WO2024033372A1 - Antiviral compound - Google Patents

Abstract

This invention relates to the use of piperlongumine compounds, such as piperlongumine, to treat viral infection or reduce susceptibility to viral infection by selectively inducing the accumulation of reactive oxygen species (ROS) in host cells infected with the virus. Methods of treatment and piperlongumine compounds for use in such methods are provided.

Description

Antiviral Compound

Field

This invention relates to compounds useful in the treatment or prevention of viral infection, for example SARS-CoV-2 infection.

Background

Following the first reported cases of COVID-19 in Wuhan, Hubei Province of China, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly and unexpectedly spread worldwide[1]. As of May 15, 2022, more than 521 million SARS-CoV-2 infections and 6.2 million deaths have been reported[2]. Despite the development and distribution of several highly effective and safe vaccines, the continued emergence of new variant strains shows that the virus is mutating over time to adapt to its new human hosts[3].

So far, the discovery of antiviral compounds against SARS-CoV-2 has mainly focused on direct-acting antivirals (DAAs), including the three drugs approved for clinical use, namely Remdesivir, PF-07321332 (paxlovid) and MK-4482/EIDD-2801 (molnupiravir) [4-6], Another class of antiviral compounds called host- directed antivirals (HDAs) or indirect-acting antivirals are believed to be more resistant to SARS-CoV-2 variants of concern (VOCs), since host genes have a low propensity to mutate[7, 8], however this is still under investigation.

Summary

The present inventors have unexpectedly found that piperlongumine and related compounds selectively induce the accumulation of reactive oxygen species (ROS) in cells infected with a virus, leading to cell death. This may be useful, for example in the treatment or prophylaxis of viral infection.

A first aspect of the invention provides a method of treating viral infection comprising; administering a piperlongumine compound to a patient in need thereof.

A second aspect provides a method of reducing susceptibility to viral infection comprising; administering a piperlongumine compound to a patient in need thereof.

A third aspect provides a method of selectively increasing the amount of ROS in a cell infected with a virus comprising; contacting the cell with a piperlongumine compound.

A fourth aspect provides a method of selectively inducing cell death in a cell infected with a virus comprising; contacting the cell with a piperlongumine compound.

Methods of the third and fourth aspect may be in vitro methods or in vivo methods.

A fifth aspect of the invention provides a piperlongumine compound for use in a method of treating viral infection or reducing susceptibility to viral infection, for example a method of the first to the fourth aspects. A sixth aspect of the invention provides the use of a piperlongumine compound in the manufacture of a medicament for use in a method of treating a viral infection or reducing susceptibility to viral infection, for example a method of the first to the fourth aspects.

Preferred piperlongumine compounds of the first to the sixth aspects include 5,6-dihydro-1-[(2E)-1-oxo-3- (3,4,5-trimethoxyphenyl)-2-propenyl]-2(1 H)-pyridinone (piperlongumine).

Preferred viral infections of the first to the sixth aspects include SARS-CoV-2.

Aspects and embodiments of the invention are described in more detail below.

Brief Description of the Figures

Figure 1 shows that piperlongumine, 1 , exhibits strong antiviral activity against SARS-CoV-2 in vitro. A) Chemical structure of piperlongumine (PL). B-C) Cytotoxicity of PL in VERO-CCL 81 cell (B) and A549- hACE2 cell (C) lines. Both cell lines were treated with indicated doses of PL with DMSO as control, and cell viability was measured after 24h. 50% cytotoxic concentration (CC50) values of PL are indicated under the curves. D-E) Dose-response antiviral activity of PL in VERO-CCL 81 cells, evaluated by qPCR assay, quantifying E gene (D) and N gene (E) of SARS-CoV-2 virus. Cells were first infected by incubation with the virus for 1 h, followed by 24 h incubation in fresh media containing PL. IC50 values of PL in VERO-CCL 81 cell line are indicated. F-G) Dose-response antiviral activity of PL in VERO-CCL 81 cells, evaluated by qPCR assay, quantifying E gene (F) and N gene (G) of SARS-CoV-2 virus. Cells were pre-treated with PL at indicated concentration for 1 h, followed by 1 h incubation with virus inoculum, then a final 24h incubation with fresh media containing PL. IC50 values of PL in VERO-CCL 81 cell line are indicated. H) Plaque assay was performed in VERO-CCL 81 cells to determine the viral titres (amount of infectious virus) produced in cells pre-treated with the indicated concentration of PL 1 h prior to infection. IC50 values are indicated. I) Representative plaques from plaque assay. The infection medium was diluted 10-fold and was indicated as 10^-1. Mock was negative control incubated with Medium. J-K) Dose-response antiviral activity of PL in A549- hACE2 cells, evaluated by qPCR assay, quantifying E gene (J) and N gene (K) of SARS-CoV-2 virus. Cells were pre-treated with PL at indicated concentration for 1 h, followed by 1 h incubation with virus inoculum, then a final 24h incubation with fresh media containing PL. IC50 values of PL in A549-hACE2 cell line are indicated.

Figure 2 shows that piperlongumine (PL), 1 , inhibits SARS-CoV-2 variant of concern (VOC) infection. A-C) In vitro antiviral activity of PL in VERO-CCL 81 cells evaluated by the qPCR assay, quantifying E gene and N gene of SARS-CoV-2 virus. Cells were pretreated with PL at indicated concentrations for 1 h, followed by addition and incubation for 1 h of alpha (A), delta (B), or omicron (C) VOCs. Finally, fresh PL media were replaced for another 24 h incubation. IC₅₀ values of PL in the VERO-CCL 81 cell line were indicated. Data are presented as mean ± SD. Two-way ANOVA test or unpaired t test. *P < 0.05, **P < 0.01 , ***P < 0.001 .

Figure 3 shows that piperlongumine (PL), 1 , shows in vivo antiviral efficacy in the K18-hACE2 mouse model. (A) Schematic of the K18-hACE2 model of SARS-CoV-2 infection. 8-12 weeks-old female K18-hACE2- transgenic mice were treated with 1 mg/kg PL or Plitidepsin for 1 h prior to intranasal infection with 5x10⁴ PFU of SARS-CoV-2 on Day 0. Daily monitoring of body weight and clinical signs was carried out until Day 5, when mice were euthanized, and lungs were harvested for virus loading quantification or histopathology analysis. (B to G) In an in vivo prophylactic treatment setting, mice were treated with indicated compound 1 h prior to infection, then challenged with different VOCs. The body weight change curve of vehicle control, PL-, or Plitidepsin-treated mice, infected with alpha VOC (B), delta VOC (C), or omicron VOC (D). The qPCR assay determined lung viral titres on Day 6 for alpha VOC (E), delta VOC (F), and omicron VOC (G), respectively. (H-l) In an in vivo therapeutic treatment setting, mice were treated with indicated compound 1 day after omicron VOC infection. The body weight curve (H) and qPCR assay (I) determined lung viral titres on Day 6. Mean ± SD. Multiple t test or one-way ANOVA test. (J) Representative images from the plaque assay quantifying virus load in left lungs of the mice. K) Score of lung pathology in K18-hACE mice inoculated with SARS-CoV-2, untreated (vehicle) and treated with PL. L) Representative microphotographs of the pathological changes in lungs of K18-hACE mice inoculated with SARS-CoV-2 at 5 DPI, untreated (a) and treated with PL (b). Depicted are the extent of the lesions (a, b), including emphysema (white arrowhead) and interstitial inflammation and haemorrhages (black arrowhead); higher magnification of the haemorrhagic foci (a’, b’); and hyperplasia (black arrowhead) and necrosis of bronchiolar epithelium (white arrowhead) (a”, b”), more severe in untreated/vehicle mice. Haematoxylin and eosin stain; original magnification 2.5x, (a, b), 20x (a’, b) and 40x’ (a”, b”).

