TECHNICAL FIELD

The present invention relates to compounds useful as modulators of sirtuins, in particular as activators of SIRT1. Furthermore, the invention refers to the medical use of such compounds, in particular in the prevention and/or treatment of cardiovascular diseases, to pharmaceutical compositions that comprise them and to an in vitro method to identify a SIRT1 modulator.

STATE OF THE ART

Sirtuins

Sirtuins are NAD ⁺ -dependent deacetylases and their activity is controlled by the cellular ratio [NAD ⁺ ]/[NADH] in which NAD ⁺ acts as an activator, while nicotinamide (NAM) and NADH as inhibitors [1] They are involved in different biological processes such as cell survival, aging, proliferation, apoptosis, DNA repair, cell metabolism, and calorie restriction [2,3]

In mammals there are seven sirtuins. Overall the family of these proteins is well represented in all phyla of eukaryotes and prokaryotes [1 ,4] Sir2 represents the progenitor of the family in Saccharomyces cerevisiae, where it controls the acetylation of histones and cellular senescence.

The targets of sirtuins are various and are determined by the subcellular localization of sirtuins themselves [5] SIRT6 and 7 are located in the nucleus, whereas SIRT3, 4 and 5 are mitochondrial proteins. SIRT1 and 2 are both cytoplasmic and nuclear proteins. This classification is actually generic, as the sub-cellular localization of some sirtuins depends on the cellular context. To date, the physiological function of sirtuins is still object of intense studies focused on SI RT1. Some sirtuins (in particular SI RT1 , 3 and 4) are able to modulate the energy metabolism, while others seem to be able to regulate cell division (SIRT2), DNA repair (SIRT6), urea catabolism (SIRT5) or ribosomal RNA transcription (SIRT7).

Table 1 : The human sirtuins [1]

SIRT1 , 2 and 3 are the most studied as they are representative of the three cell compartments

- nucleus, cytoplasm and mitochondrion - where sirtuins act.

Human sirtuins are made up of two globular subdomains - the small domain and the large domain - connected by four rings that form an active pocket between the subdomains. The secondary structure of the large domain consists of six b strands that form a parallel b-sheet and six a-helices, organized in a Rossmann-fold structure. The small domain, on the other hand, has two structural modules that fit into the Rossmann-fold structure of the large domain; the small domain is characterized by a helical motif and a zinc-binding motif. The interface between the large and small domain represents the catalytic domain with a binding site for NAD ⁺ [6]

SIRT1 is the most studied sirtuin, consisting of 747 amino acids and N- and C-terminal extensions of ~ 240 amino acids. The extensions are able to interact with regulatory proteins and substrates. At the N-terminal extension, SIRT1 contains two nuclear localization sequences (NLS) and two nuclear export sequences (NES). The coordinated balance of these signals determines the presence of SIRT 1 in the nucleus or in the cytoplasm, based on the specific cell or tissue type [7] SIRT1 located in the nucleus acts as a transcriptional repressor. SIRT1 deacetylates the histone proteins in lysine 26 of histone H1 (H1 K26ac), in lysine 16 of histone H4 (H4K16ac) and in lysine 9 of histone H3 (H3K9ac) and acts on the promoters of the target genes. SIRT1 also has non-histone targets such as the transcription factors p53, NFKB, proteins of the FOXO family, Ku70 [8]; SIRT1 is involved in many cellular processes such as differentiation, apoptosis, inflammation, glucose homeostasis, hormonal secretion, mitochondrial biogenesis, response to stress, protection from DNA damage and in many cellular signal transduction processes, such as synthesis of nitric oxide. It is involved in several pathological conditions, including cancer, metabolic diseases, cardiovascular diseases and aging [9]

SIRT1 and cancer

Sirtuins control many vital functions and are involved in different types of diseases, including neurodegenerative diseases and cancer [10-12] Some studies have highlighted an important up-regulation of SIRT1 in different types of cancer including colon, prostate and skin cancers, as well as hematological tumors such as acute myeloid leukemia.

Since H4K16 acetylation levels are known to be controlled by SIRT1/2 and some cancers are known to be associated with changes of SIRT1 , the decrease in H4K16 acetylation has been attributed to a deregulation of SIRT1 activity [13] However, the activity of SIRT1 seems contradictory since this enzyme can act both as a tumor repressor and as a tumor promoter [14, 15] In fact, as a tumor promoter SIRT1 represses the tumor suppressor p53 through deacetylation of the lysine K382. SIRT1 overexpression represses p53-dependent cell cycle arrest and apoptosis in response to DNA damage and oxidative stress [16]

In contrast, inhibition of SIRT1 increases p53-mediated apoptosis [16] SIRT1 , however, can also act as a tumor suppressor; in fact, low levels of SIRT1 have been found in glioma, tumors of bladder, prostate and ovaries; some studies have suggested that SIRTI overexpression in APC ^-/+ mice reduces, rather than increases, colon cancer formation. This action appears to be caused by SI RT1 -mediated deacetylation of b-catenin, which promotes cytoplasmic localization of the oncogenic b-nuclear chain [17] Furthermore, it has also been suggested that the tumor suppressor activity of SIRT1 is due to its ability to interact and deacetylate the MYC oncogene, strongly influencing both the stability of MYC and the oncogenic action [18-22] The expression and activity levels of SIRT1 can therefore guide the balance between repression and promotion of tumorigenesis, which in turn explains its spatial and temporal distribution in the different stages of malignant transformation.

SIRT1 and cardiovascular diseases

SIRT1 plays a cardio-protective role. In vitro, ex vivo and in vivo studies provide evidence of a protective role of SIRT1 against cardiovascular stress, inhibition of apoptosis, delayed aging and reduction of oxidative stress [23]

The overexpression of SIRT1 causes a protective effect from apoptosis for cardiomyocytes, while its down-regulation is associated with apoptotic cell death [24] Many studies show that the use of SIRT1 activators can have a cardio-protective effect in several models of cardiovascular disease. For example, resveratrol, a known SIRT1 activator, plays a cardioprotective role in diseases associated with oxidative stress. Resveratrol has been reported to reduce cell proliferation and increase doxorubicin-induced cell cycle arrest in tumor bearing mice [25] According to these results, other studies suggest that resveratrol confers anti- apoptotic effects in different models of doxorubicin-induced cardiotoxicity [26] The ability of resveratrol to eliminate free radicals and chelated copper reduces the oxidation of low-density lipoproteins (LDL) particles, a factor that contributes to the development of coronary heart disease [27] In addition, resveratrol reduces the levels of reactive oxygen species (ROS), reduces lipid peroxidation, inhibits platelet aggregation, acts as a vasodilator, increases the expression of endothelial and inducible nitric oxide synthase (eNOS and iNOS) and inhibits the proliferation of smooth muscle vascular cells (VSMC) [28] Taken together, these studies demonstrate that although the precise mechanism of action of resveratrol has not yet been clarified, the use of SIRT1 activators could represent a promising therapeutic approach to avoid doxorubicin-induced cardiotoxicity.

The activity of sirtuins can be modulated by different molecules. Sirtuins can be activated by sirtuin activating compounds (STACS), including natural molecules such as resveratrol, butein [29], curcumin [30], SRT1720 [31-37] Resveratrol is a phenol, that derives from grape skin and has an antioxidant and fluidifying action on blood. Today, resveratrol continues to be at the center of numerous controversies regarding its real activator capacity as the concentrations of the compound used in vitro and in animal models are not reasonably obtainable in vivo. To date, as regards STACs, only resveratrol has been well characterized although, most likely, its effect on SIRT1 may be indirect and not due to the binding with SIRT1 , while a class of molecules that includes SRT1460, SRT1720 and SRT2183 reported in literature by Pacholec et al. [38] as activators of SIRT 1 does not have a specific activity for SIRT1. The structures of some known activators of SIRT1 are shown below.

Y. Matsuya et al., Bioorg. Med. Chem. Lett., 2013, 23, 4907-4910 discloses a number of SIRT1 activators.

The synthesis of the compound ISIDE11 is described in Rao S. et al., 1992 [47], Iqbal R. et al. , 1982 [48] and Fedoryak SD et al., 1982 [49] The latter document also reports the antimicrobial and antituberculosis activities of ISIDE1 1. Neamati et al., 1998 [50] reports the compound ISIDE20 and indicates that it has no inhibitory activity of HIV-1 integrase.

The synthesis of ISIDE11 , compound 52, compound 54 is reported in W09320046.

Therefore, to date, there is a need to find molecules that are able to activate sirtuins, such as SIRT1.

SUMMARY OF THE INVENTION

In the present invention, the inventors have identified molecules that act as activators of sirtuins, specifically of SIRT1 , with micromolar values of AC ₅₀ of, for example, 20.11 mM for ISIDE11 , 396 mM for ISIDE20 and 5.9 pM for ISIDE11 hydrate.

In particular, the molecules of the invention interact physically with SIRT 1 and have an effect on the main targets of SIRT1 , suggesting that they improve the enzyme-mediated deacetylation. In addition, the inventors have characterized the action of the molecules against cardiovascular diseases in mouse in vivo and ex vivo models. Surprisingly, the inventors have found that such molecules are capable of recovering endothelial dysfunction and reducing the formation of thrombi in pathological models, such as MTHFR heterozygous mice. Advantageously, the molecules of the invention are able to reduce the level of acetylation of a protein, in particular of a target protein of SIRT1. For example, said protein is a histone (in particular H3 and H4), p53, NFKB, a protein of the FOXO family or Ku70. In particular, the molecules of the invention are able to reduce the level of acetylation of: H4K16, H3K9, H3K56, P53K382, p53K379 and/or H1 K26.

The CETSA assay for SIRT2 shows that ISIDE 11 does not bind to SIRT2. Furthermore, ISIDE11 has been tested in assay for other histone deacetylases such as HDAC 1 , HDAC 4 and HDAC 6 and is not active. Therefore, the compounds of the invention are selective for SIRT1.

