WO2020022961A1 - Inhibiteurs de la partie ires d'un entérovirus - Google Patents

Inhibiteurs de la partie ires d'un entérovirus Download PDF

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WO2020022961A1
WO2020022961A1 PCT/SG2019/050357 SG2019050357W WO2020022961A1 WO 2020022961 A1 WO2020022961 A1 WO 2020022961A1 SG 2019050357 W SG2019050357 W SG 2019050357W WO 2020022961 A1 WO2020022961 A1 WO 2020022961A1
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ires
heva71
prunin
enterovirus
flavonoid
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PCT/SG2019/050357
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English (en)
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Justin Jang Hann CHU
Saravanan GUNASEELAN
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Agency For Science, Technology And Research
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • the present invention relates to compositions for preventing or treating an enterovirus infection or an enterovirus induced disorder.
  • HEVA71 Human Enterovirus 71
  • CNS central nervous system
  • HEVA71 is characterized as a small, non-enveloped enterovirus with a positive-sense single stranded RNA genome. Its 7.4kb genome begins with a 5' untranslated region (UTR) and terminates with a 3' UTR, in which its open reading frame is translated into 4 capsid proteins (VP1-4), and 7 non-structural proteins (2A-C and 3A-D) 10 . Particularly of interest is the internal ribosome entry site (IRES) that lies within stem loops 2 to 6 of the 5' UTR.
  • IRES internal ribosome entry site
  • IRES substitutes the functions of some host translation initiation factors, thereby permitting cap-independent translation of viral proteins 13 . Furthermore, cap-dependent translation of host cell is decreased via HEVA71 IRES-encoded viral proteases, namely 2A pro and 3C pro , which cleave host translation factors such as elF4G to favor IRES-mediated translation 14 . As such, it is critical for competent antiviral strategies to target HEVA71 IRES-facilitated protein synthesizing machinery, in order for host cell translation to proceed normally. Over the years, IRES has been utilized in biotechnology to construct bicistronic or polycistronic vectors, where IRES was positioned in between 2 cistrons 15-17 .
  • IRES in mediating gene translation: (i) it aids in establishing simultaneous expression of the pair of cistrons in more than 90% of the cells 17 ; (ii) the ratio of protein expression by both cistrons positioned before and after IRES are not affected 17 ' 18 ; (iii) IRES allows for the translation of RNAs without the 5' cap structure, which are synthesized by RNA polymerase I and III 19-22 ; (iv) IRES cap-independent translation can be utilized in circumstances where cap-dependent host cell translation is impaired, such as during programmed cell death and cell cycle checkpoints 23 . Overall, these features of IRES enable it to be an advantageous element in genetic engineering.
  • a molecule that inhibits or prevents an interaction between at least one IRES trans-acting factor (ITAF) and an IRES portion of an enterovirus or a hepacivirus.
  • ITAF IRES trans-acting factor
  • molecule it is meant to include any purified or isolated natural or chemically-synthesised entity.
  • the term includes one more polypeptide and/or one or more small chemical molecule, wherein said polypeptide and/or small chemical molecule may or may not be modified by the ionic and/or hydrophobic and/or covalent addition of chemical groups.
  • the molecule may be amantadine, quinacrine, sorafenib and idarubicin.
  • IRES portion it is meant to include a region or regions of the molecule capable of reversibly and/or irreversibly associating with a region or regions of an ITAF and/or IRES portion of an enterovirus or a hepacivirus by covalent and/or ionic interaction.
  • the molecule inhibits the interaction between the at least one IRES ITAF and the IRES portion of the virus by protein-protein steric hindrance.
  • the term internal ribosome entry site is a nucleotide sequence that allows for translation initiation in the middle of a messenger RNA (mRNA) sequence as part of the greater process of protein synthesis.
  • mRNA messenger RNA
  • the IRES mimics the 5' cap structure, and is recognized by the 43S pre-initiation complex. IRES-containing bicistronic vectors allow the simultaneous expression of two proteins separately, but from the same RNA transcript.
  • the terms “inhibits” and “prevents an interaction” refer to the delay, repression or interference of one or more of; the activity of IRES portion of the enterovirus. More preferably, the term inhibits refers to reduction in IRES-mediated translation of the enterovirus genome. It also means that the molecule is capable of inhibiting or otherwise interfering, at least in part, with the association or binding of the at least one IRES trans-acting factor (ITAF) and an IRES portion of an enterovirus or a hepacivirus. For example, the molecule may be capable of inhibiting the binding of IRES potion defined herein by at least 10%, for example at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 35% or even by 100%.
  • ITAF IRES trans-acting factor
  • the ITAF is selected from the group consisting of hnRNPK, hnRNPAl and Sam68. More preferably, the molecule that inhibits or prevents an interaction between at least one ITAF and group 2 or group 3 IRES RNA, or the molecule targets stem loop(s) 2 and/or 4 of the IRES portion.
  • the enterovirus is HEVA71
  • the hepacivirus is Hepatitis C virus.
  • the molecule is selected from the group consisting: a flavonoid, a modified or unmodified siRNA, a phosphorothioate oligonucleotide, and vivo-MO-1 and vivo-MO-2. More preferably, the molecule is a flavonoid selected from Table 1. Still more preferably, the flavonoid is prunin.
  • a molecule that inhibits or prevents an interaction between at least one IRES trans-acting factor (ITAF) and an IRES portion of an enterovirus or a hepacivirus in the preparation of a pharmaceutical composition for preventing or treating an enterovirus or a hepacivirus infection.
  • ITAF IRES trans-acting factor
  • the ITAF is selected from the group consisting of hnRNPK, hnRNPAl and Sam68.
  • the molecule can inhibit any one of the ITAF listed in the group above or in combination with any one from the group.
  • the molecule exerts a stronger inhibitory effect on hnRNPK.
  • the enterovirus is HEVA71
  • the hepacivirus is Hepatitis C virus.
  • the molecule is selected from the group consisting: a flavonoid, a modified or unmodified siRNA, a phosphorothioate oligonucleotide, and vivo-MO-1 and vivo-MO-2. More preferably, the molecule is a flavonoid selected from Table 1. Still more preferably, the flavonoid is prunin.
  • a pharmaceutical composition for preventing or treating an enterovirus infection or an enterovirus induced disorder comprising a flavonoid that inhibits at least one IRES trans-acting factor (ITAF) association with an IRES portion of an enterovirus, together with one or more pharmaceutically acceptable diluents or carriers therefor.
  • ITAF IRES trans-acting factor
  • the composition is a unit dosage containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of the active ingredient.
  • the amount of flavonoid present in the composition may be 2C ⁇ g/ml.
  • the therapeutically effective amount of flavonoid may be 3mg per weight kg of the subject.
