WO2019004825A1 - Tomatidine and analogs thereof for use as antiviral agent - Google Patents

Abstract

The invention relates to the fields of medicine and virology, more in particular to means and methods for treating a viral disease caused by flaviviruses or alphaviruses. Provided is tomatidine or an analog thereof for use in a method of treating a viral infection caused by a flavivirus or an alphavirus, such as dengue virus or Chikungunya virus. Also provided is a pharmaceutical composition comprising tomatidine or an analog thereof, and at least one further antiviral agent.

Description

Title: Tomatidine and analogs thereof for use as antiviral agent. The invention relates to the fields of medicine and virology. More in particular, it relates to means and methods for treating a viral disease caused by flaviviruses and alphaviruses, such as dengue and Chikungunya.

Dengue virus is a member of the family Flaviviridae which also includes West Nile virus, Zika virus, Yellow Fever virus, Japanese encephalitis (JE) virus, and the Tick-borne encephalitis (TBE) virus.

Flaviviruses are small enveloped viruses with a single positive strand RNA molecule. Flaviviruses are transmitted to humans and animals via bites with chronically infected mosquito or tick vectors. Flaviviruses contain three structural proteins capsid (C), membrane (M), and envelope (E). The E and M proteins are found on the surface of the virion where they are anchored in the membrane. Mature E is glycosylated, whereas M is not, although its precursor, prM, is a glycoprotein. The E glycoprotein, the largest structural protein, contains functional domains responsible for cell surface attachment, internalization and membrane fusion. The humoral response during infection is mainly directed towards the E and prM protein.

Dengue virus is the causative agent of dengue fever and is transmitted to humans by Aedes mosquitoes, principally Aedes aegypti and Aedes albopictus. Annually, an estimated 390 million individuals are infected with dengue virus (DENV), of which 96 million individuals develop clinically apparent disease [1]. These staggering numbers make DENV the most common viral infection that is transmitted by arthropods worldwide. Clinical disease usually manifests as an acute self -limited illness with symptoms as high fever, severe headache, severe eye pain, muscle and/or bone pain and rash [2]. Approximately 0.5 to 1 million individuals, however, develop more extreme disease. Severe dengue is a potential fatal

complication due to capillary leakage, ascites, pleural effusion, severe bleeding and organ impairment [2]. Given the high endemicity of DENV in most (sub)tropical regions most cases are reported in infants and young children. Severe disease is predominantly seen in individuals experiencing a secondary DENV infection with another serotype or in infants born to dengue immune mothers [3] .

Four DENV serotypes exist in total and each serotype can lead to severe disease although not all circulating DENV strains cause severe disease. Not only virus factors but also host factors have been associated with severe disease onset. It is generally believed, however, that original antigenic SIN of T and B cells play a dominant role in the development of severe disease [4]. Low affinity T cells and high numbers of cross-reactive antibodies are postulated to limit efficient clearance of infection. In fact, these cross-reactive antibodies have been shown to enhance DENV titers in vitro and in vivo via the phenomenon of antibody-dependent enhancement of infection [3,4]. Epidemiological studies confirmed that high DENV titers early in infection correlate with an increased chance of severe disease development [5].

Researchers have attempted to identify antiviral compounds for the treatment of DENV for decades, but unfortunately with hmited success. Antiviral treatment is aimed at alleviating the viral titer , thereby decreasing the chance to develop severe disease [6]. Both direct- acting antivirals as well as host-directed antivirals have been pursued as potential candidates for dengue treatment.

However, despite the large number of compounds that exert antiviral activity in vitro, very few compounds have been further developed and evaluated in chnical trials. Moreover, none of these compounds

(chloroquine, lovastin, prednisolone, balapiravir and celgosivir,) showed a clear beneficial effect in humans [7-12]. These results emphasize the need to follow-up other and identify new compounds that intervene with DENV infection.

WO2011/002635A1 discloses methods and pharmaceutical compositions for treating viral infections such as those caused by flavivirus e.g. Dengue virus, West Nile virus, yellow fever virus, Japanese encephalitis virus, and tick-borne encephalitis virus, by administering certain 2-aryl- benzothiazole or 2-heteroaryl-benzothiazole derivative compounds.

Recently, a few other compounds with a steroid ring structure have been described as antivirals towards DENV. For example, ecdysones derived from Zoanthus spp. were found to inhibit DENV-2 replication in Huh7 cells and were predicted, by molecular docking studies, to associate with the NS5 polymerase of DENV [13]. Moreover, carbenoxolone disodium was reported to reduce DENV infectivity due to direct virucidal activity of the compound [14]. Furthermore, coumarins were shown to be potent inhibitors of both DENV as well as Chikungunya virus (CHIKV) [15].

Likewise, many other DENV inhibitors targeting viral components or directed to host cellular factors have been discovered or developed in the more than a decade hunt for specific antivirals [6]. However, none of these compounds have reached clinical trials, among other reasons because adverse effects in animals and poor pharmacokinetic properties [16].

Recognizing the major impact of dengue on global human health and huge economic burden, the present inventors set out to provide an antiviral drug available to treat the disease.

The inventors found that the compound tomatidine and analogs thereof have potent antiviral properties towards dengue virus (DENV) serotypes 1, 2, 3 and 4. For example, the effective tomatidine concentration in which a 50 and 90% reduction of infectious virus particle production is observed was 0.82 and 1.61 μΜ following infection of Huh 7 cells with

DENV-2 at MOI 1. The SI index is 97.7. Importantly, time-of-drug-addition experiments revealed that tomatidine is still active when added 12 hours post-infection, which suggests that the compound also acts at late stages of viral replication. Interestingly, the antiviral effect of tomatidine and analogs thereof was not limited to DENV. Strikingly, tomatidine was also found to have antiviral activity against Chikungunya virus (CHIKV), which is a member of the alphavirus genus.

Herewith, the invention relates to the use of tomatidine or a functional derivative thereof as antiviral compound, in particular against flaviviruses and alphaviruses.

