 6- AN Riboside as described above. % of viability (D) and cell proliferation (E) were determined after 72h of culture. Are presented means ± SEM of 3 experiments.Figure 3: 6-AN inhibits DENV infection in hamster OLiCs. OLiCs prepared as described in material and methods were infected with 103PFU of DENV-GFP and treated or not with 6-AN at the concentration of lOOpM in the subnatant culture medium for 90 h. A) observation of GFP foci by microscopy (Foci are indicated by arrows). B) DENV RNA were quantified in OLiC homogenates by specific qPCR and normalized to total cellular RNA extracted. Are presented quantification means ± SEM of 5 liver slices providing from 5 animals (n=5, Mann-Whitney test, ** / ?<0.01). C) Determination of DENV particle secretion in cell subnatants of the 5 liver slices. D) Quantification of ribosomal protein L13a (RPL13a) as an housekeeping gene in infected OLiCs at 90 h post-infection. E) Determination of tissue viability by MTT assay of 5 liver slices after 90 h treatment with 6-AN or SDS (n=5, ANOVA, **** / ?<0.0001).Fig. 4. DENV-NS3 interacts with GCKR and induces glycolysis. A) Glucose consumption and B) lactate secretion were determined in 48 h cultures of \ u 7(GCK HK2 cells transfected with plasmids 3xFlag coding or not for NS3-full length, NS3 -protease or -helicase domains. Data are normalized to control condition transfected with the empty plasmid. Data presented means ± SEM (n = 3) and p-values were determined by one-way ANOVA for multiple comparison (** p < 0.01). C) Protein-complementation assay data showing the luminescence signal resulting from interaction of DENV-NS3 full length (NS3-fl), DENV-NS3 protease (NS3-prot.) or DENV-NS3 helicase (NS3-heli.) with GCKR (grey bars). Background luminescence of non-specific interaction was determined in each experiment and presented (white bars). Data presented means ± SEM (n = 3) and p values were determined by two-way ANOVA for multiple comparison (** p < 0.01).EXAMPLE:Methods:Cell culture and reagents: Unless otherwise stated, all chemicals were from Merck Sigma- Aldrich (Saint Quentin-Fallavier, France) and cell culture reagents were from Life Technologies (ThermoFisher Scientific, Saint Aubin, France). Generation of the cell line Huh7- GCK+ / HK2- was described previously

[0029] and cells were routinely cultured in DMEM supplemented with ImM pyruvate, 1001.U. / mL of penicillin, lOOpg / mL streptomycin and 10% Fetal Calf Serum.Replicon: Plasmid containing the subgenomic replicon DENV-R2A or containing the full length genome DENV-G2A were obtained from Ralf Bartenschlager (Department of Molecular Virology, Heidelberg, Germany). Plasmid pFK-DVs-R2A was linearized with Xbal (100 pL reaction): 10 pg plasmid DNA, 10 pL 10X buffer (50 mM Potassium Acetate, 20 mM Trisacetate, 10 mM Magnesium Acetate, pH 7.9), 1 pL 100X BSA, 5 pL Xbal (20 U / pL), and H2O to 100 pL. After 2 h incubation at 37°C, the plasmid was purified using the NucleoSpin® Gel and PCR Clean-up kit (Macherey -Nagel, Hoerdt, France). RNA transcription was performed as previously described

[0030] , RNA was synthesized using the SP6 promoter using mMESSAGE mMACHINEtmSP6 Transcription Kit (Ambion, ThermoFisher Scientific) and accordingly to provider recommendations. Viral RNA was purified from reaction mix using precipitation with lithium chloride and quantified by absorbance measurement. Purified RNA was immediately frozen at -80°C until transfection. RNA was transfected into cells in suspension by electroporation as described[30, 31], Electroporation conditions were 960 pF and 270 V with a Gene Pulser system (Bio-Rad, Mames-la-Coquette, France) in a cuvette with a gap width of 0.4 cm (Bio-Rad). Immediately after electroporation, cells were resuspended in complete medium and seeded in 96 wells plates. For assaying the luciferase activity, cells were washed once with PBS, and lysed directly in the plate by incubation during 20 min at room temperature with 50 pL of ice-cold Passive Lysis Buffer (Promega, Charbonnieres-les-Bains, France). 30 pL of cell lysates were transferred to white 96 wells plates and 100 pL of Renilla-Glo® Luciferase Assay System mix added before luminescence quantification using a luminometer Tristar 5 (Berthold, Freiburg, Germany) for 5 s. To normalize RNA electroporation efficiency, basal luminescence was measured at 4 h post-electroporation. Replication at latter times was expressed as fold of RLU at 4 h.DENV infection: Virus stocks were obtained by transfecting BHK-21 cells with in vitro transcripts of the recombinant DENV genome containing the GFP (genotype 2, strain 16681)

[0030] , Infectivity titers in culture supernatants and virus stocks were quantified by determining the tissue culture infectious dose 50 (TCID50) per milliliter using a limiting dilution assay as described elsewhere

[0032] , Huh7 or Huh! -GCK+ / HK2~ cells were seeded one day before infection at a MOI=0.1. The day of infection, cells were washed twice before incubation with Opti-MEM™ I Reduced Serum Medium containing DENV for 4 h. Then, inoculum was removed and cells incubated with complete medium for the duration of the experiment.Preparation of Organotypic Liver Cultures (OLiC): Syrian golden hamsters (Mesocricetus auratus) used in this study were obtained from Janvier Labs (Le Genest-Saint-Isle, France) with clean health monitoring report. The sex of the animals was random and dependent on the litter threw by the mother. Animals were euthanized at seven to nine days old. The procedure for OLiC was adapted from the protocol for Organotypic Brain Cultures (OBC) described previously

[0033] . One day before the dissection, Millicell® cell culture inserts with PTFE membranes (Merck) were pre-activated with OLiC medium. The OLiC medium contains 375 mL of Minimal Essential Medium GlutaMAX (ThermoFisher Scientific), 125 mL of heat- inactivated horse serum (Gibco), 2.5 g of D-glucose (Sigma-Aldrich) and 1 mL of human recombinant insulin (10 mg / mL) (Sigma-Aldrich), and was sterilized with a 0.22 pm-pore size filter. The OLiC medium is identical to the OBC medium described elsewhere by Welsch et al.