The present invention provides a compound of formula I

wherein R is aryl or heteroaryl optionally substituted with a group selected from: alkoxy, phenoxy, alkyl group, amine group and halide or a salt, tautomer, solvate, stereoisomer or analogue thereof,

or of formula or a salt, tautomer, solvate, stereoisomer or analogue thereof, for use as a SIRT1 activator.

The invention also provides a SIRT1 activator of formula I

wherein R is aryl or heteroaryl optionally substituted with a group selected from: alkoxy, phenoxy, alkyl group, amine group and halide or a salt, tautomer, solvate, stereoisomer or analogue thereof,

or of formula or a salt, tautomer, solvate, stereoisomer or analogue thereof, i.e. a compound, salt, tautomer, solvate, stereoisomer or analogue as defined above being a SIRT1 activator. Preferably, said aryl is phenyl. Preferably, said heteroaryl is pyridinyl. Preferably, said alkoxy is methoxy. Preferably, R is phenyl, pyridinyl or phenyl para-substituted with methoxy.

In a preferred aspect, the compound has the formula:

or a salt, tautomer, solvate, stereoisomer or analogue thereof.

Preferably, the solvate is a hydrate such as:

In a preferred aspect, the compound, salt, tautomer, solvate, stereoisomer, analogue or SIRTI activator as defined above is for use in a method of therapy and/or prevention of a condition and/or disease selected from the group consisting of: a cardiovascular disease, a tumor, a metabolic disease, aging, a cellular senescence, a neurodegenerative disease, an endothelial dysfunction, a DNA damage, an oxidative stress, a cerebral damage, a cardiovascular damage, a coronary artery disease, thrombosis, hyperhomocysteinemia, insulin resistance, obesity, neuronal loss, an age-related cardiological condition, diabetes, preferably type 2 diabetes, dyslipidemia, hyperlipidemia, an immune disease, a complication of diabetes and/or an inflammation.

Preferably, the compound, salt, tautomer, solvate, stereoisomer, analogue or SIRT 1 activator as defined above is for use as an apoptotic, vasodilator and/or antithrombotic medicament.

Preferably, the compound, salt, tautomer, solvate, stereoisomer, analogue or SIRT 1 activator as defined above is characterized in that it is administered to a subject carrying at least one mutation in the gene of the enzyme methylene tetrahydrofolate reductase (MTHFR). Preferably, said at least one mutation is the C677T or A1298C mutation.

The invention further provides a pharmaceutical or cosmetic composition comprising the compound, salt, tautomer, solvate, stereoisomer, analogue or SIRT1 activator as defined above, a vehicle and optionally a further therapeutic agent, said pharmaceutical or cosmetic composition being for use as a SIRT1 activator.

Preferably, the composition is for use in a method of therapy and/or prevention of a condition and/or disease selected from the group consisting of: a cardiovascular disease, a tumor, a metabolic disease, aging, a cellular senescence, a neurodegenerative disease, an endothelial dysfunction, a DNA damage, an oxidative stress, a cerebral damage, a cardiovascular damage, a coronary artery disease, thrombosis, hyperhomocysteinemia, insulin resistance, obesity, neuronal loss, an age-related cardiological condition, diabetes, preferably type 2 diabetes, dyslipidemia, hyperlipidemia, an immune disease, a complication of diabetes and/or an inflammation.

Preferably, said further therapeutic agent is selected from the group consisting of: folic acid, vitamin B6 and vitamin B12.

The present invention further provides an in vitro method to identify a modulator of SIRT1 comprising activating SIRT1 with the compound, salt, tautomer, solvate, stereoisomer, analogue or SI RT 1 activator as defined above in the presence or absence of said modulator.

It is also an object of the present invention a use, in particular in vitro or ex vivo , of the compound, salt, tautomer, solvate, stereoisomer, analogue or SIRT1 activator as defined above to activate SIRT1. For instance, the invention provides an in vitro or ex vivo method of activating SIRT1 with the compound, salt, tautomer, solvate, stereoisomer, analogue or SIRT1 activator as defined above, e.g. by contacting SIRT1 or a host (such as a cell) comprising SIRT1 with the compound, salt, tautomer, solvate, stereoisomer, analogue or SIRT1 activator as defined above. It is to be understood that in the present invention any reference to“the compound”, “the compound of the invention”,“SIRT1 activator” or grammatical variants thereof also includes the salt, tautomer, solvate, stereoisomer or analogue thereof as defined above.

As used herein,“alkyl” refers to any linear or branched aliphatic hydrocarbon chain. Preferably, said alkyl comprises from 1 to 6 carbon atoms and is thus termed C1-C6 alkyl. As used herein,“alkoxy” refers to an -O-alkyl group, wherein O is an oxygen atom and alkyl is as defined above. Preferably, said alkoxy comprises a C1-C6 alkyl and is thus termed C1-C6 alkoxy.

As used herein,“aryl” refers to a monocyclic or polycyclic aromatic ring consisting solely of carbon and hydrogen atoms. Preferably, said aryl is phenyl.

As used herein,“heteroaryl” refers to an aryl ring as defined above, wherein at least one carbon atom is replaced by at least one heteroatom, in particular nitrogen, oxygen or sulfur. Preferably, said heteroaryl is pyridinyl.

As used herein, “amine group” is any group deriving from ammonia wherein one or more hydrogen atoms have been replaced by any other functionality.

It is to be understood that the expression “optionally substituted with a group” means “unsubstituted or substituted with one or more groups”, wherein said one or more groups may be the same or different and may occupy any one or more positions.

The compounds of the invention may be obtained by chemical synthesis according to standard procedures, see for instance Rao S. et al., 1992 [47], Iqbal R. et al., 1982 [48], Fedoryak SD et al., 1982 [49], Neamati et al., 1998 [50] and W09320046, all of which are herein incorporated by reference, and/or may be purchased by commercial suppliers as described in the Materials and Methods below.

In the invention,“activator of SIRT1” or “SIRT1 activator” means a molecule capable of increasing SIRTTs catalytic activity as a deacetylase. This increase can be measured with respect to any proper control by any method known in the art. Examples of methods to measure SIRT1 activation include: in vitro enzymatic assays, in which the level of substrate acetylation due to SIRT1 activity is measured in the presence of potential modulators, and Western Blotting by analyzing the acetylation of at least one target of SIRT1. Proper control against which the increase of SIRTTs activity may be measured include: the untreated enzyme, the enzyme treated with a vehicle such as DMSO, the enzyme treated with a known activator, the enzyme treated with a known inhibitor, etc. In vitro enzymatic assays may involve any technique known in the art, for instance as described in the Detailed Description of the Invention below. Targets of SIRT1 include but are not limited to: histones H1 , H3 and H4, p53, NFKB, p300, proteins of the FOXO family (such as FOXO 1 , 3a and 4), HIVTat, PGC-1a, PCAF, MyoD, peroxisome proliferator-activated receptor y, Ku70, Hif-1 a, Hif-2a, MYC, STAT3, Rb, DNMT1 , CRTC2, LXRs, AceCSl In particular, a SIRT1 activator can be defined as a molecule that decreases the acetylation level of any one or more of SIRT 1’s targets with respect to a proper control as defined above, where the acetylation level can be measured by any method known in the art including for instance Western Blotting and enzymatic assays.

Additionally or alternatively, a SIRT1 activator may be a molecule capable of increasing SIRT1 expression, which can be measured by any method known in the art. In the present invention, any reference to a protein includes the respective gene, mRNA, cDNA and the protein by them codified, including fragments, derivatives, variants, isoforms, etc. thereof. Preferably, said proteins are characterised by the following UniProt Accession Numbers. In the present invention reference is made to the following proteins:

SIRT1 , Q96EB6 (SIR1_HUMAN) (https://www.uniprot.org/uniprot/Q96EB6)

SIRT2, Q8IXJ6 (SIR2_HUMAN) (https://www.uniprot.org/uniprot/Q8IXJ6)

SIRT3, Q9NTG7 (SIR3_HUMAN) (https://www.uniprot.org/uniprot/Q9NTG7)

SIRT4, Q9Y6E7 (SIR4_HUMAN) (https://www.uniprot.org/uniprot/Q9Y6E7)

SIRT5, Q9NXA8 (SIR5_HUMAN) (https://www.uniprot.org/uniprot/Q9NXA8)

SIRT6, Q8N6T7 (SIR6_HUMAN) (https://www.uniprot.org/uniprot/Q8N6T7)

SIRT7, Q9NRC8 (SIR7_HUMAN) (https://www.uniprot.org/uniprot/Q9NRC8)

p53, P04637 (P53_HUMAN) (https://www.uniprot.org/uniprot/P04637)

NFKB, P19838 (NFKB1_HUMAN)(https://www.uniprot.org/uniprot/P19838)

proteins of the FOXO family, Q12778 (F0X01 _HUMAN)

(https://www.uniprot.org/uniprot/Q12778) - 043524 (F0X03 _HUMAN)

(https://www.uniprot.org/uniprot/043524) - P98177

(FOX04_HUMAN)(https://www.uniprot.org/uniprot/P98177)

Ku70 P12956 (XRCC6_HUMAN) (https://www.uniprot.org/uniprot/P12956)

H3, P68431 (H31_HUMAN) (https://www.uniprot.org/uniprot/P68431)

H4, P62805 (H4_HUMAN)(https://www.uniprot.org/uniprot/P62805)

MTHFR P42898 (MTHR_HUMAN) (https://www.uniprot.org/uniprot/P42898)

FOXO proteins are a family of transcription factors that play an important role in regulating the expression of genes involved in cell growth, proliferation, differentiation and longevity. The family is characterized by a preserved DNA binding domain, FOX or forked box. The family includes more than 100 members in humans, classified from FOXA to FOXR based on their sequence similarity. The distinctive feature of these proteins is FOX, a sequence of 80-100 amino acids that form a motif that binds to DNA. This motif is also known as "winged helix" because of the butterfly-shaped aspect that the loops assume.