Tomatine is a steroidal alkaloid that can be extracted from the skin and leaves of tomatoes. Unripe green tomatoes contain up to 500 mg tomatine per kg, whereas ripe red tomatoes have less than 5 mg/kg [17]. In nature, tomatine functions as an important defense mechanism for pathogens [18]. Tomatidine is an aglycon metabolite of tomatine and was shown to exert a wide array of beneficial biological activities hke anticancer, anti -inflammatory and improvement of the muscle health span by stimulating muscle hypertrophy [19-21]. See also WO2014/022772 disclosing the ability of tomatidine to promote skeletal muscle hypertrophy, increase muscle strength, increase exercise capacity, and decrease adiposity.

When consumed by animals, tomatine is hydrolyzed by stomach acid and intestinal bacteria to tomatidine, which is absorbed by the gut. Tomatidine appears to have a favorable safety profile based on several studies: 1) human consumption of indigenous variants of tomatoes with very high concentrations of a-tomatine (up to 0.05% (w/w) of dry tomato weight) appears to cause no adverse effects [22,23]); 2) tomatine content is twice as high in organically grown tomatoes compared to conventionally grown tomatoes [22]); and 3) in pregnant and non-pregnant mice, dietary

supplementation with 0.1% (w/w) tomatidine produces no adverse effects [24]). Moreover, in mouse models, tomatidine possesses anti-hyperlipidemic and anti- atherosclerotic effects without evidence of toxicity [25]).

Furthermore, anti-microbial properties of tomatidine have been described. For example, tomatidine was found to potently reduce replication of pathogenic S. aureus variants typically seen in cystic fibrosis [26].

Antiviral activity of tomatidine has been reported for the plant viruses Sunnhemp Rossette virus and Tobacco mosaic virus. In contrast, for herpex simplex virus, human respiratory syncytial and influenza virus tomatidine had no effect on virus replication [27-29]. Thus, the antiviral activity against members of the flavivirus and alphavirus families as disclosed in the present invention could not have been expected.

In one embodiment, the invention provides tomatidine or analog thereof, for use in a method of treating a viral infection caused by a flavivirus or an alphavirus, in particular Dengue Virus or Chikungunya virus. The invention also provides a method of treating a viral disease (viral infection) caused by a flavivirus or an alphavirus in an animal, comprising administering to the animal an effective amount of tomatidine or an analog thereof.

Flaviviruses are small, enveloped, positive-strand RNA viruses that are of concern in many medical and veterinary settings throughout the world. Flavivirus proteins are produced by translation of a single, long open reading frame to generate a polyprotein, which undergoes a complex series of post-translational proteolytic cleavages by a combination of host and viral proteases to generate mature viral proteins (Amberg et al., J. Virol. 73:8083- 8094, 1999; Rice, "Flaviviridae," In Virology, Fields (ed.), Raven-Lippincott, New York, 1995, Volume I, p. 937[30]). The structural proteins are arranged in the polyprotein in the order C-prM-E, where "C" is capsid, "prM" (or "pre- membrane") is a precursor of the viral envelope-bound M (membrane) protein, and Έ" is the envelope protein. These proteins are present in the N-terminal region of the polyprotein, while the non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are located in the C- terminal region of the polyprotein and these are required for replication of the viral genome.

In one aspect of the invention, said flavivirus is Dengue virus (serotype 1-4), Yellow Fever Virus (YFV), Zika virus or West-Nile virus. Preferably, the flavivirus is Dengue virus, more preferably dengue virus serotype 1, 2, 3 or 4. In a specific embodiment, the invention provides tomatidine or an analog thereof, for use in a method of treating a viral infection caused by dengue virus serotype 1, 2, 3 or 4. In another aspect, the invention provides tomatidine or an analog thereof, for use in a method of treating a viral infection caused by an alphavirus. Alphaviruses belong to the Togaviridae family viruses.

Alphaviruses, like flaviviruses, have a positive sensed, single-stranded RNA genome.

Preferably, the alphavirus is Chikungunya virus (CHIKV). It was first isolated in 1953 in Tanzania and is an RNA virus with a positive-sense single-stranded genome of about 11.6kb. It is a member of the Semliki Forest virus complex and is closely related to Ross River virus,

O'nyong'nyong virus, and Semliki Forest virus. Because it is transmitted by arthropods, namely mosquitoes, it can also be referred to as an arbovirus (arthropod-borne virus). In the United States, it is classified as a category C priority pathogen, and work requires biosafety level III precautions.

Chikungunya is a physically debilitating disease of humans mainly in Africa, Asia and the Americas. The disease is caused by CHIKV, and is spread by Aedes spp. mosquitoes, principally Aedes aegypti and Aedes albopictus. The symptoms include abrupt onset of high fever, rash or hemorrhages, arthralgia and occasional involvement of the nervous system, heart and liver. The incapacitation is due to arthralgia, which can persist for years. Treatment is only palliative and there is no commercially available vaccine. The presently disclosed antiviral activity of tomatidine or a tomatidine analog against CHIKV is therefore a major breakthrough in the management of Chikungunya.

The invention also provides a method of treating a disease caused by a flavivirus or an alphavirus in an animal, comprising administering to the animal an effective amount of tomatidine or an analog thereof. In a preferred aspect, there is provided a method of treating a disease caused by Dengue virus, preferably dengue virus serotype 1, 2, 3 or 4 in an animal, comprising administering to the animal an effective amount of tomatidine or analog thereof.

In another aspect, there is provided a method of treating a disease caused by an alphavirus, preferably Chikungunya virus, in an animal, comprising administering to the animal an effective amount of tomatidine or a analog thereof.

The invention also provides a method for inhibiting replication of Dengue virus or Chikungunya virus in a cell comprising administering tomatidine or analog thereof.

The terms "treating," "treatment," and the like are used herein to refer to obtaining a desired pharmacological and physiological effect. The effect may be prophylactic in terms of preventing or partially preventing a viral disease, symptom, or condition thereof and/or may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom, or adverse effect attributed to the disease. The term "treatment," as used herein, covers any treatment of a viral disease caused by a flavivirus or alphavirus in mammal, such as a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it, i.e., causing the clinical symptoms of the disease not to develop in a subject that may be predisposed to the viral disease but does not yet experience or display symptoms of the disease; (b) inhibiting the disease, i.e., arresting or reducing the

development of the disease or its clinical symptoms; and (c) relieving the disease, i.e., causing regression of the viral disease and/or its symptoms or conditions.