Figure 4 shows piperlongumine (PL), 1 , selectively induces reactive oxygen species (ROS) in infected cells. A) PL induced ROS elevation in infected cells, not in non-infected cells. VERO-CCL 81 cells were treated by PL with indicated concentration for 1 h, followed by 1 h incubation with virus inoculum. B-C) PL-mediated modulation of GSH and GSSG. GSH levels were determined after VERO-CCL 81 cells were pre-treated with 5mM NAC for half an hour in indicated group, then treated with PL for 1 h, followed by 1 h incubation with virus inoculum (B). GSSG levels were also determined after VERO-CCL 81 cells were treated with PL for 1 h, followed by 1 h incubation with virus inoculum (C). D) GSTP1 mRNA level quantified by qPCR assay. VERO- CCL 81 cells were pre-treated with PL at the indicated concentrations for 1 h, followed by 1 h incubation with virus inoculum, then incubated for another 24h with fresh PL media. E) PL-induced virus inhibition can be rescued by NAC. VERO-CCL 81 cells were pre-treated with 5mM NAC for half an hour, followed by PL treatment for 1 h, then 1 h incubation with virus inoculum. Anti-viral effect was evaluated by qPCR assay. All values are mean ± s.d. of three independent experiments.

Figure 5 shows the antiviral mechanism of action of piperlongumine, 1 , A) Antagonism of interferon signalling by SARS-CoV-2 and potential anti-viral mechanism of PL. B) The effect of PL on MAVS induced IFN-JAK- STAT signal pathway was determined by western blot analysis of MAVS, JAK1 , p-STAT1 proteins in A549- hACE2 cells, pre-treated with PL for 1 h, followed by 1 h incubation with virus inoculum, then another 24h incubation with fresh PL media. GAPDH expression was used as a loading control. C) The effect of PL on MAVS mRNA expression was determined by qPCR analysis in A549-hACE2 cells, pre-treated with PL for 1 h, followed by 1 h incubation with virus inoculum, then another 24h incubation with fresh PL media. D) Western blot analysis of MAVS, p-STAT1 and spike proteins from lung extracts of transgenic K18hACE2 mice treated with 1 mg/kg PL or vehicle control 1 h prior to infection. E) Fluorescence microscopy analysis of MAVS co-localization with mitochondria in A549-hACE2 cells, pre-treated with PL for 1 h, followed by 1 h incubation with virus inoculum, then another 24h incubation with fresh PL media. The percentage of MAVS co-localized with mitochondria were quantified shown in (F). Figure 6 shows the structure or molecular dynamics simulations of piperlongumine. The covalent docking shows PL fits within the binding domain of Mpro (Main Protease), but it does not bind to Mpro. (Mpro is essential for replication of SARS-CoV-2).

Figure 7 shows representative histopathology images of other organs harvested from mice in vivo study, showing heart, spleen, kidney, liver, nasal turbinates and brain histopathology.

Figure 8 shows that piperlongumine, 1 , exhibits strong antiviral activity against SARS-CoV-2 in vitro in Calu3 cells.

Detailed Description

This invention relates to the treatment or prevention of viral infections using piperlongumine compounds. Piperlongumine compounds are shown herein to be useful in selectively inducing cell death in virally infected cells compared to non-infected cells through the accumulation of reactive oxidative species (ROS). This may be useful in the treatment of viral infection and reducing inflammation associated with viral infection. In particular, as a host-directed antiviral (HDA), it may be useful in the treatment of infections with multiple different variants or strains of a virus.

Piperlongumine (Piplartine, 5,6-dihydro-1 -[(2E)-1 -oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-2(1 H)- pyridinone) (PL) is a biologically active alkaloid/amide from peppers, such as from long pepper (Piper longum L.). The piperlongumine compound may be piperlongumine or an analogue, derivative or prodrug of piperlongumine.

Suitable piperlongumine compounds may have the formula;

where Q¹ is O or S,

-Ar is an optionally substituted aryl group,

-D- is selected from -C(O)-, -C(S)-, -CH(OH)- and -CH(SH)-,and

-R¹ and -R² together with -N-D- to which they are attached, form an optionally substituted heterocyclic ring, or -R¹ and -R² are each independently selected from hydrogen and optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl and aryl, and salts, solvates and protected forms thereof.

The group Q¹ is preferably O.

The group -Ar is aryl, including carboaryl or heteroaryl, and the aryl group may be a single aromatic ring or a fused system having two or more aromatic rings.

A carboaryl group may be a Ce-14 carboaryl group, such as phenyl or naphthyl, and most preferably phenyl. A heteroaryl group may be a C5-14 heteroaryl group, such as a C5-10 heteroaryl group, such as a C5-6 heteroaryl group, such as a Ce heteroaryl group, such as pyridinyl and pyrimidinyl, or a C5 heteroaryl group, such as furanyl, thiophenyl, and pyrrolyl.

The aryl group may be an aryl group having 6 aromatic ring atoms, such as phenyl or pyridinyl.

Preferably, -Ar is carboaryl, and is most preferably phenyl.

The aryl group may be optionally substituted, such as with one or more substituent groups. The optional substituents may be selected from the group consisting of halo, cyano, -R^S1, -OH, -OR^S1, -SH, -SR^s1, - NH₂, -NHR^S1, -NR^S1R^S2, -COOH, -CONH2, -CONHR^S1, -CONR^S1R^S2, -NHCOR^S1, -N(R^S1)COR^S1, where each -R^S1 and each -R^S2 is independently alkyl, alkenyl, alkynyl, aryl or aralkyl, which are optionally substituted with halo, such as fluoro, or -R^S1 and -R^S2 may together form a heterocyclic ring.

The aryl group may be optionally substituted, such as substituted, with one or more groups, such as one, two or three groups.

The aryl group may be optionally substituted with one or more groups selected from -OH, -OR^S1, -SH, and - SR^s1, such as -SR^s1 and -OR^S1, such as -OR^S1.

Each -R^S1 and each -R^S2 is preferably selected from alkyl, alkenyl, alkynyl, which are each optionally substituted with halo, such as fluoro.

An alkyl, alkenyl or alkynyl group may be linear or branched.

Where -R^S1 or -R^S2 is alkyl, this may be C1-6 alkyl, such as C1-4 alkyl, such as methyl or ethyl, such as methyl. Where -R^S1 or -R^S2 is alkenyl, this may be C2-6 alkenyl, such as C2-4 alkyl, such as vinyl or allyl.

Where -R^S1 or -R^S2 is alkynyl, this may be C2-6 alkynyl, such as C2-4 alkynyl, such as propargyl.

Where -R^S1 or -R^S2 is aryl, this may be carboaryl, such as C6-10 aryl, such as phenyl, or heteroaryl, such as a C5-14 heteroaryl group, such as a C5-10 heteroaryl group, such as a C5-6 heteroaryl group, such as a Ce heteroaryl group, such as pyridinyl.

Where -R^S1 or -R^S2 is aralkyl, this may be an aryl group, such as described above, connected via a C1-6 alkenyl group, such as C1-2 alkylene group, such as methylene. The most preferred aralkyl group is benzyl. Where -R^S1 and -R^S2 together form a heterocyclic ring, this may be a C5-7 heterocyclic ring, and the heterocyclic ring may optionally contain a further ring heteroatom selected from O, S and N(H).

Where -Ar is a six-membered aryl group this may be substituted at one or more of the 3-, 4- and 5-positions. Here, the 2- and 6-positions may be unsubstituted. In one embodiment, two or each of the 3-, 4- and 5- positions is substituted. Here, the point of connection of the aryl radical is taken as the 1 -position.