  • compositions can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • a pharmaceutically acceptable carrier refers, generally, to materials that are suitable for administration to a subject wherein the carrier is not biologically harmful, or otherwise, causes undesirable effects.
  • Such carriers are typically inert ingredients of a medicament.
  • a carrier is administered to a subject along with an active ingredient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of a pharmaceutical composition in which it is contained.
  • Suitable pharmaceutical carriers are described in Martin, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa., (1990), incorporated by reference herein in its entirety.
  • the carrier may be selected from the group consisting of a nanoparticle, such as a polymeric nanoparticle; a liposome, such as pH-sensitive liposome, an antibody conjugated liposome; a viral vector, a cationic lipid, a polymer, a UsnRNA, such as U7 snRNA and a cell penetrating peptide.
  • the antisense oligonucleotide is administered orally, or rectal, or transmucosal, or intestinal, or intramuscular, or subcutaneous, or intramedullary, or intrathecal, or direct intraventricular, or intravenous, or intravitreal, or intra peritoneal, or intranasal, or intraocular.
  • compositions comprising therapeutically effective amounts of the flavonoid together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
  • compositions include diluents of various buffer content (e.g., phosphate, Tris-HCI, acetate), pH and ionic strength and additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol).
  • the material may be incorporated into particulate preparations of polymeric compounds such as, for example and without limitation, polylactic acid or polyglycolic acid, or into liposomes. Hylauronic acid may also be used.
  • Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the disclosed compositions.
  • the compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form.
  • compositions provided according to the disclosure may be administered by any means known in the art.
  • the pharmaceutical compositions for administration are administered by injection, orally, or by the pulmonary, or nasal route.
  • the antisense polynucleotides are, in various embodiments, delivered by intravenous, intra-arterial, intraperitoneal, intramuscular, or subcutaneous routes of administration.
  • the flavonoids of the invention may encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such pro-drugs, and other bioequivalents.
  • pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • pharmaceutically acceptable salts include, but are not limited to, (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid; (c) salts formed with organic acids such as, for exam ple, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, na
  • the pharmaceutical formulations of the disclosure may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the molecule of the present invention (flavonoid being an example) with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • the formulations may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
  • the pharmaceutical composition may further comprise a co-agent having anti-enterovirus properties.
  • the co-agent may be ribavirin, NITD0008 or 2'- C-methylcytidine.
  • the molecule or derivative or fragment thereof, or a formulation thereof may be administered by any conventional method including oral, intranasal, and parenteral (e.g. subcutaneous or intramuscular) injection. Preferred routes include oral, intravenous or subcutaneous injection.
  • the treatment may consist of a single dose or a plurality of doses over a period of time.
  • the molecule or derivative thereof may formulated in a sustained release formulation so as to provide sustained release over a prolonged period of time such as over at least 2 or 4 or 6 or 8 weeks
  • the sustained release is provided over at least 4 weeks.
  • a flavonoid for use in preventing or treating an enterovirus infection or an enterovirus induced disorder.
  • a method of preventing or treating an enterovirus infection or an enterovirus induced disorder comprising administering a therapeutically effective amount of a flavonoid to a subject in need thereof.
  • a method of inhibiting an enterovirus replication or gene expression in a cell comprising introducing to the cell a flavonoid, wherein the flavonoid inhibits or prevents an interaction between at least one IRES trans-acting factor (ITAF) and an IRES portion of an enterovirus or a hepacivirus.
  • ITAF IRES trans-acting factor
  • the enterovirus is HEVA71 and the flavonoid is prunin.
  • the present invention also includes other aspects, such as a nucleic acid construct comprising an enterovirus internal ribosome entry site (IRES) flanked by two reporter genes.
  • IRS enterovirus internal ribosome entry site
  • the IRES is a HEVA71 IRES
  • the reporter genes encode a fluorescent protein.
  • test agent may be a drug-like compound or lead compound for the development of a drug-like compound.
  • a druglike compound is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament.
  • a druglike compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons and which may be water-soluble.
  • a drug like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes or the blood:brain barrier, but it will be appreciated that these features are not essential.
  • lead compound is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, poorly soluble, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.
  • the constructs of the present invention may be useful in screening methods, such as a method for screening a candidate as a potential drug for the prevention or treatment of an enterovirus infection or an enterovirus induced disorder, or a HCV infection or HCV induced disorder, the method comprising (a) providing a population of cells comprising the nucleic acid construct disclosed in this invention ; (b) introducing or contacting the cells with a candidate obtained from a library of molecules; and (c) screening the cells for reduced or inhibited expression of one of the reporter genes, thereby identifying the candidate that reduces or inhibits expression of one of the reporter genes.
  • the library of molecules is a library of flavonoids.
  • HEVA71 mutants that may be used as platforms to find novel drugs against the particular regions of HEVA71 mutant IRES to overcome drug resistance.
  • siRNAs and/or miRNAs can be targeted against the mutant IRES to find new treatments against HEVA71, given that siRNA/miRNA therapeutics are on the rise.
  • the high-throughput screen of the present invention revealed prunin as a potent suppressor of HEVA71 in vitro and in vivo through impeding hnRNPK association with HEVA71 IRES.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), "including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • FIG. 1 Experimental design, generation and relative activities of bicistronic reporter vectors.
  • A A strategy of generating a bicistronic construct, which aids in discovering antivirals that reduce the synthesis of Protein 2 through inhibiting IRES mediated translation without concurrently affecting Protein 1 production by cap-dependent translation.
  • B Schematic illustration of the HEV71 IRES bicistronic reporter utilized in this study. HEV71 IRES is located in between two luciferase genes, namely R Luc and F Luc, which are in turn placed downstream of a CMV promoter. R Luc gene is translated into R Luc proteins (red) through cap-dependent translation while F Luc gene is translated into F Luc proteins (pink) through cap-independent translation by HEV71 IRES.
  • HEV71 IRES bicistronic hairpin used in this study.
  • HEV71 IRES hairpin is located in between two luciferase genes, namely R Luc and F Luc, which are in turn placed downstream of a CMV promoter.
  • R Luc gene is translated into R Luc proteins (red) through cap-dependent translation while F Luc gene is not translated into F Luc proteins (pink) due to the presence of the HEV71 hairpin structure.
  • FIG. 1 Heterogeneous selection and consistencies of luciferase expressions.
  • A Process of heterogeneous G418 antibiotic selection of HEV71 IRES bicistronic reporter or HEV71 IRES bicistronic hairpin.
  • B Relative cell viability profiles of RD cells treated with G418, where red dotted lines represent corresponding G418 concentration (0.25 mg/ml) for 50% cell viability (CC50 ).
