IT201900008301A1 - SIRTUINE MODULATORS - Google Patents

Description

SIRTUINE MODULATORS

FIELD OF TECHNIQUE

The present invention relates to compounds useful as sirtuins modulators, in particular as activators of SIRT1. Furthermore, the invention refers to the medical use of these compounds, in particular in the prevention and / or treatment of cardiovascular diseases, and to pharmaceutical compositions that include them.

STATE OF THE TECHNIQUE

Sirtuine

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 various biological processes such as cell survival, aging, proliferation, apoptosis, DNA repair, cell metabolism and calorie restriction [2, 3].

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

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

Table 1: Human sirtuins [1].

SIRT1, 2 and 3 are the most studied being representative of the three cellular compartments - nucleus, cytoplasm and mitochondrion - theaters of action of sirtuins.

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 β-strands forming a parallel β-sheet and six α-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 pattern and a zinc-binding pattern. The interface between the large and the small domain represents the catalytic domain with a binding site for NAD <+> [6].

SIRT1 is the most studied of the sirtuins, it is composed of 747 amino acids and with N- and C-terminal extensions of ~ 240 amino acids. Extensions are capable of interacting 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 balancing of these signals determines the presence of SIRT1 in the nucleus or in the cytoplasm, depending on the specific cell type or tissue [7]. SIRT1 located in the nucleus acts as a transcriptional repressor. SIRT1 deacetyls histone proteins in histone H1 lysine 26 (H1K26Ac), histone H4 lysine 16 (H4K16Ac) and histone H3 lysine 9 (H3K9Ac) and acts on the promoters of target genes. SIRT1 also has non-histone targets such as transcription factors p53, NFKB, FOXO family proteins, Ku70 [8]; SIRT1 is involved in many cellular processes such as differentiation, apoptosis, inflammation, glucose homeostasis, hormone secretion, mitochondrial biogenesis, stress response, protection from DNA damage and in many cell signaling processes, such as nitric oxide synthesis . 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 several types of diseases, including neurodegenerative diseases and cancer [10-12]. Some studies have shown an important up-regulation of SIRT1 in several types of cancer including colon, prostate and skin cancers, as well as haematological cancers such as acute myeloid leukemia.

As it is known that H4K16 acetylation levels are controlled by SIRT1 / 2 and that some cancers are associated with SIRT1 changes, the decrease in H4K16 acetylation has been attributed to a deregulation of SIRT1 activity [13] . However, the activity of SIRT1 seems contradictory as 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 oncosuppressant p53 through the deacetylation of lysine K382. Overexpression of SIRT1 represses p53-dependent cell cycle arrest and apoptosis in response to DNA damage and oxidative stress [16].

Conversely, 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, in cancers of the bladder, prostate and ovaries; some studies have suggested that overexpression of SIRT1 in APC <- / +> mice reduces, rather than increases, the formation of colon cancer. This action appears to be caused by the SIRT1-mediated deacetylation of β-catenin, which promotes the cytoplasmic localization of the oncogenic β-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 MYC stability and 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 cardioprotective role. In vitro, ex vivo and in vivo studies provide evidence of a protective role of SIRT1 against cardiovascular stress, inhibition of apoptosis, aging retardation and reduction of oxidative stress [23].

The overexpression of SIRT1 determines 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 cardioprotective effect in different 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]. In agreement with these results, other studies suggest that resveratrol confers antiapoptotic effects in different models of doxorubicin-induced cardiotoxicity [26]. Resveratrol's ability to eliminate free radicals and chelated copper reduces oxidation of low-density lipoprotein (LDL) particles, a contributing factor in the development of coronary heart disease [27]. Furthermore, 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 elucidated, 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 sirtuins activating compounds (STACs), including natural molecules such as resveratrol, butein [29], curcumin [30], SRT1720 [31-37]. Resveratrol is a phenol that comes from grape skin and has an antioxidant and blood thinning action. 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 far as STACs are concerned, only resveratrol has been well characterized although, most likely, its effect on SIRT1 may be indirect and not due to binding to SIRT1, while a class of molecules including SRT1460, SRT1720 and SRT2183 reported in literature by Pacholec et al. [38] as activators SIRT1 does not have a specific activity for SIRT1. The structures of some known activators of SIRT1 are shown below.