In the invention, a cardiovascular disease includes: acute myocardial infarction, angina pectoris, ischemic and hemorrhagic stroke; a tumor includes solid or hematopoietic tumors, for example leukemia, breast, liver, pancreatic, colon, colorectal, lung cancer; a metabolic disease includes diabetes mellitus, phenylketonuria, citrullinemia, dyslipidemia; a cellular senescence includes Alzheimer's disease, Parkinson's disease; a neurodegenerative disease includes Alzheimer's disease, Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis; an endothelial dysfunction includes hypertension; a DNA damage includes cancer, an oxidative stress includes: premature aging, chronic-degenerative diseases; a cerebral damage (or brain damage) includes an ischemia; a cardiovascular damage includes thrombosis, a coronary artery disease comprises cardiovascular diseases such as ischemic cardiopathy and myocardial infarction; an age-related cardiological condition includes a heart attack; an immune disease includes: rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroid disease, psoriasis; a complication of diabetes includes retinopathy, diabetic nephropathy, diabetic foot, coronopathy and cerebral vasculopathy; an inflammation includes tuberculosis and hepatitis.

In the present invention, the compound can be in the form of a salt, in particular of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts conventionally include nontoxic salts obtained by salification with inorganic acids (e.g. hydrochloric, hydrobromic, sulfuric or phosphoric acid), or with organic acids (e.g. acetic, propionic, succinic, benzoic, sulfanyl, 2- acetoxy-benzoic, cinnamic, mandelic, salicylic, glycolic, lactic, oxalic, malic, maleic, malonic, fumaric, tartaric, citric, p-toluenesulfonic, methanesulfonic, ethanesulfonic, or naphthalensulfonic acid). For information on pharmaceutically suitable salts see Berge SM et al., J. Pharm. Sci. 1977, 66, 1-19; Gould PL Int. J. Pharm 1986, 33, 201-217; and Bighley et al. Encyclopedia of Pharmaceutical Technology, Marcel Dekker Inc, New York 1996, Volume 13, page 453-497.

Furthermore, pharmaceutically acceptable salts obtained by adding a base can be formed with a suitable inorganic or organic base such as triethylamine, ethanolamine, triethanolamine, dicyclohexylamine, ammonium hydroxide, pyridine. The term "inorganic base", as used herein, has its ordinary meaning as understood by the skilled in the art, and generally refers to an organic or inorganic compound, which can act as a proton acceptor.

Other suitable pharmaceutically acceptable salts include alkali metals or alkaline earth metals, pharmaceutically acceptable salts such as sodium, potassium, calcium or magnesium salts; in particular pharmaceutically acceptable salts of one or more carboxylic acids moieties which may be present in the compound.

In addition, the compound may be administrated in non-solvated forms as well as in forms solvated with pharmaceutically acceptable solvents such as water, EtOH and similar. Compounds of the invention can exist in stereoisomeric forms (for example, they may contain one or more asymmetric carbon atoms). The single stereoisomers (enantiomers and diastereomers) and mixtures of these can be used according to the present invention. The present invention encompasses individual isomers as well as mixtures with isomers in which one or more chiral centers are reversed.

Similarly, it is understood that the compounds of the invention may exist in tautomeric forms different from those shown in the formulas and these are also included in scope of the present invention.

The invention also includes all the isotopic variants of the compounds of the invention. An isotopic variant of a compound of the invention is defined as a variant in which at least one atom of the molecule is replaced by an atom having the same atomic number but an atomic mass different from the one usually present in nature. Examples of isotopes that can be incorporated into the compound of the invention include isotopes such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ 0, ¹⁸ 0, ³¹ P, ³² P, ³⁵ S, ¹⁸ F and ³⁶ CI, respectively. Some isotopic variants of the invention, for example, those in which a radioactive isotope such as ³ H or ¹⁴ C is incorporated, can be used in the studies of tissue distribution of drugs and/or substrates.

Furthermore, substitution with isotopes such as deuterium ² H can lead to therapeutic advantages resulting from a greater metabolic stability. Isotopic variants of the sirtuin activators can generally be prepared by conventional procedures using suitable isotopic variants of suitable reagents.

As used herein, “analogue” has its ordinary meaning according to the art. In particular, an analogue is a chemical compound having a structure similar to another (primary compound) but comprising at least one different atom and/or functional group. For example, an analogue of the compound of the invention may have a different chemical structure compared to the compound of the invention, while maintaining the same pharmacophore. The analogue maintains the pharmacological activity of the primary compound.

In the present invention, the compounds can be administered as such or as pharmaceutical formulations, i.e. suitable for parenteral, oral, vaginal or rectal administration. Each of said formulations can contain excipients, fillers, additives, binders, coatings, suspending agents, emulsifying agents, preservatives and/or control release agents suitable for the selected pharmaceutical form.

The pharmaceutical compositions can be chosen on the basis of the treatment needs. Such compositions are prepared by mixing and can be administered for example in the form of tablets, capsules, oral preparations, powders, granules, pills, injectable or infusible liquid solutions, suspensions, suppositories, or preparations for inhalation. Tablets and capsules for oral administration are normally presented in unit dose and contain conventional excipients such as binders, fillers (including cellulose, mannitol, lactose), diluents, tableting, lubricants (including magnesium stearate), detergents, disintegrates (e.g. polyvinylpyrrolidone and starch derivatives such as starch sodium glycolate), coloring agents, flavoring agents, and wetting agents (e.g. sodium lauryl sulphate).

The oral solid compositions can be prepared with conventional methods of mixing, filling or tableting. The mixing operation can be repeated to distribute the active principle in all the compositions containing large quantities of fillers. These operations are conventional. Oral liquid preparations can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or they can be presented as a dry product to be reconstituted before use with water or with a suitable vehicle. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, or hydrogenated edible fats; emulsifying agents, such as lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils) such as almond oil, fractionated coconut oil, oily esters such as glycerin esters, propylene glycol, or ethyl alcohol; preservatives, such as methyl or propyl p- hydroxybenzoate or sorbic acid, and if desired, conventional flavoring or coloring agents.

Oral formulations also include conventional slow-release formulations such as gastro-resistant tablets or granules. The pharmaceutical compositions for administration by inhalation can be contained in an insufflator or in a pressurized nebulizer.

For parenteral administration, unit doses of fluid can be prepared, containing the compound and a sterile vehicle. The compound can be either suspended or dissolved, based on vehicle and concentration.

The parenteral solutions are normally prepared by dissolving the compound in a vehicle, sterilizing by filtration, filling suitable bottles and sealing.

Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle. To increase stability, the composition can be frozen after filling the vials and removing the water under vacuum. Parenteral suspensions are prepared in substantially the same way, except that the compound can be suspended in the vehicle instead of being dissolved and sterilized by exposure to ethylene oxide before suspension in the sterile vehicle. Advantageously, a surfactant or a wetting agent can be included in the composition to facilitate uniform distribution of the compound of the invention.

In order to increase bioavailability, the compounds can be formulated pharmaceutically into liposomes or nanoparticles. Acceptable liposomes can be neutral, negatively or positively charged, the charge being a function of the charge of the liposome components and of the pH of the liposomal solution. Liposomes can normally be prepared using a mixture of phospholipids and cholesterol. Suitable phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol. Polyethylene glycol can be added to improve the blood circulation time of liposomes.

Acceptable nanoparticles include albumin nanoparticles and gold nanoparticles.

For buccal or sublingual administration the compositions can be tablets, lozenges, pastilles, or gels.

The compounds can be pharmaceutically formulated as suppositories or retention enemas, for example suppositories containing conventional bases such as cocoa butter, polyethylene glycol, or other glycerides, for rectal administration.

Another means of administering the compounds of the invention regards topical treatment. Topical formulations can contain for example ointments, creams, lotions, gels, solutions, pastes and/or can contain liposomes, micelles and/or microspheres. Examples of ointments include oil- based ointments such as vegetable oils, animal fats, semisolid hydrocarbons, emulsifiable ointments such as hydroxystearyl sulphate, anhydrous lanolin, hydrophilic petrolatum, cetyl alcohol, glycerol monostearate, stearic acid, water-soluble ointments containing polyethylene glycols of various molecular weights.

The creams, as known to formulation experts, are viscous liquids or semi-solid emulsions, and contain an oily phase, an emulsifier and an aqueous phase. The oil phase generally contains petrolatum and an alcohol such as cetyl or stearic alcohol. Suitable formulations for topical administration to the eye also include eye drops, in which the active ingredient is dissolved or suspended in a suitable vehicle, especially an aqueous solvent for the active ingredient.

A further way of administering the compounds of the invention relates to transdermal delivery. Topical transdermal formulations include conventional aqueous and non-aqueous carriers, such as creams, oils, lotions or pastes or they can be in the form of medicated membranes or plasters. The formulations are known in the art (as described for example in the book by Remington "The Science and Practice of Pharmacy", Lippincott Williams & Wilkins, 2000, herein incorporated by reference).

The compounds of the present invention can be employed for use in the treatment and/or prevention of the conditions mentioned above alone as a single therapy or in combination with other therapeutic agents, both through separate administrations and by including the two or more active ingredients in the same formulation. The compounds can be administered simultaneously or sequentially. The combination can be administered as separate compositions (simultaneous, sequential) of the individual components of the treatment or as a single dosage form containing all the agents. When the compounds of this invention are combined with other active ingredients, the active ingredients may be formulated separately in single ingredient preparations of one of the forms described above, and then supplied as combined preparations, that are administered at the same time or different times, or they can be formulated together in a preparation of two or more ingredients.

The compounds of the invention can be administered to a patient in a total daily dose of, for example, 0.001 to 1000 mg/kg body weight. The unit dosage compositions may contain such quantities of submultiples thereof to compensate for the daily dose. The compounds of the invention can also be administered weekly or once a day. The determination of the optimal dosages for a particular patient is well known to an expert in the field. As is common practice, the compositions are normally accompanied by written or printed instructions for use in the treatment in question.