  • FIG. 1 HEV71 IRES inhibition profiles (brown bars) and cell viability profiles (red dots) of top 25 flavonoid hits, with 2mM of amantadine hydrochloride and HEV71 IRES bicistronic hairpin (BICIS HP) as positive controls. 7 flavonoids (underlined in pink) with more than 80% cell viability (blue dotted line) were chosen for further analysis through downstream studies. Data are expressed as the average (brown bars and red dots) with error bars (vertical lines) representing ⁇ SD from 3 independent experiments consisting of triplicates.
  • FIG. 4 Downstream validation and functional studies of flavonoids.
  • A Inhibition profiles of 7 chosen flavonoid compounds through HEV71 viral titre quantification with plaque assay at 12hpi.
  • B Cell viability profile and calculated CC50 of prunin after 24 hours. Concentrations below ImM showed more than 80% cell viability (blue dotted line). Red dotted lines represent corresponding prunin dose (2715nM) for 50% cell viability.
  • C Inhibition (bars) and cytotoxicity (grey dots) profiles of prunin (31.25 to lOOOnM) through HEV71 viral titre quantification with plaque assay at 12hpi and cell viability assay respectively. Concentrations of prunin used showed more than 80% cell viability (blue dotted line).
  • FIG. 5 In vivo studies of prunin.
  • A Survival rates, (B) mean clinical scores and (C) body masses of mice infected with HEV71 and treated with prunin at a dose of 3mg/kg. Groups of 5 to 6 mice were infected via i.p routes with HEV71 strain 41 at 2 x 107 PFU per mouse, after which prunin was administered from the day of infection to 7dpi and monitored for 14 dpi. Data are expressed as the average values with error bars representing ⁇ SD from 2 independent experiments.
  • D Quantification of viral loads in hind limb muscles of HEV71- infected mice by plaque reduction assays.
  • FIG. 6 HEV71 prunin-resistant mutant studies.
  • A Characterization of HEV71 resistant mutant phenotypes with increasing doses of pruning (red) from passages 1 to 19. Quantification of respective viral titres at passages 13, 16 and 19 were performed through plaque reduction assays. 1% DMSO (green) was used as vehicle control.
  • B Growth kinetics of wildtype and mutant HEV71 from 0 to 96hpi, which were measured through plaque reduction assays at each respective timepoint. Data are expressed as the average values with error bars representing ⁇ SD from 3 independent experiments consisting of triplicates. Statistical analyses were performed with one-way ANOVA corrected using Dunnett's post-test with Graphpad Prism 6.0.
  • Figure 7 Suppression activity of prunin against Hepatitis C virus.
  • A Inhibition (black and grey bars) and cell viabilty (red line) profiles of prunin (62.5 to 500nM) on HCV at 3dpi (black bars) and 6dpi (grey bars) through HCV viral titre quantifications via immunoflourescence assays and cell viability assays, respectively. Concentrations of prunin below 500nM showed more than 80% cell viability (blue dotted line).
  • B Disruption of HCV viral RNA synthesis by prunin (62.5 to 500nM) at 3dpi (red bars) and 6dpi (green bars) measured through qRT-PCR.
  • FIG. 8 Cell viability profile of SJ cells treated with EC50 of Prunin (115.3nM) after 24 hours. Prunin concentration at 115.3nM showed more than 80% cell viability (blue dotted line).
  • 1% DMSO was used as a vehicle control. Data are expressed as the average (grey bars) with error bars (vertical black lines) representing ⁇ SD from 3 independent experiments consisting of triplicates. Statistical analyses were performed against 1% DMSO, with one-way ANOVA tests adjusted with Dunnett's post-tests, with Graphpad Prism 6.0. ns represents not significant.
  • Figure 9 Different classifications of flavonoids, namely the isoflavones, neoflavonoids, chalcones and another huge group (F2) consisting of flavones, flavonols, flavanones, flavanonols, flavanols and anthocyanins. Adapted from [64]
  • the present invention identified potential therapeutic agents through two constructs, namely the HEVA71 IRES bicistronic reporter and HEVA71 IRES bicistronic hairpin, which were validated and screened with a 502-compound flavonoids library to shortlist potent compounds targeting HEVA71 IRES. Chosen hits were further verified with cell viability and viral plaque assays, which revealed prunin as the most potent inhibitor of HEVA71. Downstream secondary assays reaffirmed that prunin disrupted viral protein and RNA synthesis and acted as a narrow-spectrum antiviral only against Enteroviruses A and B.
  • RD Human RD
  • CCL-81 African green monkey kidney epithelial Vero cells
  • DMEM Dulbecco's Modified Eagle's Medium
  • SJ SJCRH30
  • BHK BHK-21
  • RPMI-1640 Roswell Park Memorial Institute 1640 medium
  • FCS fetal calf serum
  • HEVA71 used in this study include HEVA71 strain 41 (Accession no.
  • HEVA71 strain H VR-1432, ATCC; Accession no. AY053402.1
  • HEVA71 genogroup B5 strain accession no. FJ461781.1
  • HEVA71 genogroup C4 strain accesion no. JQ965759.1
  • Coxsackievirus A6 accesion No. KC866983.1
  • Coxsackievirus A16 Accession No. U05876)
  • Coxsackievirus A24 (Accession No. KF725085.1)
  • Coxsackievirus B5 accesion No.
  • the pCTAP ® vector (InterPlay ® Mammalian TAP System) provided the backbones for both plasmids, where it was amplified from positions 696 to 4522. Pair of primers used, included pCTAPf (forward primer): 5'- AGATCTCAGGAATTCGATATCAGG-3' and pCTAPr (reverse primer): 5'-
  • TAATAACTAATGCATGGCGGTAATAC-3' The given bicistronic construct containing HEVA71 strain 26M IRES 27 was replaced with either HEVA71 wildtype strain 41 IRES or various mutant HEVA71 IRESes.
  • Amplifications of respective target luciferase genes (CMV-R Luc-IRES-F Luc or CMV-R Luc-hairpin-F Luc) from positions 3574 to 6622 were performed with BICISf (forward primer): 5'-ATGCATTAGTTATTACGTTACATAACTTACGGTAAA-3' and BICISr (reverse primer): 5'-GAATTCCTGAGATCTTTACAATTTGGACTTTCCGC-3'.
  • RD cells were seeded at a density of 5xl0 3 to lxlO 4 cells per well in 96-well white plates or 7.5xl0 6 to lxlO 7 cells per flask in T-75 culture flasks and transfected with either HEVA71 IRES bicistronic reporter or HEVA71 IRES bicistronic hairpin by following the jetPRIME ® (Polyplus transfection ® ) recommended protocol.
  • the 502-compound flavonoids derivatives library (BML-2865, Enzo Life Sciences) with known structures was dissolved with DMSO to achieve a stock concentration of 2mg/ml. Further dilutions with serum-free DMEM were carried out to obtain a final concentration of 200pg/ml prior screening.