In one aspect, the subject is a mammal such as a primate, and, in a preferred aspect, the subject is a human. As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not hmited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, subhngual

administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra- arterial administration, intramuscular administration, and subcutaneous

administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a viral disease or condition.

The tomatidine or tomatidine analog may be administered in any

convenient vehicle which is physiologically acceptable. Such a composition may include any of a variety of standard pharmaceutically accepted carriers employed by those of ordinary skill in the art. Examples include saline, phosphate buffered saline (PBS), water, aqueous ethanol, emulsions, such as oil/water emulsions or triglyceride emulsions, tablets and capsules. The choice of suitable physiologically acceptable carrier will vary dependent upon the chosen mode of administration.

The method of the present invention may also comprise co-administration of: a) other antivirals such as Ribavirin or cidofovir; b) vaccines; and/or c) interferons or pegylated interferons. Hence, also provided herein is a pharmaceutical composition comprising tomatidine or analog thereof and at least one further antiviral agent. Preferably, the pharmaceutical composition comprises tomatidine, solasodine or sarsasapogenin. and comprising at least one further antiviral agent.

The at least one further antiviral agent is suitably selected from the group consisting of coumarins, ribavirin, cidofovir, anti-flavi virus vaccines, anti- alphavirus vaccines and (pegylated) interferons.

As used herein, the term "derivative" or "analog" refers to a compound having a structure derived from the structure of the tomatidine parent compound and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of a parent compound as disclosed e.g. WO2014/022772.

In one aspect, the tomatidine derivative or analog has a structure

represented by a formula:

wherein R-> is selected from H. C1-C6 alkyl, COR⁵³, C1-C6 alkylamino, C 1-C6 dialkylamino, C6-C 10 aryl. C3-C 10 cvcloalkyl. C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cvcloalkyl, C5-C heteroaryl. and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C 1-C6 alkyl, C 1-C6 alkoxy, C 1-C6 monohaloalkyl, C 1-C6 polyhaloalkyl, Cl- C6 alkylamino, and C1-C6 dialkylamino;

wherein R^5¾ is selected from C 1-C6 alkyl, C 1-C6 monohaloalkyl, C 1-C6 polyhaloalkyl, C6-C 10 aryl, C3-C 10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C 10 aryl, C3-C 10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C 1-C6 alkyl, C 1-C6 alkoxy, C 1-C6 monohaloalkyl, C 1-C6 polyhaloalkyl, CI- C6 alkylamino, and C 1-C6 dialkylamino;

wherein Ζ^δ1 is selected from O, S, and NR⁵⁴; preferably Z⁵¹ is O or wherein R^¾ is selected from H, C 1-C6 alkyl, COR^s, C 1-C6 alkylamino, C 1-C6 dialkylamino, C6-C 10 aryl, C3-C 10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C 10 aryl, C3-C 10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C 1-C6 alkyl, C 1-C6 alkoxy, C 1-C6 monohaloalkyl, C 1-C6 polyhaloalkyl, Cl- C6 alkylamino, and C1-C6 dialkylamino;

wherein R⁵⁵ is selected from C 1-C6 alkyl, C 1-C6 monohaloalkyl,

C 1-C6 polyhaloalkyl, C6-C 10 aryl, C3-C 10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C 10 aryl, C3-C 10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C 1-C6 alkyl, C 1-C6 alkoxy, C 1-C6 monohaloalkyl, C 1-C6 polyhaloalkyl, CI- C6 alkylamino, and C 1-C6 dialkylamino; or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

In one aspect, R⁵¹ is selected from H, C 1-C6 alkyl and COR⁵³, wherein R⁵³ is CI- C6 alkyl. In another aspect, R⁵¹ is H. In another aspect, Z⁵¹ is NR⁵'¹. In another aspect, Z⁵¹ is NR⁵⁴, wherein R⁵⁴ is selected from H, C 1-C6 alkyl, and COR∞, wherein R35 i_s C 1-C6 alkyl. In another aspect, R⁵¹ is selected from H, C1-C6 alkyl and COR⁵³, wherein R^{r !} is C1-C6 alkyl; and Z ¹ is NR⁶⁴, wherein R⁵⁴ is selected from H, C1-C6 alkyl, and COR⁵⁵, wherein R⁵⁵ is C1-C6 alkyl. In another aspect, R⁵¹ and R⁵⁴ are identical.

In one preferred embodiment, the structure is represented by the formula

wherein R⁵¹ is selected from H. C1-C6 alkyl, COR⁵³, C1-C6 alkylamino, Cl- C6 dialkylamino. C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2- C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl. cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl. C1-C6 polyhaloalkyl. C1-C6

alkylamino. and C1-C6 dialkylamino;

wherein R⁵³ is selected from C1-C6 alkyl, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl. C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl. wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy. C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl. C1-C6 alkylamino. and C1-C6 dialkylamino;

wherein R⁵⁴ is selected from H, C1-C6 alkyl, COR⁵⁵, C1-C6 alkylamino, C1-C6 dialkylamino, C6-C10 aryl. C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C 1-C6 alkyl, C 1-C6 alkoxy, C 1-C6 monohaloalkyl, C 1-C6 polyhaloalkyl, C l- C6 alkylamino, and C1-C6 dialkylamino;

wherein R⁵⁵ is selected from C 1-C6 alkyl, C 1-C6 monohaloalkyl.

