WO2020022961A1 - Inhibitors of ires portion of an enterovirus - Google Patents

Abstract

The present invention relates to compositions for treating an enterovirus infection or an enterovirus induced disorder. In one aspect of the present invention, there is provided the use of a molecule such as flavonoid that inhibits or prevents an interaction between at least one IRES trans-acting factor (ITAF) and an IRES portion of an enterovirus for treating an enterovirus infection. In particular, a composition comprising prunin (ST077105) can suppress the human enterovirus 71 (HEVA71) in cell and murine models. Similar suppressive effect is also observed in cells infected with Hepatitis C virus and Coxsackie virus.

Description

INHIBITORS OF IRES PORTION OF AN ENTEROVIRUS

The present invention relates to compositions for preventing or treating an enterovirus infection or an enterovirus induced disorder.

Human Enterovirus 71 (HEVA71) was discovered in the late 1960s in patients of California, who exhibited severe symptoms of the central nervous system (CNS) diseases¹. Since then, large-scale epidemics of HEVA71 were observed in Asia-Pacific countries, which involved millions of young children below S years old^2-4. These children were diagnosed with mild and self-limiting hand, foot and mouth disease (HFMD) that begun by the emergence of fever and blisters on their hands, feet and mouth cavities. Overtime, a substantial subset displayed serious neurological deficits in the form of aseptic meningitis, brain stem encephalitis and poliomyelitis-like acute flaccid paralysis, ultimately inducing fatality or permanent neurological sequelae in them.^5,6 With the present lack of authorized antivirals for HEVA71⁷'⁸ along with the eradication of poliovirus through triumphal vaccinations⁹, HEVA71 has qualified as the next vital non-polio neurotropic virus worldwide.

As a representative of Picornaviridae family of viruses, 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. These IRES elements were initially identified in Picornaviridae RNA viruses with long 5' UTRs lacking a 5' cap structure^11,12. Through folding into secondary RNA structures, IRES substitutes the functions of some host translation initiation factors, thereby permitting cap-independent translation of viral proteins¹³. 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 translation¹⁴. 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. This facilitated the expression of 2 proteins concurrently, a reporter gene and another gene of interest respectively. Several advantages can be obtained from using IRES in mediating gene translation: (i) it aids in establishing simultaneous expression of the pair of cistrons in more than 90% of the cells¹⁷; (ii) the ratio of protein expression by both cistrons positioned before and after IRES are not affected¹⁷'¹⁸; (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 checkpoints²³. Overall, these features of IRES enable it to be an advantageous element in genetic engineering.

Here, reverse genetics to generate a functional bicistronic reporter vector consisting of HEVA71 IRES, where cap-dependent translation could be distinguished from HEVA71 IRES- mediated translation were employed. Recently, natural products such as artemisinin and its derivatives from Artemisia plants were found to be effective against malaria parasites, which were successfully introduced into the clinical market as a FDA-approved drug for malaria²⁴. This prompted an investigation into the effects of natural compounds like flavonoids on HEVA71 IRES-mediated translation, which set us on a path to address the compelling demands for HEVA71 antiviral options through screening the HEVA71 IRES bicistronic construct with a 502-compound flavonoid derivatives library for potent antivirals opposing HEVA71 IRES without affecting host cell translation. Our results identified prunin, which effectively suppressed HEVA71 in cell culture and murine models, thus professing its potential for development into HEVA71 therapeutics. On a mechanistic point of view, the analysis of HEVA71 resistance profile displayed 5 mutations in its IRES region, hence reaffirming its IRES- inhibition properties. Further evaluations of prunin-induced IRES suppressions led us to unravel prunin as a possible antagonist of hnRNPK, an IRES-trans acting factor that is involved in HEVA71 IRES mediated translation.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. Any document referred to herein is hereby incorporated by reference in its entirety.

In an aspect of the present invention, there is provided 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.

By "molecule", it is meant to include any purified or isolated natural or chemically-synthesised entity. Preferably, 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. In addition to examples that are provided in detail below, the molecule may be amantadine, quinacrine, sorafenib and idarubicin.

By "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.

In various embodiments, the molecule inhibits the interaction between the at least one IRES ITAF and the IRES portion of the virus by protein-protein steric hindrance.

Preferably, the term internal ribosome entry site, abbreviated IRES, 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. Usually, in eukaryotes, translation can only be initiated at the 5' end of the mRNA molecule, since 5' cap recognition is required for the assembly of the initiation complex. 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.

Preferably, 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%.

Preferably, 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.

Preferably, the enterovirus is HEVA71, and the hepacivirus is Hepatitis C virus.

Preferably, 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.

In another aspect of the present invention, there is provided the use of 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.

Preferably, the ITAF is selected from the group consisting of hnRNPK, hnRNPAl and Sam68. In various embodiments, the molecule can inhibit any one of the ITAF listed in the group above or in combination with any one from the group. In a preferred embodiment, the molecule exerts a stronger inhibitory effect on hnRNPK.

Preferably, the enterovirus is HEVA71, and the hepacivirus is Hepatitis C virus.

Preferably, 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.

In another aspect of the present invention, there is provided 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.

Preferably, the composition is a unit dosage containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of the active ingredient. Preferably, 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.

In human therapy, the 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. Typically 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. In a more specific form of the disclosure there are provided pharmaceutical compositions comprising therapeutically effective amounts of the flavonoid together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such 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.

It will be appreciated that pharmaceutical compositions provided according to the disclosure may be administered by any means known in the art. Preferably, 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.

The term "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. Possible examples of 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, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

The pharmaceutical formulations of the disclosure, which may conveniently be presented in unit dosage form, 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. In addition to the ingredients particularly mentioned above, 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.

Combination therapy with an additional therapeutic agent is also contemplated by the disclosure. For example, the pharmaceutical composition may further comprise a co-agent having anti-enterovirus properties. Preferably, 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 Preferably, the sustained release is provided over at least 4 weeks.

In yet another aspect of the present invention, there is provided a flavonoid for use in preventing or treating an enterovirus infection or an enterovirus induced disorder.

In yet another aspect of the present invention, there is provided a method of preventing or treating an enterovirus infection or an enterovirus induced disorder, wherein the method comprises administering a therapeutically effective amount of a flavonoid to a subject in need thereof.

In yet another aspect of the present invention, there is provided 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.

Preferably, 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.

Preferably, the IRES is a HEVA71 IRES, and the reporter genes encode a fluorescent protein.

It is appreciated that in the method described herein, which may be drug screening methods, a term well known to those skilled in the art, the test agent may be a drug-like compound or lead compound for the development of a drug-like compound.

The term "drug-like 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. Thus, for example, 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.

The term "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.

As such, 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.

Preferably, the library of molecules is a library of flavonoids.

In another aspect of the present invention, there are provided HEVA71 mutants that may be used as platforms to find novel drugs against the particular regions of HEVA71 mutant IRES to overcome drug resistance. In terms of drugs, 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. It may be possible to generate a RNA vaccine (similar to mRNA vaccine), where mutant HEVA71 IRES can be delivered via liposomes as an adjuvant to generate a host immune response against it.

Advantageously, 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.

Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

As used in this specification and claim(s), 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.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

In order that the present invention may be fully understood and readily put into practical effect, there shall now be described by way of non-limitative examples only preferred embodiments of the present invention, the description being with reference to the accompanying illustrative figures.

In the Figures:

Figure 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. (C) Schematic illustration of the 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. Relative (D) IRES (Firefly) and (E) CMV (Renilla) activities of both HEV71 IRES bicistronic reporter (BICIS) and HEV71 IRES bicistronic hairpin (BICIS HP) at 12 and 24 hours post-transfection. Normalized relative IRES (Firefly), CMV (Renilla) and IRES:CMV (F Luc : R Luc) activities of both (F) HEV71 IRES bicistronic reporter and (G) HEV71 IRES bicistronic hairpin at 24 hours post-transfection with 1 and 2mM of amantadine hydrochloride. Statistical analyses were performed with one-way ANOVA corrected using Dunnett's post-test with Graphpad Prism 6.0. **** represents p<0.0001; *** represents p<0.001; ns represents not significant.

Figure 2. 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 ). Relative (C,E) IRES (Firefly) and (D,F) CMV (Renilla) activities of HEV71 IRES bicistronic reporter (BICIS) or HEV71 IRES bicistronic hairpin at 3 time-points (24, 48 and 72 hours) after 0, 4, 6 and 8 days selection with 0.25 mg/ml of G418. Relative IRES:CMV (F Luc : R Luc) activities and calculated Z-factors of (G) BICIS and 2mM amantadine-treated BICIS; and (H) BICIS and BICIS HP at 48 hours after 6 days post-selection. Data are expressed as the averages with error bars representing ±SD from 3 independent experiments consisting of triplicates. Statistical analyses were performed accordingly with one-way or two-way ANOVA corrected using Dunnett's post-test or Tukey's post-test with Graphpad Prism 6.0.**** represents p<0.0001; *** represents p<0.001; ns represents not significant.