  • the positive control namely amantadine hydrochloride, was dissolved in water to yield a stock dose of 5mM.
  • HEVA71 IRES bicistronic reporter or HEVA71 IRES bicistronic hairpin transfected RD cells were trypsinized and seeded into 96-well white or transparent plates accordingly.
  • Luciferase assay Respective luciferase activities of HEVA71 IRES bicistronic reporter or HEVA71 IRES bicistronic hairpin or various HEVA71 mutant IRES bicistronic reporters were determined by following the Dual-Glo ® Luciferase Assay (Promega ® ) protocol. In short, 100mI of Dual-Glo ® Reagent was added per well of 96-well white plates and measured for F Luc activities. Following this, 100mI of Dual-Glo ® Stop & Glo ® Reagent was added to each well and determined for R Luc activities. Each step required an incubation time of 10 minutes at room temperature (25°C). All measurements were performed in a Promega ® Glomax ® -Multi Detection System with InstinctTM Software (luminometer).
  • Prunin (ST077105) was purchased from Timtec while ribavirin (R9644) was obtained from Sigma Aldrich respectively, where they were diluted with 100% DMSO to obtain corresponding 25mM and 1M stock solutions. Further dilutions to working concentrations of prunin and ribavirin were achieved with respective maintenance media or RNase-free water (Qiagen) used in various experiments.
  • RD cells were seeded at a density of 5xl0 3 to lxlO 4 cells per well in 96-well transparent plates or 5xl0 4 to 1x10 s cells per well in 24-well plates. These plates were subsequently infected with HEVA71 virus at MOI of 1 diluted in 50mI (96-well) or 100mI (24- well) of DMEM with 2% FCS, respectively. Negative controls without viral infections were only added with maintenance media. To allow for viral adsorption, HEVA71 infected plates were incubated for 1 hour with regular rocking at 15-minute intervals.
  • HEVA71-infected cells in 96-well plates were treated with 50mI of DMEM with 2% FCS containing each of the 7 top hits of the flavonoid drugs library at 20pg/ml while infected cells in 24-well plates were treated with a range of concentrations of prunin (31.25nM to ImM) diluted in 1ml of DMEM with 2% FCS.
  • Non-drug treated samples only contained of appropriate maintenance media with vehicle control (1% DMSO).
  • RD cells were also separately seeded in 96-well transparent plates and treated with the above doses of prunin for 24 hours, before relevant cell viabilities for each concentration were quantified through cell viability assays.
  • RD or Vero cells seeded in 24-well plates were infected with Enteroviruses (HEVA71, CA6, CA16, CA24, CB5, ECH07, HRV10, clinical isolates) at MOIs of 1 while CHIKV or HSV infection studies were conducted in respective SJ cells or BHK cells in 24-well plates at MOIs of 1. Plates with infections of each Enterovirus or HSV were incubated for 1 hour, while those with CHIKV infections were incubated for 1.5 hours prior prunin treatments at 115.3nM for 12 hours.
  • Enteroviruses HEVA71, CA6, CA16, CA24, CB5, ECH07, HRV10, clinical isolates
  • CHIKV or HSV infection studies were conducted in respective SJ cells or BHK cells in 24-well plates at MOIs of 1. Plates with infections of each Enterovirus or HSV were incubated for 1 hour, while those with CHIKV infections were incubated for 1.5 hours prior prunin treatments at 115.3nM for 12 hours.
  • the drug was diluted in specific maintenance media (lml) such as RPMI with 2% FCS for infections in SJ and BHK cells; and DMEM with 2% FCS for infections in RD or Vero cells. After 12hpi, corresponding supernatants were collected and subjected to plaque reduction assays.
  • specific maintenance media lml
  • RPMI 2% FCS for infections in SJ and BHK cells
  • DMEM 2% FCS for infections in RD or Vero cells
  • Viral plaque assays RD or SJ or BHK or Vero cells were seeded in 24-well plates at seeding density of 5xl0 4 to 1x10 s cells per well prior plaque reduction assays. Respective supernatants from infection studies were diluted in 10-fold dilutions ranging from 10 1 to 10 7 with appropriate maintenance media (DMEM with 2% FCS or RPMI with 2% FCS), which were then incubated with respective cells, ranging from 1 to 1.5 hours. Following viral adsorptions, plates were washed twice with PBS and an overlay medium of either lml of DMEM or RPMI containing 1% carboxymethylcellulose (CMC) and 2% FCS was added to replace the corresponding maintenance media.
  • DMEM 2% FCS
  • RPMI RPMI with 2% FCS
  • CHIKV or HSV supernatants were incubated for 3 days while those with supernatants from Enteroviruses were incubated for 4 days for plaque formations, after which they were subjected to fixation and staining with 10% paraformaldehyde/1% crystal violet (Sigma-Aldrich) solution at 25°C overnight. Average numbers of plaques were enumerated through the visualization of clear patches in monolayers of respective cells. These numbers were then multiplied to their dilution factors to accurately determine respective viral titres, which were represented as plaque forming units per millilitre (PFU/ml).
  • Prunin-resistant HEVA71 viruses (P19) were selected through plaque purification assays, where 10 random individual plaques were chosen from 10 5 to 10 6 dilutions via pipette tips. Respective agarose plugs were each then transferred to and eluted in 500mI of serum-free DMEM in order to obtain mutant HEVA71, which was further subjected to RNA isolation.
  • nitrocellulose membranes were first blocked with 5% of skim milk (Anlene) for an hour and further treated with blocking reagent consisting of mouse anti-HEVA71 antibody (1:2500, MAB979, Millipore) or rabbit polyclonal anti-Sam68 antibody (1:1000, 10222-1-AP, ProteinTech) or rabbit polyclonal anti-hnRNPK antibody (1:300, 11426-1-AP, ProteinTech) or rabbit polyclonal anti-hnRNPAl antibody (1:400, 11176-1-AP, ProteinTech) or mouse anti- actin antibody (1:5000, MAB1501, Millipore).
  • blocking reagent consisting of mouse anti-HEVA71 antibody (1:2500, MAB979, Millipore) or rabbit polyclonal anti-Sam68 antibody (1:1000, 10222-1-AP, ProteinTech) or rabbit polyclonal anti-hnRNPK antibody (1:300, 11426-1-AP, ProteinTech) or rabbit polyclonal anti-hnRNPAl antibody (1:400, 11
  • RNA from cells were extracted using a Total RNeasy kit (Qiagen). All RNA samples were treated with DNAse (Promega) prior quantifications in the StepOne Plus Real-time PCR system (Applied Biosystems) via a SYBR green-based RT-PCR kit (Maxima, Thermo Scientific).
  • Primers used targeted HEVA71 5' UTR 100 which were MD90 (5'-ATTGTCACCATAAGCAGCCA-3') and MD91 (5'-CCTCCGGCCCCTGAATGCGGCTAAT-3').