The present invention will now be described with non-limiting examples, referring to the following figures:

Figure 1 : In vitro SIRT1 assay. Figure 2: Identification of ISIDE11 and ISIDE20 compounds. (A) Flowchart of the screening procedure in high-throughput screening (HTS) mode to identify new SIRT1 modulators. (B) The primary enzyme assay was performed using the library molecules at a fixed concentration of 10 mM, compared to the control without any modulator, the known activator (STAC-2) [39] and the known inhibitor (EX527). In this assay the percentage of activity for ISIDE11 is about 140% and for ISIDE20 of 138%. (C) Counter-assay performed to evaluate whether ISIDE11 and ISIDE20 at 10 mM modulate the activity of the enzyme nicotinamidase (NMase), compared to the control in which no modulator is present. (D, E) Dose curve (from 1 to 250 mM) for enzyme activity to evaluate the AC ₅₀ of ISIDE11 (D) and ISIDE20 (E).

Figure 3: ISIDE11 interacts with SIRT1 enzyme. (A) The CETSA assay was performed in MCF7 cells treated with DMSO (vehicle - Ctrl) or treated with 50 pM of ISIDE11 at the indicated temperatures. The extracts were then loaded onto acrylamide gel and incubated with the SIRT1 antibody. The band relative to SIRT1 signal is still visible at the highest temperature of 67°C only in the sample treated with ISIDE 1 1 and not in the control, indicating that the molecule protects the protein SIRT1 from degradation. (B) SIRT 1-ISIDE11 interaction monitored by fluorescence spectroscopy. The fluorescence emission of tyrosine as the F0/F ratio was evaluated for both SIRT1 and free tyrosine after the addition of ISIDE11 at different concentrations (0.5, 1 , 5, 10, 20, 30, 50 pM). The working concentrations were 10 pM for both SIRT1 and free tyrosine. Other experimental details are described in the Materials and Methods.

Figure 4: (A) The cell permeability of ISIDE11 at 50 pM was assessed in CACO-2 cells (B) Stability profile of ISIDE11 in human serum. ISIDE11 (1 mg/ml) in 20% DMSO solution and 90% DMEM at T 37°C. ISIDE11 was quantified at 375 nm. The residual amounts of the compound were determined after 15, 30, 60 and 90 minutes by RP-HPLC in 20% DMSO solution and 90% human serum at 37°C.

Figure 5: ISIDE11 modulates histones. (A) HepG2 cells were treated for 24 hours with DMSO (Ctrl) and with ISIDE11 and ISIDE20 at two concentrations (5 and 50 pM). The protein extracts have been used for Western blotting for the detection of acetylated histone H4 in lysine 16 (H4K16ac). The histone H3 (H3) signal was used as a loading control. (B) MCF7, HCT116, and U937 cells were treated with DMSO (Ctrl), ISIDE11 at 50 pM, ISIDE20 at 50 pM and EX- 527 (SIRT 1 inhibitor) at 5 pM for 24 hours. Protein extracts have been used for Western blotting for the detection of acetylated histone H3 in lysines 9 and 14 (H3K9/14ac). The histone H4 (H4) signal was used as a loading control.

Figure 6: ISIDE11 counteracts the effects of cell damage. (A) Western blotting for p53 acetylated in lysine 382 (p53K382ac) in HepG2 cells pretreated for 6 h with etoposide at 2 pM and subsequently treated with ISIDE11 and ISIDE20 at 50pM. The signal related to Erk1/2 has been used as a loading control. (B) Hep-G2 cells were treated with Etoposide at 20pM in combination with ISIDE11 or ISIDE20 at 50 pM for 6 h; (C) Hep-G2 cells were treated with Etoposide at 20 mM and ISIDE11 at 50 mM in combination for 6 h. Western blotting for total p53 (p53) and its forms acetylated in lysine 382 (p53K382ac) and in lysine 379 (p53K379ac) and for histone H3 acetylated in lysine 56 (H3K56ac). The signal related to Erk1/2 and the red Ponceau were used as loading controls. (D-E) Evaluation of the expression of mRNA of SIRT1 and p53 in Hep G2 after treatment with ISIDE11 at 50 pM for 6 h.

Figure 7: MTT assay. (A) A2058 cells were treated with ISIDE11 from 50 pM to 0.39 pM for both 24 hours and 72 hours. (B) MRC-5 cells were treated with ISIDE11 from 50 pM to 0.39 pM for both 24 hours and 72 hours. (C) A549 cells were treated with ISIDE1 1 from 50 pM to 0.0975 pM for 24 hours. (D) HT-29 cells were treated with ISIDE11 from 50 pM to 0.0975 pM for 24 hours. (E) MiaPaCa cells were treated with ISIDE11 from 50 pM to 0.0975 pM for 72 hours. Figure 8: Dose-response curves to acetylcholine (ACh) of mesenteric arteries of Wild-type ^+/+ or MTHFR ^+/- mice pre-contracted with phenylephrine in basal (untreated) conditions and incubated ex-vivo with ISIDE11. The ex-vivo incubation of mesenteric arteries of mice with ISIDE11 induces the restoration of normal vasodilation after stimulation with ACh. (A-B) The vessels were pre-exposed to resveratrol (RSV) at 25 pM for 1 hour. (C-D) The vessels were pre-exposed to ISIDE11 at 50 or 100 pM for 1 hour. *** p<0.001 ; ** p <0.01 ; * p <0.05; ### p <0.001 ; ## p <0.01 ; # p <0.05.

Figure 9: ISIDE1 1 normalizes the bleeding time in MTHFR ^+/- mice. Bleeding times of the tail of Wild-type(+/+) or MTHFR ^+/- mice determined after in vivo treatment with resveratrol (RSV; 10 mg/Kg intraperitoneal administration (ip.) for 21 days) or ISIDE11 (ISIDE11 ; 10 mg/Kg intraperitoneal administration (ip.) for 21 days) alone or in combination with SIRT 1 inhibitor, EX- 527 (5 mg/Kg intraperitoneal administration (ip.). ** p <0.01 ; * P <0.05.

Figure 10: ISIDE11 in vivo reduces thrombus formation in MTHFR ^+/- mice. (A) Percentage reduction of superficial femoral artery blood flow measured by ultrasound analysis in Wild-type and MTHFR ^+/- mice treated in vivo for 21 days with ISIDE11 (10 mg/Kg) or resveratrol (RSV; 10 mg/Kg), alone or in combination with EX- 527 (5 mg/Kg). (B) Hematoxylin and eosin staining of the femoral arteries of mice split after 21 days by different groups of animals treated with ISIDE11 (10 mg/kg for 21 days) or resveratrol (RSV; 10 mg/kg for 21 days) alone or in combination with EX-527(5 mg/Kg). 100mm scale.

Figure 11 : In vivo treatment with ISIDE11 (10 mg/kg for 21 days) recovers the altered endothelial function of MTHFR ^+/- mice. Vascular response of mesenteric arteries excised from Wild-type or MTHFR+/- mice and pre-contracted with phenylephrine and evaluated by dose-response to (A) acetylcholine (endothelium-dependent) or (B) nitroglycerin (independent on the endothelium). The mice were treated in vivo for 21 days with ISIDE11 (10 mg/kg) or resveratrol (RSV; 10 mg/kg), alone or in combination with EX-527 (5 mg/kg). * P <0.05. Figure 12: SIRT1 enzymatic assay for the compounds ISIDE11 , cmp52, cmp54 and ISIDE11 hydrate (ISIDE11 H20). All compounds were tested at 10 mM compared to the control (without modulator), the known activator (STAC-2) and the known inhibitor (EX-527).

Figure 13: ISIDE11 hydrate (ISIDE11 H20) structure (top) and dose curve (from 0.001 to 250 mM) for enzyme activity to evaluate AC50 which is equal to 5.9 pM (bottom).

DETAILED DESCRIPTION OF THE INVENTION

Materials and methods

Cell lines

Human hepatocellular carcinoma cells (HepG2) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA, HB-8065) and cultured in Dulbecco's Modified Eagle's Medium (DMEM; Euroclone) supplemented with 10% fetal bovine serum heat inactivated (FBS; Sigma-Aldrich) and antibiotics (100 Ul/ml penicillin, 100 mg/ml streptomycin and 250 mg/ml amphotericin-B). The cells were maintained in an incubator at 37°C and 5% CO ₂ in a completely humidified environment. HCT-116 (ATCC No. CCL-247), HT-29 (ATCC No. HTB-38) MCF7 (ATCC No. HTB-22), A549 (ATCC No. CCL-185), MiaPaCa (ATCC No. CRL-1420) and A2058 (ATCC No. CRL-11 147) were grown in Dulbecco's Modified Eagle's Medium (Euroclone, Milan, Italy) with 10% fetal bovine serum (FBS) (Euroclone), 2 mM L-glutamine (Euroclone) and antibiotics (100 Ul/ml of penicillin, 100 mg/ml of streptomycin, Euroclone).

The MRC5 cells (ATCC No. CCL-171) were cultured in the Eagle minimum essential medium (EMEM; Euroclone) supplemented with 10% FBS (Euroclone) and 10 mg/ml of gentamicin solution (Euroclone). U937 (ATCC No. CRL-1593.2) were grown in RPMI 1640 MEDIUM (Euroclone) supplemented with heat-inactivated 10% fetal bovine serum (FBS; Sigma-Aldrich) and antibiotics (100 Ul/ml of penicillin, 100 mg/ml of streptomycin and 250 mg/ml of amphotericin-B). Caco-2 (ATCC No. HTB-37) were grown in the Eagle Minimum Essential Medium (EMEM; Euroclone) supplemented with 10% FBS (Euroclone) and 10 mg/ml of gentamicin solution (Euroclone).

Compounds

All compounds were purchased from ChemBridge Corporation and dissolved in DMSO (Sigma- Aldrich No. D2650), EX527 (Sigma; No E7034), resveratrol (Santa Cruz; CAS No. 501-36-0), etoposide (Teva).