Figure 3. 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.

Figure 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). (D) Tabulated EC50 of prunin. Red dotted lines represent corresponding prunin dose (115.3nM) for 50% inhibition in HEV71 viral titre. Disruption of HEV71 viral protein production by prunin (31.25 to lOOOnM) measured at (E) 6hpi and (F) 12hpi via Western blot and band intensities of VP2 (red solid lines) and VP0 (green dotted lines). Disruption of HEV71 viral RNA synthesis by prunin (31.25 to lOOOnM) at (G) 6hpi (red bars) and (H) 12hpi (green bars) measured through qRT-PCR. (I) Inhibition profiles of prunin (31.25nM) against HEV71 clinical isolates, HEV71 strain H, HEV71 strain B5 genotype, HEV71 strain C4 genotype, CA6, CA16, ECH07, HSV and CHIKV through respective viral titre quantifications with plaque assays at 12hpi.l% DMSO were used as vehicle controls accordingly. Data are expressed as the average values with error bars representing ±SD from 3 independent experiments consisting of triplicates. Statistical analyses were performed against 1% DMSO, with one-way ANOVA corrected using Dunnett's post-test with Graphpad Prism 6.0. **** represents p<0.0001; *** represents p<0.001; ns represents not significant.

Figure 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. (E) H&E staining showing minimal muscle tissue damage in prunin-treated infected mice at 7dpi. (F) IHC staining indicating little viral antigen presence in prunin-treated infected mice at 7dpi. PBS was used as treatment controls.

Figure 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. **** represents p<0.0001; *** represents p<0.001; ns represents not significant. (C) Differences in IRES RNA secondary structures of stem loops 2 and 4, between wildtype and mutant HEV71. Sites of mutations (T164C, G165C, G368C, T370G and C177T) and causative variations in structures are marked in boxes.

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. Only HCV RNA quantifications at 6dpi were signficant relative to the vehicle control. 1% DMSO were used as vehicle controls accordingly. Data are expressed as the average values with error bars representing ±SD from 3 independent experiments consisting of triplicates. Statistical analyses were performed against 1% DMSO, with one-way ANOVA corrected using Dunnett's post-test with Graphpad Prism 6.0. **** represents p<0.0001; *** represents p<0.001; ** represents p<0.01; and * represents p<0.05.

Figure 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]

Due to a lack of effective antivirals against HEVA71, 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. Continuous HEVA71 passaging with prunin yielded HEVA71-resistant mutants, in which 5 mutations were mapped to the IRES region. Knockdown studies revealed that the mutations allowed HEVA71 to overcome drug-induced suppression via differentially regulating recruitments of IRES-trans acting factors (ITAFs), namely Sam68 and hnRNPK, without affecting hnRNPAl interaction. Furthermore, prunin effectively impeded HEVA71- associated clinical symptoms and mortality in HEVA71-infected BALB/c mice. Interestingly, prunin suppressed Hepatitis C virus (HCV) at higher concentrations, suggesting a similar mechanism of drug-mediated IRES inhibition on both viruses. These discoveries establish prunin as a suitable clinical candidate for further development into a HEVA71 therapeutic agent.

Example 1

1. Methods and materials

Cells and viruses. Human RD (CCL-136, ATCC) and African green monkey kidney epithelial Vero cells (CCL-81, ATCC) were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich) while cell lines including SJCRH30 (SJ) (CRL-2062, ATCC) and BHK-21 (BHK) (CCL-10, ATCC) were cultured in Roswell Park Memorial Institute 1640 medium (RPMI-1640; Sigma-Aldrich). Medias were supplemented with 10% fetal calf serum (FCS; PAA) and 2g of sodium hydrogen carbonate. HEVA71 used in this study include HEVA71 strain 41 (Accession no. AF316321.2), HEVA71 strain H (VR-1432, ATCC; Accession no. AY053402.1), HEVA71 genogroup B5 strain (Accession no. FJ461781.1), HEVA71 genogroup C4 strain (Accession no. JQ965759.1), and clinical isolates belonging to KK woman's and Children's hospital. Coxsackievirus A6 (Accession No. KC866983.1), Coxsackievirus A16 (Accession No. U05876), Coxsackievirus A24 (Accession No. KF725085.1), Coxsackievirus B5 (Accession No. JX843811.1), Echovirus 7 Wallace strain (Eo7-Wallace; Accession no. AF465516), Human Rhinovirus A10 (Accession No. JN541269.1), Chikungunya virus (Accession No. FJ445502.2), and Herpes Simplex Virus 1 (Accession No. JQ673480.1) were also utilized in this study. Apart from CHIKV and HSV that were propagated in SJ and BHK cells respectively in RPMI with 2% FCS, other viruses were cultivated in RD or Vero cells in DMEM with 2% FCS. All live cell culture experiments were incubated in humidified incubator with growth settings of 37°C and 5% C0₂, except HRV10 (33°C and 5% C0₂).

Plasmids. HEVA71 IRES bicistronic reporter, HEVA71 IRES bicistronic hairpin and various HEVA71 mutant IRES bicistronic plasmids were modified from the bicistronic constructs that were kindly gifted by Professor Peter C McMinn of University of Sydney. 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 IRES²⁷ 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'. All PCR amplifications were carried out with the Q5^® Hot Start High-fidelity 2X Master Mix. The linearized pCTAP vector and respective target luciferase genes were fused together through In-Fusion^® Cloning reactions (ClonTech^® Laboratories, 2014). Generated plasmids were further verified via automated DNA sequencing with different sets of primers (See Table 5 below).

* All are forward primers

^L Primers were designed to overlap regions to increase accuracy of DNA sequencing

Table 5. The primers (H EV1-16) used for DNA sequencing of the modified H EV71 I RES bicistronic reporter construct. The primer (H EVh4, shaded in grey) replaced H EV4 for the DNA sequencing of the modified H EV71 I RES bicistronic hairpin.

Transfection and antibiotic selection. RD cells were seeded at a density of 5xl0³ to lxlO⁴ cells per well in 96-well white plates or 7.5xl0⁶ to lxlO⁷ 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. Briefly, 500ng (96-well white plate) or 50pg (T-75) of respective plasmids were dissolved in 5mI or 500mI of jetPRIME^® buffer, after which ImI or IOOmI of jetPRIME^® reagent was added to the corresponding mixtures. These mixtures were then added with IOOmI per well or 10ml per flask of fresh growth medium (DMEM with 10% FCS). Transfections in 96-well white plate formats were carried out for 12 or 24 hours while transfections in T-75 flasks were performed for 48 hours. Following 48 hours, cells in T-75 flasks were treated and selected with 10ml of fresh growth media containing 0.25mg/ml (per flask) of G418 antibiotic for 0, 4, 6 and 8 days, respectively. These cells were later trypsinized and seeded onto 96-well white plates at a density of 5xl0³ to lxlO⁴ cell per well with appropriate growth media for 24, 48 and 72 hours, respectively. All transfected RD cells were measured for their R Luc and F Luc activities via luciferase assays.

Compound screening. 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. After 6 days of post-G418 selection, HEVA71 IRES bicistronic reporter or HEVA71 IRES bicistronic hairpin transfected RD cells were trypsinized and seeded into 96-well white or transparent plates accordingly. Following 12 hours of incubation, the growth medium of each well was replaced with 50mI of DMEM with 2% FCS containing 20pg/ml of each flavonoid or 2mM of amantadine. Plates were subsequently measured for their respective cell viabilities and luciferase activities at 36 hours post-treatments.

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).

Cell viability assay. Cytotoxicity profiling was performed in 96-well transparent plates with alamarblue^® (Invitrogen^®) cell viability reagent. Phosphate buffered saline (PBS) was used to wash each well twice prior to the addition of 100mI of alamarblue^® mixture, which consisted of 10mI of alamarblue^® reagent and 90mI of DMEM with 2% FCS. After 2 to 4 hours of incubation in the dark, these plates were measured for corresponding cell viabilities with the TECAN™ Reader (Infinite^® 200 PRO series) at excitation and emission wavelengths of 570nm and 585nm, respectively.

Preparation of drugs. 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.