  • HEVA71 resistant mutants RD cells were seeded at a density of 5xl0 4 to 1x10 s cells per well in a 24-well plate and subsequently infected with WT HEVA71 at a MOI of 1 for an hour. Following PBS washes, HEVA71-infected cells were exposed to EC50 of prunin (115.3nM) and incubated for 1 to 3 days till virus-induced extensive cytopathic effects were observed in 50% of RD cells. Plates were then subjected to freeze-thaw, where supernatants were collected for plaque reduction assays and infection of another 24-well plate of RD cells (passage 1).
  • RNA from mutant viruses at passage 19 were plaque purified and extracted using a Total RNeasy kit (Qiagen). These RNA were further amplified via RT-PCR with Superscript III one-step RT-PCR kit (Invitrogen) by 4 pairs of primers 101 , which spanned the entire HEVA71 viral genome. 1 agarose plug consisting of mutant HEVA71 genome was further verified via automated DNA sequencing and analyzed for mutations.
  • RD cells were seeded in a 24-well plate with approximately 1x10 s cells per well and subjected to either WT or mutant HEVA71 infection at a MOI of 1 for an hour. Following removal of excess viruses with PBS washes, HEVA71-infected cells were treated with 115.3nM of prunin and/or ImM of ribavirin for 12 hours, after which supernatants were collected for subsequent plaque reduction assays.
  • HEVA71 IRES mutagenesis The pSMART ® LC ampicillin plasmid (Addgene) including the full length of HEVA71 strain 41 genome was constructed in our laboratory. Various HEVA71 IRES mutations were then generated via In-Fusion ® Cloning reactions (ClonTech ® Laboratories, 2014), where generated plasmids were further verified via automated DNA sequencings prior respective in-vitro transcriptions via MEGAscript ® T7 kit (Invitrogen). Following this, transcribed RNAs were reverse transfected into RD cells in 24-well plates with DharmaFECTTM transfection reagents for 12 hours before collection of viral supernatants consisting of corresponding mutated HEVA71 viruses.
  • bicistronic plasmids with distinct combinations of mutated HEVA71 IRES were transfected into RD cells in T-75 flasks for 48 hours. Following G418 selection after 6 days, RD cells containing each respective HEVA71 mutant IRES were reseeded into 96-well white plates (lxlO 4 cell per well), where they were treated with prunin (ImM) or vehicle control (1% DMSO) for 36 hours prior to respective luciferase measurements. siRNA and reverse transfection.
  • siRNAs targeting Sam68, hnRNPAl and hnRNPK were purchased from Dharmacon RNA Technologies (Thermo Scientific) and dissolved in diethyl pyrocarbonate (DEPC)-treated reverse osmosis (RO) water to obtain a stock concentration of IOOmM. Further dilutions to working concentrations were attained with DharmaFect Cell Culture Reagent (DCCR) and DharmaFect-1 transfection reagent. Reverse transfections of respective siRNA into RD cells in a 24-well plate format were carried out for 72 hours prior to WT or mutant HEVA71 infections.
  • DCCR DharmaFect Cell Culture Reagent
  • DharmaFect-1 transfection reagent Reverse transfections of respective siRNA into RD cells in a 24-well plate format were carried out for 72 hours prior to WT or mutant HEVA71 infections.
  • siRNA sequences are as follows: Sam68 (5'- GGACCACAAGGGAAUACAAUC-3'), hnRNPAl (5'-GGAGGGUUGAGCUUUGAAAUU-3') and hnRNPK (5'-AAUUCCUCCUGCUAGACUCUGAUGA- 3').
  • Respective wildtype and mutant HEVA71 IRES were biotinylated via in vitro transcriptions through MEGAscript ® T7 kit (Invitrogen). Briefly, 2mI of lOx MEGAscript ® T7 buffer, 2mI of MEGAscript ® T7 enzyme mix, ImI of RiboLock, ImI of 75mM ATP, ImI of 75mM GTP, ImI of 75mM CTP, 0.8mI of 75mM UTP and 1.5mI of lOmM Biotin-16- UTP (Thermo Scientific) were added with lOOng of respective T7 DNA templates (mutant and wildtype HEVA71 IRES) and incubated at 37°C for 4 hours.
  • RNA samples were subjected to RNA purification via Total RNeasy kit (Qiagen). Resultant HEVA71 mutant and wildtype IRES RNAs were stored at -80°C. Biotin-RNA pulldown assay. RD cells were disrupted with Nonidet P-40 (NP-40) lysis buffer [50mM Tris-HCI, 120mM sodium chloride and 1% NP-40] supplemented with Halt phosphatase, protease inhibitor cocktail (100X) and 0.5M EDTA (100X).
  • NP-40 Nonidet P-40
  • biotinylated RNA HEVA71 wildtype IRES, mutant IRES, GFP or actin
  • DTT 2mM 1,4 dithiothreitol
  • 111 RNAaseOUTTM Ribonuclease inhibitor Thermo Scientific
  • lpg/pl yeast tRNA Invitrogen
  • RNA-protein complexes were further added to 50mI of streptavidin magnetic beads (NEB; S1420) and incubated for another 30 minutes at room temperature to allow for binding. After washing thrice carefully with TBST, 60mI of 2.5X SDS-PAGE loading buffer was added to the beads in order to dissociate the proteins bound to the specific RNAs. The samples were then boiled at 95°C for 5 minutes prior to Western blot analyses via 10% SDS-PAGE gels.
  • Huh-7.5 cells 102 were cultured in Dulbecco's modified Eagle's Medium (DMEM, Wako) supplemented with 10% fetal bovine serum (FBS, Biowest), O.lmM non-essential amino acids (Invitrogen), 100 III penicillin per ml and 100 pg streptomycin per ml (Invitrogen), where they were infected with HCV J6-JFH1 strain (Accession no. JF343793.1) at a MOI of 1.
  • DMEM Dulbecco's modified Eagle's Medium
  • FBS fetal bovine serum
  • Invitrogen O.lmM non-essential amino acids
  • HCV J6-JFH1 strain accesion no. JF343793.1
  • HCV-infected cells were then treated with 2-fold increments of prunin from 62.5 to 500nM and a vehicle control (1% DMSO) for 3 and 6 days post infection, where cells were subsequently fixed with 3.7% paraformaldehyde for 10 minutes at room temperature, followed by permeabilization in 0.1% Triton X-100 in PBS for another 10 minutes at room temperature. Following this, cells were blocked with 5% goat serum and incubated with human monoclonal antibodies of a HCV-infected patient, where flourescein isothiocyanate- conjugated goat anti-human IgG (MBL) was utilized as a secondary antibody.