The group -Ar may be trialkoxy phenyl, such as 3,4,5-trialkoxyphenyl. The group -Ar may be trimethoxy phenyl, such as 3,4,5-trimethoxyphenyl. The group -D- is preferably selected from -C(O)- and -C(S)-, and is preferably -C(O)-. Here, the group -D- forms an imide or thioimide together with the nitrogen to which it is attached and -C(Q¹)-.

The groups -R¹ and -R², together with -N-D- to which they are attached, form an optionally substituted heterocyclic ring. The heterocyclic ring may be a single ring, or it may be a fused ring system having at least one heterocyclic ring fused to a further ring. The further ring may be another heterocyclic ring, a cycloalkyl ring or an aryl ring.

Preferably the heterocyclic ring is a single ring, and it is not fused to another ring. The heterocyclic ring may be a 4- to 9-membered ring, such as a 4- to 6-membered ring, such as 5- or 6-membered ring, such as a 6- membered ring.

The heterocycle may contain a further ring heteroatom, which may be selected from O, S and N(H). Where a further heteroatom is present, this is separated from the nitrogen atom in -N-D- by at least one carbon ring atom. Typically, no further heteroatoms are present, and the remaining ring atoms are carbon ring atoms.

The heterocyclic ring may be saturated, or partially or fully unsaturated. The heterocyclic ring may be an aromatic ring, but this is not preferred.

Preferably, the heterocyclic ring is partially unsaturated. For example, the heterocyclic ring may contain one double bond, which is a carbon-carbon double bond. For the avoidance of doubt the double bond is endo to the heterocycle (that is, within the ring). Where the group -D- is -C(O)- or -C(S)- it is preferred that the double bond is conjugated with the group -D-.

The heterocyclic ring may be a lactam when -D- is -C(O)- or -C(S)-. This is preferred.

The lactam may be partially or fully unsaturated. The lactam may be a,p-unsaturated, and may be further y,8-unsaturated where a second double bond is present.

Preferably, -R¹ and -R², together with -N-D- to which they are attached, form an a,p-unsaturated 8-lactam (5,6-dihydropyridin-2-one-1-yl). Alternatively, -R¹ and -R², together with -N-D- to which they are attached, form an a, p-unsaturated 8-lactam.

The heterocyclic ring formed by R¹ and -R² together with -N-D- may be optionally substituted, such as optionally substituted with alkyl or halo, such as alkyl. Preferably, the heterocyclic ring is not further substituted.

Alternatively, each of -R¹ and -R² is independently selected from hydrogen, alkyl, alkenyl, alkynyl, and optionally substituted cycloalkyl, heteroalkyl, heterocyclyl and aryl. Preferably one of -R¹ and -R² is not hydrogen. For example, -R² may be hydrogen, whilst -R¹ is other than hydrogen.

Both of -R¹ and -R² may not be hydrogen. The cycloalkyl, heteroalkyl, heterocyclyl and aryl groups may be optionally substituted with alkyl, such as C1-6 alkyl, such as methyl.

The group -R¹ may be alkyl or alkenyl, for example when -R² is hydrogen.

Where -R¹ or -R² is an alkyl group, this may be a straight or branched alkyl group, such as a C1-10 alkyl group, such as a C1-6, such as a C1-4 alkyl group, such as C1-2 alkyl. Examples include methyl, ethyl and propyl.

Where -R¹ or -R² is an alkenyl group, this may be a straight or branched alkenyl group having one or more, preferably one carbon-carbon double bond, such as a C2-10 alkenyl group, such as a C2-6 alkenyl group, such as a C2-4 alkenyl group. Examples include vinyl and allyl.

Where -R¹ or -R² is an alkynyl group, this may be a straight or branched alkynyl group having one or more, preferably one carbon-carbon triple bond, such as a C2-10 alkynyl group, such as a C2-6 alkynyl group, such as a C2-4 alkynyl group. An example is propargyl.

A reference to a cycloalkyl group, this may be a cyclic alkyl group such as C3-7 cycloalkyl group, such as a C5-6 cycloalkyl group. The cycloalkyl group may be saturated, or partially or fully saturated, but it is not aromatic. An example is cyclohexyl.

Where -R¹ or -R² is an heteroalkyl group, this may be an alkyl group where one or two of the carbon atoms is replaced with a heteroatom selected from O, S and N(H). The heteroalkyl group may be linear or branched, and may be a C2-10 heteroalkyl group, such as C2-6 heteroalkyl group, such as C2-4 heteroalkyl group. The heteroalkyl group may be connected via a carbon atom, or a nitrogen atom, where present. An example is methoxymethyl.

Where -R¹ or -R² is an a heterocyclyl group, this may be a cyclic heterocyclic group having one or two heteroatoms each independently selected from O, S and N(H). The heterocyclyl group may be a C3-10 heterocyclyl group, such as a C5-7 heterocyclyl group, such as a C5-6 heterocyclyl group. The heterocyclic group may be saturated, or partially or fully saturated, but it is not aromatic. The heterocyclyl group may be connected via a carbon ring atom, or a nitrogen ring atom, where present. The heterocyclic group may be pyrrolidinyl, morpholinyl and piperidinyl.

Where -R¹ or -R² is an aryl group this may be an aromatic group that may be carboaryl or heteroaryl. The carboaryl group may be phenyl or naphthyl. The heteroaryl group may be C5-10 heteroaryl, such as C5-6 heteroaryl. Examples include pyridinyl, imidazoyl and thiazoyl.

The compound may be provided as a solvate, such as a hydrate.

The compound may be provided in salt form, where appropriate, for example where carboxy (-COOH) or amino (such as -NH- or -NH2) functionality is present within the compound. The compound may be provided as a protected from, where appropriate. For example, where amino functionality (such as -NH- or -NH2) is present this may be protected with a carbamate group, such as a Boc group, and where hydroxyl functionality (-OH) is present this may be protected with a silyl group, such as TBDMS. The use of protecting groups is well known in the art and the skilled person can appreciate that other functionality may be protected, where required, and other protecting groups may be used, as required.

Tautomers of the compounds of formula 1 are also within the scope of the invention.

Prodrug forms of the compound of formula 1 are also within the scope of the invention. For example, where the compound has carboxy (-COOH) or hydroxy (-OH) functionality, these groups may be provided in ester form, where such esters are labile under physiological conditions.

Most preferably, the piperlongumine compound is piperlongumine (piplartine, 5,6-dihydro-1-[(2E)-1-oxo-3- (3,4,5-trimethoxyphenyl)-2-propenyl]-2(1 H)-pyridinone) or an analogue, derivative or prodrug of piperlongumine. In the compound of formula 1 , piperlongumine is a compound where Q¹ is O, -Ar is 3,4,5-triemethoxyphenyl, and -R¹ and -R², together with -N-D- to which they are attached, form an a,p- unsaturated 8-lactam (5,6-dihydropyridin-2-one-1-yl). Piperlongumine (CAS 20069-09-04) may be obtained from commercial suppliers, synthesised using standard techniques or isolated from the Piper longum plant in accordance with standard methods. For example, Seo et al. [9] describe the preparation of piperlongumine derivatives. Other suitable piperlongumine compounds may include (E)-3-chloro-1-(2-methyl-3-(3,4,5- trimethoxyphenyl) acryloyl)-5,6-dihydro-pyridin-2(1 H)-one (Cmpd 11 h; Wu et al (2014) Eur J Med Chem 82 545-551) and the compound of Formula 2 (Cmpd 11 ; Qian et al (2021) J. Nat. Prod. 84 12 3161-3168)

Formula 2

While it is possible for a piperlongumine compound, such as piperlongumine, to be administered to a patient or individual alone, it is preferable to present the compound in a pharmaceutical composition or formulation.