ISIDE11 (Chembridge, ID: 5132326), ISIDE20 (Chembridge, ID: 5246926), COMPOUND 52 (Chembridge, ID: 5545852), COMPOUND 54 (Chembridge, ID: 5550454), hydrated form of ISIDE11 (Chembridge, ID: 5131383).

Purification of recombinant Sirt1-GST

The recombinant SI RT1 -GST (glutathione S-transferase) enzyme was purified from E. Coli BL21 bacteria after transfection with plasmid pGEX-SIRT1 (Addgene). A selected colony of bacteria was grown in LB Broth (Lennox) medium supplemented with antibiotics (100 ug/mL of ampicillin) in a shaking incubator overnight.

When the OD was between 0.6 and 0.8, the expression of the protein was induced by isopropyl- b-D-1-thiogalactopyranoside (IPTG, AppliChem) at a concentration of 200 mM for 5 hours. The bacteria were centrifuged at 3000 rpm (Beckman centrifuge), then the pellet was lysed with the sonication procedure (Sonic Diagenode). The lysis buffer was composed of phosphate buffer saline (PBS), 1 mM of dithiothreitol (DTT) (Applichem), 0.5 mM of phenylmethylsulfonylfluoride (AppliChem) and 1 tablet of protease Inhibitor complex (PIC) for every 10 mL (Roche). The bacteria were sonicated for 10 cycles for 45 seconds at 14,000 MHz with 30 second intervals between each sonication. Then, 0.1% Triton X100 (Acros) was added and incubated for 15 minutes on ice. Subsequently, the sonicate was centrifuged at 13,000 rpm for 30 minutes and filtered with a 0.45 mm filter.

The bacterial lysate was purified using columns of GE Healthcare Life Sciences (GSTrap 4B). The columns were equilibrated with 20 mL of lysis buffer. Then, the lysate was loaded onto columns and subsequently washed with the lysis buffer. The elution was carried out with 20 mL of elution buffer consisting of 50 mM Tris-HCI at pH 8.0, 1 mM of DTT, 20 mM reduced L- glutathione (AppliChem) and H20dd. The SIRT1-GST protein was detected using colorimetric methods - Bradford protein Assay (Biorad).

Dialysis was performed using a buffer consisting of 50 mM Tris-HCI pH 8.0, 100 mM NaCI (Sigma), 1 mM DTT, 1 tablet of PIC (for every 10 mL) and H20dd overnight at 4°C. After overnight, the samples were cryopreserved in 20% glycerol (SigmaAldrich).

NMase-His tag purification

The histidine-labeled nicotinamidase enzyme (NMase-His) was expressed in E.Coli BL21 bacteria. The bacteria were grown in LB Broth medium supplemented with antibiotics (100 mg / mL of ampidllin) in an overnight incubator. When OD was 0.7, isopropyl-b-D-1- thiogalactopyranoside (IPTG) was added to 1 mM concentration for 5 hours. The bacteria were centrifuged at 3000 rpm for 20 minutes and were lysed using H20 lysis buffer (50 mM NaH2P04 pH 8, 300 mM NaCI, 10 mM imidazole (Applichem), 100 mg / 50 mL lysozime (Applichem), 1 PIC tablet per 10 ml. The bacterial lysate was incubated on ice for 30 minutes and then subjected to ultrasound for 10 cycles for 45 seconds at 14,000 MHz with 30 second intervals between each sonication. After sonication, the lysate was filtered with a 0.45 mm filter and incubated for 3 hours at 4 ° C with 1 mL of NTA nickel agarose pre-equlibrate (ABT) with equilibration buffer (EQ) (50 mM NaH2P04 pH8, 300 mM, NaCI, 20 mM imidazole in H20dd).

Subsequently, the column was washed with EQ and then the enzyme was eluted with a buffer consisting of 50 mM NaH2P04 pH8, 300 mM NaCI and 250 mM imidazole, 1 PIC tablet for every 10 mL and the samples were cryopreserved in glycerol at 20%.

SIRT1 enzymatic assay The test was performed on a 96-well microplate reader with fluorescent reading (Corning 96 Flat Bottom Black Polystyrol). The reaction volume was 25 mL. The reaction buffer was composed of PBS and 1 mM DTT. All library compounds, including known inhibitors and activators (used as reference compounds), were dissolved in DMSO and tested with a concentration of 10 mM. The purified SIRT1 enzyme (5 mL) was used with a concentration of 1 mg/ml, and incubated for 15 minutes at 37 °C with 5 mL of intermediate dilution (50 mM) of compounds. Then, a mixture consisting of 5 mL of enzyme purified with NMase, 5 mL of intermediate dilution b-NAD (1 mM) and 5 mL diluted p53K382 acetylated peptide (250 mM) (synthesized by INBIOS) were added and the whole mixture was incubated for 40 minutes at 37 ° C.

Subsequently, the reaction stop buffer was added (70% of PBS, 30% of ethanol, 10 mM of DTT and 10 mM of 1 ,2-Phthalic dicarboxaldehyde OPT) (Acros organics, No 131080250) followed by a second incubation of 30 minutes at room temperature. The detection of the fluorescent signal was performed with a TECAN INFINITE M200 microplate reader at 420/460 nm.

SIRT1 enzymatic assay in high-throughput mode

Primary screening was conducted using a Tecan Robot Freedom EVO 150. The standard assay (SIRT1 Fluorescence assay) was miniaturized in plates with 384 wells (Corning 384 Flat Bottom Black Polystyrene) with a final reaction volume of 15 mL. Plate 384 had previously been loaded with the known activator (STAC-2) and the known inhibitor (EX-527) used as references in the top left and bottom right wells by using HP D300 TECAN digital dispenser, and a control without any modulator. 160 compounds were tested in duplicate per plate.

The reaction buffer was placed on the robot worktable (PBS, DTT 1 mM and 0.6% DMSO, compounds at 10 mM, mixture of SIRT1 enzymes and nicotinamidase (NMase), which will be used for the assay). First, the robot produced dilutions of compounds (from 10 mM to 10 mM) and added the mixture of the SIRT 1 enzyme to the whole plate except for the negative control wells. After incubation for 15 minutes at 37°C, the NMase mixture was added and incubated for 40 minutes at 37 °C. After the reaction stop buffer was added for 30 minutes at room temperature, the fluorescence reading was performed with a TECAN INFINITE M1000 microplate reader.

Counter-assay for NMase enzyme

The compounds were re-tested in a fluorescence assay in presence of only NMase enzyme, to exclude the potential activity of the molecules on this enzyme. The assay was performed in a 96-well plate with the same positive and negative reaction controls as the SIRT1 fluorescence assay. Each compound was always tested at 10 mM and then 5 mL of intermediate dilution of the compound (50 mM) was added to 5 mL of the reaction buffer. After 40 minutes of incubation at 37 °C, the reaction stop buffer was added and the plate was incubated again in the dark for 30 minutes at room temperature. The fluorescence reading took place at 420/460 nm and was performed with the TECAN INFINITE M200 microplate reader. Evaluation of intrinsic fluorescence

The compounds, dissolved in the reaction buffer, were placed in a 96-well black plate and incubated for 40 minutes at 37°C. The fluorescence reading took place at 420/460 nm and was performed with the TECAN INFINITE M200 microplate reader.

AC50 / IC50 evaluation

AC50 or IC50 are the concentration at which a compound (activator or inhibitor, respectively) shows 50% of its maximum activity to respectively activate or inhibit the SIRT1 enzyme. The compounds were loaded (0.01 mM to 100 mM) into a 96-well plate with the TECAN HP D300 Digital Dispenser. The test was conducted under the same conditions as the SIRT1 enzyme assay. The AC50 / IC50 values were calculated using the GraPhpad Prism 6 program.

Cellular Thermal Shift Assay (CETSA)

MCF7 cells were treated with ISIDE1 1 at 50 pM or with DMSO (vehicle) for 1 hour. Cells were collected and washed with PBS. The respective samples were suspended in PBS (1.5 ml), divided into aliquots (100 pi) and heated to different temperatures (RT, 4, 37, 47, 57 and 67 ° C) for 3 minutes using the Thermo Mixer (Eppendorf, Milan, Italy), followed by cooling for 3 minutes at 4 °C. After incubation, 50 pi of RIPA buffer (50 mM Tris-HCI pH 7.4; 1% NP40; 0.5% Na- deoxycholate; 0.1 % SDS; 150 mM NaCI; 2 mM EDTA; 50 mM NaF; a tablet of protease / phosphatase inhibitors) were added to the samples and incubated at 37 °C for 15 minutes. The samples were then centrifuged at 13,000 rpm for 30 minutes at 4 °C, the supernatant was recovered and the protein content was determined using a Bradford assay (Bio-Rad, No. 5000006). Of the total protein extract, 20 mg were loaded onto 10% SDS-PAGE. The nitrocellulose filters were colored with Ponceau red (Sigma) as the loading control. The filters were then hybridized with SIRT1 Abeam antibody.

Evaluation of Fluorescence quenching

Fluorescence measurements were performed on a LS 55 spectrometer by Perkin Elmer Life Sciences. The fluorescence emission of tyrosine (Aex 275 nm /Aem 305 nm) was assessed both for SIRT 1 (after cleavage of thrombin to remove GST) and for free tyrosine a few seconds after adding ISIDE1 1 at different concentrations (0, 0.5, 1 , 5, 10, 20, 30, 50 pM) in the PBS and DTT solution (SIRT1 protein buffer). The extinction of the tyrosine fluorescence was monitored by estimating the F0 / F ratio considering the intensity of the fluorescence at 305 nm of the sample before (F0) and after (F) the addition of ISIDE 11. The working concentrations were 10 pM for both SIRT1 and free tyrosine.