  • MBL flourescein isothiocyanate- conjugated goat anti-human IgG
  • RNA samples were counterstained with Hoechst 33342 solution (Molecular Probes) at room temperature for 10 minutes, after which they were mounted on glass slides and examined under a fluorescence microscope (BX51, Olympus) for HCV viral quantifications. Infected cells with drug treatments were also examined for their RNA contents, where total RNA was extracted via RNAiso (TaKaRa) according to the manufacturer's instructions. QuantiTect reverse transcription kit (Qiagen) was used to reverse transcribe isolated RNAs with random primers, and RT-qPCR analyses were followed with.
  • Hoechst 33342 solution Molecular Probes
  • BX51 fluorescence microscope
  • Primers used to quantify HCV RNA amounts included the sense (5'-TCTGCGGAACCGGTGAGTA-3') and antisense (5'- TCAGGCAGTACCACAAGGC-3') versions. The amount of HCV transcripts were then tabulated with respect to a standard curve consisting of serial dilutions of the Huh-7.5 transfected HCV J6/JFHl cDNA plasmid.
  • mice clinical scoring system was utilized to record the survival and severity of clinical manifestations observed in the infected mice that comprised of four criteria, namely activity, diarrhea, movement and body mass change. Scores of each criteria per mouse were added up to determine the total score on each day, where an absolute score of 6 was defined as the endpoint.
  • HEVA71-infected mice hind limb muscle tissues were harvested for histopathology studies and viral load quantifications via plaque reduction assays.
  • Prior histopathology studies harvested muscle tissues were subjected to fixation with 4% paraformaldehyde for 7 days at 4°C, followed with 2 hours decalcification at 25°C. After this, fixed muscle tissues were paraffin-embedded and sectioned into 4pm slices for respective detections of tissue damage by H&E stainings and HEVA71 antigen via IHC stainings using bondmax system.
  • Mouse anti-HEVA71 antibody (1:200, MAB979, Millipore) was utilized for IHC stainings.
  • FIG. 1A illustrates the possible model from which the HEVA71 IRES containing bicistronic luciferase reporter vector was generated. This model has shown efficacy in searching for potent antivirals against IRES mediated protein-synthesizing machinery without simultaneously affecting host translation 26 .
  • a HEVA71 IRES bicistronic luciferase reporter consisting of a human cytomegalovirus promoter sequence (CMV promoter) at its start point, with downstream Renilla Luciferase (R Luc) and Firefly Luciferase (F Luc) genes, which in turn flank the HEVA71 IRES site.
  • CMV promoter human cytomegalovirus promoter sequence
  • R Luc Renilla Luciferase
  • F Luc Firefly Luciferase
  • this reporter assay could likely determine compounds that exclusively inhibit IRES-mediated F Luc expression without influencing cap-dependent R Luc translation.
  • Another strategy of replacing the HEVA71 IRES element of the bicistronic construct with an IRES hairpin structure was also included, which was found to interrupt IRES activity 27 . This construct serves as a positive control, where cap-independent IRES translation of F Luc will be prevented with no effects on cap-facilitated R Luc protein synthesis.
  • HEVA71 IRES bicistronic reporter exhibited higher amount of IRES mediated F Luc production and similar amounts of cap-dependent R Luc expression in comparison to HEVA71 IRES bicistronic hairpin at both time points: 3.91 > 2.61 logioRLU at 12 hours and 4.57 > 2.71 logioRLU at 24 hours for F Luc whilst 3.38 « 3.22 logioRLU at 12 hours and 3.88 « 3.74 logioRLU for R Luc at 24 hours correspondingly ( Figures ID & IE).
  • heterogeneous G418 selection aided in (i) extending the drug-treatment window by 6 days for useful observation of downstream drug effects; (ii) reducing the heterogeneity of transfected population of RD cells, and thereby; (iii) increasing the reliability of the assay for drug-screening purposing, particularly for detecting HEVA71 IRES inhibitors.
  • Flavonoids are considered to be part of an omnipresent group of secondary metabolites in the plant kingdom, which are regularly consumed in the human diet 35 . Although flavonoids have similar structural morphologies, they are reported to perform a broad scope of biological activities such as antiviral, antioxidant, anti-cancer, anti- bacterial, anti-inflammatory activities and enzyme inhibition in eukaryotes 35-38 . Recent in vitro studies over the years have shown that flavonoids have anti-viral activity against a wide plethora of viruses such as Dengue virus type 2, human immunodeficiency virus type 1, influenza virus and many human enteroviruses 37,39-42 . Considering flavonoids as abundant and ubiquitous sources in plants, the 502-compound flavonoids library made an excellent choice for the initial screening of useful antivirals via the luciferase-screening assay.
  • HEVA71 I RES bicistronic reporter-transfected cells were exposed to flavonoids at a concentration of 20pg/ml and were subsequently measured for their respective cell viabilities and luciferase activities.
  • the normalized ratios of I RES to CMV expressions were calculated and converted to percentage inhibitions of HEVA71 I RES activity for each flavonoid.
  • Positive controls such as the HEVA71 I RES bicistronic hairpin and amantadine were also included in this study.
  • the activity of prunin against HEVA71 through a series of downstream validations were examined and profiled.
  • the cytotoxicity profile of prunin by treating RD cells with a wide range of doses, ranging to a maximum concentration of 5mM were characterised. After 24 hours of drug treatment, the relative cell viabilities for each dose were measured using cell viability assay (Figure 4B). With a threshold of 80% cell viability, we found only concentrations of prunin below ImM to be well tolerated by the RD cells.
  • prunin displayed potent antiviral activity against HEVA71.
  • prunin was discovered from a luciferase- based assay targeting IRES elements, it was hypothesized that prunin would be able to suppress other members of Enteroviruses, due to the similarities in IRES structures shared among them 10 . Therefore, the antiviral spectrum of pruning was characterised, if its effects were specific to Enteroviruses only or to other viruses as well.
  • RD or SJ cells were initially infected with each of the Enteroviruses such as low passage HEVA71 clinical isolates, HEVA71 strain H, HEVA71 strain B5 genotype, HEVA71 strain C4 genotype, CA6, CA16, ECH07, CB5, CA24, HRV10; and other viruses such as HSV and CHIKV with an appropriate vehicle control (1% DMSO).
  • Enteroviruses such as low passage HEVA71 clinical isolates, HEVA71 strain H, HEVA71 strain B5 genotype, HEVA71 strain C4 genotype, CA6, CA16, ECH07, CB5, CA24, HRV10; and other viruses such as HSV and CHIKV with an appropriate vehicle control (1% DMSO).
  • EC50 of Prunin 115.3nM
  • respective HEVA71 viral titres were quantified for each virus through plaque reduction assays ( Figure 41).