A pharmaceutical composition may comprise, in addition to the piperlongumine compound, one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well-known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active compound. The precise nature of the carrier or other material will depend on the route of administration, which may be by bolus, infusion, injection or any other suitable route, as discussed below. Suitable materials will be sterile and pyrogen free, with a suitable isotonicity and stability. Examples include sterile saline (e.g. 0.9% NaCI), water, dextrose, glycerol, ethanol or the like or combinations thereof. The composition may further contain auxiliary substances such as wetting agents, emulsifying agents, pH buffering agents or the like. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington’s Pharmaceutical Sciences, 18^th edition, Mack Publishing Company, Easton, Pa., 1990.

The term “pharmaceutically acceptable” as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. Such methods include the step of bringing into association the active compound 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 compound with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, lozenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols.

The piperlongumine compound or pharmaceutical compositions comprising the piperlongumine compound may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); and parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example, subcutaneously or intramuscularly. In some preferred embodiments, the piperlongumine compound may be administered by an intranasal or inhalation route.

The pharmaceutical compositions comprising the piperlongumine compounds may be formulated in a dosage unit formulation that is appropriate for the intended route of administration.

Formulations suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or oily solutions of the active compound.

Formulations suitable for administration by inhalation include those presented as an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichorotetrafluoroethane, carbon dioxide, or other suitable gases. Optionally, other therapeutic or prophylactic agents may be included in the pharmaceutical composition or formulation.

Piperlongumine compounds as described herein is useful in the treatment of viral infection. Viral infection may include infection with a respiratory virus, for example a coronavirus, such as HCoV-NL63, Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (Sars-Cov), or severe acute respiratory syndrome coronavirus 2 (Sars-Cov2); or an influenza virus, such as an influenza A, B, C or D virus; a DNA virus, for example monkeypox (Mpox); or an RNA virus, for example a flavivirus, such as Zika virus.

In some preferred embodiments, the virus is SARs-CoV-2. Viral infections may include for example SARS- CoV-2 infection.

A method may comprise intranasal administration or administration by inhalation of the piperlongumine compound.

An individual or patient suitable for treatment as described above may be a mammal, such as a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), a primate, simian (e.g. a monkey or ape), a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orang-utan, gibbon), or a human. In some preferred embodiments, the individual or patient is a human. In other preferred embodiments, non-human mammals, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g. murine, primate, porcine, canine, or leporid) may be employed.

The individual or patient may be vaccinated against the virus or non-vaccinated against the virus.

The term “treatment”, as used herein in the context of treating a condition, pertains generally to treatment and therapy in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress and amelioration of the condition, and cure of the condition.

In particular, treatment of a viral infection as described herein may, for example, achieve one or more of (i) a reduction in susceptibility to infection with a virus, (ii) a decrease in the extent or severity of infection with an virus, (iii) inhibition of the progress of infection with an virus, (iv) a reduction or halt in the rate of infection with an virus, (v) prevention, delay, reduced risk of or reduced severity of the acute phase of infection with an virus; (vi) reduced production, amplification, propagation or transmittal of virus following infection with an virus, (vii) prevention or reduction of the shedding of virus following infection with an virus, for example shedding into surrounding tissue, the lumen of hollow lumen organs or bodily fluids/secretions, (viii) amelioration of the one or more symptoms of infection with a virus, and (ix) cure of infection with an virus.

In some preferred embodiments, treatment may be prophylactic or preventative treatment (i.e. prophylaxis). Prophylactic or preventative treatment may be provided to an individual before or after exposure to a virus. For example, a method described herein may be a prophylactic method for preventing or reducing the risk of viral infection in an individual. The treated individual may be uninfected with the virus and the risk of infection of the individual may be reduced. For example, an individual susceptible to or at risk of infection with a virus may be treated as described herein. A method of reducing the susceptibility or increasing the resistance of an individual to a viral infection or reducing the risk of an infection in an individual may for example comprise increasing the amount or concentration of ROS in virally infected cells, as described herein. This treatment may prevent or delay infection in the individual, reduce the susceptibility of the individual to infection, increase the resistance or reduce the risk of the individual to infection.

An individual or patient suitable for treatment as described above may be at risk of viral infection or might be exposed to a viral infection. Treatment as described herein may prevent or reduce the risk of the occurrence of viral infection in the patient or individual. Other suitable patients or individuals may be suffering from a viral infection. Treatment as described herein may decrease in the extent or severity of infection of the viral infection in the patient or individual.

A piperlongumine compound may be administered as described herein in a therapeutically-effective amount.

The term “therapeutically-effective amount” as used herein, pertains to that amount of an active compound, or a combination, material, composition or dosage form comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio.

The appropriate dosage of a piperlongumine compounds may vary from individual to individual. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the administration. The selected dosage level will depend on a variety of factors including, but not limited to, the route of administration, the time of administration, the rate of excretion of the active compound, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the individual. The amount of active compounds and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve therapeutic plasma concentrations of the active compound without causing substantial harmful or deleterious side-effects.

In general, a suitable dose of the active compound is in the range of about 100 pg to about 400 mg per kilogram body weight of the subject per day, preferably 200 pg to about 200 mg per kilogram body weight of the subject per day. Where the active compound is a salt, an ester, prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.

Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals).

Methods of determining the most effective means and dosage of administration are well known in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the physician. Multiple doses of the piperlongumine compound may be administered, for example 2, 3, 4, 5 or more than 5 doses may be administered. The administration of the piperlongumine compound may continue for sustained periods of time. For example, treatment with the piperlongumine compound may be continued for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month or at least 2 months. Treatment with the piperlongumine compound may be continued for as long as is necessary to reduce viral infection symptoms or achieve complete curing.

The piperlongumine compound may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the individual circumstances. For example, a piperlongumine compound as described herein may be administered in combination with one or more additional active compounds.

The piperlongumine compound may be administered in combination with a second therapeutic agent. For example, the piperlongumine compound may be administered in combination with vaccines and other drugs approved for pre-exposure or post-exposure treatment or prophylaxis. In some embodiments, the piperlongumine compound may be administered in combination with an anti-viral compound, for example, an anti-viral compound selected from plitidepsin, remdesivir, paxlovid and molnupiravir.

The amount or level of ROS in a cell may be increased as described herein to induce or increase the cell death of virally infected cells. A method of increasing the amount or level of ROS in a virally infected cell comprises contacting the virally infected cell with a piperlongumine compound as described herein.

Piperlongumine compounds which can induce ROS accumulation in a host cell to selectively induce virally infected cell death are described above and may include for example l-3-chloro-1-(2-methyl-3-(3,4,5- trimethoxyphenyl) acryloyl)-5,6-dihydro-pyridin-2(1 H)-one.

The cell may be an isolated cell, for example a cell line or a cell from a sample obtained from an individual; or may be within a cellular assembly, organoid, tissue or organ.

The amount or level of ROS in a cell may be increased in vitro or in vivo. For example, the virally infected cell may be in a patient or individual.

Methods described herein may also be useful in determining the efficacy of a piperlongumine compound for the treatment of a viral infection. For example, a method of determining the efficacy of a piperlongumine compound in the treatment of a viral infection may comprise determining the effect of a piperlongumine compound on the amount or concentration of ROS in one or more cells. An increase in the amount or level of ROS in the one or more cells may be indicative that the piperlongumine compound may be efficacious in the treatment of a viral infection. The one or more cells may be isolated cells, for example a cell line or cells from a sample obtained from an individual; or may be a cellular assembly or organoid. In some embodiments, the cells may be from a sample obtained from an individual being treated with the piperlongumine compound. Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of’ and the aspects and embodiments described above with the term “comprising” replaced by the term ’’consisting essentially of’.

It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention.

All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.

“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above.

Experimental

Methods

Cell lines and culture

Vero CCL-81 cells and human lung adenocarcinoma epithelial A549 cells expressing hACE2 cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium, Life Technologies), supplemented with 10% (v/v) Fetal bovine serum (FBS), 1 % penicillin-streptomycin and 1 % glutamax (ThermoFisher). All cell lines were cultured at 37°C and 5% CO2.