Post-treatment assays. RD cells were seeded at a density of 5xl0³ to lxlO⁴ cells per well in 96-well transparent plates or 5xl0⁴ 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. After washing twice with PBS, 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). Following 12hpi, respective plates were subjected to 2 cycles of freeze-thawing (-80°C; 37°C) and the respective supernatants were transferred to 24-well plates with seeded RD cells for viral titre quantifications through plaque reduction assays. 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.

For spectrum activity studies of prunin, 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. 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.

Viral plaque assays. RD or SJ or BHK or Vero cells were seeded in 24-well plates at seeding density of 5xl0⁴ 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. Plates with 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).

Plaque purification. 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.

Cell extract preparations and western blot analyses. Following post-treatment assays of HEVA71-infected RD cells with a range of prunin concentrations (31.25nM to ImM) and siRNA knockdown studies for 6 and 12 hours, plates were subjected to 2 ice-cold PBS washes prior cell lysis. Cell extracts were prepared with ice-cold cell lysis buffer cocktails, which comprised of Halt phosphatase, protease inhibitor cocktail (100X), 0.5M EDTA (100X) and Mammalian Protein Extraction Reagent (mPER) (Thermo Scientific). These lysates were then quantified for their protein amounts through Bradford protein assays (Thermo Scientific) before subsequent loading into polyacrylamide gels along with the designated protein ladder (Thermo Scientific). Loading dye (4X) or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (4X) was added to each sample and incubated at 95°C for 5 minutes, for protein denaturation. SDS-PAGE was performed for 2 to 3 hours and the divided protein constituents of the polyacrylamide gels were transferred onto respective nitrocellulose membranes through the Trans-Blot^® Turbo™ Transfer System (Bio-Rad).

Following this, 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). Three 10-minute washes with Tris-buffered saline/0.1% Tween-20 (TBST) were subsequently performed on the membranes prior to another hour incubation with blocking reagent containing polyclonal goat anti-mouse or anti rabbit IgG (H+L) antibody conjugated with horseradish peroxidase (1:2500, HRP, Thermo Scientific). Following washing again, membranes were later exposed to an enhanced chemiluminescent substrate (Thermo Scientific) for 5 minutes. Separated protein bands were viewed via the C-DiGit Chemiluminescence Western Blot Scanner (LI-COR). In the case of reprobing, bound antibodies were effectively removed with Restore PLUS stripping buffer (Thermo Scientific).

Quantitative RT-PCR (qRT-PCR). Following post-treatment assays of HEVA71-infected RD cells with a range of prunin concentrations (31.25nM to ImM) for 6 and 12 hours, 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' UTR¹⁰⁰, which were MD90 (5'-ATTGTCACCATAAGCAGCCA-3') and MD91 (5'-CCTCCGGCCCCTGAATGCGGCTAAT-3'). A housekeeping gene, namely actin, was also detected with forward primer (5'-AGCGCGGCTACAGCTTCA-3') and reverse primer (5'- GGCGACGTAGCACAGCTTCT-3'), which quantified total RNA in cell lysates.

HEVA71 resistant mutants. RD cells were seeded at a density of 5xl0⁴ 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). Continuous culturing of WT HEVA71 with 115.3nM of prunin was performed till passage 13, after which drug doses were increased to 500nM from passages 14 to 16 and lOOOnM from passages 17 to 19, respectively. Negative control of non-drug treated HEVA71 (1% DMSO) and positive control of WT HEVA71 were also included. Viral titres of WT and mutant HEVA71 from every passage were quantified via plaque reduction assays.

In order to discover mutations that were vital for HEVA71 prunin-resistance, 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 primers¹⁰¹, 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.

Synergistic studies. 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 DharmaFECT™ transfection reagents for 12 hours before collection of viral supernatants consisting of corresponding mutated HEVA71 viruses. Meanwhile, 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 (lxlO⁴ 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. The 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. Following 12hpi, corresponding viral supernatents and cell lysates were collected for plaque reduction assays and Western blot analysis, respectively. The siRNA sequences are as follows: Sam68 (5'- GGACCACAAGGGAAUACAAUC-3'), hnRNPAl (5'-GGAGGGUUGAGCUUUGAAAUU-3') and hnRNPK (5'-AAUUCCUCCUGCUAGACUCUGAUGA- 3').

Generation of biotinylated RNAs. 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. After removing excess DNA via MEGAscript^® Dnase (ImI) for 15 minutes at 37°C, 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). Following sonication of cell lysates at 20kHz for 30 seconds on ice, harvested cell extracts were incubated with respective O.ImM of biotinylated RNA (HEVA71 wildtype IRES, mutant IRES, GFP or actin) for 30 minutes at room temperature in a RNA binding buffer consisting of 2mM 1,4 dithiothreitol (DTT; Sigma Aldrich), 111 RNAaseOUT™ Ribonuclease inhibitor (Thermo Scientific) and lpg/pl yeast tRNA (Invitrogen). Non-biotinylated RNAs were used as negative controls to affirm the specificity of biotin pulldown assays. The mixtures of respective 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.

HCV studies. Huh-7.5 cells¹⁰² 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. 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. After washing with PBS, stained cells 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. 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.

In vivo studies. Animal work was implemented according to previously approved protocols by IACUC, where they were conducted in Animal Biosafety Level 2 (ABSL-2) facilities. 1-day old suckling BALB/c mice (Invivos), weighing approximately 4 to 6g were infected with HEVA71 strain 41 (2 x 10⁷ PFU of HEVA71 per mouse) via i.p injections. At 1 or 6hpi, HEVA71- infected mice were either treated with prunin at doses of 1, 3 or lOmg/kg or vehicle control (PBS) in separate cages on a daily basis till 7 dpi, in which they were monitored and scored daily throughout 14dpi. A 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.

Following 7dpi, 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.

Statistical analysis. Robustness of the compound screening assay was determined by Z- factors⁴⁵, which measures the intervals among the standard deviations (SDs) of the signals against the background noise of an assay. Z-factor experiments were carried out in HEVA71 IRES bicistronic reporter (negative control) or HEVA71 IRES bicistronic hairpin (positive control) transfected RD cells seeded in 96-well plates. Another positive control, namely amantadine, was treated at 2mM with HEVA71 IRES bicistronic reporter transfected cells. Z- factors of 60 samples were then calculated according to the equation: 1 - [(3 x SD of positive control + 3 x SD of negative control) / | (mean positive control— mean negative control) | ]. For studies involving heterogenous selections, two-way ANOVA tests were conducted and subsequently corrected with Tukey's post-tests while one-way ANOVA tests adjusted with Dunnett's post-tests were performed for other studies. Results were considered to be significant if p < 0.05.

2. Results

Rationale of experimental design. A few antibiotics have abilities to differentiate variations in translational efficiencies amongst prokaryotes and eukaryotes, thus precisely targeting translation in a group of cells. Antibiotics such as hygromycin and tetracycline suppress prokaryotic translation, which are successes of modernized medicine²⁵. Therefore, it is plausible to discover compounds that discriminate between host mediated cap-dependent protein synthesis and IRES facilitated cap-independent translation. Figure 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 translation²⁶.

In order to screen a 502-compound flavonoids library, a HEVA71 IRES bicistronic luciferase reporter (Figure IB) 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. The human CMV promoter facilitates initial transcription of R Luc and F Luc genes and the transcript generated is subsequently translated into R Luc and F Luc proteins, in cap-dependent and cap-independent manners respectively. Hence, 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 (Figure 1C) was also included, which was found to interrupt IRES activity²⁷. 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.

Functional validations of HEVA71 IRES bicistronic reporter and HEVA71 IRES bicistronic hairpin. Prior to screening, respective luciferase expressions of both bicistronic reporters that were transiently transfected into human rhabdomyosarcoma (RD) cells individually at 2 different time points (12 and 24 hours post -transfection) were measured. This is to ensure the validity of our downstream studies involving these constructs. As predicted, 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). Moreover, we observed significant (p = 0.0008; p=0.00006) increases in IRES and CMV facilitated F and R Luc synthesis in HEVA71 IRES bicistronic reporter-transfected RD cells from 12 to 24 hours, thus indicative of its high translation efficiency till 24 hours: 3.91 to 4.57 logioRLU for F Luc and 3.38 to 3.88 logioRLU for R Luc from 12 to 24 hours (Figures ID & IE). The HEVA71 IRES bicistronic hairpin showed only an expected rise in R Luc expression (3.22 to 3.74 logioRLU) with no significant change (p=0.28) in F Luc synthesis (2.61 « 2.71 logioRLU) from 12 to 24 hours due to the presence of its IRES hairpin structure (Figures ID & IE).