  • Enterovirus A HEVA71, CA6 and CA16
  • Enterovirus B EH07 and CB5
  • distantly related Enteroviruses such as CA24 and HRV10, which belong to respective Enterovirus C and Rhinovirus A species were not affected by prunin treatments. This may suggest that the mechanistic action of prunin on IRES could be primarily due to its indirect effects on certain ITAFs rather than its direct effects on IRES structure.
  • CHIKV is an alphavirus of the Togaviridae family, consisting of an RNA genome that is capped and expressed only through cap-dependent translation 43 while HSV contains a double-stranded DNA genome with no IRES elements 44 .
  • cytotoxicity assays were also performed to ensure that SJ cells used for CHIKV infections were not vulnerable to prunin at a dose of 115.3nM (see Figure 8). These results therefore classify prunin as a limited spectrum class of Enterovirus suppressor against Enterovirus A (HEVA71, CA6 and CA16) and Enterovirus B (ECH07 and CB5) species.
  • the above treatment was also applied to the negative control group, which comprised of HEVA71 infected suckling mice treated with the vehicle control (PBS) instead of prunin.
  • PBS vehicle control
  • These HEVA71 infected mice were then monitored on a daily basis for 14 days post infection (dpi), where survival rate (Figure 5A) and clinical manifestations (Figure 5B) were recorded. It was observed that the infected mice treated with the vehicle control and lmg/kg of prunin exhibited a 100% mortality rate by 7 dpi, whilst those with higher doses of prunin treatments (3 and lOmg/kg) had extended lifespans till 14 dpi.
  • mice infected with HEVA71 were presented with severe clinical symptoms such as ruffled hair, huddling up, sedentary appearance, limb weakness, rapid body weight loss and hind limb paralysis over time. These symptoms were quantified via the mice clinical scoring system (see Table 6), where mean clinical scores in the vehicle and prunin (lmg/kg) treated infected mice accelerated rapidly to a higher value as compared to that of the prunin treated (3 and lOmg/kg) infected mice by 7 dpi.
  • mice inactivity, hunched back, ruffled fur and limb weakness
  • 3mg/kg and lOmg/kg of prunin inactivity, hunched back, ruffled fur and limb weakness
  • mice completely recovered from HEVA71 infection and were healthy by 13 dpi (3mg/kg) and 9 dpi (lOmg/kg).
  • body weights ( Figure SC) of mice exposed to prunin (1 to lOmg/kg) continuously on a per day basis via i.p. injections for 14 days were measured, which were used as indicators for possible cytotoxic effects that could be caused by prunin.
  • mice with prunin treatments showed progressive increases of approximately 5.61 to 5.83g from 0 to 14 days, which was similar to the ones treated with PBS, where rise in body weight of 5.10g was seen till 14 days post-treatment. Overall, these data indeed proved that prunin at all doses had negligible cytotoxicity on the suckling BALB/c mice and possessed protective efficacy in HEVA71 infected mice.
  • mice clinical scoring system in EV71 BALB/c mice model mice clinical scoring system in EV71 BALB/c mice model.
  • H&E stainings Figure 5E
  • mice treated with PBS displayed loss of muscle fibers leading to severe damage of their hind limb muscle tissues.
  • mice were subjected to serious inflammation issues following HEVA71 infection as recognized by massive infiltration of immune cells into their hind limb muscles.
  • the mutant version of HEVA71 increased steadily from approximately 5.8 to 7 logio PFU/ml, even though WT HEVA71 exhibited a dose- dependent reduction (4.3 to 3.4 logio PFU/ml) with increasing concentrations of prunin (115.3 to lOOOnM).
  • ribavirin a broad-spectrum nucleoside analogue, namely ribavirin, which was elucidated as a potent HEVA71 3D polymerase suppressor both in vitro and in v/Vo 46-49 .
  • ribavirin was first established as an inhibitor of viral RNA replication via causing lethal mutagenesis in vitro 50 , which further showcased anti-HEVA71 properties in vivo, where it reduced HEVA71- facilitated paralysis and mortality events 51 .
  • Prunin displayed inhibitory activity against HCV that consists of a different type of IRES element. Due to the exemplary activity of prunin against HEVA71 IRES both in vitro and in vivo, we wanted to investigate if these effects were exclusive to a particular group of IRES RNAs or could be observed against diverse viral IRES structures. Scientific literature has categorized viral IRESs into 4 major groups, namely groups 1 to 4, according to their respective requirements for various host factors, hypothesized secondary structures, positions of the start codon relative to IRESs and competence of IRESs to function in rabbit reticulocyte extracts with or without supplementations 52 .
  • HCV eukaryotic initiation factors
  • prunin can suppress HCV infection, probably through employing a similar anti-IRES strategy against HCV, since both viruses contain IRES elements. This also demonstrates the ability of prunin to be a broad-spectrum IRES inhibitor, at least for group 2 and 3 IRESs.
  • Prunin-resistant HEVA71 differentially regulate IRES-mediated activity via Sam68 and hnRNPAl without hnRNPK. Given that earlier results demonstrated that prunin induced major folding changes in HEVA71 mutant IRES secondary structure, the differences in ITAF recruitments between the mutant and WT HEVA71 IRES were investigated. Specifically, since stem loops 2 and 4 of mutant HEVA71 IRES showcased considerable structural modifications that played roles in prunin resistance, it was questioned which of the reported ITAFs have been validated to bind to those regions and also interact with each other.
  • 3 ITAFs were uncovered, namely heterogeneous nuclear ribonucleoprotein A1 (hnRNPAl), hnRNPK and Src-associated in mitosis 68-kDa protein (Sam68).
  • hnRNPAl heterogeneous nuclear ribonucleoprotein A1
  • hnRNPK hnRNPK
  • Src-associated in mitosis 68-kDa protein (Sam68).
  • all 3 ITAFs have been well documented as RNA-binding proteins due to their dual RNA-binding domains, including an RGG box 54 and a KH domain 55 ' 56 , which also enable them to act as nuclear-cytoplasmic shuttlers for various molecular and cellular functions 57 .
  • these 3 ITAFs have been classified as positive regulators of HEVA71 IRES facilitated translation 58-60 , where they were found to interact with different regions of HEVA71 5' UTR.
  • hnRNPAl was elucidated to bind to stem loop 2 of IRES 58
  • Sam68 was recently discovered to specifically interact with stem loop 4 of IRES 59
  • the KH2 domain and proline-rich domain with a neighboring KH domain of hnRNPK was found to maintain contacts with both stem loops 2 and 4 of HEVA71 IRES 60 .
  • both mutant and WT HEVA71 viral titres in siRNA-treated RD cells were quantified via plaque reduction assays (Figure 8E).
  • knockdown of hnRNPAl resulted in both WT and mutant HEVA71 viral titres to drop equally by approximately 4.0 logio PFU/ml.