The synthesis of the compound ISIS 11 is described in Rao S. et al., 1992 [47], Iqbal R. et al., 1982 [48] and Fedoryak S.D. et al., 1982 [49]. This latest document also reports the antimicrobial and antituberculosis activity of ISIDE 11. Neamati et al., 1998 [50] reports the compound ISIDE20 and indicates that it has no inhibitory activity of HIV-1 integrase.

The synthesis of Isis11, compound 52, compound 53 is reported in WO9320046.

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

SUMMARY OF THE INVENTION

In the present invention, the authors have identified molecules that act as activators of sirtuins, specifically of SIRT1, with micromolar values of AC50, for example, of 20.11 μM for ISIDE 11, of 396 μM for ISIDE20 and of 5.9 μM for ISIDE hydrate.

In particular, the molecules of the invention physically interact with SIRT1 and have an effect on the main targets of SIRT1, suggesting that they improve enzyme-mediated deacetylation. Furthermore, the authors characterized the action of molecules against cardiovascular disease in in vivo and ex vivo mouse models. Surprisingly, the authors found that such molecules are able to recover endothelial dysfunctions and reduce thrombus formation in pathological models, such mice as heterozygous for MTHFR.

Advantageously, the molecules of the invention are able to reduce the level of acetylation of a protein, in particular of a SIRT1 target protein. For example, said protein is a histone (in particular H3 and H4), p53, NFKB, a protein of the FOXO or Ku70 family. In particular, the molecules of the invention are able to reduce the level of acetylation of: H4K16, H3K9, H3K56, p53K382, p53K379 and / or H1K26.

The CETSA assay for SIRT2 shows that ISIS 11 does not bind SIRT2. Furthermore, ISIDE11 has been tested for other histone deacetylases such as HDAC1, HDAC 4 and HDAC 6 and is not active. Therefore, the compounds of the invention are selective for SIRT1.

The present invention provides a SIRT1 activator of formula I

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

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

Preferably said aryl is phenyl. Preferably said heteroaryl is pyridinyl. Preferably said alkoxy is methoxy. Preferably, R is phenyl, pyridinyl or para-substituted phenyl with methoxy.

In a preferred form, the activator of SIRT1 has the formula:

N '- (1,3-dioxo-1,3-dihydro-2H- N'-benzoyl-3,4-dichlorobenzohydrazide

inden-2-diene) isonic nohydrazide

N '- (1,3-dioxo-1,3-dihydro-2H-inden-2- N' - (1,3-dioxo-1,3-dihydro-2H-diene) -4-methoxybenzohydrazide inden-2- diene) benzohydrazide or a salt, tautomer, solvate, stereoisomer or analogue thereof.

Preferably the solvate is a hydrate.

In a preferred form, the SIRT1 activator, salt, tautomer, solvate, stereoisomer or analog thereof 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 from: cardiovascular disease, tumor, metabolic disease, aging, cellular senescence, neurodegenerative disease, endothelial dysfunction, DNA damage, oxidative stress, brain damage, cardiovascular damage, coronary heart 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 inflammation.

Preferably the activator of SIRT1, salt, tautomer, solvate, stereoisomer or analogue thereof as defined above is for use as an apoptotic drug, vasodilator and / or antithrombotic.

Preferably the activator of SIRT1, salt, tautomer, solvate, stereoisomer or analogue thereof of the invention 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 the mutation is the C677T or A1298C mutation.

The invention further provides a pharmaceutical or cosmetic composition comprising the activator of SIRT1, salt, tautomer, solvate, stereoisomer or analogue thereof of the invention, a vehicle and optionally a further therapeutic agent for use as an activator of SIRT1.

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 disease neurodegenerative, endothelial dysfunction, DNA damage, oxidative stress, brain damage, cardiovascular damage, coronary artery disease, thrombosis, hyperhomocysteinaemia, 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 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 for identifying a SIRT1 modulator comprising activating SIRT1 with the activator of SIRT1, salt, tautomer, solvate, stereoisomer or analogue thereof as defined above in the presence or absence of said modulator.

In the invention, the activator of SIRT1 means a molecule capable of increasing its catalytic activity. This activity is measured by in vitro enzymatic assays, in which the level of acetylation of the substrate due to the SIRT1 enzyme is measured in the presence of potential modulators or by Western Blotting by analyzing the acetylation of specific targets of SIRT1.