Further attempts were carried out to evaluate the functionalities of both constructs via a known anti-HEVA71 IRES compound, such as amantadine hydrochloride²⁶. Being discovered in the mid 1960s, this compound had a broad spectrum of antiviral actions against influenza A replicative activity via binding its matrix (M2) protein^28,29, Hepatitis A, Hepatitis C and HEVA71 IRES translational activity by possible binding to ITAFs^26,30-32. We incubated HEVA71 IRES bicistronic reporter and HEVA71 IRES bicistronic hairpin-transfected RD cells independently with amantadine (1 and 2mM) at 12 hours post -transfection, for a duration of 24 hours. After normalizing to transfected RD cells without drug treatment, we found that amantadine significantly reduced HEVA71 IRES activity (F Luc) by 52% (p=0.00006) and 82% (p=0.00004) in a dose-dependent manner (Figure IF) with minimal effects on CMV activity (R Luc) of the HEVA71 IRES bicistronic reporter. This also led to corresponding declines in its F Luc to R Luc expression ratios by 47% and 80% with increasing concentrations of amantadine (Figure IF). In contrast, we observed no significant changes (p=0.23; p=0.44) in F Luc and R Luc expressions and ratios of HEVA71 IRES bicistronic hairpin with amantadine (Figure 1G). These events affirm: (i) amantadine as an effective HEVA71 IRES inhibitor as described in scientific literature; and therefore (ii) its suitability as another positive control along with HEVA71 IRES bicistronic hairpin for downstream novel drug screening and (iii) the strong performance potential of both reporter constructs being able to pinpoint HEVA71 IRES suppressors during high-throughput compound screening.

Heterogeneous antibiotic selection enhances luciferase expression that is highly consistent and reliable. Although the functionalities both bicistronic constructs were validated through transient transfections, genes that are transiently transfected tend to lose their expressions overtime as they are not stably integrated into the genome³³. This would limit the duration of intended drug studies to a very short period and may produce unreliable results through introducing bias into gene expression profiles. This problem was approached via studying respective luciferase expressions of HEVA71 IRES bicistronic reporter or HEVA71 IRES bicistronic hairpin-transfected RD cells with geneticin (G418) selection overtime (0, 4, 6 and 8 days) at 24, 48 and 72 hours (Figure 2A). Preceding this, the working G418 concentration was determined as 0.25mg/ml, at which 50% of RD cells survived after a week with continuous G418 treatment (Figure 2B).

Intriguingly, relative IRES and CMV activities of HEVA71 IRES bicistronic reporter appeared to gradually escalate from 0 to 6 days post-G418 selection, where they peaked at 5.41 and 4.81 logioRLU respectively after 48 hours (Figures 2C & 2D). Beyond 8 days of G418 selection, the luciferase expressions were seen to decrease rapidly, till they were significantly lower than the onset of G418 treatment at all time points: 3.79 < 4.57 logioRLU (24 hours; p=0.00003); 4.42 < 4.80 logioRLU (48 hours; p=0.00003) and 3.33 < 4.18 logioRLU (96 hours p=0.00005) for F Luc while 3.21 < 3.89 logioRLU (24 hours p=0.00003); 3.92 < 4.26 logioRLU (48 hours p=0.00002) and 2.89 < 3.62 logioRLU (96 hours p=0.00002) for R Luc (Figures 2C & 2D). Meanwhile, HEVA71 IRES bicistronic hairpin did not exhibit convincing increments in its IRES activity over the time course of antibiotic selection due to its IRES hairpin structure (Figure 2E), albeit showing similar progressive elevations in its CMV activity, where it reached a maximum reading of 4.57 logioRLU at 48 hours after 6 days post-G418 selection (Figure 2F). Furthermore, its CMV and IRES facilitated luciferase expressions also displayed identical pattern of deterioration above 8 days of G418 selection, where measurements hit below the start of G418 treatment at all time points: 3.07 < 3.67 logioRLU (24 hours); 3.85 < 3.97 logioRLU (48 hours) and 2.85 < 3.37 logioRLU (96 hours) for R Luc while 2.48 < 2.81 logioRLU (24 hours); 2.51 < 2.83 logioRLU (48 hours) and 2.20 < 2.53 logioRLU (96 hours) for F Luc (Figures 2E and 2F). These common accelerated depletion of luciferase signals in both constructs could be attributed to the lost of introduced plasmids overtime due to the lack of plasmid integration into the host genome³³, as affirmed by their large sizes (7.5kb). Nevertheless, 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.

Following the establishment of a suitable extended therapeutic window for downstream large-scale screening, the robustness of the assay via calculating the Z-factors between the HEVA71 IRES bicistronic vector and the associated positive controls, namely the HEVA71 bicistronic hairpin and HEVA71 IRES bicistronic reporter with 2mM of amantadine were also assessed. Based on the ratios of IRES to CMV activities (n=60), Z-factors were tabulated to be 0.859 between the HEVA71 IRES bicistronic reporter and bicistronic hairpin (Figure 2G); and 0.646 between the HEVA71 IRES bicistronic reporter and its drug-treated counterpart (Figure 2H). Since these Z-factors fell between robust cutoff values³⁴ of 0.5 to 1.0, it is illustrated that the luciferase-screening assay was an excellent, consistent and reliable platform to be used for downstream drug studies.

Screening of flavonoids derivatives library identified 7 novel compounds that actively suppress HEVA71 IRES activity. Flavonoids are considered to be part of an omnipresent group of secondary metabolites in the plant kingdom, which are regularly consumed in the human diet³⁵. 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.

Following 6 days of G418 selection, 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. We observed 25 top flavonoid hits to suppress 55 to 62% of HEVA71 I RES activity, which was more than amantadine treatment (40%), though only IS of them exhibited low cytotoxicity profiles of more than 80% cell viability (Figure 3 and Table 1). Further literature analysis classified only a subset of the hits to be novel, namely ST077105, ST024699, ST024368, ST002086, ST024702, ST066904 and ST024081, with no prior publications and patents pertaining to HEVA71 (Table 1). Therefore, these 7 compounds were chosen for a series of downstream validation assays to confirm its anti-HEVA71 activities.

Table 1. 13 flavonoid hits ranked with cell viability profiles (>80%); characterized with remarks and patents. 7 flavonoids (highlighted) were chosen for further evaluation. Functional validation assays identified and characterized prunin as a potent inhibitor of HEVA71 with limited spectrum activity. In order to further pinpoint one suitable candidate for downstream characterizations, the effects of the 7 chosen flavonoids on infectious HEVA71 through quantifying HEVA71 viral titres with plaque reduction assays at 12 hours post-infection (hpi) were first explored. Interestingly, 6 out of the 7 hits significantly (p=0.0008) reduced HEVA71 viral titres, with prunin (ST077105) most effectively suppressing

HEVA71 viral titres by approximately 1.8 logio PFU/ml (Figure 4A). Only 1 flavonoid, namely ST002086, showed no significant change (p=0.52) in HEVA71 viral titre. 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. Doses above ImM reduced cell viability drastically below 80% cell viability. Using these data, the cytotoxic concentration that resulted in 50% of cell death (CC50) was calculated to be at a high concentration of 2715nM. Based on the cytotoxicity profile of prunin, it was decided to utilize a range of prunin concentrations from 31.25 to lOOOnM, in order to determine the effectiveness of prunin on HEVA71 viral titres. After 12hpi and treatment with the specified range of prunin doses, significant dose-dependent reductions (p=0.00003 to 0.00008) in HEVA71 viral titres from doses between 62.5 to lOOOnM were noted, which were quantified by plaque reduction assays (Figure 4C). Cytotoxicity assays that were re-performed ensured that the selected concentrations did not exert any negative effects on cell viability; where all doses resulted in cell viabilities to be above 80%. In addition, lOOOnM of prunin treatment significantly (p=0.00007) reduced HEVA71 viral titre by approximately 3.5 logio PFU/ml in comparison to 1% DMSO (vehicle control), though 31.25nM of prunin was not seen to inflict any significant changes (p=0.69) in HEVA71 viral titre. These data determined the effective concentration of Prunin that resulted in 50% inhibition of HEVA71 viral titre (EC50) to be at a low concentration of 115.3nM (Figure 4D).