Viral strains and stocks

The Wuhan-like early European SARS-CoV-2 B.1 Lineage was isolated from a Portuguese patient (internal reference: 606JMM ID_5452) at approximately 1.7x10⁶ PFU/mL. The alpha variant (NR-54000; lineage B.1.1 .7, Isolate hCoV-19/England/204820464/2020) was obtained through BEI Resources, NIAID, NIH, contributed by Bassam Hallis. The delta variant (NR-55611 ; lineage B.1.617.2; Isolate hCoV- 19/USA/PHC658/2021) was obtained through BEI Resources, NIAID, NIH, contributed by Dr. Richard Webby and Dr. Anami Patel. The original and alpha variant virus stocks were propagated using Vero CCL-81 cells, 1 .4 x10⁷ cells were seeded in different T175 culture flasks and infected the following day at a 0.005 multiplicity of infection (MOI) in 10 mL of Maintenance Medium (MM; DMEM medium supplemented with 2.5% FBS, 1 % penicillin-streptomycin and 1 % glutamax). After 1 h inoculation, the culture medium was replaced with another fresh 25 mL Maintenance Medium, and virus propagation was continued until 4-day post infection. To isolate the virus, cell supernatants were collected from the T175 flasks, centrifuged at 300 x g for 5min to remove the cell debris, aliquoted and stored at -80°C. The titers of the generated stock virus were quantified by plaque assay. All work with infectious SARS-CoV-2 was conducted at Level 3 Biosafety Laboratory (BSL3) facility of Instituto de Medicina Molecular where all procedures follow Directive 2000/54/- C - on the protection of workers from risks related to exposure to biological agents at work, Directive (EU) 2020/7-9 - as regards the inclusion of SARS-CoV-2 in the list of biological agents known to infect humans, and WHO (World Health Organization) guidelines.

SARS-CoV-2 infection experiments

All the antiviral studies were performed in animal biosafety level 3 (BSL3) facility at institute of molecular medicine (iMM) in Lisbon, Portugal. Vero CCL-81 cells or A549-hACE2 cells were seeded in 24 well plates at 1 .6 x 10⁵/well the day before infection. A series of concentrations of piperlongumine were added 1 h prior to or after infection. SARS-CoV-2 virus were thawed, vortexed, centrifuged, and used to infect the cells at different multiplicity of infection (MOI), 0.035 of MOI for Vero-CCL 81 cells and 0.1 of MOI for A549-hACE2 cells. After 1 h inoculation, the inoculum was replaced by fresh media containing piperlongumine. Supernatants were collected for plaque assay after 24h treatment, and cells were collected for qPCR quantification to quantify virus load.

Plaque assay (detect viral plaque-forming units)

Vero CCL-81 cells were seeded in 6 well-plates at 8 x 10⁵/well and allowed to grow to around 80% confluence after 24h. Supernatants of cultures treated with compounds were 1 :10 serially diluted in Maintenance Medium to obtain 10^-1 and 10^-2 dilutions, added to pre-seeded 6-well plate cells, and incubated at 37 °C for 1 h, shaken every 15min. Then replaced with 1.25% carboxymethylcellulose (CMC) and incubated at 37 °C for 4 days. The CMC was removed after the incubation, cells were fixed with 4% formaldehyde/PBS and stained with 0.1 % toluidine blue. Viral plaques were counted to determine the infectious titers [PFU (plaque forming units)/mL],

Cytotoxicity Assay

Vero CCL-81 cells or A549-hACE2 cells were seeded in 96 well-plates at 1 x 10⁴/well the day before treatment. An increasing concentration of piperlongumine was added to cells for another 24h incubation. CellTiter Blue viability assay (Promega, Cat#G8080) was used to assess the viability of cells after treatment, according to manufacturer’s protocol.

Viral RNA isolation and quantitative PCR

The viral pellet was suspended in NVL buffer and extracted using NZY Viral RNA Isolation Kit (NZYtech, Cat#MB40701). 1 ug of total RNA was used for RT-PCR using the NZY First-Strand cDNA Synthesis Kit (NZYtech, Cat#MB12502). The qPCR amplification was performed with a dilution 1 :10 of cDNA using the iTaq Universal SYBR Green Supermix (Bio-rad, Cat#1726124) according to the manufacturer’s instructions, and analysed on Quantstudio™ 5 real-time PCR machine (Applied Biosystems). The relative quantification of target gene expression was performed using comparative cycle threshold (CT) method.

Western Blotting analysis

For cell lysis analysis, cells were lysed using whole cell lysis buffer (50 mM Tris-HCI pH = 8.0, 450 mM NaCI, 0.1 % NP-40, 1 mM EDTA), supplemented with 1 mM DTT, protease inhibitors (Sigma), and phosphatase inhibitors (Sigma). For in vivo experiments, the left lung from mice was homogenized in 3mL of DMEM and 750uL was transferred to an equal volume of whole cell lysis buffer, supplemented as above. Protein concentrations were quantified using Bradford Assay (Biorad). 30ug of protein was loaded per lane and separated on SDS-PAGE gels, then transferred onto Polyvinylidene Difluoride (PVDF) membranes (GE Healthcare). Membranes were blocked for 1 h with 5% skim milk or 5% BSA in TBS supplemented with 0.05% Tween-20 (TBST) at room temperature for 1 h, then probed with any of the following specific primary antibodies in 0.05% TBST at 4 °C overnight. After three washes with 0.05% TBST, secondary antibodies, Antimouse (1 :5000) or Antirabbit (1 :5000) (Jackson ImmunoResearch) were added to the membrane in 0.05% TBST for 1 h at room temperature. All membranes were washed three times and exposed using ECL substrate (Biorad, Cat#170-5060) and Amersham 800 Imaging System (Cytiva) The primary antibodies included: MAVS (sc-166583), JAK1 (sc-376996), phos-STAT1 (sc-8394), STAT1 (sc-464), phos-STAT3 (sc- 8059), STAT3 (sc-8019), GAPDH (sc-47724), goat anti-mouse IgG H&L (HRP) (Abeam, ab205719) and goat-anti rabbit HRP (Abeam, ab6721).

Animal models of SARS-CoV-2 in vivo experiments

Animal studies were conducted in the BSL-3 Facility in strict compliance with the relevant EU and national legislation and were approved by the Portuguese official veterinary department for welfare licensing - Direqao Geral de Alimentaqao e Veterinaria - (license number 01878/2021) and the Institute de Medicina Molecular Animal Ethics Committee. Eight to twelve week-old specific pathogen-free hemizygous for Tg(K18-ACE2)2Prlmn mice (Strain B6.Cg-Tg(K18-ACE2)2Prlmn/J, the Jackson laboratory strain 034860) were used in this study. Mice were treated with Vehicle (n=8), piperlongumine at 1 mg/kg (n=18) or Plitidepsin at 1 mg/kg (n=6) 1 hour prior to infection. Vehicle and piperlongumine were administered intranasally, and Plitidepsin was given by subcutaneous route. Then mice were intranasally infected with 1 x 10⁴ PFU of SARS-CoV-2 in 50 p.l of Maintenance medium. The body weight of mice and signs of illness were monitored daily, on day 5 post infection, all mice were humanely euthanized and lung tissues were harvested. All left lungs were homogenized by ULTRA-TURRAX® Tube Drive control and frozen at - 80 °C for viral quantification by qPCR and plaque assay, and protein analysis. All right lungs were fixed in 10% neutral buffered formalin and sent for histology analysis. We also included a toxicity study group where we treated mice with 1 mg/kg piperlongumine at the same time, without infection with virus, to test piperlongumine toxicity on mice.