In the invention, a cardiovascular disease includes: acute myocardial infarction, angina pectoris, ischemic and hemorrhagic stroke, a tumor includes solid or hematopoietic tumor, for example leukemia, breast, liver, pancreatic, 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. Endothelial dysfunction includes hypertension, DNA damage includes cancer, oxidative stress includes: premature aging, chronic degenerative disease, brain damage includes ischemia, cardiovascular damage includes thrombosis, coronary artery disease includes cardiovascular diseases such as heart disease ischemic and myocardial infarction. An age-related cardiological condition includes heart attack. An immune disease includes rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroid, psoriasis. A complication of diabetes includes retinopathy, diabetic nephropathy, diabetic foot, coronary artery disease, and cerebral vascular disease. An inflammation includes tuberculosis and hepatitis.

In the present invention, the sirtuins activator can be in the form of salt, in particular of pharmaceutically acceptable salt. Pharmaceutically acceptable salts conventionally include non-toxic salts obtained by salification with inorganic acids (for example hydrochloric, hydrobromic, sulfuric or phosphoric acid), or with organic acids (for example acetic, propionic, succinic, benzoic, sulphanilic, 2-acetoxy-benzoic acids , cinnamic, mandelic, salicylic, glycolic, lactic, oxalic, malic, maleic, malonic, fumaric, tartaric, citric, p-toluenesulfonic, methanesulfonic, ethanesulfonic, or naphthalenesulfonic acids). For information on pharmaceutically suitable salts see Berge S. M. et al., J. Pharm. Sci. 1977, 66, 1-19; Gould P. L. 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 addition of 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 one of ordinary skill in the art, and generally refers to an organic or inorganic compound that 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 portions of carboxylic acids which may be present in the activators of sirtuins.

Furthermore, the activators of sirtuins can be administered in unsolvated forms as well as in solvated forms with pharmaceutically acceptable solvents such as water, EtOH and the like.

Activators of sirtuins can exist in stereoisomeric forms (for example, they can contain one or more asymmetric carbon atoms). The single stereoisomers (enantiomers and diastereomers) and mixtures thereof can be used according to the present invention. The present invention covers single isomers as well as mixtures with isomers in which one or more chiral centers are reversed.

Similarly it is understood that sirtuins activators may exist in tautomeric forms other than those shown in the formulas and these are included within the scope of the present invention.

The invention also includes all isotopic variants of sirtuins activators. An isotopic variation of an activator of the invention is defined as a variation 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 atomic mass usually present in nature. Examples of isotopes that can be incorporated in the activators of the invention include isotopes such as <2> H, <3> H, <13> C, <14> C, <15> N, <17> O, <18> O, <31> P, <32> P, <35> S, <18> F and <36> Cl, 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 tissue distribution studies of drugs and / or substrates.

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

An analogue is a chemical compound having a structure similar to another (primary compound) but inside it there can be different atoms and functional groups. The analog maintains the pharmacological activity of the primary compound.

In the present invention, the activators can be administered as pure or as pharmaceutical formulations, i.e. suitable for parenteral, oral, vaginal or rectal administrations. Each of said formulations can contain excipients and / or fillers and / or additives and / or binders, coatings and / or suspending agents and / or 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 doses and contain conventional excipients such as binders, fillers (including cellulose, mannitol, lactose), diluents, tableting agents, lubricants (including magnesium stearate), detergents, disintegrants (e.g. polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate), coloring agents, flavoring agents, and wetting agents (e.g. sodium lauryl sulfate). The solid oral compositions can be prepared by conventional mixing, filling or tableting methods. The mixing operation can be repeated to distribute the active ingredient in all compositions containing large quantities of fillers. Such 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 by reconstitution with water or a suitable vehicle prior to use. 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, oil 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 via inhalation can be contained in an insufflator or in a pressurized nebulizer.

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

Parenteral solutions are normally prepared by dissolving the activator 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. The parenteral suspensions are prepared substantially in the same way, except that the activator 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 the uniform distribution of the activator of the invention.

In order to increase bioavailability, activators can be pharmaceutically formulated 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 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, phosphotidylglycerol, 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, lozenges, or gels.

Activators may be pharmaceutically formulated as suppositories or retention enemas, e.g., suppositories containing conventional bases such as cocoa butter, polyethylene glycol, or other glycerides, for rectal administration.