C 1-C6 polyhaloalkyl, C6-C 10 aryl, C3-C 10 cycloalkyl. C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C 10 aryl. C3-C 10 cycloalkyl. C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C 1-C6 alkyl, C 1-C6 alkoxy, C 1-C6 monohaloalkyl, C 1-C6 polyhaloalkyl, C 1-C6 alkylamino, and C 1-C6 dialkylamino; or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

Preferably, the structure is represented by the formula:

For example, the formula has the structure:

or a salt thereof, in particular the HC1 salt. This compound is referred to as tomatidine (25<S -226N-5a-spirolane). as the structure

or a salt thereof, in particular the HC1 salt. This tomatidine analog is referred to as solasodine ((25i?)-22aN-spiroal-5-ene). Solasodine is a main active component isolated from Solatium incanum L. that has been reported to perform a wide range of functions containing anti-oxidant, anti -infection, and neurogenesis promotion. Furthermore, Zhuang et al. (Cancer Sci. 2017 Nov; 108(11): 2248-2264) showed that solasodine prohibited human colorectal cancer cell proliferation dose- and time-dependently, suggesting that solasodine may be a therapeutic drug for CRC treatment.

In another aspect, the tomatidine analog has a structure represented by the formula

wherein Z⁵¹ is O and wherein R⁵¹ is defined as herein above. For example, the analog is sarsasapogenin of the formula

Sarsasapogenin and its C-25 epimer smilagenin lowered blood sugar and reversed diabetic weight gain in experiments within mice with a mutant diabetes gene (db).Both steroids also halted the decline in muscarinic acetylcholine receptors (niAChRs) in animal models of Alzheimer's disease. However, an antiviral effect has heretofore never been suggested. In one embodiment, the invention provides a method for treating a viral infection caused by Dengue Virus or Chikungunya virus in a subject, comprising administering to the subject an effective amount of tomatidine, solasodine or sarsasapogenin. Preferably, the invention provides a method for treating a viral infection caused by Dengue Virus, comprising

administering to the subject an effective amount of tomatidine or solasodine. In another embodiment, the invention provides a method for treating a viral infection caused by Chikungunya Virus, comprising administering to the subject an effective amount of tomatidine, solasodine or sarsasapogenin. As used herein, the terms "effective amount" and "amount effective" refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a "therapeutically effective amount" refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. The dose will be adjusted to the individual requirements in each particular case.

The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration.

Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a "prophylactically effective amount"; that is, an amount effective for prevention of a disease or condition.

For oral administration, a daily dosage of between about 0.01 and about 1000 mg/kg body weight per day should be appropriate in

monotherapy and/or in combination therapy. A preferred daily dosage is between about 0.1 and about 500 mg/kg body weight, more preferred 0.1 and about 100 mg/kg body weight and most preferred 1.0 and about 10 mg/kg body weight per day. Thus, for administration to a 70 kg person, the dosage range would be about 7 mg to 0.7 g per day. The daily dosage can be administered as a single dosage or in divided dosages, typically between 1 and 5 dosages per day. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect for the individual patient is reached. One of ordinary skill in treating diseases described herein will be able, without undue experimentation and in reliance on personal knowledge, experience and the disclosures of this application, to ascertain a therapeutically effective amount of the

compounds of the present invention for a given disease and patient.

Time-of-drug-addition experiments show that tomatidine is still active when added 12 hours post-infection, suggesting that tomatidine predominantly acts at a late stage of viral replication. This is strengthened by the observation that only a minor reduction in the number of infected cells (up to 4-fold, Fig. 2) is observed when compared to the overall reduction in infectious virus particle production (up to 100-fold, Fig. 1A). Tomatidine (analog) does not control the infectious properties of progeny virions as the number of infectious particles was equally reduced to that of the absolute number of particles (Genome equivalent particles, GEC). Collectively, this suggests that tomatidine (analog) predominantly intervenes with steps downstream of protein translation/replication but prior to the secretion of progeny virions. Tomatidine (analog) might act directly on the viral proteins or indirectly by controlling the expression of a cellular factor that is important in the late stages of viral replication. Hence, a use or method of the invention advantageously allows to treat subjects that have acquired or are suspected of having acquired the viral disease. In a further aspect, the disclosed compounds (i.e. tomatidine or analog thereof) have antiviral activity when administered at an oral dose of greater than about 5 mg per day in a human. In a further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 10 mg per day in a human. In a further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 25 mg per day in a human. In a further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 50 mg per day in a human. In a further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 75 mg per day in a human. In a further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 100 mg per day in a human. In a further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 150 mg per day in a human. In a further aspect, the disclosed have antiviral activity when administered at an oral dose of greater than about 200 mg per day in a human. In a further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 250 mg per day in a human. In a yet further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 300 mg per day in a human. In a still further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 400 mg per day in a human. In an even further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 500 mg per day in a human. In a further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 750 mg per day in a human. In a yet further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 1000 mg per day in a human. In a still further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 1500 mg per day in a human. In an even further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 2000 mg per day in a human. Legend to the Figures

Figure 1. Tomatidine reduces the production of infectious DENV particles. (A) Huh7 cells were infected with DENV serotype 2 at MOI 1 and 10. Simultaneously with the infection, cells were treated with 10 μΜ of tomatidine, the equivalent volume of EtOH or left un-treated (NT). (B)

Dose-response curve showing the inhibition of DENV infection at increasing concentrations of tomatidine in relation to the equivalent EtOH-treated control. EC50 values were calculated with GraphPacl Prism software. (C) 1χ10^δ PFU of DENV was incubated for 2 h at room temperature RT or 37 °C with 10 μΜ of tomatidine. The infectivity was determined by plaque assay on BHK-15 cells. Data is presented as mean ± SEM from three independent experiments.

Figure 2. Tomatidine decreases the percentage of DENV-infected Huh7 cells. (A, B) Huh7 cells were infected with DENV serotype 2 at MOI 1 and 10 in the presence of 1 or 10 μΜ tomatidine as indicated. At 2 hpi, DENV inoculum was removed and incubation was continued in the presence of the compound until harvesting of cells at 24 hpi. As control, cells were infected with DENV in presence of an equal volume of EtOH. (A)

Representative dot plots. (B) Quantification of the percentage of infected cells and mean fluorescence activity (MFI). (C, D) Tomatidine was added 2 hpi of Huh7 cells with DENV at MOI 1. (C) Representative dot plot. (D) Quantification of the percentage of infected cells and MFI. (A, C) Red numbers indicate the percentage of infected cells and grey numbers the MFI. Data is presented as mean ± SEM from three independent

experiments.