Following this, the effects of prunin on HEVA71 viral protein synthesis through Western blot and SDS-PAGE were investigated. After infection of RD cells with HEVA71 (MOI:l), those cells were treated with a range of Prunin doses (31.25 to lOOOnM), including 1% DMSO (vehicle control) and subsequently lysed at 6 and 12hpi. The respective cell lysates were then collected and detected for VP2, a known HEVA71 structural protein along with other precursor HEVA71 viral proteins, such as PI, VP0 and (VP4+VP2+VP3). In contrast to the vehicle control, we noticed dose-dependent reductions in all probed viral proteins [VP2, PI, VP0 and (VP4+VP2+VP3)] from 62.5 to lOOOnM of prunin treatment at both 6 (Figure 4E) and 12hpi (Figure 4F) respectively. Importantly, no visible viral proteins could be detected with lOOOnM of Prunin treatment at 6 and 12hpi, albeit prunin at a dose of 31.25nM did not seem to impact HEVA71 viral protein production at both time points. With reference to actin and 1% DMSO, the blots were further analysed via quantifying the band intensities of VP2 and precursor VP0 at 6 and 12hpi. The observed dose-dependent decreases of VP2 and VP0 due to prunin (62.5 to lOOOnM) were tabulated as significant (p=0.00004 to 0.00008) at both time points, when compared to the vehicle control. ImM of prunin caused declines in band intensities of VP2 and VP0 by 0.99 and 0.97 units respectively at 6hpi and 0.99 and 0.98 units respectively at 12hpi. However, it should be noted that 31.25nM of prunin did not significantly (p=0.55) reduce band intensities (VP2 and VP0) in contrast to the vehicle control at both time points. These results were not surprising, as similar results were obtained from the effects of prunin on HEVA71 viral titres.

Due to the strong interplay between HEVA71 RNA and protein synthesis machineries, the effects of prunin on viral RNA production were studied. Infected RD cells treated with identical doses of prunin (31.25 to lOOOnM) exhibited similar trend of significant (p=0.00003 to 0.0004) dose-dependent reductions in HEVA71 viral RNA amounts from 62.5 to lOOOnM of prunin treatment, in relative to 1% DMSO (vehicle control), at both 6 (Figure 4G) and 12hpi (Figure 4H). Specifically, ImM of prunin significantly (p=0.00003; p=0.00008) diminished viral RNA amounts from: (1) 8.4 to 6.45 logio viral RNA copy number at 6hpi and (2) 9.45 to 8 logio viral RNA copy number at 12hpi. Consistent with the previous datasets, 31.25nM of prunin could not result in any significant (p=0.88) changes in HEVA71 RNA amounts at both time points.

Through examining the anti-HEVA71 profile of prunin, it is evident that prunin displayed potent antiviral activity against HEVA71. Given that 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 them¹⁰. 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). Following infection, EC50 of Prunin (115.3nM) was used as an antiviral treatment for 24 hours and respective HEVA71 viral titres were quantified for each virus through plaque reduction assays (Figure 41). Strikingly, prunin managed to significantly (p=0.00002 to 0.00009) decrease corresponding viral titres of members of Enterovirus A (HEVA71, CA6 and CA16) and Enterovirus B (ECH07 and CB5) species by approximately 3.0 to 3.5 logio PFU/ml in contrast to the vehicle control. However, 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. Moreover, infections with HSV or CHIKV did not yield any significant (p=0.55) changes in their respective viral titres despite being treated with prunin. This was expected, as CHIKV is an alphavirus of the Togaviridae family, consisting of an RNA genome that is capped and expressed only through cap-dependent translation⁴³ while HSV contains a double-stranded DNA genome with no IRES elements⁴⁴. As a precaution, 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.

Administration of prunin in HEVA71 murine model yielded success with minimal cytotoxicity effects. Following the triumph of prunin against HEVA71 in in vitro cell culture, we progressed on to evaluate the in vivo efficacy of prunin in suckling BALB/c mice. 1-day old suckling BALB/c mice were first injected with 2 x 10⁷ PFU of HEVA71 per mouse for 1 hour or 6hours, after which prunin (1, 3 or lOmg/kg) was administered via intraperitoneal injections (i.p.) consecutively for 7 days once daily. 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. 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. Moreover, 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. Even though we did observe an initial gradual increase in the clinical scores of the mice (inactivity, hunched back, ruffled fur and limb weakness) treated with 3mg/kg and lOmg/kg of prunin till 8 and 7 dpi respectively, those mice completely recovered from HEVA71 infection and were healthy by 13 dpi (3mg/kg) and 9 dpi (lOmg/kg). In addition, the 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.

Table 6. Mice clinical scoring system in EV71 BALB/c mice model.

Table 2. Nucleotide substitutions revealed in IRES region of HEVA71 prunin-resistant mutant, of which 4 (highlighted) affected IRES RNA secondary structure folding.

To further examine the in vivo protective efficacy of prunin against HEVA71, the viral loads in the hind limb muscles were quantified from both groups of BALB/c mice (prunin or vehicle control) that were sacrificed on 7 dpi via plaque reduction assays (Figure 5D). Viral titres measured in the infected mice treated with 3mg/kg of prunin were significantly (p=0.0054) lower by approximately 10⁴ PFU per gram than those treated with the vehicle control, demonstrating the competence of prunin against HEVA71. Moreover, a trend of dose dependency was observed, where a higher dose of prunin (lOmg/kg) resulted in a 10⁵ PFU per gram decrease in viral load in comparison to PBS treated mice. In accordance to this, we also affirmed HEVA71 infection in the muscle tissues of hind limbs from both groups in terms of tissue deterioration and viral antigen presence by haematoxylin and eosin (H&E) and immunohistochemistry (IHC) stainings respectively at 7 dpi. H&E stainings (Figure 5E) evidently showed that mice treated with PBS displayed loss of muscle fibers leading to severe damage of their hind limb muscle tissues. Moreover, these mice were subjected to serious inflammation issues following HEVA71 infection as recognized by massive infiltration of immune cells into their hind limb muscles. This was foreseen, as neutrophils and macrophages were shown to accumulate and secrete many pro-inflammatory cytokines in sites of HEVA71 infection, which would worsen muscle tissue destruction⁴⁵. Interestingly, these effects of muscle tissue damage; immune cells infiltration and subsequent tissue inflammation induced by HEVA71 infection were effectively suppressed by prunin as revealed by the healthy tissues seen via H&E staining. Consistent with the H&E stainings, the IHC studies (Figure 5F) detected extensive viral antigen distribution in hind limb muscles of infected mice treated with the vehicle control, thus indicating progressive HEVA71 infection. However, prunin treated mice showed strong reduction of viral antigens in their hind limb muscles, thus reemphasizing the in vivo potency of the drug.

Development of HEVA71 resistant mutants against prunin mapped 5 mutations to the viral IRES region. In order to reaffirm the mechanism of action of prunin against HEVA71 IRES, we successfully grew prunin-resistant viruses via repeated culturing of the wildtype (WT) virus for 19 passages with increasing doses of prunin, which comprised of: (i) first 13 rounds with EC50 of prunin (115.3nM), (ii) following 3 rounds (passages 14 to 16) with 500nM and (iii) last 3 rounds till passage 19 with lOOOnM of prunin. For every round of passaging, proper controls of non-drug treated HEVA71 (1% DMSO) and WT HEVA71 were also included. The viruses from the controls and drug treatment were then tabulated through plaque reduction assays, where selected representative results were plotted for passages 13, 16 and 19 (Figure 6A). Despite prunin treatment, HEVA71 was noticed to exhibit significant (p=0.0007) initial resistance to 115.3nM of prunin at passage 13 in comparison to WT HEVA71. This resistance significantly (p=0.00006) improved over passages 16 and 19, when prunin concentrations was increased to 500 and lOOOnM respectively. 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). More importantly, significant (p=0.00008; p=0.0006) differences in viral titres that were observed between non-drug and drug-treated HEVA71 at passages 13 (7.0 versus 5.9 logio PFU/ml) and 16 (7.0 versus 6.2 logio PFU/ml) were lost at passage 19; where no significant (p=0.76) disparity could be seen between their viral titres (7.0 logio PFU/ml), thus indicating the successful generation of HEVA71 resistant mutant against prunin.

After obtaining HEVA71 resistant mutant from via plaque purification, we wanted to characterize the mutant via 3 approaches: (i) examining its growth replication kinetics through plaque reduction assays, (ii) investigating if the acquired resistance could be overcome via treatment with a secondary anti-HEVA71 drug and (iii) finding mutations that were responsible for conferring prunin resistance through sequencing its whole viral genome. As for (i), with reference to WT HEVA71, there were no significant (p=0.73) differences in viral titres of mutant HEVA71 observed from 0 to 96hpi in RD cells (Figure 6B). Both viruses achieved peaks of around 7 logio PFU/ml by 96hpi, indicating no replicative variations between them and their comparable reproducibilities in cell cultures. With respect to (ii), we utilized 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. Apparently, ribavirin was first established as an inhibitor of viral RNA replication via causing lethal mutagenesis in vitro⁵⁰, which further showcased anti-HEVA71 properties in vivo, where it reduced HEVA71- facilitated paralysis and mortality events⁵¹. Interestingly, ribavirin exposure reduced viral titres of both WT and mutant HEVA71 equitably of approximately 1.2 logio PFU/ml, whereby antiviral effects of prunin were exclusively limited to WT HEVA71 (Figure 6C). Moreover, synergistic treatment studies involving ribavirin and prunin resulted in significant (p=0.00006 to 0.00008) declines in both WT and mutant viruses, albeit greater comprehensive inhibitory effect on WT HEVA71 was observed. Notably, there were no significant (p=0.53 to 0.66) differences seen in mutant HEVA71 viral titres amongst either ribavirin or ribavirin with prunin treatments, hence suggesting ribavirin as the sole contributor of the detected inhibition. Since prunin resistance was conquered by ribavirin, this emphasized that the mutant HEVA71 functioned similarly to WT HEVA71 in terms of its replication fidelity, particularly facilitated by 3D polymerase.