[0033] , Seven to nine-day suckling hamsters were sacrificed and their abdominal cavity was opened. Livers were collected and placed into a solution of Hibemate®-A medium (Sigma- Aldrich) supplemented with 100 I.U. / mL of penicillin and lOOpg / mL streptomycin (Corning). The biggest lobe of the liver was isolated and was placed on 3 layers of Whatman paper with their longitudinal axis perpendicular to the tissue chopper blade and sliced transversely using the McIlwain® tissue chopper (Campden Instruments) at 500 pm thickness for all experiments. The slices were dissociated under a dissection microscope. Undamaged and homogenous slices were selected and maintained on an air-liquid interface provided by PTFE membranes preactivated with OLiC medium.OLiC infection: A 2 pL drop containing 1000 viral plaque-forming units (pfu) of the DENV2- EGFP was placed on each organotypic liver slice. Infected slices were incubated at 37°C until collection. For treatment with 6-AN, the compound was diluted at the appropriate concentrationin the culture medium (100 pM), and slices were then treated by adding a 2 pL drop of this medium on the top of each slice daily until the end of the experiment; For Mock treatments, OLiC were treated with medium containing DMSO at the equivalent concentration to the 6-AN treated samples. Viral progression was followed by epifluorescence microscopy. Pictures were obtain using a Nikon Eclipse Ts2R optical microscope and stitched using the Stitching plugin in ImageJ.ORF cloning into Gateway-compatible plasmids: NS3 full length, NS3 helicase or NS3 protease ORFs were cloned from DENV genotype 2 (strain 16681), using the Gateway recombination-based cloning system

[0036] . Viral ORFs were PCR-amplified (with Novagen KOD polymerase, Merck-Millipore) from a DNA template using sequence-specific primers fused to attBl.l and attB2.1 recombination sites. PCR products were subsequently cloned into pDONR223 to generate entry plasmids (BP Clonase II Enzyme mix; ThermoFisher Scientific). Entry plasmid containing the ORF of GCKR was picked from the Human ORFeome v3.1 collection (Open Biosystem, Huntsville, AL, USA

[0037] ). Each ORF was transferred by in vitro recombination into the different Gateway-compatible destination vectors used in this study (LR Clonase II Enzyme mix; Life Technologies). LR reaction products were subsequently transformed into DH5a competent bacterial cells and grown overnight on LB-agarose plates containing ampicillin before amplification into liquid LB medium also containing ampicillin. Plasmids were purified using a NucleoSpin Plasmid kit from Macherey -Nagel and validated by sequencing (Eurofins, Nantes, France).Glucose and lactate quantification: Metabolites were quantified from cell supernatants using the Glucose Oxidase (GO) assay kit and the Lactate assay kit from Millipore Sigma-Aldrich. Assays were performed according to the manufacturer’s instructions and results were normalized to protein quantity (DC Protein Assay; Bio-Rad) or number of cells per sample.Reverse Transcription-quantitative PCR (RT-qPCR): Total RNA from tissue slices was extracted using the NucleoSpin RNA extraction kit (Macherey-Nagel) according to manufacturer instructions. Viral RNA was extracted from culture supernatant using the QIAmp viral RNA extraction kit with AVL Buffer (Qiagen, Les Ulis, France) according to manufacturer instructions. cDNA was obtained by reverse transcription of 250 ng of the extracted RNA using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Thermo Fisher Scientific) according to manufacturer protocol, in a final reaction volume of 20pL. 0.4 pL of the cDNA reaction product was used for qPCR using QuantiNova SYBR Green RT-PCR PCR was performed with the StepOnePlus Quantitative PCR System (Applied Biosystems, ThermoFisher Scientific) and analyzed with the StepOne v2.3 software (Applied Biosystems).Protein-Complementation Assay (PCA): NS3 interaction with GCKR was determined by NanoLuc Two-Hybrid (N2H) assay, a recently developed split-luciferase complementation assay

[0038] . In this system, two complementary fragments of NanoLuc, Fl and F2, are fused to proteins of interest. The reconstitution of NanoLuc activity by trans-complementation of Fl with F2 is dependent on the physical interaction of candidate proteins. This assay was used to test the interaction of GCKR with either NS3 full-length or its helicase or its protease domains. The NS3 coding sequences were cloned by in vitro recombination (Gateway system; Thermo Fisher) from entry plasmids into pDEST-N2H-Nl, whereas the GCKR sequence was cloned into the pDEST-N2H-C2 vector. The obtained constructs were co-transfected in HEK-293T cells with the JetPrime reagent (Polyplus-Transfection, Illkirch-Graffenstaden, France). This allowed for the co-expression of NS3 N-terminally tagged with fragment Fl of NanoLuc together with GCKR C-terminally tagged with fragment F2 of NanoLuc. After 48 h of culture, cells were lysed and NanoLuc activity was determined as previously described[39, 40], The bioluminescent signal obtained when co-expressing N1-NS3 and GCKR-C2 was compared to the sum of the signals obtained when co-expressing N1-NS3 with F2 or GCKR-C2 with Fl (background signal).Western-blot analysis: Cell lysates were prepared in lysis buffer (1% Triton X-100, 5 mM EDTA in PBS with 1% protease inhibitor cocktail (Merck Sigma-Aldrich)). After elimination of insoluble material, proteins were quantified, separated by SDS-PAGE and analyzed by western-blot on PVDF membrane. After saturation of the PVDF membrane with PBS-0.1% Tween 20 supplemented with 5% (w / v) non-fat milk powder, blots were incubated 1 h at room temperature with the primary antibody in PBS-0.1% Tween 20 (1 :2000 dilution). After washing, incubation with the secondary HRP -labeled antibody (1 : 10,000 dilution) was performed for 1 h at room temperature and detected by enhanced chemiluminescence reagents according to the manufacturer’s instructions (SuperSignal Chemiluminescent Substrate, Thermo Fisher Scientific).Statistics and reproducibility: All statistical analyses were performed with GraphPad Prism software. The confidence interval was set to 95% in all statistical tests. Details of statistical analyses can be found in the figure legends. The p values and sample size (n) are indicated either directly in the figure or in the legend. The mean ± standard error of the mean (SEM) is displayed unless otherwise stated.Results:DENV-NS3 protein interacts with GCKR and enhances glycolysisDENV-NS3 protein was previously reported to bind GAPDH and inhibit its activity

[0026] , potentially enabling the accumulation of glycolysis intermediates essential for connected anabolic pathways. As part of a prospective study to identify new protein-protein interactions (PPIs) between viral factors and cellular enzymes of the glycolytic pathway, we found that the NS3 protein of DENV binds GCKR, the negative regulator of GCK. This was achieved by NanoLuc Two-Hybrid (N2H) technology, a split-luciferase complementation assay, developed for PPIs detection

[0036] . Two complementary fragments of the bioluminescent enzyme NanoLuc were fused to GCKR and DENV-NS3, respectively. Once the two constructs were co-expressed in HEK293T cells, the luciferase activity which depends on reconstitution of the NanoLuc enzyme, demonstrated an interaction between NS3 and GCKR (Figure 4C). Although full- length NS3 (NS3-fl) or its isolated protease domain (NS3-prot) showed an interaction with GCKR, the strongest signal was observed with the helicase domain of NS3 (NS3-heli) suggesting that this region plays a preponderant role in the interaction with GCKR.Since GCKR is an important regulator of GCK hexokinase activity in the liver, we investigated the effect of NS3 expression on glycolysis in Huh7-GCK+ / HK2‘ hepatocytic cells. This cell line was previously described in details and corresponds to the hepatocellular carcinoma cell line Huh7 where the cancer-associated hexokinase HK2 was knocked-out and replaced by GCK, the isoenzyme expressed in primary hepatocytes

[0029] . As opposed to parental Huh7, these cells respond to extracellular glucose concentrations and produce lipids similarly to primary hepatocytes. NS3-fl expression increased both glucose consumption (Figure 4A) and lactate secretion (Figure 4B) in Huh7-GCK+ / HK2‘ cells, suggesting an increase in glycolytic activity. In contrast, the isolated protease or helicase domains of NS3 had no effect, thus demonstrating that full length NS3 protein is required for a functional effect on glycolysis. Finally, we observed that DENV replication in Huh7-GCK+ / HK2‘ cells increased by 2-fold glucose consumption (data not shown), which is in agreement with previous observations in humanforeskin fibroblasts