Figure 3. Tomatidine reduces DENV infectivity when added up to 12 hpi. (A) Outhne of the experimental set-up. (B) Infectious virus particle production following the conditions presented in (A). The EtOH control was added to all experimental conditions and the average titer is depicted. For the tomatidine samples, data is presented as mean ± SEM from three independent experiments.

Figure 4. Tomatidine reduces the number of secreted DENV genome-equivalent copies. Huh7 cells were infected with DENV serotype 2 at MOI 1 and treated with 10 μΜ tomatidine at 12 hpi. The number of DENV genome-equivalent copies per ml (GEC/ml) was determined in the cell culture supernatant at 24 hpi. Data is presented as mean ± SEM from three independent experiments.

Figure 5. Tomatidine also reduces the number of secreted genome- equivalent copies of DENV serotype 1 and 4. Huh7 cells were infected with DENV- 1 at MOI 1 (panel A) or DENV-4 at MOI 0.1 (panel B) and treated with 10 μΜ tomatidine. The number of DENV genome-equivalent copies per ml (GEC/ml) was determined in the cell culture supernatant at 24 hpi. Data is presented as mean ± SEM from three independent experiments. "NT" denotes non-treated and EtOH served as an ethanol solvent control.

Figure 6. Dose-response curve showing the inhibition of DENV-1, DENV-3 and DENV-4 infection at increasing concentrations of tomatidine in relation to the equivalent EtOH-treated control. Huh7 cells were infected with DENV serotype 1 (MOI 1), serotype 3 (MOI 0.5) and serotype 4 (MOI 1). The supernatants were harvested at 24 hpi for DENV-1 and DENV -3 and at 30 hpi for DENV-4. The number of infectious virus particles was determined by an immunofocus assay on HK-15 cells. EC50 values were calculated with GraphPad Prism software. Data is presented as mean ± SEM from three independent experiments.

Figure 7. Tomatidine reduces the number of secreted genome- equivalent copies of Chikungunya (CHIKV) virus. Huh7 cells were infected with CHIKV (MOI 1) and treated with 10 μΜ tomatidine. The number of CHIKV genome-equivalent copies per ml (GEC/ml) was determined in the cell culture supernatant at 24 hpi. Data is presented as mean ± SEM from three independent experiments. "NT" denotes non- treated and EtOH served as an ethanol solvent control.

Figure 8. Anti-viral activity of tomatidine analogs. Huh7 cells were infected with (panel A) DENV serotype 2 or (panel B) CHIKV at MOI 1. Test compound was added to the cells at the time-point of infection at the highest non-toxic dose. Tomatidine 10 μΜ; Solasodine 5 μΜ; Sarsasapogenin 20 μΜ. In control conditions, the equivalent concentration of the solvent (96% w/v ethanol; end concentration <0.05%) was added to the cells at the time point of infection. The supernatants were harvested at (panel A) 24 hpi or (panel B) 9 hpi. For panel A, the number of produced DENV genome- equivalent copies were determined by Q-RT-PCR. For panel B, the number of produced infectious CHIKV particles was determined by plaque assay on BHK -15 cells. EXPERIMENTAL SECTION Materials and Methods Cell culture

Baby hamster kidney-21 cells clone 15 (BHK-15) was a kind gift from Richard Kuhn (Purdue University). BHK-15 cells were grown in Dulbecco's minimal essential medium (DMEM) (Gibco, the Netherlands) supplemented with 10% fetal bovine serum (FBS) (Lonza, Basel,

Switzerland), lOOU/mL penicillin and lOOmg/mL streptomycin (PAA

Laboratories, Pasching, Austria), 100 μΜ of non-essential aminoacids (Gibco) and lOmM of HEPES (Gibco). Human hepatocarcinoma (Huh 7) cells (JCRB0403) were a kind gift from Tonya Colpitts (University of South Carolina) and cultured in DMEM/Glutamax supplemented with 10% FBS, lOOU/mL penicillin and lOOmg/mL streptomycin. Vero WHO cells (WHO Reference Cell Bank 10-87) were grown in DMEM supplemented with 10% FBS, lOOU/mL penicillin and lOOmg/mL streptomycin. Aedes albopictus C6/36 cells (ATCC: CRL-1660) were maintained in minimal essential medium (Invitrogen, Carlsbad, California, USA) supplemented with 10%> FBS, 25 mM HEPES, 7.5% sodium bicarbonate, lOOU/mL penicillin and lOOmg/mL streptomycin, 200 mM glutamine, and 100 μΜ nonessential amino acids. All mammalian cells were cultured at 37°C and 5% CO2 and C6/36 cells were cultured at 28°C and 5% CO₂. Virus stocks and titration.

DENV serotype 2 strain 16681, DENV serotype 1 strain 16007, DENV serotype 3 strain H87 and DENV serotype 4 strain 1036 were propagated on C6/36 cells as described before [28]. CHIKV strain OPY LR2006 was propagated on Vero cells. The number of infectious particles was determined by plaque assay on BHK-15 cells[31]. For plaque assays, BHK-15 cells were seeded in 12-well plates at a cell density of 9.0xl0 cells per well. At 24 h post-seeding, cells were infected with 10-fold serial dilutions of the sample. At 2 h post-infection (hpi), an overlay of 1% seaplaque agarose (Lonza, Swiss) prepared in MEM was added and plaques were counted 2 (for CHIKV) and 5 (for DENV) days post-infection. Titers are reported as plaque forming units (PFU) per ml. The number of genome equivalent copies (GEC) in a solution was determined by Q-RT-PCR as described previously [31]. Briefly, viral RNA was extracted using a QIAamp viral RNA mini kit (QIAGEN, Venlo, The Netherlands) following

manufacturer's instructions. cDNA was synthesized from viral RNA using Omniscript (QIAGEN) and the primers and probes (Eurogentec, Maastricht, The Netherlands) are listed in Table 1.