Examining (iii) identified numerous silent mutations that were found in VP1, VP2, VP3, 2B, 2C and 3D polymerase regions (see Table 3), along with 5 nucleotide substitutions in its IRES site (Table 2) with comparison to WT HEVA71. Intriguingly, while 2 of these mutations (T164CIRES and G 165CIRES) were mapped to stem loop 2, another 2 were located in stem loop 4 (G368CIRES and T370GIRES) with the last one confined to a connecting loop between stem loops 2 and 3 (C177TIRES). Functional analyses of these point mutations on WT HEVA71 IRES via plaque reduction assays (Figure 6D) and bicistronic luciferase assays (Figure 6E) revealed individual mutations such as C177TIRES, G368CIRES and T370Gi_REs to be devoid of playing active roles in prunin resistance. Interestingly, either of stem loop 2 mutations (T164CIRES or G165CIRES) were sufficient in triggering similar resistances to prunin resistant mutant HEVA71, thus suggesting that stem loop 2 might play greater roles than stem loop 4 in prunin resistance. However, our efforts in generating a plethora of WT HEVA71 IRES mutated combinations led us to realize that both stem loop(s) 2 and/or 4 mutations are equally vital in prunin resistance. To understand these mutations further, the IRES RNA secondary structures between HEVA71 mutant and WT viruses were closely examined, where they revealed some major folding changes (Figure 6F): (1) Mutations T164Ci_REsand G165CIRES shortened stem loop 2 of IRES with a removal of an irregular 5-sided polygon (GTATC) and (2) mutations G368CIRES and T370GIRES eliminated an additional hairpin loop (GCGCTGGC) in stem loop 4 of IRES. Consistent with our results from viral plaque assays, mutation C177TIRES did not affect the loop attached amid stem loop 2 and 3 of HEVA71 IRES, thus confirming its lack of part in conferring resistance to prunin. Overall, these data comprehensively demonstrate that the four mutations seen in both IRES stem loops 2 and 4 could maintain IRES functionality and play roles in resistance to prunin.

Table 3. Sequencing revealed other silent mutations discovered in the genome of H EV71 prunin-resistant mutant.

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 supplementations⁵². These findings have placed HEVA71 IRES as a member of the group 3 IRES RNA, which require canonical eukaryotic initiation factors (elFs) and other ITAFs for their translational functions. In order to test our hypothesis, we tested prunin against HCV, which belongs to group 2 IRESs that interact with the 40S ribosomal subunit and a subset of canonical elFs⁵². HCV is a positive-sense, single-stranded RNA virus that resides in the genus Hepacivirus of the Flaviviridae family, which has been responsible for chronic hepatitis, liver cirrhosis and hepatocellular carcinoma⁵³.

Doses of prunin, ranging from 62.5 to 500nM, were treated with HCV-infected Huh-7.5 cells, where the cytotoxicity and effectiveness profiles of prunin on Huh 7.5 cells and HCV viral titres respectively, were evaluated. After 3dpi and 6dpi with various prunin dosages, we on ly observed significant (p=0.011 to 0.022) declines in HCV viral titres at a dose of 500nM (Figure 7A), which were quantified by immunofluorescence assays. Cytotoxicity assays also revealed that the above selected concentrations had no negative effects on cell viability; in which all doses resulted in more than 80% cell viability. Furthermore, 500nM of prunin significantly (p=0.011 to 0.022) reduced HCV viral titres by approximately 0.5 and 0.7 logio PFU/ml in comparison to 1% DMSO (vehicle control) at 3dpi and 6dpi correspondingly, though other concentrations of prunin (62.5 to 250nM) were not seen to inflict any significant changes in HCV viral titres.

In order to affirm the suppression of prunin on HCV, the effects of prunin on HCV RNA generation were also studied. T reatment of infected Huh-7.5 cells with similar doses of prunin (62.5 to 500nM) resulted in significant (p=0.005 to 0.00008) dose-dependent fold reductions in HCV RNA expressions from 62.5 to 500nM of prunin treatments, in relative to 1% DMSO (vehicle control), though only seen at 6dpi (Figure 7B). Particularly, 500nM of prunin significantly (p=0.00008) diminished HCV RNA amounts by 0.7 fold at 6dpi, which fits the data obtained for HCV viral titre quantifications. These data hence showcase that 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). Evidently, all 3 ITAFs have been well documented as RNA-binding proteins due to their dual RNA-binding domains, including an RGG box⁵⁴ and a KH domain⁵⁵'⁵⁶, which also enable them to act as nuclear-cytoplasmic shuttlers for various molecular and cellular functions⁵⁷. More importantly, 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. Notably, via biotin RNA pulldown assays, hnRNPAl was elucidated to bind to stem loop 2 of IRES⁵⁸, whereas Sam68 was recently discovered to specifically interact with stem loop 4 of IRES⁵⁹. Interestingly, 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 IRES⁶⁰. Further functional interaction studies involving these 3 ITAFs conducted over the past decades have demonstrated Sam68 as a multifunctional SH3 and SH2 adaptor protein⁶¹, thus enabling itself to link with hnRNPs via its 5 proline-rich motifs⁶²'⁶³, which in turn can form an ITAF complex that drives IRES mediated cap-independent translation. Intriguingly, hnRNPAl, hnRNPK and Sam68 were also established as proviral factors for HCV replication⁵⁷'⁶⁴'⁶⁵, either by their direct interactions with viral core proteins⁶⁴ or via indirect associations with other cellular scaffold proteins⁵⁷'⁶⁵. Altogether, this information suggested that the activities of prunin towards WT HEVA71 and HCV IRES might decrease or prevent one or more ITAFs, particularly, Sam68, hnRNPK and/or hnRNPAl, from binding to those respective ITAFs. In order to affirm this, knockdown studies were carried out where RD cells were treated with a range of siRNA concentrations targeting each ITAF, namely Sam68, hnRNPK and hnRNPAl, along with non-targeting siRNA control (NTC) for 72 hours. Following this, infection studies were carried out with either WT HEVA71 or mutant HEVA71 at a MOI of 1, in which they were lysed at 12hpi for Western blot and SDS PAGE analyses. Dose dependent reductions in protein bands were seen for Sam68 (Figure 8A), hnRNPK (Figure 8B) and hnRNPAl (Figure 8C) after respective siRNA treatments regardless of WT or mutant HEVA71 infections, where low amounts of visible proteins could be observed with 25nM of Sam68 or hnRNPAl or 35nM of hnRNPK siRNA. Notably, these phenomenons were not seen in NTC treatments (Figure 8D), thus attributing the observed reductions in corresponding protein amounts to siRNA treatments. Moreover, tabulated band intensities of Sam68, hnRNPK and hnRNPAl showcased significant (p=0.00005 to 0.00009) siRNA dose-dependent declines in comparison to the mock control (OnM), hence suggesting that the magnitudes of siRNA dosages utilized in this study were effective in silencing Sam68, hnRNPK and hnRNPAl genes respectively.