[0023] . Overall, these results shed light on how DENV controls glycolytic activity in infected liver cells, in line with glucose requirement for DENV replication

[0023] ,DENV replication depends on cellular hexokinase activityResults presented above show that glycolysis is a pathway tightly regulated by DENV, suggesting that glucose is essential for viral replication. Whether glucose can be substituted by other sources of carbon is unknown. To address this question, we first analyzed DENV replication in Huh7-GCK+ / HK2‘ cultured in the presence of glucose or other sources of carbon such as glutamine or galactose, another hepatocyte-metabolizable hexose. We showed that the highest replication of DENV subgenomic replicon was observed with glucose. Neither supplementation with glutamine, galactose or both of them enabled strong DENV replication (data not shown), whereas cell proliferation was not significantly different according to carbon source (data not shown). Moreover, viral replication was proportional to glucose concentration in the culture medium (data not shown). DENV replication thus appeared particularly dependent on the presence of glucose in cell culture medium which is fueling glycolysis.To further support this conclusion, we compared the replication in Huh7-GCK+ / HK2- vs Huh7 cells expressing HK2. Indeed, we previously found that Huh7 -GCK / HK2~ cells had a higher glycolytic activity than Huh7 cells from which they are derived, with restored lipogenesis and glycogenesis

[0029] , A recombinant strain of DENV type 2 expressing GFP as a viral growth reporter was used (DENV-GFP; strain 16681). Interestingly, we observed that an increased infection of GCK-expressing cells compared to Huh7 cells expressing HK2 (data not shown). This was associated with a higher secretion of viral particles by Huh7 -GCK / HK2~ infected cells (data not shown). Accordingly, replication of a DENV-R2A replicon expressing luciferase as a reporter instead of structural proteins

[0037] was enhanced in transfected Huh7- GCK+ / HK2~ cells compared to Huh7 cells (data not shown) and confirmed by quantifying NS3 protein expression by western-blot (data not shown). Therefore, Huh7 -GCK / HK2~ cells better replicate DENV than parental Huh7 cells expressing HK2.To further establish the role of GCK in DENV replication, standard Huh7 cells were transduced with increasing amounts of a lentiviral vector expressing GCK. We observed that viral replication was proportional to the amount of GCK expressed in the cells (data not shown), and correlated with hexokinase activity measured in homogenates of transduced cells (data notshown). Taken together, these observations confirm that DENV depends on glucose for its replication, and that the level of GCK controls the level of viral replication.DENV replication is repressed by GCKR expression in Huh7-GCK+ / HK2- cellsAs mentioned above, GCKR is the natural repressor of GCK. In a feedback loop slowing down cellular glycolysis, fructose-6-phosphate acts as an allosteric effector of the interaction of GCKR with GCK, forcing GCK into a weakly active form

[0027] . Since DENV replication depends on glycolysis, we tested whether modulation of GCK-GCKR interaction was able to control viral replication. We observed that addition of fructose-6-phosphate at the moment of cell electroporation with DENV replicon, inhibited by about 50% viral replication (data not shown). Conversely, addition in the cell culture medium of a GCK-GCKR interaction inhibitor (AMG-3969) increased DENV replication (data not shown), further supporting the idea that GCK-GCKR interaction modulates viral replication. To confirm these observations, GCKR was overexpressed in order to limit GCK activity in Huh7 -GCK / HK2~ cells, resulting in reduced DENV replication (data not shown). Mutations that impact GCKR’s capacity to interact with GCK have been described in the literature. In particular, mutation D413A in GCKR, was reported to decrease its capacity to inhibit GCK

[0038] , As expected, overexpression of the GCKRD413Amutant in YiuNI -GCK+ / HK2~ cells had no effect on DENV replication (data not shown). Interestingly, overexpression of wild-type GCKR or its mutant GCKRD413Ahad no effect on DENV replication in Huh7 cells that do not express GCK (data not shown), confirming that it is the specific inhibition of GCK by GCKR that inhibits viral replication.DENV replication is repressed by NAD synthesis inhibitor 6-Aminonicotinamide (6-AN) NAD+ is essential for glycolysis as it is used by GAPDH and lactate dehydrogenase (LDH) as a coenzyme. 6-Aminonicotinamide (6-AN) is an inhibitor of NAD+ biosynthesis that affects several metabolic pathways including glycolysis and pentose phosphate pathway (PPP), as depicted in Figure 1A. Since DENV-NS3 interferes with GCKR activity, the potential increase of G6P (product of glucose phosphorylation by hexokinases), could feed PPP to enhance the production of ribose-5-phosphate (R5P; Figure 1A), a key precursor in the synthesis of purine and pyrimidine nucleotides. 6-AN is well known as a potent inhibitor of G6PD, which is the first enzyme controlling the entry of G6P into the PPP and the production of R5P. We thus tested the effect of 6-AN on DENV replication in Huh7 -GCK / HK2~ cells. As shown in Figure IB, DENV replication was suppressed by 80% in the presence of 6-AN. Surprisingly, this inhibition was not reverted by D-ribose (Figure IB), which can be phosphorylated by theribokinase RBKS into R5P to sustain nucleotide biosynthesis [39, 40], This suggested that inhibition of the PPP was not at the origin of DENV inhibition by 6-AN despite DENV’s high dependence for nucleotides biosynthesis pathways in this model (see Figure 1C showing that DENV replication is strongly suppressed by pyrimidine biosynthesis inhibitor Brequinar).In contrast, DENV inhibition by 6-AN was reverted by NAM riboside, a precursor of the NAD salvage pathway (Figure IB). Once internalized, 6-AN is used instead of nicotinamide (NAM) by nicotinamide phosphoribosyl transferase (NAMPT), the first enzyme of the Nicotinamide Adenine Dinucleotide (NAD) salvage pathway. It is thus metabolized into 6-Amino- Nicotinamide Adenine Dinucleotide (6ANAD) and 6ANAD Phosphate (6ANADP), which are non-reducible forms of NAD and NADP and thus inhibitors of enzymes using these metabolites as cofactors. We confirmed that DENV replication was indeed dependent on the activity of the NAD-salvage pathway, using the specific NAMPT inhibitor FK-866, whose effect was also reverted by the addition of NAM-riboside in the culture medium (Figure ID). This suggests that 6-AN metabolization into 6ANAD(P) decreases the pool of NAD(P) that is available for metabolic functions, in particular glycolytic NAD-dependent enzymes (Figure 1A).This was confirmed in Huh7-GCK+ / HK2‘ cells treated with 6-AN where a decrease in NAD+ correlated with a lower consumption of glucose and addition of NAM riboside restored both the pool of NAD+ and the consumption of glucose (Figure 2A and B). As a consequence, intracellular ATP is decreased by 6-AN and restored by NAM riboside, revealing its indirect impact on CCM (Figure 2C). At the same time, cell viability was not impacted by 6-AN treatment and cellular growth only weakly affected (Figure 2D and 2E). It was further confirmed by the positive correlation between viral replication and intracellular ATP levels which are progressively restored by increasing doses of NAM riboside in presence of 6-AN (data not shown). Overall, these observations indicate that inhibition of NAD biosynthesis by 6-AN is efficient to block glycolysis and inhibit DENV replication in cell cultures.6-AN inhibits DENV replication in hamster-derived organotypic liver culturesIn this study, we used the Huh7 -GCK / HK2~ cell line, which is quite unique in that it enables analysis of both GCK and GCKR activities. However, it is important to note that although these cells express GCK instead of HK2, they remain cancer cells with an anabolism-oriented metabolism associated with high proliferation, unlike primary hepatocytes that do not proliferate. Therefore, we wondered whether 6-AN efficiently inhibited DENV replication inprimary liver cells, ex-vivo. To achieve that, we infected hamster-derived organotypic liver cultures (OLiC) as previously performed for measles virus and SARS-CoV-2 infection of organotypic lung and kidney cultures[33-35], 350 pm-thick liver slices from hamsters were cultured at the air / liquid interface on porous polytetrafluoroethylene (PTFE) membranes as described in Material and Methods. The day of slicing, OLiCs were infected with 103PFU of DENV-GFP and treated or not with 100 pM 6-AN in the subnatant culture medium. 90 h postinfection, we were able to observe infectious GFP foci in liver slices cultured in control condition, whereas no infection was detected in liver slices treated by 6-AN (Figure 3A). Total cellular RNA was extracted from liver slices and DENV genomes were quantified. We observed a 2 log10decrease of intracellular DENV genomes in presence of 6-AN (Figure 3B) and a total inhibition of DENV particle secretion in cell subnatants (Figure 3C). Quantification of ribosomal protein L13a (RPL13a) as a housekeeping gene revealed that 6-AN treatment had no impact on the quantity of extracted RNA (Figure 3D), as well as on global metabolic activity determined by MTT assay (Figure 3E). Altogether, these results confirm that 6-AN effectively inhibits DENV replication in primary liver cells.The targeting o f the NAD biosynthesis pathway inhibits the DENV replication.The inventors demonstrated that the DENV NS3 protein interacts with the cellular GCKR protein, therefore increasing the activity of GCK (figure 4)