Determination of the number of RNA copies was performed with a standard curve (correlation co-efficient >0.995) of quantified DENV plasmids constructed with standard DNA techniques. The plasmids were: DENV-1: pcDNA3 encoding the M protein sequence of DENV- 1 strain 16007; DENV-2: pSINDENCprME encoding the CprME sequence of DENV- 2 strain 16681; and DENV-4: pcDNA3 encoding the E protein sequence of DENV-4 strain 1036.

Table 1 Primer and probes used for cDNA generation and quantitative PCR

Serotype Forward primer Keverse primer TaqMan Probes

DENV- 1 5^'-TGCTCTC AAACT 5^'-TCCAAGCACCTTC o -FAM-CCGTCGCATT

GGCGAACA-3' AGAGG AC AT- 3' GGCCCCACA-TAMRA-3^'

DENV-2 f - AC AGGCTATGG C D'-TGCAGCAACAC o'-F l-AGTGCTCTCCAAGA

ACTGTTACGAT-3^' CATCTCATTG-3 AC G GG C C T C G - T AMR A- 3 '

DENV-4 5^?-CATGGGTCGAT 5^' -C CTTGG CTGTT o'-FAM- CATGGCCCAGGG

CTGGTGCTA-3' GTCTTAGTCA-3' AAAACCAACCTT-TAMRA-3^' A StepOne Real-Time PCR instrument (Applied Biosystems, Carlsbad, CA) was used (pSINDENCprME).

CHIKV La Reunion 2006 OPY-strain was propagated on Vero -WHO cells, as described before (Richter et al. 2015. J. Gen. Virol., 96: 2122-2132 [32]). Plaque assay was performed to determine the infectious titer and Q-RT-PCR was performed to determine the number of genome equivalent copies (GEC). For Q-RT-PCR, viral cDNA was synthesized by reverse transcriptase (RT) PCR using the forward primer 5'-AGCTCCGCGTCCTTTACCA-3' and the reverse primer 5'-GCCAAATTGTCCTGGTCTTCCT-3'. For the qPCR, the TaqMan probe 5'-FAM-CAC TGTAACTGCCTATGCAAACGGCGAC-

TAMRA-3' was added. For the standard curve, a CHIKV plasmid containing the E l sequences (pCHIKV-LS3 IB) was used.

Chemicals

Tomatidine hydrochloride, solasodine and sarsasapogenin were purchased from Sigma-Aldrich (St. Louis, Missouri, USA) and dissolved in absolute ethanol (EtOH) to the desired final concentration. Aliquots were stored for no longer than three months

at -20°C. The final concentration of EtOH was below 0.01% in all infectivity experiments.

Cytotoxicity assay

Cytotoxicity of tomatidine was assessed in vitro by the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazohum bromide (MTT) assay. Huh7 cells were seeded in 96-well plates at a density of 1.0x10⁴ and 7.0xl0³ cells per well, respectively. At 24 h post-seeding, cells were treated with

increasing concentrations of tomatidine ranging from 1 to 200 μΜ. At 24 h,

MTT was added at a final concentration of 0.45mg/ml and incubated for 3 h.

Subsequently, media was removed and cells were incubated for 1 h at room temperature (RT) with acidic-2-2-propanol. The absorbance was measured with a microplate reader (Biotek, Sinergy, HT, Vermont, USA) at 570 nm. Cell viability was expressed according to the following formula: sample)— (Abs blank)

% Cell viability = * 100

(Abs negative control)— (Abs blank)

Antiviral assays

Huh7 cells were infected with the indicated viruses

multiplicity of infection (MOI) of 1 or 10. Tomatidine, tomatidine analogor the equivalent volume of EtOH, was added at different stages of infection. In most experiments, increasing concentrations of tomatidine (analog) was added together with the virus to the cells. At 2 hpi, the virus inoculum was removed, cells were washed three times and drug-containing medium was added for the duration of the experiment. In case of pre-treatment experiments, tomatidine (analog) was added 1 or 2 h prior to infection. At the time of infection, cells were washed three times before the virus inoculum was added. The condition "during" relates to the presence tomatidine during the infection for 2 h. Also, tomatidine was added 2, 4, 6, 12, 16, 20 hpi. In all experiments, the virus inoculum was removed at 2 hpi, cells were washed three times and incubation was continued. At 24 hpi, cell supernatants were harvested and the titer was determined by plaque assay or Q-RT-PCR.

Virucidal effect

DENV serotype 2 (lxlO⁵ PFU) was incubated for 2 h at room temperature or 37°C in the absence or presence of 10 μΜ tomatidine in a final volume of 250 μΐ. Upon incubation, the infectious titer was determined by plaque assay.

Flow cytometry

Huh7 cells were trypsinized using IX Trypsin/EDTA (Gibco). Cells were fixed with 2% paraformaldehyde and permeabilized with 0.5% saponin. Staining was performed with 4G2 antibody and a rabbit anti- mouse IgG coupled to AF647 (Molecular probes, Eugene, Oregon, USA). Flow cytometry was carried out in a FACSCalibur cytometer (BD

Biosciences) and analysis was performed with Kaluza 1.1.

Statistical analysis.

The tomatidine concentration at which 50 and 90% reduction in virus particle production is seen is referred to as EC50 and EC90,

respectively. The concentration of tomatidine that caused 50 and 90% cellular cytotoxicity is referred to as EC50 and EC90, respectively. Dose- response curves were fitted by non-linear regression analysis employing a sigmoidal model. The selectivity index (SI) was determined by the ratio of CC50 to EC50. All data were analyzed in GraphPad Prism software (La Jolla, CA, USA). Data is presented as mean ± SEM. Student T test was used to evaluate statistical differences and a p value <0.05 was considered significant with *p<0.05 , **p<0.01 and ***p<0.001.

EXAMPLE 1: Tomatidine inhibits the production of progeny infectious virus particles.