After successive knockdowns of respective ITAFs, the experiments proceeded to detect for VP0, a HEVA71 precursor protein, in conjunction with VP2, a known HEVA71 structural protein via Western blots. In comparison to mock-treated cells, dose-dependent reductions in both viral proteins for Sam68 (Figure 8A) and hnRNPAl (Figure 8C) knockdown RD cells were detected, disregarding WT or mutant HEVA71 infections. Through analyzing the results further by determining VP0 and VP2 band intensities, it was discovered that Sam68 knockdown had a greater effect on mutant HEVA71 than WT HEVA71, albeit decreasing hnRNPAl protein amounts caused constant downturns of HEVA71 viral protein synthesis during both WT and mutant HEVA71 infections. In particular, 5nM of Sam68 siRNA reduced VP0 and VP2 of WT HEVA71 by 0.37 and 0.50 units whilst decreasing VP0 and VP2 of mutant HEVA71 by 0.82 and 0.88 units respectively. However, 15nM of hnRNPAl siRNA treatments led to similar significant (p=0.00006 to 0.00008) decreases in VP0 and VP2 protein of both viruses by 0.98 and 0.94 units, correspondingly. Apart from Sam68 and hnRNPAl, hnRNPK knockdown also resulted in dose-dependent declines of both VP0 and VP2, which however were limited to only WT HEVA71 (Figure 8B). Interestingly, based on the graphical representations of band intensities, even though 35nM of hnRNPK siRNA significantly (p=0.00006 to 0.00008) knocked out productions of VP0 and VP2 proteins during WT HEVA71 infection, relative band intensities of those viral components following hnRNPK knockdown were not significantly (p=0.55 to 0.78) affected amidst mutant HEVA71 progeny virion synthesis, which were akin to NTC treatments (Figure 8D).

In order to verify whether infectious viral particle productions were further affected, both mutant and WT HEVA71 viral titres in siRNA-treated RD cells were quantified via plaque reduction assays (Figure 8E). Coinciding with the knockdown data from Western blots, 55nM of Sam68 siRNA treatments decreased viral titres of mutant HEVA71 by approximately 5.8 logio PFU/ml, which was significantly (p=0.008) greater than the 3.5 logio PFU/ml reduction observed in WT HEVA71 titres. Moreover, knockdown of hnRNPAl resulted in both WT and mutant HEVA71 viral titres to drop equally by approximately 4.0 logio PFU/ml. Furthermore, hnRNPK siRNA mediated knockdown only caused significant (p=0.0006) declines in mutant HEVA71 titres of about 3.8 logio PFU/ml, without exerting any effects on WT HEVA71 titres. It is imperative to note that these observed effects were not due to cellular toxicities as siRNA- treated cells displayed cell viabilities of above 80% (Figure 8E). Overall, since the knockdowns of Sam68 and hnRNPK had varying impacts on mutant and WT HEVA71 productions, it can be implied that prunin might be suppressing Sam68 and/or hnRNPK from binding to WT HEVA71 IRES during IRES-facilitated translation of WT HEVA71.

In order to affirm the above notion, we performed 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. 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). In particular, band intensities of hnRNPK significantly (p=0.00097) decreased by 0.67 (65kDa) and 0.71 (51kDa) units while band intensity of Sam68 increased by 0.59 units respectively for the pulldown of mutant HEVA71 IRES versus WT HEVA71 IRES. Overall, these results hence showcase that prunin is able to suppress the binding of hnRNPK to a great extent, which provides a selection pressure for WT IRES to evolve into a mutant IRES form that allows for Sam68 to bind with greater affinity, so as to facilitate the IRES translation of mutant HEVA71 in the absence of hnRNPK activity.

3. Discussion

With recent elevations in HEVA71-related morbidities and mortalities observed in humans, there are pressing demands for the development of effective anti-HEVA71 therapies. In the present invention, these needs were addressed through targeting a potential target of HEVA71, namely IRES, so that its protein synthesizing machinery, such as IRES-mediated cap- independent translation could be reduced without affecting host cell translation, which would then potentially create more avenues in the spectrum of antiviral strategies against HEVA71⁸. To start off, functional simplified cell-based bicistronic vector systems were designed, which comprised of HEVA71 IRES bicistronic reporter and its positive control known as HEVA71 IRES bicistronic hairpin. Since amantadine hydrochloride was identified as a HEVA71 IRES inhibitor through a similar bicistronic reporter construct approach²⁶, 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-factors⁶⁶, which determined the qualities of the systems to be excellent platforms for downstream drug studies.

Naturally occurring chemical substances have consistently been abundant and divergent sources for antiviral discoveries and possess increased biological activities based on their organic roots⁶⁷. Therefore, the present bicistronic assays were utilised to screen a 502- compound flavonoid derivatives library, which revealed 13 "true" hits after accounting for the false positives generated by toxicity-related cell death prior protein synthesis. Although these hits exhibited higher IRES inhibition profiles in comparison to amantadine, only 7 were classified as novel, amongst which 6 effectively reduced HEVA71 viral titres. The reason for choosing prunin for further evaluations stemmed from its anti-HEVA71 potency, where it reduced HEVA71 viral titre by approximately 1.8 logio PFU/ml at 20pg/ml (=34.5mM). Further characterization of prunin in vitro displayed a low EC50 of 115.3nM, a high CC50 of 2715nM, with a selectivity index (CC50/EC50) of 23.6 towards HEVA71. These results were surprising as CC50 and EC50 of prunin were much lower in comparison to the screening concentration used, which could be due to interbatch variability in production and extraction of prunin from various companies. Nevertheless, the present flavonoid library hits (Table 1) also included other drugs such as fisetin (=69.9mM), apigenin (=74.1mM) and luteolin (=69.9mM), which inhibited HEVA71 at the expected doses, hence indicative of possibly only prunin being affected. Advancing on, we also noticed significant dose-dependent declines in HEVA71 structural proteins (VP0 and VP2) and RNA amounts at 6 and 12hpi after prunin treatments. Other than HEVA71, 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. Importantly, treatment of HEVA71-infected BALB/c mice with prunin at a dose of 3mg/kg reduced clinical manifestations, hind limb muscle tissue damage, viral titre and subsequent inflammation, thus completely preventing fatality and body weight loss in vivo. Interestingly, thorough investigations of the mechanism of IRES inhibition by prunin mapped 5 resistant mutations to the IRES region; of which 4 of them were hypothesised (T164CIRES, G 165CIRES, G 368CIRES and T370G IRES) to play active roles in HEVA71 drug resistance properties.

Prunin is a natural compound that originated from immature citrus fruits, specifically from the Citrus aurantium and Citrus paradise⁶⁸'⁶⁹ comprising of oranges, tangerines, limes, lemons and grapefruits⁷⁰; and is also found in minute amounts in tomatoes⁷¹. Usually found at a range of 19 to 47mg per lOOg of citrus fruits, an intrinsic advantage showcased by prunin is the ease of extraction, purification and processing for large-scale productions⁷⁰. Currently, 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 purposes⁷²'⁷³. The activity of prunin suppression on HEVA71 IRES is not fascinating, as it is not the first flavonoid to exhibit inhibition properties against IRES. Research in past years had revealed a few potent flavonoids against IRES, like apigenin⁷⁴ and kaempferol⁴¹, which functioned at EC50 values in the micromolar ranges of 30.5 and 31.8mM respectively. Other than flavonoids, there are also other non-natural substances that act against IRES, for instance, amantadine that acts at 2mM²⁶. Interestingly, based on the numerical values of concentrations of existing drugs utilized against IRES, prunin has the lowest EC50 located within the nanomolar range, which establishes its degree of high potency. This feature of prunin allows for its administration at a lower dosage to achieve full anti-IRES activity that might reduce the probability and severity of side effects seen with high drug dosages.

Many drugs unveiled over the years have been designed or discovered to target either HEVA71 structural or non-structural proteins independently⁷'⁸. For instance, pleconaril was found to perturb the functions of VP1 capsid structural proteins through stabilizing them, which prevented corresponding uncapping and discharge of HEVA71 genome into host cells⁹'⁷⁵'⁷⁶. Other examples would include fistein and rutin, where they were classified as 3C^pro suppressors, thus further interfering with HEVA71 polyprotein processing and subsequent virion assembly⁷⁷. However, by using IRES as its target, prunin is able to "kill two birds with one stone" specifically by preventing the cap-independent translations of both HEVA71 structural and non-structural proteins. Evidently, the drop in non-structural proteins was shown indirectly via 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 synthesis⁷⁸ and development of replication complexes⁷⁹; HEVA71 3D protein that codes for a RNA polymerase that produces numerous positive RNA strands⁸⁰; and HEVA71 3A protein which has been reported to induce RNA polymerase activity⁸¹. The action of prunin inhibiting HEVA71 through a two-pronged approach is useful for avoiding combination therapy involving HEVA71 structural and non-structural suppressors, which forces the effective dosages of drugs to be reduced in order to avoid adverse and cytotoxic effects. This was observed in combinatorial therapies comprising of well-validated compounds such as NITD008, ALD and 1-acetyllycorine (suppressors of 3D polymerase, VP1 and 2A^pro) or gemcitabine and ribavirin (suppressors of 3D polymerase)^82-84.