[0052] , Simple expression of the virus NS3 protein is sufficient to induce an increase of the glycolysis in hepatocytic cells (Figure 4A & Figure 4B). Thus, the virus NS3 protein interacts specifically with the GCKR protein (Figures 4C and data not shown) and prevents the inhibition of GCK by GCKR (data not shown) releasing the hepatic glycolysis and inducing a cellular metabolism suitable for the virus replication. Thus, the inventors targeted the NAD biosynthesis to inhibit the glycolysis and the virus replication. The inhibition of the NAD biosynthesis affecting the pentosephosphate synthesis pathway (PPP) involved in cell multiplication and viral replication, the inventors tested whether this pathway was involved. To do this, in the presence of 6-AN, the inventors compensated for the inhibition of the PPP by adding D-Ribose to the medium that can be phosphorylated by cellular RBKS ribokinase. Under these conditions, 6-AN induces a decrease of the viral replication whether there is the presence of D-ribose or not (Figure IB), thus showing that the effect of 6-AN on the PPP was not involved in the inhibition of the virus

[0034]

[0035] , These results demonstrate that the inhibition of the PPP is not the cause of the DENV inhibition by 6-AN. However, the inhibition of the DENV replication has been reversed by the nicotinamide riboside (NAM Riboside) (Figure IB), a precursor of the NAD recovery pathway.These results confirm that the targeting of the NAD biosynthesis pathway inhibits the DENV replication.Discussion:The studies analyzing how viruses control cellular metabolism to replicate have often overlooked the unique metabolic characteristics of the infected cell type. Liver cells, especially, have unique metabolic functions, playing a pivotal role in the regulation of systemic metabolism. Therefore, viruses infecting hepatocytes have evolved dedicated strategies to manipulate their specific metabolic functions. Here we demonstrate that DENV interferes with glycolytic activity in the hepatocyte by interacting with GCKR, the negative regulator of the liver-specific hexokinase GCK. We have also shown that inhibition of NAD biosynthesis dampens cell glycolysis and inhibits DENV replication in hepatocellular carcinoma Huh7- GCK+ / HK2‘ cells and in liver tissue from ex-vivo hamster-derived OLiCs. These findings provide clues for a better understanding of the molecular mechanisms by which DENV hijacks cell metabolism, and pave the way for potential therapies targeting hepatocyte metabolism to control DENV replication in liver.Hepatocellular injury is observed in majority of patients with dengue hemorrhagic fever, manifesting as hepatomegaly, jaundice, elevated aminotransferase enzymes and a critical state in the form of acute liver failure

[0028] . Although the incidence of acute liver failure in patients with dengue hemorrhagic fever is very low, it is associated with a relatively high mortality rate and therefore specific strategies to control replication of DENV in liver and therefore limit potential severe clinical manifestations are needed. Albeit DENV can replicate in many cell types in-vitro, a few studies have already described how infection in hepatocellular carcinoma cells led to altered CCM. The first observation was made by El-Bacha et al, who showed in HepG2 cells that DENV infection induced a metabolic stress, associated with mitochondrial dysfunction

[0041] , Following infection, intracellular ATP levels fell, highlighting the energy requirements and consumption associated with viral replication. Heaton et al, demonstrated in Huh7.5 cells (derived from Huh7 cells) that the NS3 protein induced recruitment of the FASN protein at the replication site, thereby increasing intracellular lipid biosynthesisfl 6], Femandes- Siqueira et al. subsequently demonstrated that DENV replication in Huh7 cells increased glucose metabolism, that play an anaplerotic role in the oxidation of endogenous fatty acids

[0020] , Our results, obtained in cells expressing GCK, provide another molecular mechanism explaining how DENV specifically interfere with hepatocyte metabolism. Indeed, NS3 byinteracting with GCKR, can interfere with the first step of glycolysis controlled by GCK in the hepatocyte. One of the consequences of NS3 interaction with GCKR could be to prevent GCKR interaction with GCK due to steric hindrance. To investigate this hypothesis, we have carried out molecular docking simulation to build a three-dimensional model of the complex formed between NS3 and GCKR. The only crystal structure available for full length NS3 in the PDB database is for the DENV4 NS3 protein at a resolution of 3.15 A (PDB code 2VBC)

[0042] , We submitted to ClusPro server the search of protein-protein docking between this NS3 structure and GCKR (PDB code 4BBA), using standard parameters, as previously described[43-46]. The obtained model of GCKR-NS3 complex (data not shown), was compared to the published 3D structure of the GCKR-GCK complex (PDB code 4LC9). The superimposition of the two complexes on the GCKR backbone revealed steric interference between GCK and NS3. Indeed, the site of NS3 interaction on GCKR is close to the site of interaction with GCK, as revealed by the tangle of NS3 and GCK if we try to make them both interact with GCKR (data not shown). It suggests that NS3, by interacting with GCKR, can compete with GCK and therefore potentially inhibit the formation of GCK-GCKR complex. Therefore NS3 may limit GCKR capacity to control glycolysis explaining why NS3 expression induced the glycolytic activity of Huh7-GCK+ / HK2- cells (data not shown).This interaction is of importance for hepatic infection, since GCKR inhibitory activity on glycolysis is intimately linked to the expression of the liver-specific hexokinase GCK. Hence, NS3 interference with GCKR can maintain a high glucose consumption of hepatocytes, which is essential for viral replication, as described in this study (data not shown). Reciprocally, overexpression of GCKR or inducing its interaction with GCK inhibits viral replication (data not shown This further argues for DENV dependence on glycolysis in hepatocytes and NS3 interaction with GCKR appears to be specifically useful in the liver for controlling this essential pathway for viral replication. Globally, our observations are complementary of previous work showing that NS3 activates fatty acid biosynthesis in hepatocytes