The effect of tomatidine on DENV infectivity was determined in human hepatocarcinoma (Huh 7) cells. This cell line was chosen as

hepatocytes are important target cells during DENV infection. Furthermore, this cell line is permissive to DENV infection. Indeed, at 24 hpi, on average 8.3xl0⁴ progeny infectious particles per ml are produced following infection at MOI 1. Fig. 1A shows that tomatidine has potent antiviral activity towards DENV serotype 2. At a concentration of 10 μΜ tomatidine, infectious virus particle production was reduced 2.02 log when compared to DENV-infected cells treated with equivalent volumes of EtOH. The final concentration of EtOH was below 0.01% and had no effect on virus particle production when compared to non-treated cells. The EC50 and EC90 values are 0.82 and 1.61 μΜ for MOI 1, respectively. At MOI 10, the EC50 and EC90 values are 0.97 and 5.72 μΜ, respectively (Fig. IB). Viability assays were performed in parallel and revealed a CC50 value of 80.1 μΜ for tomatidine in Huh7 cells (Figure 7). This implies that the SI index is 97.7 at MOI 1 and 82.6 at MOI 10. To analyze if the compound has a direct negative (virucidal) effect on the virion, we next incubated lxlO⁵ PFU of DENV with 10 μΜ tomatidine for 2 h at room temperature or 37°C and determined the infectious titer by plaque assay. Figure 1C shows that tomatidine does not influence DENV infectivity, indicating that tomatidine is not virucidal.

EXAMPLE 2: Tomatidine decreases the number of infected cells.

To better understand the potent antiviral effect of tomatidine, we investigated whether, next to reducing the production of infectious particles, the compound also reduces the number of infected cells. The percentage of infection was determined by flow cytometry using the 4G2 antibody which recognizes the viral E-protein. At MOI 1, the percentage of infection in EtOH-treated control cells was on average 21.9% whereas at tomatidine concentrations of 1 and 10 μΜ, the percentage of infected cells were 12.7 and 5.1%, respectively (Fig. 2A and 2B). At MOI 10, the percentage of infection in EtOH-treated cells was on average 74.2% while in cells treated with 1 and 10 μΜ tomatidine we observed on average 64.2 and 43.2% of infected cells, respectively. At higher MOI values, less robust effect of tomatidine is observed as, compared to EtOH, treatment with 10 μΜ tomatidine reduced the percentage of infection by 76 and 41% at MOI 1 and 10, respectively. The mean fluorescent intensity (MFI) of infected cells treated with 10 μΜ tomatidine was also decreased in comparison with the EtOH-treated DENV-infected control cells (Fig. 2B), indicating that E protein expression is reduced by the treatment with tomatidine. This suggests that tomatidine reduces both, the number of infected cells and the level of E protein expression in infected cells. Less pronounced inhibition of infection was observed when the compound was added at 2 hpi, thus, after virus-cell binding, entry and removal of the virus inoculum (Fig. 2C). At 10 μΜ tomatidine, however, a significant (Fig. 2C) and comparable (Fig. 2B vs Fig. 2C) decrease in the number of infected cells and MFI was observed, suggesting that tomatidine predominantly acts at a late stage of infection.

EXAMPLE 3: Tomatidine has a potent antiviral effect when added post-infection.

To study the observations of Example 2 in more detail we next performed time of addition experiments (Fig. 3A). In the re-treatment experiments, cells were incubated with tomatidine for 2 or 1 h after which the compound was washed out and DENV serotype 2 infection was initiated. Alternatively, the compound was added together with the virus to Huh7 cells and at 2 hpi the medium was removed, cells were washed and incubation was continued without tomatidine. For the post-treatment experiments, the compound was added at 2, 4, 6, 12, 16 and 20 hpi. In all experiments, cells were infected at MOI 1 and tomatidine was added at a final concentration of 10 μΜ. At each time point, EtOH-treated cells were included. We found that tomatidine reduces DENV infectivity when added pre, during and up to 12 hpi (Fig. 3B). This suggests that tomatidine may interfere with multiple stages of infection. Nevertheless, the strongest antiviral effect was seen when the compound was added post-infection.

Indeed, addition of tomatidine to cells at 2, 4, 6, and 12 hpi reduced infectious virus particle production by on average 98.4, 97, 95.5 and 96.6%, respectively. No antiviral effect was observed when the compound was added at 16 hpi. Detailed growth kinetic analysis of DENV in Huh7 cells revealed that initial virus particle production is seen at 18 hpi (Fig. 8).

Hence, the above results suggest that tomatidine interferes with a step prior to progeny virus secretion. To study this, we next analyzed the number of genome-equivalent copies (GEC) secreted by DENV-infected Huh7 cells treated with 10 μΜ tomatidine at 12 hpi.

Fig. 4 shows that the number of GEC is reduced by 1 Log (90.2%) when compared to EtOH-treated control cells. Thus, the number of secreted infectious particles (Fig. 3B) and GEC (Fig.4) is correspondingly reduced, thereby confirming that tomatidine acts at a step prior to virion secretion.

EXAMPLE 4: Antiviral activity of tomatidine is independent of the DENV serotype.

The effect of tomatidine on DENV serotype 1 and 4 was

determined in human hepatocarcinoma (Huh 7) cells. This cell line was chosen since hepatocytes are important target cells during DENV infection. Furthermore, this cell line is permissive to DENV infection. Tomatidine was added at a concentration of 10 μΜ and remained present for the duration of the experiment (24 hr). For DENV serotype 1, cells were infected at MOI 1 and for DENV serotype 4 MOI 0.1 was used. Fig. 5 shows that tomatidine exerts antiviral activity towards DENV serotype 1 and 4. At a concentration of 10 μΜ tomatidine more than 75% reduction in GEC production is seen. Figure 6 shows a dose-response curve showing the inhibition of DENV- 1, DENV-3 and DENV-4 infection at increasing concentrations of tomatidine.

EXAMPLE 5: Tomatidine also has antiviral activity against alphavirus.

The effect of tomatidine on CHIKV was determined in human hepatocarcinoma (Huh 7) cells. Tomatidine was added at a concentration of 10 μΜ and remained present for the duration of the experiment (16 hr). Fig. 7 shows that tomatidine exerts potent antiviral activity towards CHIKV- LR. At a concentration of 10 μΜ tomatidine, more than 99% reduction in GEC production is seen. EXAMPLE 6: Anti-viral activity of tomatidine analogs.