Another means of administering the activators of the invention concerns topical treatment. Topical formulations may contain for example ointments, creams, lotions, gels, solutions, pastes and / or may 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 sulfate, anhydrous lanolin, hydrophilic petrolatum, cetyl alcohol, glycerol monostearate, stearic acid, water-soluble ointments containing polyethylene glycols of various molecular weights.

Creams, as known to formulation experts, are viscous liquids or semi-solid emulsions, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase generally contains petrolatum and an alcohol such as cetyl or stearic alcohol. Formulations suitable for topical administration of 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 activators of the invention concerns transdermal delivery. Topical transdermal formulations include conventional aqueous and non-aqueous carriers, such as creams, oils, lotions or pastes or may be in the form of medicated membranes or patches.

The formulations are known in the art (Remington Remington "The Science and Practice of Pharmacy", Lippincott Williams & Wilkins, 2000).

The activators of the present invention can be employed for use in the treatment and / or prevention of the aforementioned conditions alone as a sole therapy or in combination with other therapeutic agents, either through separate administrations or 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 (simultaneous, sequential) compositions of the individual treatment components or as a single dosage form containing all agents. When the activators of this invention are in combination with other active ingredients, the active ingredients can be formulated separately in single ingredient preparations of one of the forms described above and then provided as combined preparations, which are administered at the same time or several times, or they can be formulated together in a two or more ingredient preparation.

Activators can be administered to a patient in a total daily dose of, for example, 0.001 to 1000 mg / kg body weight daily. The unit dosage compositions can contain such amounts of submultiples thereof to compensate for the daily dose. Activators can also be administered weekly or once a day. Determining optimum dosages for a particular patient is well known to one skilled in the art. As is common practice, the compositions are normally accompanied by written or printed instructions for use in the treatment in question.

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) : //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/Q9NRC46) p53 (P53_HUMAN) (https://www.uniprot.org/uniprot/P04637)

NFKB, P19838 (NFKB1_HUMAN) (https://www.uniprot.org/uniprot/P19838) FOXO family proteins, Q12778 (FOXO1_HUMAN) (https://www.uniprot.org/uniprot/Q12778) - O43524 (FOXO3_HUMAN) (https://www.uniprot.org/uniprot/O43524) - P98177 (FOXO4_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 conserved DNA-binding domain, the FOX or fork box. The family includes more than 100 human members, classified from FOXA to FOXR on the basis of their sequence similarity. The defining feature of these proteins is FOX, a sequence of 80-100 amino acids that form a pattern that binds to DNA. This motif is also known as a "winged helix" due to the butterfly-shaped appearance of the loops.

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 the compounds ISIDE11 and ISIDE20. (A) The flowchart of the high-throughput screening (HTS) procedure to identify the new SIRT1 modulators. (B) The primary enzyme assay was performed using the library molecules at a fixed concentration of 10 μM, compared to the control without any modulator, of the known activator (STAC-2) [39] and the known inhibitor (EX527 ). In this assay, the percentage of activity for ISIDE11 is approximately 140% and for ISIDE20 it is 138%. for I (C) Counter-test performed to assess whether ISIDE11 and ISIDE20 at 10 μM modulate the activity of the nicotinamidase enzyme (NMase), compared to the control in which no modulator is present. (D, E) Dose enzymatic assay (from 1 to 250 μM) for the evaluation of the AC50 of ISIDE11 (D) and ISIDE20 (E).