Cell permeability test

Caco-2 cells were grown in Dulbecco's Modified Eagle Medium (DM EM) containing 10% of FBS, 1 % of nonessential amino acids (Euroclones), 100 U / mL of penicillin-streptomycin and 2 mM of L-glutamine. The cells were pre-incubated with culture medium for 1 hour at 37 °C and then 20,000 cells were resuspended in 100 pL of complete DMEM and placed in the upper chamber of the transwell (0.33 cm2 per insert) on permeable inserts. Then, 600 mL of medium were applied to the bottom of the transwell. The medium was changed only 24 hours after seeding and then 3 times a week. Caco-2 monolayers were grown for 21 days before use. When the monolayer was ready, the cells were washed with PBS solution and subsequently the ISIDE11 compound 100 mg / ml and lucifer yellow (Sigma Aldrich, No. 67769-47-5) 100 pM were added, the negative control and propanolol 100 mg / ml were dissolved in 0.1% PBS and DMSO. Lucifer yellow is a fluorescent marker used to verify the integrity of the test. The permeability assay was conducted using 200 pL of apical donor solution, 200pL of ISIDE11 compound in PBS and 600 pL of basal acceptor solution of PBS. All compounds were tested in triplicate. The monolayers were incubated with all the compounds in oscillation at room temperature (25 °C) for 2 hours. The concentration of lucifer yellow was evaluated by TECAN M-200 at 480/530 nm. The concentrations of the other compounds were evaluated by HPLC analysis by taking the acceptor solution after two hours of incubation. The fluorescence was determined without any fluorescence interference. The experiments were carried out in triplicate.

Half-life

The compound ISIDE11 at 50 mM in DMSO was diluted to 10 mM in DMSO then again to 50 mM in human serum and left to incubate at room temperature for 6 hours. The residual amount of the compound was determined over time by RP-HPLC using an ONYX 50 x 2 mm IDY-18 column, applying a gradient of CH3CN, 0.1% of TFA on H20, 0.1 % of TFA from 1 % to 35% in 4 minutes. Total duration: 9 minutes. The compound was monitored by the DAD (Diode Array HPLC Detectors) between 200 and 400 nm. ISIDE11 was quantified in chromatograms extracted at 375 nm. The percentage of compound is given by the ratio between the area of the chromatographic peak of the compound after 6 hours in human serum and the area of the chromatographic peak of the compound in human serum at time 0, multiplied by 100.

Histone protein extraction

After treatment, the cells were collected, washed with PBS (Euroclone) and lysed in extraction buffer with Triton (TEB; PBS containing 0.5% Triton X 100 (v / v), 2 mM PMSF, 0.02% (p / v) NaN3) at a cell density of 10 ⁷ cells / ml for 10 minutes on ice, with gentle stirring. After centrifugation (2000 rpm at 4 °C for 10 min), the supernatant was removed and the pellet was washed at half the volume of TEB and centrifuged again. The pellet was suspended in 0.2 N HCI at a cell density of 4 x 10 ⁷ cells / mL overnight at 4 °C on a mobile table. The samples were centrifuged at 2000 rpm for 10 minutes at 4 °C, the supernatant was recovered and the protein content was determined using a Bradford assay (Bio-Rad, No. 5000006).

Total protein extraction

After treatment, the cells were collected, washed with PBS (Euroclone; No. ECM4053XL) and lysed for 15 minutes at 4 °C in lysis extraction buffer with protease and phosphatase inhibitors (complete, Mini Protease Inhibitor Cocktail, ROCHE, No. 11836153001): 50 mM Tris-HCI pH 8.0, 150 mM NaCI, 1% NP40, 10 mM sodium fluoride, 0.1 mM sodium orthovanadate (AppliChem No. 13721-39-6), 40 mg / ml of phenylmethylsulfonyl fluoride (PMSF). The cells were then centrifuged at 13,000 rpm for 30 minutes at 4 °C and the concentration of the protein content of the supernatant was determined by colorimetric assay (Biorad, No. 5000006). The cell extracts were diluted 1 : 1 in 2X Laemmli buffer (0.217 M Tris-HCI pH 8.0, 52.17% SDS, 17.4% glycerol, 0.026% bromo-phenol blue, 8.7% beta-mercapto-ethanol), then boiled for 3 min. Equal quantities of protein (20 mg) were analyzed and separated by SDS-PAGE gel (acrylamide gel).

Cell viability

MTT assay: quantification of cell viability was performed by reducing 3-(4,5-dimethyl-thiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) at a concentration of 0,5 mg / ml in PBS for 4 hours. The absorbance of each well was determined with a TECAN INFINITE M200 microplate reader at 570 nm with a reference filter at 630 nm. The cell viability values were expressed as a percentage with respect to the control (100% cell viability) treated with DMSO. All experiments were performed in triplicate.

Cell counting

The cells were incubated with trypan blue (Euroclone) with a 1 : 1 ratio. Blue trypan positive cells (dead cells) and the total cell population were counted using an optical microscope to calculate the percentage of viable cells.

Western blotting

Western blotting analysis was performed following the recommendations of the antibody suppliers and loading 20 mg of protein extracts obtained by total extraction of proteins on 10% polyacrylamide gel and 8 mg of histone extracts obtained by extraction of histone proteins on 15% polyacrylamide gel and subsequently transferred onto nitrocellulose membranes (Trans- Blot Turbo Mini Nitrocellulose Transfer No. 1704158, BioRad). The membranes were blocked with 5% milk in a saline solution buffered with 0.5% TrisHCI of Tween-20 (TBST) for at least 1 hour at room temperature. The membranes were then incubated overnight at 4 ° C with primary antibody diluted in TBST. After washing, the membranes were incubated for 1 hour at room temperature with secondary anti-rabbit antibodies (Amersham No. NA934) or secondary antimouse antibodies (Amersham No. NA931) diluted in 3% milk in TBST. The antibodies used included: Anti-SIRT1 (Abcam, ab12193), Anti-p53 (acetyl K382) (Abeam, ab75754), Anti-Acetyl- p53 (K379) (Cell signaling No. 2570), Anti-P53 (Santa Cruz No. sc-126); Anti-H4K16ac (Diagenode No. C15410300), Anti-H3K9 / 14ac (Diagenode, No. C15410200), Anti.H3K56ac (Cell Signaling, No. 4243); ERK1 / 2 (Santa Cruz No. sc-514302), were used as a loading control; anti-H4 antibodies (Abeam, No. ab16483) and anti-H3 (Abeam, No. ab213257) were used as loading controls.

Extraction of RNA and RT-PCR RNA extraction was performed using RNase-free material and the solutions were prepared with diethyl pyrocarbonate (DEPC) (Sigma, No. 1609-47-8) to prevent degradation of RNA by ribonucleases. Cell lysis was achieved by the TRIzol method (Invitrogen; No. 15596026) using 1 ml of TRIzol / 10 ⁷ cells, according to the protocol. After centrifugation at 12,000 rpm for 15 minutes, Bromo-1-chloro-3-propane was added in a 1 :10 ratio with TRIzol. Once the RNA was recovered it was precipitated in isopropanol at -80 ° C for 30 minutes. The RNA samples were centrifuged at 12,000 rpm for 10 minutes and cold washed with 75% ethanol. Eventually, they were dried at 42 °C and suspended in DEPC water. For mRNA expression, total RNA (1 mg) was retrotranscribed using Superscript VILO cDNA synthesis kit (Invitrogen, No. 11754050) according to the manufacturer's instructions. Primers used for qRT-PCR were: SIRT1 FW: 5'- GCCGGAAACAATACCTCCAC-3 '(SEQ ID NO: 1), RV: 51-ACCCCAGCTCCAGTTAGAAC-3' (SEQ ID NO: 2); P53 FW5'-CAGCCATTCTTTTCCTGC-3 '(SEQ ID NO: 3), RV 5'- GCTCGACGCTAGGATCTG-3' (SEQ ID NO: 4) (produced by Bio-Fab Research s.r.l., Rome). Vascular reactivity study

Animals

Heterozygous mice with modification of the MTHFR gene were generated at the Montreal Children's Hospital Research Institute by Dr. R. Rozen [40] Procedures involving animals and their care comply with institutional guidelines. Every effort was made to minimize the number of animals used and their suffering. The vessels used for ex-vivo experiments were obtained from wild-type and Mthfr heterozygous animals following terminal anesthesia using isoflurane and were subsequently explanted and mounted in the organ bath of the pressure myograph (DMT - Denmark).

For in vivo experiments, Wild-type (C57BL/6) (Jackson Laboratory, STRAIN - 000664) and MTHFR ^+/- mice, at 8 weeks of age, were treated by intraperitoneal injection with resveratrol (10 mg / Kg) or ISIDE11 (10 mg / kg), alone or after 30 minutes following the injection with EX- 527 (5 mg / Kg) for 21 days. Resveratrol and EX- 527 (sigma-aldrich) were prepared daily in a solution of DMSO (2%) in saline solution; ISIDE11 was prepared in a solution of DMSO (2%) in sesame oil (Sigma-Aldrich, No. 1612404).

The animals used as controls were treated in a similar way, but with the drugs replaced by vehicle only (DMSO 2% in saline solution). At the end of the in vivo treatments, the mesenteric arteries were explanted to perform the ex vivo vascular reactivity studies. The femoral arteries were cryopreserved in OCT (Vector Labs) and then dissected to the cryostat (Leica) in 10 mm sections and analyzed by hematoxylin/eosin staining.

Vascular reactivity

Second-order branches of the mesenteric arterial tree were removed from C57BL6 mice (controls) and MTHFR +/- mice for vascular studies. In detail, after isolating the vessels, the adventitious fat was carefully removed and the arteries were cut into segments (length ~ 2 mm) and placed in a pressure myograph filled with Krebs solution maintained at pH 7.4 at 37 ° C (chamber size 5 mL DMT, Danish Myosystem). After an equilibrium period of 60 minutes, the arteries were pre-contracted with potassium chloride (80 mM KCI) until reaching a plateau. The vessels were then washed and this was repeated at least three times to stabilize the tissue. Endothelium-dependent and independent relaxations were assessed by measuring the dilatory response of the mesenteric arteries at cumulative concentrations of acetylcholine (from 10 ^-9 M to 10 ^-5 M) or nitroglycerin (from 10 ^-9 M to 10 ^-5 M), respectively, in vessels pre-contracted with phenylephrine at a dose necessary to obtain a similar level of pre-contraction in each ring (80% of the initial contraction induced with KCI). Vascular relaxation is reported as a negative percentage, considering the basal tension, i.e. the one before the stimulus with phenylephrine as -100% of vascular relaxation and the phenylephrine-induced tension (vasoconstriction) as 0% of vascular relaxation. For ex-vivo studies, vascular reactivity was measured before (basal) and after 1 hour of incubation in the organ bath with 25 mM of resveratrol or 50/100 mM of ISIDE 11 as shown in the figure.