Solasodine and Sarsasapogenin are two exemplary tomatidine analogs showing a high structural similarity to tomatidine.

Tomatidine

Solasodine

.Sarsasapogenin

In this example, solasodine and sarsasapogenin were tested for their antiviral activity against different viruses.

To determine the effect on dengue virus, Huh7 cells were infected with DENV serotype 2 strain 16681 at MOI 1. Test compound was added to the cells at the time-point of infection at the highest non-toxic dose. Tomatidine 10 μΜ; Solasodine 5 μΜ; Sarsasapogenin 20 μΜ. In control conditions, the equivalent concentration of the solvent (96% w/v ethanol; end concentration <0.05%) was added to the cells at the time point of infection. The

supernatants were harvested at 24 hpi and the number of produced genome- equivalent copies was determined by Q-RT-PCR. N=3.

To determine the effect on Chikungunya virus, Huh7 cells were infected with Chikungunya virus OPY LR2006 at MOI 1. Test compound was added to the cells at the time-point of infection at the highest non-toxic dose. Tomatidine 10 μΜ; Solasodine 5 μΜ; Sarsasapogenin 20 μΜ. The supernatants were harvested at 9 hpi and the number of produced infectious particles was determined by plaque assay on BHK -15 cells. Q-RT-PCR. In control conditions, the equivalent concentration of the solvent (96% w/v ethanol; end concentration <0.05%) was added to the cells at the time point of infection (N= 2).

As shown in Figure 8A, solasodine showed a strong antiviral activity to dengue virus, whereas for sarsasapogenin a negative trend in virus particle production was observed.

As shown in Figure 8B, both solasodine and sarsasapogenin showed a good antiviral activity against Chikungunya virus.

This example demonstrates that the anti-viral activity is not confined to tomatidine, but can also be observed for structurally related tomatidine analogs. REFERENCES

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Claims

Claims

1. Tomatidine or analog thereof for use in a method of treating a viral infection caused by Dengue Virus or Chikungunya virus.

2. Tomatidine or analog thereof for use according to claim 1, wherein said viral infection is caused by Dengue virus, preferably Dengue virus serotype 1, 2, 3 or 4.

3. Tomatidine or analog thereof for use according to claim 1, wherein said viral infection is caused by Chikungunya virus.

4. Tomatidine or analog thereof for use according to any one of the preceding claims, wherein said tomatidine or tomatidine analog has a structure represented by a formula:

wherein R ¹ is selected from H, C1-C6 alkyl, COR⁵³, C 1-C6 alkylamino, C1-C6 dialkylamino. C6-C 10 aryl, C3-C 10 cycloalkyl. C5-C9 heteroaryl, and C2-C9 heterocyclyl. wherein C6-C10 aryl, C3-C 10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxy!, cyano. amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkvl, C1-C6 polyhaloalkyl, Cl- C6 alkylamino, and C1-C6 dialkylamino; wherein R⁵³ is selected from C 1-C6 alkyl, C 1-C6 monohaloalkyl, C 1-C6 polyhaloalkyl, C6-C 10 aryl, C3-C 10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C 10 aryl, C3-C 10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C 1-C6 alkyl, C 1-C6 alkoxy, C 1-C6 monohaloalkyl, C 1-C6 polyhaloalkyl, CI- C6 alkylamino, and C 1-C6 dialkylamino;

wherein Z⁵¹ is selected from O, S, and NR⁵⁴; preferably wherein Z⁵¹ is O or NR⁵⁴;

wherein R⁵⁴ is selected from H, C 1-C6 alkyl, COR⁵⁵, C 1-C6 alkylamino, C 1-C6 dialkylamino, C6-C 10 aryl, C3-C 10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C 10 aryl, C3-C 10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C 1-C6 alkyl, C 1-C6 alkoxy, C 1-C6 monohaloalkyl, C 1-C6 polyhaloalkyl, Cl- C6 alkylamino, and C1-C6 dialkylamino;

wherein R⁵⁵ is selected from C 1-C6 alkyl, C 1-C6 monohaloalkyl, C 1-C6 polyhaloalkyl, C6-C 10 aryl, C3-C 10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C 10 aryl, C3-C 10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C 1-C6 alkyl, C 1-C6 alkoxy, C 1-C6 monohaloalkyl, C 1-C6 polyhaloalkyl, CI- C6 alkylamino, and C 1-C6 dialkylamino; or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

5. Tomatidine or a derivative thereof for use according to claim 4, wherein the tomatidine or tomatidine analog has a structure represented by the general formula

or pharmaceutically acceptable salt thereof.

6. Tomatidine or analog thereof for use according to claim 5, wherein tomatidine represented by the structure

or solasodine represented by the structure

is used.

is used.

8. Tomatidine or analog thereof for use according to any one of the preceding claims, wherein tomatidine or a salt thereof, preferably the HCl salt, is used. 9. A pharmaceutical composition comprising tomatidine, solasodine or sarsasapogenin, and at least one further antiviral agent.

10. Pharm aceutical composition according to claim 9, wherein said at least one further antiviral agent is selected from the group consisting of coumarins, ribavirin, cidofovir, anti-flavivirus vaccines, anti-alphavirus vaccines and (pegylated) interferons.

11. A method for treating a viral infection caused by Dengue Virus or Chikungunya virus in a subject, comprising administering to the subject an effective amount of tomatidine or a tomaticline analog.

12. Method according to claim 11, comprising administering tomatidine, solasodine or sarsasapogenin.

13. Method according to claim 12, comprising administering pharmaceutical composition according to claim 9 or 10.

14. Method according to claim 12 or 13 for treating a viral infection caused by Dengue Virus, comprising administering to the subject an effective amount of tomatidine or solasodine.

15. Method according to claim 12 or 13 for treating a viral infection caused by Chikungunya Virus, comprising administering to the subject an effective amount of tomatidine, solasodine or sarsasapogenin.