  • biotin-RNA pulldown assays involving either the WT or mutant HEVA71 IRES, where we probed for hnRNPK, Sam68 and hnRNPAl protein interactions with the respective biotinylated RNAs. Though all 3 ITAFs were seen to specifically interact with both types of biotinylated IRES but not control RNAs (non- biotinylated IRES, GFP and actin), differences in interactions of hnRNPK and Sam68 with mutant HEVA71 IRES were observed.
  • hnRNPK associated with the mutant IRES In comparison to WT IRES, hnRNPK associated with the mutant IRES to a lesser extent, which allowed Sam68 to compensate for the above lack of interaction via increasing its association with mutant IRES RNA (Figure 8F).
  • amantadine hydrochloride was identified as a HEVA71 IRES inhibitor through a similar bicistronic reporter construct approach 26 , which was as a second positive control, which further affirmed the functional characteristics of our bicistronic constructs. It should be noted that amantadine has not been yet validated in murine models or clinical trials for its anti-HEVA71 efficacy. In order to extend the limitations of our transiently transfected bicistronic systems, their durations were extended and increased their consistencies of gene expressions via a process known as heterogeneous selection. Following the establishment of optimal time settings for the assay, its robustness was evaluated via statistical criteria known as Z-factors 66 , which determined the qualities of the systems to be excellent platforms for downstream drug studies.
  • HEVA71 structural proteins VP0 and VP2
  • RNA amounts at 6 and 12hpi after prunin treatments.
  • prunin was discovered to have equivalent potencies against other closely related Enteroviruses A and B such as CA6, CA16, ECH07, CB5 that predominantly cause HFMD, with no effects exerted on distantly related Enteroviruses (CA24 and HRV10) and other viruses including CHIKV and HSV.
  • Prunin is a natural compound that originated from immature citrus fruits, specifically from the Citrus aurantium and Citrus compassion 68 ' 69 comprising of oranges, tangerines, limes, lemons and grapefruits 70 ; and is also found in minute amounts in tomatoes 71 .
  • an intrinsic advantage showcased by prunin is the ease of extraction, purification and processing for large-scale productions 70 .
  • prunin has not been extensively studied in scientific literature, albeit related compounds such as phloridzin and naringenin have been researched for their respective anti-diabetes and chondroprotective purposes 72 ' 73 .
  • HEVA71 RNA amounts as those proteins play critical roles in catalyzing HEVA71 RNA replication.
  • examples include HEVA71 2C protein that has been shown to aid in negative RNA strand synthesis 78 and development of replication complexes 79 ; HEVA71 3D protein that codes for a RNA polymerase that produces numerous positive RNA strands 80 ; and HEVA71 3A protein which has been reported to induce RNA polymerase activity 81 .
  • the present screen managed to pick up 5 other significant hits (ST024699, ST024368, ST024702, ST066904 and ST024081) along with prunin that reduced infectious HEVA71 viral titre, although ST002086 from the initial screen was found not to exhibit any effects on HEVA71. Further understanding of the individual structures and classifications of these flavonoids aided us in determining the possibility of the above-mentioned difference.
  • flavonoids There are 4 main classifications of flavonoids (see Figure 9), namely the isoflavones, neoflavonoids, chalcones and another huge group (F2) consisting of flavones, flavonols, flavanones, flavanonols, flavanols and anthocyanins 85 .
  • F2 flavonols
  • ST002086 was placed in the chalcones group of flavonoids that consisted of open carbon ring structures, while the other 6 flavonoids belonged to the large F2 group, comprising of B rings attached to position 2 of the carbon on another C ring.
  • ST077105 (highlighted), also known as prunin, is a flavanone that was chosen for further downstream assays.
  • Prunin could possibly suppress HEVA71 IRES via directly intercalating between the bases of the IRES RNA at certain regions or stem loops, thus rendering an incompetent IRES secondary RNA structure, which would indirectly inhibit one or more ITAFs from IRES interactions, given that ITAFs are prudent for the facilitation of cap-independent translation 13 .
  • This concept was first demonstrated by a drug known as quinacrine 86 that was capable of suppressing Hepatitis C virus IRES-facilitated translation in vitro via intercalating into IRES RNA and affecting downstream ITAF interactions.
  • the drug In line with its in vitro properties, the drug also established high in vivo efficacies in mice models of HEVA71 infection, which could be further characterized for preclinical development.
  • HEVA71 in murine models, namely pleconaril 92 and lactoferrin 93 against HEVA71 VP1 protein, rupintrivi r 94 and chrysin 95 targeting HEVA71 3C pro , lycorine and 1-acetyllycorine 96 inhibiting HEVA71 2A pro and lastly ribavirin 51 and NITD008 97 suppressing HEVA71 3D polymerase.
  • prunin together with the cell-based bicistronic vector systems can be utilized as a gene translational regulation system in vitro via the inhibitory effects of prunin on IRES-mediated translation.
  • Traditional inducible gene reporter systems consist of 3 components: (i) a promoter that can be easily activated akin to a lac promoter 98 or a TRE-CMVmin promoter 99 ; (ii) a protein activator and/or repressor of transcription such as the tTa transactivator, which aids in TRE-CMVmin activation or the lad repressor that inhibits the activity of the lac promoter respectively; and (iii) small-molecule regulators including IPTG or tetracycline, which mediates interactions among protein transcription activators or repressors and their associated promoters.
  • the problem of this system is its stringent requirement and need for unique promoters along with specific transcription factors and regulators.
  • This can be resolved by exploiting the simplified IRES repressor assay, which takes into account the exclusive properties of prunin, HEVA71 IRES bicistronic reporter and HEVA71 IRES bicistronic hairpin, where no definitive promoters or protein factors are needed.
  • this bicistronic gene regulation system can concurrently express reporter and antibiotic selection genes via cap-dependent translation, which can be used for reaffirming transfection efficiencies and selecting heterogeneous cell- lines respectively.
  • the IRES repressor system can function as a high- throughput screening platform against numerous drug libraries, where more HEVA71 antivirals like prunin can be identified in order to meet the demands for potent HEVA71 therapies.
  • antiviral flavonoids such as prunin

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Abstract

La présente invention concerne des compositions destinées au traitement d'une infection à entérovirus ou d'un trouble induit par un entérovirus. Selon un aspect, la présente invention concerne l'utilisation d'une molécule telle qu'un flavonoïde qui inhibe ou empêche une interaction entre au moins un facteur trans-activateur IRES (ITAF) et une partie IRES d'un entérovirus pour traiter une infection à entérovirus. En particulier, une composition comprenant de la prunine (ST077105) permet de supprimer l'entérovirus humain 71 (HEVA71) dans des modèles cellulaires et murins. Un effet suppresseur similaire est également observé dans des cellules infectées par le virus de l'hépatite C et le virus Coxsackie.
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