[0016] . Indeed, the activation of glycolysis by DENV NS3 protein, is increasing the production of pyruvate that can fuel downstream metabolic pathways, such as TCA cycle and lipid biosynthesis. From a pathophysiological point of view, liver damage is a component of the natural history of the disease. Hepatomegaly is encountered in 10 to 80% of cases, depending on the study, and elevation of hepatitis markers such as AST is observed in 80 to 90% of cases

[0028] . Although little clinical data exist on DENV replication in patients' liver, several studies have found the virus in this organ and shown its ability to replicate in hepatocytes, with a potential cytopathiceffect[37, 47, 48], Hepatic steatosis was observed in patients in some studies[47, 48], Even if we cannot exclude inflammation as a contributor to the induction of liver steatosis, the ability of the virus to directly induce and deregulate hepatocyte glucido-lipidic metabolism appears as a potential enhancer of clinical manifestations.Another major observation of our study is that inhibition of the cell's glycolytic capacity by down-regulation of NAD metabolism strongly inhibits DENV replication both in vitro and ex vivo. We demonstrated the importance of NAD metabolism in maintaining a high level of glucose metabolism and ATP synthesis to support viral replication (Figures 1-3). Moreover, chemical inhibition of NAD biosynthesis both in HCC cell lines and in liver slices inhibits viral replication (Figure 1 and 3). Altogether, these observations highlight the importance of the intracellular pool of NAD coenzyme to sustain the glycolytic flux induced during infection. Interestingly, NS3 was described as a direct interactor of GAPDH, another glycolytic enzyme, reducing its activity

[0026] . However, our results indicate that NS3 as well as DENV subgenomic replicon expression in hepatoma cells expressing GCK both result in enhanced activity of the glycolysis pathway, supporting the viral need of glucose metabolism (Figure 4A, Figure 4B and data not shown). Thus, NS3 protein may be able to modulate the carbon flow in glycolysis in a complex manner, by interfering directly with glycolytic enzymes such as GAPDH or indirectly as a decoy for modulators of glycolysis such as GCKR. This multiple interference of the virus with glycolysis should enable adaptation to the infected host cell to maintain a high level of glycolysis. Finally, it emphasizes the capacity of DENV to enhance glycolysis in a multiple approach, according to metabolic specificity of the cell type and potentially whether certain isoenzymes such as hexokinases are expressed or not.As previously observed for Hepatitis B

[0039] and Zika

[0039] viruses, we observed that 6-AN was a potent inhibitor of DENV replication. However, the inhibitory effect of 6-AN on viral replication was not due to the inhibition of ribose-5-phosphate synthesis as suspected, but to its inhibitory effect on global NAD metabolism. Indeed, we observed that 6-AN reduced the amount of intracellular NAD+ and that viral replication was restored by the addition of NAM riboside, the precursor of NAD synthesis by the salvage pathway. This could be linked to reduced glucose consumption and intracellular amounts of ATP upon 6-AN treatment that is restored by addition of NAM riboside. To our knowledge we demonstrate for the first time an inhibition of DENV replication by 6-AN. both in Huh7-GCK+ / HK2‘ cells, and ex-vivo in hamster liver slices (OLiCs). This latter model consists of slices of liver tissue obtained bycutting of the organ from animals immediately after sacrifice. Slices are cultured and infected at the air / liquid interface on microporous membranes as previously described for other organs and with other viruses[34, 35], Organotypic cultures allow the tissue physiological structure and organization to be preserved with all cell types and the intercellular connections within the tissue. This culture system is therefore able to bridge the gap between the simplicity of cell cultures and the excessive complexity of an animal model. It enables infection of liver cells in a 3D culture system characteristic of the tissue. Under these conditions, we observed a 2-log inhibition of viral replication with 6-AN and total inhibition of infectious particle secretion with no appreciable effect on cell viability (Figure 3). Indeed, in the liver NADH metabolism is central in both catabolic and anabolic pathways such as glucogenesis, glycogenesis or lipogenesis. The antimetabolite 6-AN has been used for decades in preclinical trial in combination with other chemicals to increase the effectiveness of treatment in many cancers, including liver cancer[49, 50], Recently, in the light of a better-provided rational, 6-AN has been re-purposed in preclinical studies because of its ability to inhibit PPP[51-53], Induction of reactive oxygen species (ROS)-mediated apoptosis by ER stress in lung cancer cells was involved

[0052] . Moreover, 6-AN was associated with impaired NAD(H)-dependent glycolytic steps and inhibition of Zika virus replication

[0039] , Our study strongly suggest that targeting NAD metabolism to inhibit glycolysis, and thus the capacity of cells to replicate the virus, could be an efficient therapeutic approach to control dengue infection in liver. This is all the more relevant as hepatocytes have the intrinsic capacity to increase glycolysis when blood glucose concentration rises which could allow increased viral replication.The use of therapeutic molecules targeting host rather than viral proteins is a promising strategy, since it limits the risk of developing resistance due to a higher genetic barrier, and has a broader potential of action since it can render the host refractory to infection. Identification of the interaction between NS3 and GCKR (Figure 4C and data not shown), that is regulating glycolysis activity in the liver, should therefore pave the way to develop host metabolism directed strategies to limit hepatic infection and associated complications.Several studies have demonstrated the presence of DENV in the liver, showing its ability to replicate in primary human hepatocytes

[0053]

[0045] , In DENV infection, hepatomegaly is observed in 10-80% of cases. Then, blood markers of hepatic inflammation such as transaminases are increased in 80-90% of cases

[0054] , Moreover, hepatic steatosis is observed in infected patients

[0045] , Finally, acute liver failure is also observed in severe forms of infection