Interestingly, 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. 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 anthocyanins⁸⁵. Based on Table 4, 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. This tells us the importance of having closed carbon ring structures, with carbon backbones similar to the F2 group in order to effectively inhibit HEVA71 IRES activity, which can therefore serve as lead molecules for future antiviral design or as effective tools in helping to understand IRES inhibition mechanisms better. Moreover, HEVA71 structural and non-structural proteins that were not present in the initial screening assay could play various roles in sequestering open carbon ring structures, thus attenuating the anti-IRES activity of susceptible ST002086.

Table 4. Structures and classification of 7 chosen flavonoids. 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 translation¹³. This concept was first demonstrated by a drug known as quinacrine⁸⁶ that was capable of suppressing Hepatitis C virus IRES-facilitated translation in vitro via intercalating into IRES RNA and affecting downstream ITAF interactions. By combining results from the limited-spectral activity of prunin and 4 resistance mutations (T164CIRES, G165CIRES, G368CIRES and T370GIRES) seen in HEVA71 IRES against prunin, we confirmed if this notion was correct, through comparing the IRES sequences of Enteroviruses such as HEVA71, CA6, CA16, ECH07 and CB5 that were affected by prunin, in order to identify if those 4 non-mutated nucleotides (T164, G165, G368 and T370) were shared commonly among them. Although this was shown to be negative³, it should be noted that those mutated nucleotides were analyzed from only a single mutated genome (1 agarose plug) instead of many genomes. More full-genome sequencing of 2 or more purified plugs could aid in identifying more mutations or affirming the current mutations present in prunin-resistant HEVA71, which can eventually assist in IRES sequence comparison of Enteroviruses. Furthermore, many recent reports have suggested that IRES consist of evolutionary conserved motifs, which have the tendency to only preserve sequences affecting their structure and subsequent protein interactions for their functions, despite the lack of conservations in their primary sequence and secondary RNA structure^{87- 89}. This supports the notion of prunin directly inhibiting conserved motifs of IRES that could vary in sequence and structure. Moreover, significant declines in HCV RNA amounts were observed and viral titres at 500nM of prunin, thereby suggesting that the mechanism of prunin could be affecting common conserved regions shared between distinct HEVA71 and

HCV IRESs.

In order to understand whether the effect of prunin on IRES structures in turn differentially regulates ITAF recruitments, the S ITAFs (Sam68, hnRNPAl and hnRNPK) that have been reported in current literature were investigaed, which positively regulate HEVA71 IRES- mediated translation^58-60 along with HCV replication⁵⁷'⁶⁴'⁶⁵. Apparently, knockdown studies and biotin-RNA affinity assays revealed distinct recruitment patterns of ITAFs by mutant HEVA71, where associations with hnRNPK were forfeited so as to gain higher reliance on Sam68 for maintaining its IRES facilitated translation. It was also shown that prunin suppressed hnRNPK interaction, which in turn compromised the plethora of essential functions and interactions of hnRNPK during IRES-mediated translation⁹⁰, thus contributing to a selection pressure for the mutation of WT HEVA71 IRES. Interestingly, Sam68 compensated for the loss of hnRNPK activity in mutant HEVA71, hence revealing that the conformational change by prunin in mutated stem loop 4 allowed for a greater binding affinity to Sam68 in one way or another. Given that Sam68 interacts with hnRNPAl during WT HEVA71 infection⁹¹, similar interactions can be maintained without hnRNPK amidst mutant virus production, so as to form the ITAF complex that is necessary in driving mutant IRES dependent translation. However, it should be noted that current literature has not identified many ITAFs that interact with various IRESs, therefore suggesting a need for more research in order to uncover more ITAFs for a higher understanding of IRES-mediated translation and drug-associated suppression mechanisms.

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. Currently, only a small population of drugs has been effective against HEVA71 in murine models, namely pleconaril⁹² and lactoferrin⁹³ against HEVA71 VP1 protein, rupintrivi r⁹⁴ and chrysin⁹⁵ targeting HEVA71 3C^pro, lycorine and 1-acetyllycorine⁹⁶ inhibiting HEVA71 2A^pro and lastly ribavirin⁵¹ and NITD008⁹⁷ suppressing HEVA71 3D polymerase. Based on the above list, it should be noted that no antiviral against HEVA71 IRES has been effective in murine models, thus placing prunin as the first potent anti-HEVA71 IRES compound in vivo. However, a critical caveat to futuristic prunin administration for HEVA71 therapy would be the development of resistance as shown in our study. Given that prunin resistance was surmounted with ribavirin, combinatorial therapies of prunin with other known anti-HEVA71 drugs that target distinct points in HEVA71 life cycle could be ultimately employed. Nevertheless, accurate and careful preclinical profiling should be carried out to define the feasibility of prunin for clinical development against HEVA71 infections. Hopefully, the lag time for the occurrence of prunin resistance will be used to develop a potential vaccine or an improved drug design from lead molecules like prunin.

Other than its potential clinical applications, 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 promoter⁹⁸ or a TRE-CMVmin promoter⁹⁹; (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. Notably, 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. Moreover, 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. Most importantly, 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. In conclusion, it is anticipated that this research on antiviral flavonoids such as prunin, will extensively impact the drug discovery, research and development sectors, as natural compounds, which were previously neglected over man-made "designer compounds", do have potential therapeutic functions that should be further investigated in order to increase the spectrum of effective drugs available against viral infections.

Whilst there has been described in the foregoing description preferred embodiments of the present invention, it will be understood by those skilled in the technology concerned that many variations or modifications in details of design or construction may be made without departing from the present invention.

Claims

1. Use of 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 manufacture of a medicament for preventing or treating an enterovirus or a hepacivirus infection.

2. The use according to claim 1, wherein the molecule 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.

3. The use according to any one of claims 1 or 2, wherein the ITAF is selected from the group consisting of hnRNPK, hnRNPAl and Sam68.

4. The use according to any one of claims 1 to 3, wherein the enterovirus is HEVA71, and hepacivirus is Hepatitis C virus.

5. The use according to any one of the preceding claims, wherein the molecule is selected from the group consisting: a flavonoid, an antibody, a modified or unmodified siRNA, a phosphorothioate oligonucleotide, and vivo-MO-1 and vivo-MO-2.

6. The use according to claim 5, wherein the flavonoid inhibits the translation of the IRES portion.

7. The use according to claim 5, wherein the flavonoid is selected from Table 1.

8. The use according to claim 7, wherein the flavonoid is prunin.

9. A pharmaceutical composition for preventing or treating an enterovirus infection or an enterovirus induced disorder, comprising 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 together with one or more pharmaceutically acceptable diluents or carriers therefor.

10. The pharmaceutical composition according to claim 9, wherein the molecule 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.

11. The pharmaceutical composition according to any one of claim 9 or 10, wherein the ITAF is selected from the group consisting of hnRNPK, hnRNPAl and Sam68.

12. The pharmaceutical composition according to any one of claims 9 to 11, wherein the molecule is selected from the group consisting: a flavonoid, an antibody, a modified or unmodified siRNA, a phosphorothioate oligonucleotide, and vivo-MO-1 and vivo-MO-2.

13. The pharmaceutical composition according to any one of claim 9 to 12, wherein the flavonoid is selected from Table 1.

14. The pharmaceutical composition according to claim 13, wherein the flavonoid is prunin.

15. The pharmaceutical composition according to any one of claims 9 to 14, further comprising a co-agent having anti-enterovirus properties.

16. The pharmaceutical composition according to any one of claim 15, wherein the co agent is selected from the group consisting of: ribavirin, NITD0008 and 2'-C-methylcytidine.

17. The pharmaceutical composition according to any one of claims 9 to 16, wherein the composition is suitable for orally, or rectal, or transmucosal, or intestinal, or intramuscular, or subcutaneous, or intramedullary, or intrathecal, or direct intraventricular, or intravenous, or intravitreal, or intraperitoneal, or intranasal, or intraocular administration to a patient.

18. The pharmaceutical composition according to any one of claim 9 to 17, wherein the carrier is 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.

19. The pharmaceutical composition according to any one of claims 9 to 18, wherein the amount of flavonoid present in the composition is 2C^g/ml.

20. A flavonoid for use in preventing or treating an enterovirus infection or an enterovirus induced disorder.

21. The flavonoid according to claim 20, wherein the enterovirus is HEVA71 and the flavonoid is prunin.

22. A method of preventing or treating an enterovirus infection or an enterovirus induced disorder, wherein the method comprises administering a therapeutically effective amount of a flavonoid to a subject in need thereof.

23. The method according to claim 22, wherein the flavonoid is prunin.

24. The method according to any one of claims 22 or 23, wherein the therapeutically effective amount of flavonoid is 3mg per weight kg of the subject.

25. 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.

26. The method according to claim 25, wherein the flavonoid is prunin.