Figure 3: ISIDE11 interacts with the SIRT1 enzyme. (A) The CETSA assay was performed in MCF7 cells treated with DMSO (vehicle - Ctrl) or treated with 50 μM of ISIDE11 at the indicated temperatures. The extracts were then loaded onto acrylamide gel and incubated with the SIRT1 antibody. The SIRT1 signal band is still visible at the highest temperature of 67 ° C only in the sample treated with ISIDE11 and not in the control, indicating that the molecule protects the SIRT1 protein from degradation. (B) SIRT1-ISIDE11 interaction monitored by fluorescence spectroscopy. The fluorescence emission of tyrosine as an F0 / F ratio was evaluated for both SIRT1 (black line) and free tyrosine (gray line) after the addition of ISIDE11 at different concentrations (0.5, 1, 5, 10, 20 , 30, 50 μM). Working concentrations were 10 μM 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 μM was evaluated in CACO-2 cells (B) Stability profile of ISIDE11 in human serum. ISIDE11 (1 mg / ml) in 20% DMSO solution in 90% DMEM at T 37 ° C. ISIDE11 was quantified at 375 nm. The residual quantities of the compound were determined after 15, 30, 60 and 90 minutes by RP-HPLC in a 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 μM). The protein extracts were used for Western blotting to detect acetylated histone H4 in lysine 16 (H4K16ac). The signal relating to histone H3 (H3) was used as a loading control. (B) MCF7, HCT116 and U937 cells were treated with DMSO (ctrl), ISIDE11 at 50 μM, ISIDE20 at 50 μM and EX-527 (SIRT1 inhibitor) at 5 μM for 24 hours. The protein extracts were used for Western blotting for the detection of acetylated histone H3 in lysines 9 and 14 (H3K9 / 14ac). The signal relating to histone H4 (H4) 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 20 µM and subsequently treated with ISIDE11 and ISIDE20 at 50µM. The signal related to Erk1 / 2 was used as a loading control. (B) Hep-G2 cells were treated with Etoposide at 20µM and ISIDE11 AND ISIS 20 at 50µM in combination for 6 hours; (C) Hep-G2 cells were treated with Etoposide at 20 µM and ISIDE11 at 50 µM in combination for 6h. Western blotting for total p53 (p53) and its acetylated forms 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 control. (D-E) Evaluation of the mRNA expression of SIRT1 and p53 in HepG2 after treatment with ISIDE11 at 50 µM for 6 h.

Figure 7: MTT assay. (A) A2058 cells were treated with ISIDE11 from 50 μM to 0.39 μM at both 24 hours and 72 hours. (B) MRC-5 cells were treated with ISIDE11 from 50 μM to 0.39 μM at both 24 hours and 72 hours. (C) A549 cells were treated with ISIDE11 from 50 μM to 0.0975 μM for 24 hours. (D) HT-29 cells were treated with 50 μM to 0.00975 μM ISIDE11 for 24 hours. (E) MiaPaCa cells were treated with 50 μM to 0.0975 μM ISIDE11 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 conditions (untreated) 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) Vessels were pre-exposed to resveratrol (RSV) at 25 μM for 1 hour. (C-D) The vessels were pre-exposed to ISIDE11 at 50 or 100 μM for 1 hour.

Figure 9: ISIDE11 normalizes bleeding time in MTHFR / - mice. Tail bleeding times of Wild-type (+ / +) or MTHFR / - mice determined after in vivo treatment with resveratrol (RSV; 10 mg / kg intraperitoneal administration (i.p.) for 21 days) or ISIDE11 (ISIS 11; 10 mg / kg intraperitoneal administration (i.p. for 21 days) alone or in combination with SIRT1 inhibitor, EX-527 (5 mg / kg intraperitoneal administration (i.p.). ** p <0.01; * P <0.05.

Figure 10: ISIDE11 in vivo reduces thrombus formation in MTHFR / - mice. (A) Percentage reduction in 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 femoral arteries of mice cleaved after 21 days from 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). 100μm scale.

Figure 11: In vivo treatment with ISIDE11 (10 mg / kg for 21 days) recovers the impaired endothelial function of MTHFR <+/-> mice. Vascular response of cleaved mesenteric arteries from Wild-type or MTHFR / - mice pre-contracted with phenylephrine (A) acetylcholine (endothelium-dependent) or (B) nitroglycerin (endothelium-independent). 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 enzyme assay for the compounds ISIDE11, cmp52, cmp54 and ISIDE 11 HYDRATE (ISIDE11 H2O). All compounds were tested at 10μM in comparison with the control (without modulator), the known activator (STAC-2) and the known inhibitor (EX-527).

Figure 13: ISIDE 11 hydrate (ISIDE11 H2O) structure (top) and dose enzymatic assay (from 0.001 to 250 μM) for the evaluation of AC50 which is equal to 5.9 μM (bottom).

DETAILED DESCRIPTION OF THE INVENTION

Materials and methods

Cell lines

Human hepatocellular carcinoma (HepG2) cells 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 of penicillin, 100 mg / ml of streptomycin and 250 mg / ml of amphotericin-B). The cells were kept in an incubator at 37 ° C and 5% CO2 in a completely humidified environment. HCT-116 (ATCC No. CCL-247) and 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-11147) 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).