For the analysis of statistical relevance, the effects of different treatments on vasodilation were measured using ANOVA for repeated 2-way measurements followed by the post-hoc Bonferroni test for multiple comparisons. A p value of < 0.05 was considered statistically significant. All statistical analyses were carried out with the Prism statistical software (GraphPad, La Jolla, CA).

Mouse tail bleeding

The time of bleeding of the mouse tail was measured with the following specifications. The mice were anesthetized with inhalation of gas composed of 30% oxygen and 70% nitrous oxide (0.7 I / min). The gas was passed through an isoflurane vaporizer (VetEquip,) set to deliver 3-4% of isoflurane during initial induction and 1.5-2% during surgery. After anesthesia, the mice were placed on a heating plate at 37 °C. A cut was made with a disposable surgical blade at about 2- 4 mm from the tip of the mouse's tail (approximately 1 mm in diameter). After resection, the tail was immediately placed in a 50 ml tube filled with saline solution at 37 °C and the bleeding time was recorded for up to 10 minutes. For the analysis of independent groups the inventors used the 1-way ANOVA followed by the post-hoc Bonferroni test. A p value of £ 0.05 was considered statistically significant. All statistical analyses were carried out with the Prism statistical software (GraphPad, La Jolla, CA).

Thrombosis method

The mice were anesthetized and a longitudinal skin incision performed on the right paw to expose both common and superficial femoral arteries. Distal portions of common and superficial femoral arteries have been temporarily tied with a nylon suture. A small incision was made on the distal portion of the superficial femoral artery proximal to the previously described surgical circuit. A 0.4 mm wire was then introduced by incision through the superficial femoral artery to its proximal portion, 2-3 mm below the bifurcation with the common femoral artery. The thread was then retracted and reintroduced 5 times to induce endothelial damage. Subsequently, the thread was extracted and the distal portion of the superficial femoral artery was permanently tied proximal to the incision used to introduce the thread. On the other hand, the surgical ring previously placed in the distal portion of the common femoral artery was removed and the blood flow was consequently restored. After a set period of time, the mice were subjected to ultrasound analysis with a 30 MHz electronic probe (Vevo 3100). After visualization of the superficial femoral artery of the right paw using the color-Doppler technique, the peak of the blood flow velocity was measured at the level of the central portion of the artery. The same procedure was then performed on the left paw which was considered as the internal control (non-thrombotic). In detail, the reduction of the flow was calculated by measuring the difference in the flow velocity between the femoral artery of the left and right paw, and then calculating the percentage of reduction obtained from the ratio between the measured difference and the healthy femoral flow expressing it as a negative value.

The statistical analysis of the independent groups was conducted using the 1-way ANOVA followed by the Bonferroni post-hoc test. A p value of < 0.05 was considered statistically significant. All statistical analyses were conducted with the Prism statistical software (GraphPad, La Jolla, CA).

After ultrasound analysis, the mice underwent euthanasia and the femoral arteries of the two legs were collected for histological analysis.

Histological preparation

The isolated femoral arteries were incubated in Krebs solution for 15 minutes, cleaned and then the adventitial layer was removed mechanically to reveal the smooth muscle layer and to avoid non-specific cross reactions. Then, the arteries were washed three times with Krebs solution and incubated for 2 hours in 4% PFA. Subsequently, they were included in an OCT (Vector Labs) cryopreservative solution and stored at -20°C and then dissected to a thickness of 10 mm to perform the hematoxylin and eosin staining in order to visualize the vascular structure and extension of the thrombotic process. The image was scanned by a Fujix digital camera (HC-300/ OL, Fuji film Co.) on AXA-phot ZAISS microscope (morphometric not available).

EXAMPLES

Example 1 : Identification of ISIDE11 in HTS mode

The SIRT1 fluorescence assay was used to develop a HTS method in order to examine a library of small molecules for the identification of SIRT1 modulators. Specifically, an in vitro test developed in the laboratory was optimized in HTS mode, using a TECAN robotic station. This assay correlates the activity of the SI RT 1 deacetylase with the production (and quantification) of ammonia, coupling two reactions catalyzed by SIRT1 and nicotinamides (NMase).

In the first reaction, SIRT1 removes the acetyl group from the lysine in position 382 of the p53 peptide (aa 374-389) (SEQUENCE: GQSTSRHKKacLMFKTEG SEQ ID NO: 5) by reaction with its NAD ⁺ cofactor which is cleaved to form O-acetyl-ADP-ribose and nicotinamide (NAM) (Fig. 1). In the second reaction, the NMase enzyme converts NAM into nicotinic acid and free ammonia (NH3). At the end, ammonia is detected as a fluorescent adduct at the wavelength 420/460 nm, in the presence of optaldehyde (OPT), present in the stop solution of the reaction (Fig. 1).

Both enzymes were produced through the expression of SIRT1-GST and NMase-His recombinant in E. Coli bacteria by FPLC as indicated in the Materials and Methods.

Using a TECAN robotic station, the enzymatic test was used in HTS mode. This procedure allows to conduct hundreds of reactions simultaneously and to evaluate the modulation capacity of the related compounds. In particular, the screening was performed in 384-well plates, each plate loaded with 160 compounds (in duplicate), using a reaction volume of 15 ml. The optimization of the test for high-throughput screening (HTS) is based on several experimental factors: enzymatic kinetic parameters, which include the enzyme concentration, Km and Vmax, the optimal substrate concentration, response to known compounds, referents of reaction; concentration of DMSO in which the compounds to be tested have been diluted. The optimal substrate concentration was determined by the Michaelis constant (Km): the scalar substrate concentrations (from 0 to 50 mM) were tested in the presence and absence of SIRT1 enzyme (as a control) and therefore fluorescence was detected (420 nm - 460 nm).

This work in HTS mode made it possible to obtain highly reproducible data in terms of z' and standard deviation.

Furthermore, a compound is identified as a SIRT1 activator when its activity was ³ 120% and as a SIRT1 inhibitor when it is £ 70%. The enzymatic activity expressed as a percentage is calculated as the ratio between the fluorescence value (420 nm - 460 nm) of each compound and that of the control ([Fluo cmpd] / [Fluo Ctrl] * 100).

In the primary screening, all compounds were tested at 10 mM. The HTS station was used massively for screening allowing the identification of potentially active molecules. From the massive screening of the entire library, 70 active molecules were identified both as activators and as inhibitors. (Fig. 2A). Once the activity was reconfirmed, each identified compound was subjected to intrinsic fluorescence assessments and counter-assay for the NMase enzyme. From these steps, about 20 compounds were selected, the 50 discarded compounds showed intrinsic fluorescence at the wavelength of the SIRT 1 assay. None of the compounds was active on NMase. For each of the 20 selected compounds, a concentration-response curve was performed for the determination of AC50 / IC50. Of these 20 molecules ISIDE11 and ISIDE20 (Fig. 2A) showed an optimal dose-response curve for the determination of AC50 / IC50. As shown in Fig. 2B: both compounds ISIDE11 and ISIDE20 showed an enzymatic activity on SIRT1 higher than the reference activator, about 140% for ISIDE11 and 138% for ISIDE20. The two molecules did not show significant activity on the recombinant NMase enzyme (Fig. 2C), proving to be SIRT1 modulators.

As reported in figures 2D and E, the activity of ISIDE11 and ISIDE20 has been shown to be dose dependent. By processing the dose-response curves with the GraphPad Prism6 program, the AC50 values were evaluated, resulting to be of 20.11 mM for ISIDE11 and 396 mM for ISIDE20 (Fig. 2D-E). The chemical structures of the ISIDE1 1 and ISIDE20 compounds are shown below.

Example 2: Characterization of the SIRT1 -ISIDE11 interaction and stability of ISIDE11

A CETSA assay was performed to evaluate the interaction between ISIDE1 1 and the SIRT1 protein in a physiological cell environment. Compared to the control, ISIDE11 protected SIRT1 from thermal degradation in the MCF7 protein extract treated with the drug. The SIRT1 signal remained at the maximum temperature of 67 °C while no signal was detected in the control extract (Fig. 3A). This result represented the first evidence of a physical interaction between ISIDE11 and the SIRT1 enzyme.

The molecular interaction between the SIRT1 protein under native conditions (the form in which a protein folds naturally is called "native state") and ISIDE1 1 has also been studied by fluorescence spectroscopy. SIRT1 contains several tyrosine residues that emit fluorescence and its spectrum is characterized by the typical 305 nm emission. The emission spectra of SIRT1 fluorescence were recorded in the absence and presence of ISIDE11 with different molar ratios and the change in fluorescence intensity was reported in Figure 3B. The intensity of the fluorescence decreases regularly as the concentration of ISIDE11 increases, thus indicating that ISIDE11 interacts directly with SIRT1 and induces the decrease in the fluorescence emitted by tyrosine. To exclude a collisional effect by ISIDE11 , the same experiment was also performed on the monomeric tyrosine residue (N-acetyl-L-tyrosine ethyl ester) and the variation of fluorescence is shown in Fig. 3B. The F0 / F values recorded for free tyrosine are significantly higher than those recorded for SIRT1 at each molar ratio, indicating the formation of a SIRT1- ISIDE11 complex, as no collisional effect is involved. In addition, the cell permeability of the drug was assessed. As shown in Fig. 4A, approximately 21% of the molecule was found to be absorbed by Caco-2 cells after 3 hours of treatment. This result indicates a direct physical link between the enzyme and the molecule.