Mouse lung histological analysis Lungs were fixed in formalin, embedded in paraffin, and tissue sections cut and stained with H&E. Scoring of lung pathology was performed taking into account features described in Table 1 , adapted from previously published critetia [10], Briefly, haemorrhage, congestion, necrosis and hyperplasia of bronchiolar epithelium, inflammation and proteinaceous debris were scored according to a 5-tier scale: 0, absent; 1 , minimal; 2, mild; 3, moderate; 4, marked. For scoring thickness of alveolar wall: 0, <2x; 1 , 2-4x; 2, 5-1 Ox; 3, 11-20x; 4, no airspace. For percentage of area affected: 0, none; 1 , <25% of total lung area; 2, 26-50%; 3, 51-75%; 4, >76%. Final score for each lung/animal was calculated by dividing the final score per number of features assessed (total of 8) and multiplying by % of area affected (multiplication factor for score 1 was 0,25; for 2, 0,5; for 3, 0,75 and for 4, corresponding to 76 to 100% of lung area affected, multiplication factor was 1). Representative hematoxylin and eosin pictures were obtained using NDP.view2 software (Hamamatsu) in slides digitally scanned in the Hamamatsu NanoZoomerSQ. Measurement of ROS level

The level of Reactive Oxygen Species (ROS) was measured by ROS-ID Total ROS detection kit (Enzo, Cat#ENZ-51011), according to manufacturer’s instructions. Briefly, Vero CCL-81 cells were seeded in 96- well black wall/clear bottom plates at a density of 1x10⁴ cells per well one day before the assay. Cells were treated with piperlongumine for 1 h prior to the infection, then incubated for 1 h with virus inoculum in combination the ROS detection reagent at 37 °C. Subsequently, the ROS levels were measured using a fluorescence microplate reader (TECAN Infinite M200, Switzerland) and a standard fluorescein (Ex=488nm, Em=520nm) filter set. N-acetyl cysteine (NAC, 5mM) was used as ROS inhibitor for the assay.

Glutathione (GSH) and glutathione disulfide (GSSG) levels

Measurement of GSH/GSSG was performed following Rahman et al, with minor adjustments. Cells were seeded in a 6-well plate at a density of 4.5 x 10⁵ cells per ml and subjected to the different treatments and inoculations. During harvesting, cells were collected in ice cold 1x PBS, centrifuged and resuspended in ice- cold extraction buffer (0.1 % Triton-X and 0.6% Sulfosalicyclic acid in KPE). After homogenization, samples were sonicated in ice for 3 min and subjected to 2 freeze-thaw cycles to ensure proper lysis. Then the samples were centrifuged at 3000g for 4 min at 4°C, and the supernatant was collected and stored at -80°C until further procedures. For GSH determination, 20 pd of each sample was mixed with freshly prepared DTNB and glutathione reductase, incubated for 30 seconds and further mixed with p-NADPH. Absorbance was measured at 412 nm for 2 min to determine the rate of 2-nitro-5-thiobenzoic acid formation. For GSSG quantification, 100 pd of the sample was incubated with 2 pd of 2-vinylpyridine for 1 hour at room temperature. For neutralization, 6 pd of triethanolamine was added and incubated for 10 min. GSSG samples were then subjected to the same protocol as for the GSH determination. The GSH and GSSG levels were determined from comparisons with a linear GSH or GSSG standard curve, respectively. [11]

Fluorescence Microscopy

The day before the beginning of the experiment, A549-hACE2 cells were seeded at a confluence of 3x10⁴ per well in a p.-Slide 8-well Ibidi plate. Subsequently, cells were treated with PL 1 hour before infection, followed by SARS-CoV-2 inoculation at a 0.1 multiplicity of infection (MOI) for another hour. Then the medium was replaced for fresh Maintenance medium (MM) containing PL for 24 hours. Cells were then washed with 1x PBS and fixed with 4% paraformaldehyde in 1x PBS for 30 min, and then washed 3 times with 1x PBS. After fixation, cells were permeabilized with 0.3% triton in 1x PBS for 10 min, washed 3 times with 1x PBS and blocked in 3% BSA in PBS for 1 hour at RT. Cells were then incubated with the primary antibody against MAVS (Santa Cruz, sc-166583) and Citrate Synthase (Proteintech, #16131 -1 -AP) in blocking buffer for 3h at RT, followed by secondary antibody (AF-488 and AF-568, respectively) in 1x PBS for 1 hour at RT. Nuclei were stained with Hoechst 33342 for 10 min. Images were acquired in z-stacks with the 63x objective of the LSM880 Airyscan setup (Zeiss) and processed and analyzed in Image J, using JaCoP Macro. [12]

Data analysis

The data quantification was performed using the GraphPad Prism software. Data are presented as mean±SD. A one-way ANOVA was used to compare differences in more than two groups. A Student’s t test was used to compare differences between two groups. In all circumstances, P-values <0.05 were considered significant (*P<0.05, **P<0.01 , ***P<0.001).

Results

Here we observed piperlongumine, 1 , also shows anti-SARS-CoV-2 activity, with low micromolar potency in vitro. By inducing the accumulation of ROS in infected cells selectively, piperlongumine, 1 , leads to anti-viral effect. Since piperlongumine, 1 , didn’t target the virus directly, instead it affects the ROS level in infected cells, therefore it may be the pan-SARS-CoV-2 therapeutic. Finally, we validated the anti-viral effect of piperlongumine, 1 , by using K18-hACE2 mouse model (transgenic expression of human ACE2 (hACE2) under a cytokeratin 18 promotor). Since only a few studies have been tested successfully to inhibit SARS- CoV-2 in this animal model, we included Plitidepsin as a benchmark and piperlongumine, 1 , seems to be more potent in reducing viral lung titers. [13] Overall, piperlongumine, 1 , is a high potential antiviral compound against COVID-19 and emerging SARS-CoV-2 VOCs.

Piperlongumine, 1, is a potent inhibitor of SARS-CoV-2 in vitro

In an effort to explore the effect of piperlongumine, 1 , (Fig 1 A) against SARS-CoV-2, we evaluated the antiviral activity of piperlongumine, 1 , in vitro by using two different cell lines. One is VERO-CCL 81 cell, which was isolated from Cercopithecus aethiops kidney, widely used in virus study due to its improved host cells for virus propagation and viral vaccine production, production of high-titer viral stocks and rescue of clinical viral isolates. [14] Another one is human lung carcinoma cells (A549) expressing angiotensin-converting enzyme 2 (HA-FLAG) (A549-hACE2) cell. The cytotoxicity of piperlongumine, 1 , was first examined in both cell lines, we found both cells are tolerant to piperlongumine, 1 , with half-maximal cytotoxic concentration (CC50) values at 51.16 pM in VERO-CCL 81 cell (Fig 1 B) and 627.1 pM in A549-hACE2 cell (Fig 1C) respectively. This allowed us to test the anti-viral activity of piperlongumine, 1 , in a range of concentration under 10 |j.M. Then we tested piperlongumine, 1 , inhibition of SARS-CoV-2 replication using both cell types. With piperlongumine, 1 , given 1 h after infection and incubate for 24h in VERO-CCL 81 cell, it can greatly inhibit Envelop (E) gene and Nucleocapsid (N) gene of SARS-CoV-2 virus in a dose-dependent manner with half-maximal inhibitory concentration (IC50) at 1 .518 |j.M for E gene (Fig 1 D) and 2.112 |j.M for N gene (Fig 1 E), quantified by qPCR assay. With piperlongumine, 1 , treated prophylactically as 1 h prior to infection and incubation for another 24h after infection, we observed that piperlongumine, 1 , inhibit the E gene and N gene of SARS-CoV-2 virus in a dose-dependent manner with IC50 at 1.103 |j.M for E gene (Fig 1F) and 1.176 |j.M for N gene (Fig 1G) in VERO-CCL 81 cell, which is even more potent than the therapeutic treatment. It was also supported by plaque assay that piperlongumine, 1 , can significantly reduce virus plaque forming with IC50 at 1 .349 |j.M (Fig 1 H-l), which also indicated the observed effect on viral replication was a result of the specific antiviral activity of piperlongumine, 1 . In A549-hACE2 cell line, we observed the same inhibition effect of piperlongumine, 1 , with IC50 at 3.315 |j.M for E gene (Fig 1 J) and 3.558 |j.M for N gene (Fig 1 K), quantified by qPCR assay. We observed the same dose-dependent inhibition effect of piperlongumine, 1 , in Calu3 cells using a plaque assay, with an IC50 of about 10.7 |j.M (Fig 8).