MRC5 cells (ATCC No. CCL-171) were cultured in Eagle minimal essential medium (EMEM; Euroclone) supplemented with 10% FBS (Euroclone) and 10 μg / ml gentamicin solution (Euroclone). U937 (ATCC No. CRL-1593.2) cultured in RPMI1640 MEDIUM (Euroclone) supplemented with heat inactivated 10% fetal bovine serum (FBS; Sigma-Aldrich) and antibiotics (100 Ul / ml penicillin, 100 mg / ml streptomycin and 250 mg / ml of amphotericin-B). Caco-2 (ATCC No. HTB-37) were cultured in Eagle minimal essential medium (EMEM; Euroclone) supplemented with 10% FBS (Euroclone) and 10 μg / ml 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).

Recombinant Sirt1-GST purification

The recombinant SIRT1-GST enzyme (glutathione S-transferase) was purified from E.Coli BL21 bacteria after transfection with pGEX-SIRT1 plasmid (Addgene). A selected colony of bacteria was grown in LB Broth (Lennox) medium supplemented with antibiotics (100 µg / mL ampicillin) in a tilting 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 μM 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 dithiothreitol (DTT) (Applichem), 0.5 mM phenylmethylsulfonyl fluoride (AppliChem) and 1 tablet of protease inhibitor (PIC) for every 10 mL (Roche ). Bacteria were sonicated for 10 cycles for 45 seconds at 14,000 MHz with 30 second intervals between each sonication. Then, Triton X100 0.1% (Acros) was added and followed by incubation for 15 minutes on ice. Subsequently, the sonicate was centrifuged at 13,000 rpm for 30 minutes and filtered with a 0.45 μm filter.

The bacterial lysate was purified using GE Healthcare Life Sciences (GSTrap 4B) columns. The columns were equilibrated with 20 mL of lysis buffer. Then, the lysate was loaded onto columns and subsequently washed with the lysis buffer. Elution carried out with 20 mL of elution buffer consisting of 50 mM Tris-HCl at pH 8.0, 1 mM of DTT, 20 mM reduced L-glutathione (AppliChem) and H2Odd. The SIRT1-GST protein was detected using colorimetric methods - Bradford protein Assay (Biorad).

Dialysis was performed using a buffer consisting of 50 mM Tris-HCl ph 8.0, 100 mM NaCl (Sigma), 1 mM DTT, 1 PIC tablet (for each 10 mL) and H2Odd overnight at 4 ° C. Afterwards overnight, the samples were cryopreserved in 20% glycerol (SigmaAlrich).

NMase-His tag purification

The histidine-labeled nicotinamidase enzyme (NMase-His) was expressed in E.Coli BL21 bacteria. Bacteria were grown in LB Broth medium supplemented with antibiotics (100 µg / mL ampicillin) in a tilting incubator overnight. When OD was 0.7, isopropyl-b-D-1- thiogalactopyranoside (IPTG) was added at 1 mM concentration for 5 hours. Bacteria were centrifuged at 3000 rpm for 20 minutes and lysed using lysis buffer A in H2O (50 mM NaH2PO4 pH 8, 300 mM NaCl, 10 mM imidazole (Applichem), 100 mg / 50 mL lysozyme (Applichem) , 1 PIC tablet for each 10 mL. The bacterial lysate was incubated on ice for 30 minutes and then sonicated for 10 cycles for 45 seconds at 14,000 MHz with 30 second intervals between each sonication. After sonication, the lysate was filtered through a 0.45 μm filter and incubated for 3 hours at 4 ° C with 1 mL of NTA (ABT) nickel agarose pre-equilibrated with equilibration buffer (EQ) (50 mM NaH2PO4 pH8, 300 mM , NaCl, 20 mM imidazole in H2Odd).

Subsequently, the column was washed with EQ and then the enzyme was eluted with a buffer consisting of 50 mM NaH2PO4 pH8, 300 mM NaCl and 250 mM imidazole, 1 PIC tablet for every 10 mL and the samples were cryopreserved in glycerol. to 20%.