[0054] , These studies suggest that the virus could directly modify the metabolism of the hepatocyte carbohydrate and lipid suggesting that the infection of the liver by DENV could have a decisive impact in the progression of the disease. Here, the inventors have identified a mechanism by which DENV NS3 protein interferes with the activity of the hepatic GCKR protein, suggesting that DENV has selected a specific interaction that allows it to control glycolysis specifically in the hepatocyte, an interaction that has never been demonstrated for other members of the Flaviviridae family.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) Sanchez, E. L.; Lagunoff, M. Viral Activation of Cellular Metabolism. Virology 2015, 479-480, 609-618. https: / / doi.Org / 10.1016 / j.virol.2015.02.038.(2) Mayer, K. A.; Stockl, J.; Zlabinger, G. J.; Gualdoni, G. A. Hijacking the Supplies: Metabolism as a Novel Facet of Virus-Host Interaction. Front Immunol 2019, 10, 1533. https: / / doi.org / 10.3389 / fimmu.2019.01533.(3) Thaker, S. K.; Ch’ng, J.; Christofk, H. R. Viral Hijacking of Cellular Metabolism. BMC Biology 2019, 17 (1), 59. https: / / doi.org / 10.1186 / sl2915-019-0678-9.(4) Diaz, O.; Vidalain, P.-O.; Ramiere, C.; Lotteau, V.; Perrin-Cocon, L. What Role for Cellular Metabolism in the Control of Hepatitis Viruses? Frontiers in Immunology 2022, 13.(5) Girdhar, K.; Powis, A.; Raisingani, A.; Chrudinova, M.; Huang, R.; Tran, T.; Sevgi, K.; Dogus Dogru, Y.; Altindis, E. Viruses and Metabolism: The Effects of Viral Infections and Viral Insulins on Host Metabolism. Annu Rev Virol 2021, 8 (1), 373-391. https : / / doi . org / 10.1146 / annurev-virology-091919-102416.(6) Diaz, O.; Cubero, M.; Trabaud, M. A.; Quer, J.; Icard, V.; Esteban, J. I.; Lotteau, V.; Andre, P. Transmission of Low-Density Hepatitis C Viral Particles during Sexually Transmitted Acute Resolving Infection. Journal of Medical Virology 2008, 80 (2), 242-246. https: / / doi.org / 10.1002 / jmv.21037.(7) Piver, E.; Boyer, A.; Gaillard, J.; Bull, A.; Beaumont, E.; Roingeard, P.; Meunier, J.-C. Ultrastructural Organisation of HCV from the Bloodstream of Infected Patients Revealed by Electron Microscopy after Specific Immunocapture. Gut 2017, 66 (8), 1487-1495. https: / / doi.org / 10.1136 / gutjnl-2016-311726.(8) Scholtes, C.; Ramiere, C.; Rainteau, D.; Perrin-Cocon, L.; Wolf, C.; Humbert, L.;Carreras, M.; Guironnet-Paquet, A.; Zoulim, F.; Bartenschlager, R.; Lotteau, V.; Andre, P.; Diaz, O. High Plasma Level of Nucleocapsid-Free Envelope Glycoprotein-Positive Lipoproteins in Hepatitis C Patients. Hepatology 2012, 56 (1), 39-48. https: / / doi.org / 10.1002 / hep.25628.(9) Shimotohno, K. HCV Assembly and Egress via Modifications in Host Lipid MetabolicSystems. Cold Spring Harb Perspect Med 2021, 11 (1), a036814. https : / / doi . org / 10.1101 / cshperspect. a036814.(10) Dubuisson, J.; Cosset, F.-L. Virology and Cell Biology of the Hepatitis C Virus Life Cycle - An Update. Journal of Hepatology 2014, 61 (1, Supplement), S3-S13. https: / / doi.Org / 10.1016 / j.jhep.2014.06.031.(11) Diamond, D. L.; Syder, A. J.; Jacobs, J. M.; Sorensen, C. M.; Walters, K.-A.; Proll, S.C.; McDermott, J. E.; Gritsenko, M. A.; Zhang, Q.; Zhao, R.; Metz, T. O.; Camp, D. G.; Waters, K. M.; Smith, R. D.; Rice, C. M.; Katze, M. G. Temporal Proteome and Lipidome Profiles Reveal Hepatitis C Virus-Associated Reprogramming of Hepatocellular Metabolism and Bioenergetics. PLoS Pathog. 2010, 6 (1), el000719. https : / / doi . org / 10.1371 / j ournal .ppat.1000719.(12) Ramiere, C.; Rodriguez, J.; Enache, L. S.; Lotteau, V.; Andre, P.; Diaz, O. Activity of Hexokinase Is Increased by Its Interaction with Hepatitis C Virus Protein NS5A. J. Virol. 2014, 88 (6), 3246-3254. https: / / doi.org / 10.1128 / JVI.02862-13.(13) Martin-Acebes, M. A.; Jimenez de Oya, N.; Saiz, J.-C. Lipid Metabolism as a Source of Druggable Targets for Antiviral Discovery against Zika and Other Flaviviruses. Pharmaceuticals (Basel) 2019, 12 (2). https: / / doi.org / 10.3390 / phl2020097.(14) Martin-Acebes, M. A.; Merino-Ramos, T.; Blazquez, A.-B.; Casas, J.; Escribano- Romero, E.; Sobrino, F.; Saiz, J.-C. The Composition of West Nile Virus Lipid Envelope Unveils a Role of Sphingolipid Metabolism in Flavivirus Biogenesis. J Virol 2014, 88 (20), 12041-12054. https: / / doi.org / 10.1128 / JVI.02061-14.(15) Kao, Y.-T.; Chang, B.-L.; Liang, J. -I; Tsai, H.-J.; Lee, Y.-L.; Lin, R.-J.; Lin, Y.-L. Japanese Encephalitis Virus Nonstructural Protein NS5 Interacts with Mitochondrial Trifunctional Protein and Impairs Fatty Acid P-Oxidation. PLoS Pathog 2015, 11 (3), el004750. https: / / doi.org / 10.1371 / journal.ppat.1004750.(16) Heaton, N. S.; Perera, R.; Berger, K. L.; Khadka, S.; LaCount, D. J.; Kuhn, R. J.; Randall, G. Dengue Virus Nonstructural Protein 3 Redistributes Fatty Acid Synthase to Sitesof Viral Replication and Increases Cellular Fatty Acid Synthesis. PNAS 2010, 107 (40), 17345- 17350. https: / / doi.org / 10.1073 / pnas.1010811107.(17) Jordan, T. X.; Randall, G. Dengue Virus Activates the AMP Kinase-mTOR Axis To Stimulate a Proviral Lipophagy. J Virol 2017, 91 (11). https: / / doi.Org / 10.l 128 / JVI.02020-16.(18) Jordan, T. X.; Randall, G. Flavivirus Modulation of Cellular Metabolism. Curr Opin Virol 2016, 19, 7-10. https: / / doi.Org / 10.1016 / j.coviro.2016.05.007.(19) Lee, Y.-R.; Wu, S.-Y.; Chen, R.-Y.; Lin, Y.-S.; Yeh, T.-M.; Liu, H.-S. Regulation of Autophagy, Glucose Uptake, and Glycolysis under Dengue Virus Infection. The Kaohsiung Journal of Medical Sciences 2020, 36 (11), 911-919. https: / / doi.org / 10.1002 / kjm2.12271.(20) Fernandes-Siqueira, L. O.; Zeidler, J. D.; Sousa, B. G.; Ferreira, T.; Poian, A. T. D. Anaplerotic Role of Glucose in the Oxidation of Endogenous Fatty Acids during Dengue Virus Infection. mSphere 2018, 3 (1), e00458-17. https: / / doi.org / 10.1128 / mSphere.00458-17.(21) Perrin-Cocon, L.; Diaz, O.; Jacquemin, C.; Barthel, V.; Ogire, E.; Ramiere, C.; Andre, P.; Lotteau, V.; Vidalain, P.-O. The Current Landscape of Coronavirus-Host Protein-Protein Interactions. J Transl Med 2020, 18 (1), 319. https: / / doi.org / 10.1186 / sl2967-020-02480-z.(22) Hafirassou, M. L.; Meertens, L.; Umana-Diaz, C.; Labeau, A.; Dejarnac, O.; Bonnet- Madin, L.; Kummerer, B. M.; Delaugerre, C.; Roingeard, P.; Vidalain, P.-O.; Amara, A. A Global Interactome Map of the Dengue Virus NS1 Identifies Virus Restriction and Dependency Host Factors. Cell Rep 2017, 21 (13), 3900-3913. https: / / doi.Org / 10.1016 / j.celrep.2017.l l.094.(23) Fontaine, K. A.; Sanchez, E. L.; Camarda, R.; Lagunoff, M. Dengue Virus Induces and Requires Glycolysis for Optimal Replication. J. Virol. 2014. https: / / doi.org / 10.1128 / JVI.02309-14.(24) Allonso, D.; Andrade, I. S.; Conde, J. N.; Coelho, D. R.; Rocha, D. C. P.; da Silva, M. L.; Ventura, G. T.; Silva, E. M.; Mohana-Borges, R. Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3 -Phosphate Dehydrogenase Activity. J. Virol. 2015, 89 (23), 11871-11883. https: / / doi.org / 10.1128 / JVI.01342-15.(25) Perrin-Cocon, L.; Kundlacz, C.; Jacquemin, C.; Hanoulle, X.; Aublin-Gex, A.; Figi, M.; Manteca, J.; Andre, P.; Vidalain, P.-O.; Lotteau, V.; Diaz, O. Domain 2 of Hepatitis C Virus Protein NS5A Activates Glucokinase and Induces Lipogenesis in Hepatocytes. International Journal of Molecular Sciences 2022, 23 (2), 919. https: / / doi.org / 10.3390 / ijms23020919.(26) Silva, E. M.; Conde, J. N.; Allonso, D.; Ventura, G. T.; Coelho, D. R.; Cameiro, P. H.; Silva, M. L.; Paes, M. V.; Rabelo, K.; Weissmuller, G.; Bisch, P. M.; Mohana-Borges, R. Dengue Virus Nonstructural 3 Protein Interacts Directly with Human Glyceraldehyde-3 -Phosphate Dehydrogenase (GAPDH) and Reduces Its Glycolytic Activity. Sci Rep 2019, 9 (1), 1-19. https: / / doi.org / 10.1038 / s41598-019-39157-7.