Piperlongumine, 1, inhibits SARS-CoV-2 VOC infection (both in vitro and in vivo)

Given the continuous evolution of the virus that leads to SARS-CoV-2, more and more variants of concern (VOC) emerged. Based on that, we further tested piperlongumine, 1 , against three VOCs in VERO-CCL 81 cells: Alpha (B.1 .1 .7), Delta (B.1 .617.2) and Omicron (B.1 .1 .529). We observed piperlongumine, 1 , can inhibit Alpha in a dose-dependent manner with IC50 at 0.15 |j.M for E gene and 0.07 |j.M for N gene (Fig 2A), quantified by qPCR assay. Also, piperlongumine, 1 , can inhibit Delta in a dose-dependent manner with IC50 at 0.22 |j.M for E gene and 0.49 |j.M for N gene (Fig 2B), quantified by qPCR assay. Also, piperlongumine, 1 , can inhibit Omicron, quantified by qPCR assay (Figure 2C). This underlines the potential of piperlongumine, 1 , to act as a pan-variant, host-directed antiviral against emerging SARS-CoV-2 VOCs.

Piperlongumine, 1, shows in vivo antiviral efficacy in mouse models of SARS-CoV-2 infection

After testing the efficacy of piperlongumine, 1 , in vitro, we investigated whether intranasal administration would improve morbidity and survival in vivo, using transgenic K18-hACE2 mice expressing the human ACE2 receptor driven by the keratin 18 promotor, an established mouse model of severe SARS-CoV-2 disease. [15, 16]

The preclinical anticancer potential of piperlongumine, 1 , has been extensively investigated, with a well- established safety profile and pharmaco-kinetics, supported by several publications and patents for the treatment of cancer using piperlongumine, 1 .[17-22] Dosing regimens and drug concentration were chosen based on the solubility of piperlongumine, 1 , and on previous study about piperlongumine, 1 , treatment in cancer. [23]

We firstly evaluated the desired dose of piperlongumine, 1 , toxicity on our mouse model, with single dose 1 mg/kg piperlongumine, 1 , administered on Day 0, we didn’t observe any body weight lose nor side effect, which indicate 1 mg/kg piperlongumine, 1 , is safe to the animals. Meanwhile we included a benchmark in this in vivo study named Plitidepsin, as it showed a great reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment in mouse models of SARS-CoV-2 infection. [24] The mice were administered with 1 mg/kg piperlongumine, 1 , or Plitidepsin 1 h prior to infection, challenged with 5x10⁴ PFU/mouse of SARS-CoV-2 on Day 0, and monitored daily until Day 5 or 6 when the vehicle mice reached 80% of initial body weight and showed severe disease signs including back arching, hard breathing, and decreased mobility, then the mice were euthanized, and lungs were collected for virus loading quantification or histopathology analysis (Fig 3A).

We found mice infected with Alpha VOC (Fig 3B), Delta VOC (Fig 3C) and Omicron (Fig 3D) and pre-treated with piperlongumine, 1 in an in vivo prophylactic setting showed a delay on body weight losing and onset of symptoms comparing to vehicle group or Plitidepsin-treated group (Fig 3B to D). Both qPCR and plaque assay used for virus loading quantification indicated that piperlongumine, 1 , significantly reduced the viral lung titres (over 60% reduction) at Day 5 and was even more potent than single dose 1 mg/kg Plitidepsin treatment (Fig 3E-G).

In an in vivo therapeutic treatment setting, mice were treated with piperlongumine or plitidepsin 1 day after omicron VOC infection. No significant change in body weight was observed over 6 days (Fig 3H) but a qPCR assay showed that piperlongumine significant reduced lung viral titers on Day 6 compared to vehicle group or Plitidepsin-treated group (Fig 3I). Histopathology analysis (Fig 3J-L) also showed a reduction of lung inflammation in piperlongumine-treated mice (histopathology score of 1 .40/4) over vehicle-treated (histopathology score of 2.26/4) at Day 5, as well as less pulmonary edema, hyaline membranes, proliferative bronchiolar epithelium, haemorrhage and neutrophil infiltration in piperlongumine-treated group.

Taken together, these results showed that piperlongumine, 1 , can reduce the replication of SARS-CoV-2 and reduce lung inflammation in vivo, which has compelling potential for clinical efficacy for the prophylactic treatment of COVID-19.

Piperlongumine, 1, anti-viral effect is related to its selectively induction of ROS in infected cells Given that piperlongumine, 1 , selectively kills cancer cells but not normal cells by inducing reactive oxygen species (ROS) in cancer cells, [23] we were wondering if piperlongumine, 1 , could also induce ROS level in infected cells. We firstly determined the effect of piperlongumine, 1 , on total cellular ROS level and found piperlongumine, 1 , can selectively induce the ROS level in infected cell, but not in non-infected cell (Fig 4A). More specifically, we observed that piperlongumine, 1 , treatment led to a decrease in reduced glutathione (GSH) level and an increase in oxidized glutathione (GSSG) levels in infected cell but didn’t affect GSH or GSSG levels in non-infected cells (Fig 4B-C). GSH is one of the most important scavengers of ROS, and the reaction change from GSH to GSSG may be used as a marker of oxidative stress. [25]Additionally, qPCR assay showed the reduction of pi-class glutathione S-transferase (GSTP1) in piperlongumine-treated infection cells (Fig 4D). Known as protein to regulate oxidative stress, the reduction of GSTP1 expression in cells is believed to increase the generation of ROS level [26], which also support that piperlongumine, 1 , enhance ROS accumulation in infected cells.

Furthermore, co-treatment with piperlongumine, 1 , and the reducing agent N-acetyl-L-cysteine (NAC, 5mM), which quenches ROS, prevented piperlongumine-mediated GSH depletion (Fig 4B). The increased dependence of infected cells on the ROS stress-response pathway may be the basis for the piperlongumine- induced anti-viral effect. To prove this hypothesis, we treated the cells with NAC prior to piperlongumine, 1 ,- treatment and virus inoculum, it showed that piperlongumine-induced virus inhibition is rescued by the antioxidant NAC (Fig 4E), suggested by both E gene and N gene of SARS-CoV-2 quantified by qPCR assay.

Piperlongumine, 1, potently triggers MAVS-induced IFN-JAK-STAT pathway

ROS modulate various inflammatory processes and it’s reported that increased cellular ROS amplifies retinoic acid inducible gene 1 (RIG-1) signaling and mitochondrial antiviral-signalling protein (MAVS) function at the mitochondria. [27-29] Once coronavirus RNA in the cytoplasm sensed by the cytoplasmic RNA sensors RIG-1 and melanoma differentiation associated protein 5 (MDA5), which will trigger conformational changes in theses sensors and interacts with MAVS, recruiting the downstream effector proteins, including IFN-JAK- STAT pathway for further antiviral response (Fig 5A). [30] We then performed western blot assay to quantify the proteins levels in vitro to test whether the anti-viral effect of piperlongumine, 1 , related to above pathway. We observed that MAVS, JAK1 , p-STAT1 are upregulated in piperlongumine-treated infection sample comparing to DMSO infection control (Fig 5B). The upregulation of MAVs can also be validated by qPCR assay (Fig 5C). Furthermore, we also tested it in vivo by using the homogenized lung sample and found MAVS and p-STAT1 are upregulated in piperlongumine-treated infected mice, along with decrease in spike protein level of SARS-CoV-2 (Fig 5D). The fluorescence microscopy also showed the percent of MAVS co- localized with mitochondria increased in PL-treated infected cells, which indicated more translocation of MAVS to mitochondria comparing to vehicle group (Fig 5E-F).