The stability of ISIDE11 has been studied in vivo (Fig. 4B). The compound was tested at a concentration of 50 mM and the stability profile was performed in DMEM with incubation at RT for 6 hours. The residual quantities of compound were determined over time by RP-HPLC. ISIDE11 was quantified in chromatograms extracted at 375 nm. The residual stable quantity of the compound after 1 hour was 5%.

In conclusion, these data confirmed that the compound is an excellent SIRT1 activator.

Example 3: The effects of ISIDE11 on the targets of SIRT1

To better characterize the molecules, the biological effects of ISIDE11 and ISIDE20 in tumor cell systems have been evaluated. Several cell lines have been untreated and treated with the ISIDE11 and ISIDE20 compounds to evaluate their effects on histone modifications. HepG2 cells were treated with ISIDE11 and ISIDE20 at two different concentrations (5 and 50 pM) for 24 hours or were left untreated. Western blotting analyses were then conducted to investigate the main targets of SIRT1 , these analyses showed that ISIDE11 is able to induce a dose-dependent decrease in the acetylation level of lysine K16 of histone H4 (H4K16ac) (Fig.5A).

ISIDE11 was also able to reduce the level of histone H3 acetylation in both solid and hematological tumor cell lines, as revealed by Western blotting analyses performed on MCF7, HCT1116 and U937 cells (Fig. 5B). These data confirm that ISIDE11 is an activator of SIRT1 acting on the histone targets of the enzyme. This confirms its good enzymatic activity with an AC50 value of 20.11 pM.

Example 4: ISIDE11 improves the effects mediated by SIRT1 in the response to stress

Many genotoxic stresses induce acetylation of p53 in the carboxy-terminal region, increasing the activity of p53 with consequent arrest of cell growth. SIRT1 is capable of deacetylating p53, attenuating p53-dependent apoptosis in response to DNA damage and oxidative stress. To study the role of ISIDE11 and ISIDE20 in modulating the functions mediated by SIRT1 in these responses, the deacetylation activity of SIRT1 on p53 was assessed. Western blotting analyses were performed by pre-treating HepG2 cells with a genotoxic drug, etoposide, for 6 hours, and subsequently with ISIDE11 or ISIDE20 at 50 pM for 18 hours. The effect of the molecules on the deacetylation activity of SIRT1 was monitored following the signal of p53 acetylated in K382 (p53K382ac) (Fig. 6A-B). The data showed that ISIDE11 was able to reduce the acetylation of p53 in lysine 382 (p53K382ac) induced by etoposide. This experiment shows the ability of ISIDE11 , as an activator of SIRT1 , to reduce genotoxic damage.

With the activation of SIRTI , ISIDE11 could strongly mitigate the effects of DNA damage. Under the same experimental conditions p53K379ac and H3K56ac were decreased. In particular, ISIDE11 was able to restore the normal level of total p53 protein, increased by etoposide (Fig. 6C).

At the transcriptional level, ISIDE11 activated the expression of SIRT1 and did not alter the expression of p53 (Fig. 6D, E), while the compound EX-527 as SIRT1 inhibitor decreases the expression level of both SIRT1 and of p53. This indicates that ISIDE1 1 not only increases the activity, but also the expression of SIRT1 while it does not modulate the transcriptional expression of p53 but only its activity. Together these data confirm that the compound ISIDE11 is able to increase the deacetylase activity of SIRT 1 and of counteracting the genotoxic effects on DNA.

Example 5: Cellular effects of ISIDE11

The biological effects of ISIDE11 were assessed at different doses in a panel of A2058 (melanoma), MRC-5 (normal lung fibroblast), HT-29 (colon cancer), A549 (lung cancer), MiaPaCa (pancreas carcinoma) cell lines after 24 and / or 72 hours of treatment (Fig. 7). In particular, A2058 shows a survival of over 100% at 25 mM both at 24 hours and at 72 hours (Fig. 7A); MRC-5 shows a survival rate greater than 100% after 24 hours and 80% after 72 hours at 50 mM (Fig. 7B). Fig. 7C-D-E show the results in A549, HT-29, and MiaPaCa cells where ISIDE11 does not significantly modulate cell viability after 24 hours in A549 cells and 72 hours in HT-29 and MiaPaCa cells, compared to control (cells treated with DMSO). This means that ISIDE11 does not induce the death of cancer and normal cells. Therefore, the compounds of the invention have no toxic effects.

Example 6: ISIDE11 treatment recovers the endothelial dysfunction in MTHFR ^+/- heterozygous mice

The enzyme methylene tetrahydrofolate reductase (MTHFR) plays a key role in the methylation cycle by converting homocysteine into methionine [41]

A common variant in MTHFR, C677T, is associated with a decreased enzymatic activity that leads to an increase in plasma homocysteine levels in humans. Generally, patients with heterozygous MTHFR are predisposed to develop cerebral and cardiovascular events [41] In fact, although the combined administration of folic acid, vitamin B6 and vitamin B12 represents the only therapeutic approach to low homocysteine levels, it has been shown that these supplements do not reduce the risk of vascular events [42], laying the foundations for investigating a possible direct correlation between MTHFR mutation and vascular alteration, regardless of homocysteine levels. In this context, the down-regulation of SIRT1 has been reported in MTHFR heterozygous animal models associated with dysfunction of the nitric oxidase enzyme and thrombotic events [41 , 43, 44] Therefore, in order to formulate a new therapeutic strategy capable of reducing endothelial dysfunction and preventing the typical cerebral and cardiovascular damages associated with the mutation, ex vivo and in vivo experiments were performed on MTHFR ^+/- heterozygous mice. Preliminary data obtained on dysfunctional resistant arteries of MTHFR ^+/- mice demonstrate that ex-vivo treatment of vessels with resveratrol (RSV) or ISIDE11 improves nitric oxide-dependent endothelial vasodilation.

For this purpose, the ex vivo vascular function in wild-type mesenteric arteries and MTHFR ^+/- in the presence of ISIDE11 or RSV was evaluated using the pressure myography system (Fig. 8). These results show the ability of ISIDE11 to induce a significant improvement in endothelial function in vessels originating from MTHFR ^+/- animals (Fig. 8D). This beneficial effect is similar to that evoked by the treatment with resveratrol (RSV) (Fig. 8B) known activator of SIRT1 with numerous pleiotropic effects [45-46] Interestingly, both compounds have no effect on vascular function in vessels from wild-type animals (MTHFR ^+/+ ) (Fig. 8A, 8C), thus suggesting that under physiological conditions the hyper-stimulation of SIRT1 does not induce further action on vascular reactivity.

Example 7: In vivo treatment with ISIDE11 reduces pro-thrombotic phenotype of MTHFR ^+/- mice

An important key vascular phenotype observed in MTHFR mutant animals and humans is the increase in thrombotic events [41] In order to test the effect of ISIDE11 on platelet function and induced thrombosis, the inventors performed in vivo treatment of MTHFR ^+/- and wild type mice for 21 days with ISIDE11 or RSV, alone or in combination with a SIRT1 inhibitor, EX-527.

The mouse tail bleeding time is a useful test to detect abnormalities involving platelets and the integrity of blood vessels which represent an important step towards the thrombotic event. The data showed that in vivo treatment with ISIDE11 (10mg / Kg) is able to significantly increase the bleeding time, reaching values similar to those observed in control mice (Fig. 9). Furthermore, concomitant treatment with EX- 527 completely abolished the beneficial effect of ISIDE11. These results demonstrate that ISIDE1 1 is able to contain the pro-thrombotic phenotype observed in MTHFR ^+/- animals, thus contributing to the reduction of the high cardiovascular risk associated with the mutation.

Based on ISIDE 1 1's ability to regulate the homeostatic process, the inventors have tested its effect on thrombosis induced in the femoral artery using ultrasound and histological approaches. It is interesting to note that the evaluation of the rate of reduction of the blood flow velocity of the superficial femoral artery of the MTHFR ^+/- mice treated with ISIDE11 appears to be significantly reduced compared to the heterozygous mice treated with the vehicle only. In fact, it is possible to observe a reduction in the flow rate similar to that observed in wild-type mice or in MTHFR heterozygous animals treated with RSV. Furthermore, the concomitant treatment with the SIRT1 inhibitor is able to block the protective effect of ISIDE1 1 (Fig. 10A).

Subsequently, a histological analysis was performed to visualize the extent of the thrombosis damage and, as shown in Fig. 10B, the treatment with ISIDE11 counteracts the thrombosis damage more efficiently than the RSV. To validate the efficacy of ISIDE1 1 on endothelial function, the vascular reactivity of the mesenteric arteries of the same groups of animals treated in vivo was assessed to evaluate the impact on the formation of thrombi. The analysis of endothelium-mediated vascular reactivity, evoked by the administration of increasing doses of acetylcholine which represents the gold standard for the evaluation of endothelial function, clearly shows that treatment with ISIDE1 1 is able to induce a significant improvement in endothelial function compared to vessels from animals treated with a vehicle only which instead show an important endothelial dysfunction (Fig. 11A) which contributes to the cardiovascular risk associated with the mutation.

Smooth muscle vasodilation induced by nitroglycerin is not influenced by the administration of ISIDE11 (Fig. 11 B), suggesting its specific endothelial effect.

Example 8: ISIDE11’s analogues compounds

In an attempt to improve the modulation activity of SIRT1 , two synthetic analogues of ISIDE11 as well as ISIDE 11’s hydrate were tested, indicated as cmp52 and 54 (purchased from ChemBridge Corp.). The structures are shown below.

The two compounds were able to induce an increase in SIRT1 activity in the same way as ISIDE11 (Fig. 12). In addition, the hydrated form of ISIDE11 was also able to induce an increase in the activity of SI RT1 (Fig. 12), with a dose/response curve with an AC50 of 5.9 mM (Fig. 13). BIBLIOGRAPHY

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