(27) Beck, T.; Miller, B. G. Structural Basis for Regulation of Human Glucokinase byGlucokinase Regulatory Protein. Biochemistry 2013, 52 (36), 6232-6239. https: / / doi.org / 10.1021 / bi400838t.(28) Leowattana, W.; Leowattana, T. Dengue Hemorrhagic Fever and the Liver. World Journal of Hepatology 2021, 13 (12), 1968-1976. https: / / doi.org / 10.4254 / wjh.vl3.il2.1968.(29) Choi, S. G.; Olivet, J.; Cassonnet, P.; Vidalain, P.-O.; Luck, K.; Lambourne, L.; Spirohn, K.; Lemmens, I.; Dos Santos, M.; Demeret, C.; Jones, L.; Rangarajan, S.; Bian, W.; Coutant, E. P.; Janin, Y. L.; van der Werf, S.; Trepte, P.; Wanker, E. E.; De Las Rivas, J.; Tavernier, J.; Twizere, J.-C.; Hao, T.; Hill, D. E.; Vidal, M.; Calderwood, M. A.; Jacob, Y. Maximizing Binary Interactome Mapping with a Minimal Number of Assays. Nat Commun 2019, 10. https: / / doi.org / 10.1038 / s41467-019-11809-2.(30) Perrin-Cocon, L.; Vidalain, P.-O.; Jacquemin, C.; Aublin-Gex, A.; Olmstead, K.; Panthu, B.; Rautureau, G. J. P.; Andre, P.; Nyczka, P.; Hutt, M.-T.; Amoedo, N.; Rossignol, R.; Filipp, F. V.; Lotteau, V.; Diaz, O. A Hexokinase Isoenzyme Switch in Human Liver Cancer Cells Promotes Lipogenesis and Enhances Innate Immunity. Communications Biology 2021, 4 (1), 1-15. https: / / doi.org / 10.1038 / s42003-021-01749-3.(31) Chatel-Chaix, L.; Cortese, M.; Romero-Brey, I.; Bender, S.; Neufeldt, C. J.; Fischl, W.; Scaturro, P.; Schieber, N.; Schwab, Y.; Fischer, B.; Ruggieri, A.; Bartenschlager, R. Dengue Virus Perturbs Mitochondrial Morphodynamics to Dampen Innate Immune Responses. Cell Host Microbe 2016, 20 (3), 342-356. https: / / doi.Org / 10.1016 / j.chom.2016.07.008.(32) Choi, J. M.; Seo, M.-H.; Kyeong, H.-H.; Kim, E.; Kim, H.-S. Molecular Basis for the Role of Glucokinase Regulatory Protein as the Allosteric Switch for Glucokinase. PNAS 2013, 110 (25), 10171-10176. https: / / doi.org / 10.1073 / pnas.1300457110.(33) Nong, S.; Han, X.; Xiang, Y.; Qian, Y.; Wei, Y.; Zhang, T.; Tian, K.; Shen, K.; Yang, J.; Ma, X. Metabolic Reprogramming in Cancer: Mechanisms and Therapeutics. MedComm 2023, 4 (2), e218. https: / / doi.org / 10.1002 / mco2.218.(34) Sahoo, B. R.; Crook, A. A.; Pattnaik, A.; Torres-Gerena, A. D.; Khalimonchuk, O.; Powers, R.; Franco, R.; Pattnaik, A. K. Redox Regulation and Metabolic Dependency of Zika Virus Replication: Inhibition by Nrf2-Antioxidant Response and NAD(H) Antimetabolites. Journal of Virology 2023, 97 (2), e01363-22. https: / / doi.org / 10.1128 / jvi.01363-22.(35) Pauly, D. F.; Pepine, C. J. D-Ribose as a Supplement for Cardiac Energy Metabolism. J Cardiovasc Pharmacol Ther 2000, 5 (4), 249-258. https: / / doi.org / 10.1054 / JCPT.2000.18011.(36) Ferren, M.; Favede, V.; Decimo, D.; lampietro, M.; Lieberman, N. A. P.; Weickert, J - L.; Pelissier, R.; Mazelier, M.; Terrier, O.; Moscona, A.; Porotto, M.; Greninger, A. L.; Messaddeq, N.; Horvat, B.; Mathieu, C. Hamster Organotypic Modeling of SARS-CoV-2 Lung and Brainstem Infection. Nat Commun 2021, 12 (1), 5809. https: / / doi.org / 10.1038 / s41467-021- 26096-z.(37) Shyfrin, S. R.; Ferren, M.; Perrin-Cocon, L.; Espi, M.; Charmetant, X.; Brailly, M.; Decimo, D.; lampietro, M.; Canus, L.; Horvat, B.; Lotteau, V.; Vidalain, P.-O.; Thaunat, O.; Mathieu, C. Hamster Organotypic Kidney Culture Model of Early-Stage SARS-CoV-2 Infection Highlights a Two-Step Renal Susceptibility. J Tissue Eng 2022, 13, 20417314221122130. https: / / doi.org / 10.1177 / 20417314221122130.(38) Welsch, J. C.; Lionnet, C.; Terzian, C.; Horvat, B.; Gerlier, D.; Mathieu, C. OrganotypicBrain Cultures: A Framework for Studying CNS Infection by Neurotropic Viruses and Screening Antiviral Drugs. Bio Protoc 2017, 7 (22), e2605. https: / / doi.org / 10.21769 / BioProtoc.2605.(39) El-Bacha, T.; Midlej, V.; Pereira da Silva, A. P.; Silva da Costa, L.; Benchimol, M.; Galina, A.; Da Poian, A. T. Mitochondrial and Bioenergetic Dysfunction in Human Hepatic Cells Infected with Dengue 2 Virus. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 2007, 1772 (10), 1158-1166. https: / / doi.Org / 10.1016 / j.bbadis.2007.08.003.(40) Luo, D.; Xu, T.; Hunke, C.; Gruber, G.; Vasudevan, S. G.; Lescar, J. Crystal Structure of the NS3 Protease-Helicase from Dengue Virus. Journal of Virology 2008, 82 (1), 173-183. https: / / doi.org / 10.1128 / jvi.01788-07.(41) Desta, I. T.; Porter, K. A.; Xia, B.; Kozakov, D.; Vajda, S. Performance and Its Limits in Rigid Body Protein-Protein Docking. Structure 2020, 28 (9), 1071 -1081 ,e3. https: / / doi.Org / 10.1016 / j.str.2020.06.006.(42) Vajda, S.; Yueh, C.; Beglov, D.; Bohnuud, T.; Mottarella, S. E.; Xia, B.; Hall, D. R.; Kozakov, D. New Additions to the ClusPro Server Motivated by CAPRI. Proteins 2017, 85 (3), 435-444. https: / / doi.org / 10.1002 / prot.25219.(43) Kozakov, D.; Beglov, D.; Bohnuud, T.; Mottarella, S. E.; Xia, B.; Hall, D. R.; Vajda, S. How Good Is Automated Protein Docking? Proteins 2013, 81 (12), 2159-2166. https: / / doi.org / 10.1002 / prot.24403.(44) Kozakov, D.; Hall, D. R.; Xia, B.; Porter, K. A.; Padhorny, D.; Yueh, C.; Beglov, D.; Vajda, S. The ClusPro Web Server for Protein-Protein Docking. Nat Protoc 2017, 12 (2), 255- 278. https: / / doi.org / 10.1038 / nprot.2016.169.(45) Aye, K. S.; Chamgkaew, K.; Win, N.; Wai, K. Z.; Moe, K.; Punyadee, N.; Thiemmeca, S.; Suttitheptumrong, A.; Sukpanichnant, S.; Prida, M.; Halstead, S. B. Pathologic Highlights of Dengue Hemorrhagic Fever in 13 Autopsy Cases from Myanmar. Hum Pathol 2014, 45 (6), 1221-1233. https: / / doi.Org / 10.1016 / j.humpath.2014.01.022.(46) Pagliari, C.; Quaresma, J. A. S.; Fernandes, E. R.; Stegun, F. W.; Brasil, R. A.; de Andrade, H. F.; Barros, V.; Vasconcelos, P. F. C.; Duarte, M. I. S. Immunopathogenesis of Dengue Hemorrhagic Fever: Contribution to the Study of Human Liver Lesions. J Med Virol 2014, 86 (7), 1193-1197. https: / / doi.org / 10.1002 / jmv.23758.(47) Dewey, D. L.; Hawes, C. 6-AMINONICOTINAMIDE AND THE RADIOSENSITIVITY OF HUMAN LIVER CELLS IN CULTURE. Nature 1963, 200, 1176- 1178. https: / / doi.org / 10.1038 / 2001176a0.(48) Dietrich, L. S.; Muniz, O.; Farinas, B.; Franklin, L. 6-Aminonicotinamide-14C Utilization by the 755 Tumor and Host Liver Tissue. Cancer Res 1968, 28 (8), 1652-1654.(49) Li, Y.; Zheng, F.; Zhang, Y.; Lin, Z.; Yang, J.; Han, X.; Feng, Y.; Pei, X.; Li, F.; Liu, Q.; Yan, L.; Li, T.; Zhang, Y.; Li, D.; Fu, Z.; Wang, C.; Sun, Q.; Li, C. Targeting Glucose-6- Phosphate Dehydrogenase by 6-AN Induces ROS-Mediated Autophagic Cell Death in Breast Cancer. FEBS J 2023, 290 (3), 763-779. https: / / doi.org / 10.l l l l / febs.16614.(50) Kaushik, N.; Kaushik, N. K.; Choi, E. H.; Kim, J. H. Blockade of Cellular Energy Metabolism through 6-Aminonicotinamide Reduces Proliferation of Non-Small Lung Cancer Cells by Inducing Endoplasmic Reticulum Stress. Biology (Basel) 2021, 10 (11), 1088. https: / / doi.org / 10.3390 / biologyl0111088.(51) Arbe, M. F.; Agnetti, L.; Breininger, E.; Glikin, G. C.; Finocchiaro, L. M. E.; Villaverde, M. S. Glucose 6-Phosphate Dehydrogenase Inhibition Sensitizes Melanoma Cells to Metformin Treatment. Transl Oncol 2020, 13 (11), 100842. https: / / doi.Org / 10.1016 / j.tranon.2020.100842.(52) Ogire E et al., 2024, Antiviral Res, DOI : 10.1016 / j. antiviral.2024.105939(53) L. Suksanpaisan, A. Cabrera-Hernandez, et D. R. Smith, « Infection of human primary hepatocytes with dengue virus serotype 2 », Journal of Medical Virology, vol. 79, no 3, p. 300-307, 2007, doi: 10.1002 / jmv.20798.(54) W. Leowattana et T. Leowattana, « Dengue hemorrhagic fever and the liver », World Journal of Hepatology, vol. 13, no 12, p. 1968-1976, dec. 2021, doi: 10.4254 / wjh.vl3.il2.1968.