In the present study, we report on piperlongumine, 1 , a small molecule extracted from natural plant, shows great antiviral effect both in vitro and in vivo. The potential for broad SARS-CoV-2 antiviral activity makes piperlongumine, 1 , an intriguing candidate for further exploration as a treatment for viral infection and may offer a complement to current antiviral drugs. Importantly, piperlongumine, 1 , acts as a kind of host-targeted antiviral by inducing ROS levels in infected cells, may be more resistant to naturally occurring viral mutants comparing to those viral-targeted therapeutics and vaccines.

The K18-hACE2 mouse model we employed in this study is shown to be a robust model to recapitulate COVID-19 disease, due to high morbidity and mortality. [31] The SARS-CoV-2 infection on this model provides severe viral replication in the lung, leads to severe immune cell infiltration, inflammation, and pulmonary disease, which is highly useful to evaluate compound that reduce virus infection or associated pathological inflammatory responses. [32] With this severe model, despite piperlongumine, 1 , not fully maintaining the body weight of the infected mice, it delayed the disease progression and greatly reduce viral lung titers with over 60% inhibition. Our benchmark Plitidepsin, is a very promising antiviral compound with nanomolar potency, and is going on to clinical trials. By comparing to Plitidepsin antiviral activity in mouse model, our piperlongumine, 1 , shows three advantages: 1) the effect of Plitidepsin on mortality was not mentioned in the paper [13], From our data, Plitidepsin cannot protect the mice from losing weight, while piperlongumine, 1 , could delay the body weight losing, which means it reduce the disease symptoms. 2) piperlongumine, 1 , was administered intranasally while Plitidepsin was given subcutaneously. This route may be useful to maximize airway and lung exposure and limit the systemic exposure [33], especially for SARS- CoV-2 which enters the human body through nasal epithelial cells. [34] More importantly, it may also facilitate self-administration. 3) The viral lungs titer in piperlongumine-treated group is less than Plitidepsin- treated group through qPCR quantification for the E gene of SARS-CoV-2 virus, which indicates that piperlongumine, 1 , is even more potent in this mouse model.

For the mechanism of action (MOA) of piperlongumine, 1 , anti-viral activity, we show above that piperlongumine, 1 , selectively induces ROS level in infected cells, but not in non-infected cells. The ROS accumulation explains how piperlongumine, 1 , upregulate MAVS expression and further activate IFN-JAK- STAT pathway. This pathway plays an essential role in the regulation of local and systemic inflammation in response to viral infections. JAKs are responsible for the phosphorylation and activation of STATs. STAT1 after phosphorylation forms a complex with other proteins and travels to the nucleus where it interacts with the interferon-stimulated response element (ISRE) promoter to drive the expression of downstream interferon-stimulated genes (ISGs) which perform different antiviral functions. [35, 36]

Overall, we identified and validated piperlongumine, 1 as a potent antiviral compound against SARS-CoV-2 and VOCs, by selectively induce ROS level in host cells and further trigger MAVS-induced IFN-JAK-STAT pathway.

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Claims

Claims:

1 . A method of treating or reducing susceptibility to a viral infection comprising; administering a piperlongumine compound to a patient in need thereof.

2. A method according to claim 1 wherein the piperlongumine compound selectively increases the amount or concentration of ROS in the virally infected cells.

3. A method according to claim 2 wherein the piperlongumine compound has the formula 1 :

where Q¹ is O or S,

-Ar is an optionally substituted aryl group,

-D- is selected from -C(O)-, -C(S)-, -CH(OH)- and -CH(SH)-, and

-R¹ and -R², together with -N-D- to which they are attached, form an optionally substituted heterocyclic ring, or -R¹ and -R² are each independently selected from hydrogen and optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl and aryl, and salts, solvates and protected forms thereof.

4. The method according to claim 3, wherein Q¹ is O.

5. The method according to claim 2 or claim 4, wherein -D- is -C(O)- or -C(S)-, such as -C(O)-.

6. The method according to any one of claims 3 to 5, wherein -R¹ and -R², together with -N-D- to which they are attached, form an optionally substituted heterocyclic ring, such as a heterocyclic ring.

7. The method according to claim 6, wherein the heterocyclic ring is partially unsaturated.

8. The method according to claim 7, wherein -D- is -C(O)- or -C(S)-, and the unsaturation is conjugated with the -C(O)- or -C(S)-.

9. The method according to any one of claims 6 to 8, wherein the heterocyclic ring is single ring, such as a 4- to 9-membered ring, such as a 4- to 6-membered ring, such as 5- or 6-membered ring, such as a 6- membered ring.

10. The method according to any one of claims 6 to 9, wherein -R¹ and -R², together with -N-D- to which they are attached, form an a,p-unsaturated 8-lactam, such as wherein -R¹ and -R², together with -N-D- to which they are attached is 5,6-dihydropyridin-2-one-1-yl.

11 . The method according to any one of claims 3 to 10, wherein Ar- is an optionally substituted carboaryl group, such as phenyl, or an optionally substituted heteroaryl group, such as pyridinyl, pyrimidinyl, furanyl, thiophenyl, and pyrrolyl.

12. The method according to claim 11 , wherein Ar- is optionally substituted phenyl.

13. The method according to any one of claims 3 to 12, wherein Ar- is an aryl group optionally substituted with one or more groups, such as one, two or three groups, selected from halo, cyano, -R^S1, -OH, -OR^S1, -SH, -SR^s1, -NH₂, -NHR^S1, -NR^S1R^S2, -COOH, -CONH₂, -CONHR^S1, -CONR^S1R^S2, -NHCOR^S1, - N(R^S1)COR^S1, where each -R^S1 and each -R^S2 is independently alkyl, alkenyl, alkynyl, aryl or aralkyl, which are optionally substituted with halo, or -R^S1 and -R^S2 may together form a heterocyclic ring.

14. The method according to claim 13, wherein Ar- is an aryl group optionally substituted with one or more groups, such as one, two or three groups, selected from -OH, -OR^S1, -SH, and -SR^s1, such as -SR^s1 and -OR^S1, such as -OR^S1.

15. The method according to any one of claims 3 to 14, wherein Ar- is trialkoxy phenyl, such as trimethoxy phenyl.

16. A method according to any one of claims 1 to 15, wherein the piperlongumine compound is piperlongumine.

17. A method according to any one of claims 1 to 16 wherein the viral infection is caused by a respiratory virus.

18. A method according to any one of claims 1 to 17 wherein the viral infection is caused by an RNA virus.

19. A method according to claim 18 wherein the RNA virus is a coronavirus.

20. A method according to claim 19 wherein the coronavirus is SARS-CoV-2.

21 . A method according to any one of claims 1 to 20 wherein the compound is administered intranasally.

22. A method according to any one of claims 1 to 21 further comprising identifying the patient as having or at risk of viral infection before said administration.

23. A method according to claim 22 wherein the patient is identified as having or at risk of viral infection before said administration.

24. A piperlongumine compound for use in a method of treating or preventing a viral infection according to any one of claims 1 to 23.

25. Use of the piperlongumine compound in the manufacture of a medicament for use in a method of treating or preventing a viral infection according to any one of claims 1 to 23.