Claims

CLAIMS:

1. A method of treating a dengue virus infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of NAD biosynthesis.

2. The method of claim 1 wherein the inhibitor of NAD biosynthesis is 6- Aminonicotinamide (6-AN) (IUPAC name: 6-aminopyridine-3-carboxamide).

3. The method of claim 1 wherein the inhibitor of NAD biosynthesis is a NAMPT inhibitor.

4. The method of claim 3 wherein the NAMPT inhibitor is selected from the group consisting of:- FK866 ((E)-N-[4-(l-benzoyl-4-yl)-butyl]-3-(pyridin-3-yl) acrylamide),CHS-828 (N-[6-(4-chlorophenoxy)hexyl]-N?-cyano-N?-4-pyridinyl-guanidine,GNE-617 (N-(4-((3,5-difluorophenyl)sulfonyl)benzyl)imidazo[l,2-a]pyridine-6- carboxamide),GNE-618 (N-[[4-[[3-(Trifluoromethyl)phenyl]sulfonyl]phenyl]methyl]-lH- pyrazolo[3,4-b]pyridine-5-carboxamide),STF118804 (4-[5-Methyl-4-[[(4-methylphenyl)sulfonyl]methyl]-2-oxazolyl]-N-(3- pyridinylmethyljbenzamide),KPT-9274 ((E)-3-(6-aminopyridin-3-yl)-N-[[5-[4-(4,4-difluoropiperidine-l- carbonyl)phenyl]-7-(4-fluorophenyl)-l-benzofuran-2-yl]methyl]prop-2-enamide), and,LSN3154567 (2-hydroxy-2-methyl-N-[l,2,3,4-tetrahydro-2-[2-(3-pyridinyl oxy)acetyl]-6-isoquinolinyl]-l-propane-sulfonamide.

Images