SIRT1 enzyme assay

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

Subsequently, the reaction stop buffer (70% of PBS, 30% of ethanol, 10 mM of DTT and 10 mM of 1,2-Phthalic dicarboxaldehyde OPT) (Acros organics, No 131080250) was added followed by a second incubation 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 enzyme assay in high-troughput mode

Primary screening was conducted using a Tecan Robot Freedom EVO 150. The standard assay (SIRT1 Fluorescence assay) was miniaturized in 384-well plates (Corning 384 Flat Bottom Black Polystyrene) with a final reaction volume of 15 μL. Plate 384 was previously loaded with the known activator (STAC-2) and known inhibitor (EX-527) used as references in the upper left and lower right wells with the HP D300 TECAN Digital Dispenser, and a control without any modulator. 160 compounds were tested in duplicate for each plate.

The reaction buffer (PBS, DTT 1mM and 0.6% DMSO, compounds at 10mM, mixture of enzymes SIRT1 and nicotinamidase (NMase), which will be used for the assay) was placed on the working table of the robot. First, the robot produced dilutions of compounds (10 mM to 10 μM) and added the SIRT1 enzyme mixture to all of the plate except 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 retested in a fluorescence assay in the presence of NMase enzyme alone, to exclude the potential activity of the molecules on this enzyme. The assay was run 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 μM and then 5 μL of intermediate compound dilution (50 μM) was added to 5 μL of reaction buffer. After 40 minutes of incubation at 37 ° C, the reaction stop buffer was added and the plate was again incubated 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 black 96-well plate and incubated for 40 minutes at 37 ° C. The fluorescence reading wavelength is 420/460 nm and performed with the microplate reader. TECAN INFINITE M200.

AC50 / IC50 rating

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

Cellular Thermal Shift Assay (CETSA) MCF7 cells were treated with 50 μM ISIDE11 or DMSO (vehicle) for 1 hour. The cells were collected and washed with PBS. The respective samples were suspended in PBS (1.5 ml), divided into aliquots (100 μl) and heated to different temperatures (RT, 4, 37, 47, 57 and 67 ° C) for 3 minutes using Thermo Mixer (Eppendorf , Milan, Italy), followed by cooling for 3 minutes at 4 ° C. After incubation, 50 μl of RIPA buffer (50 mM Tris-HCl pH 7.4; 1% NP40; 0.5% Na-deoxycholate; 0.1% SDS; 150 mM NaCl; 2 mM EDTA; 50 mM NaF ; one 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 μg were loaded onto 10% SDS-PAGE. The nitrocellulose filters were stained with Ponceau red (Sigma) as a loading control. The filters are then hybridized with SIRT1 Abcam antibody.

Fluorescence quenching evaluation

Fluorescence measurements were performed on a Perkin Elmer Life Sciences LS 55 spectrofluorimeter. The fluorescence emission of tyrosine (λex 275 nm / λem 305 nm) was evaluated both for SIRT1 (after cleavage of thrombin to remove GST) and for free tyrosine a few seconds after the addition of ISIDE11 at different concentrations (0, 0.5, 1, 5, 10, 20, 30, 50 μM in PBS and DTT solution (SIRT1 protein buffer) The extinction of the tyrosine fluorescence was monitored by estimating the F0 / F ratio considering the fluorescence intensity at 305 nm of the sample before (F0) and after (F) the addition of ISIDE11 The working concentrations were 10 μM for both SIRT1 and free tyrosine.

Cell permeability assay

Caco-2 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) containing 10% of FBS, 1% of non-essential amino acids (Euroclones), 100 U / mL of penicillin-streptomycin and 2 mM 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 μL of complete DMEM and placed in the upper chamber of the transwell (0.33 cm2 per insert) on permeable inserts. Then, 600 μL of medium was applied to the bottom of the transwell. The medium was changed only 24 hours after sowing and then 3 times a week. Caco-2 monolayers were grown for 21 days prior to use. When the monolayer was ready, the cells were washed with PBS solution and subsequently added the compound ISIDE11 100 μg / ml, and the lucifer yellow (Sigma Aldrich, No. 67769-47-5) 100 μM, the negative control and propanolol 100 μg / mL were solubilized in 0.1% PBS and DMSO. Lucifer yellow is a fluorescent marker used to verify the integrity of the assay. The permeability assay was performed using 200 μL of apical donor solution, 200 μL of compound ISIDE11 in PBS and 600 μL of PBS basal acceptor solution. All compounds were tested in triplicate. The monolayers were incubated with all 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 are carried out in triplicate.

Half-life

The compound ISIDE11 at 50 mM in DMSO was diluted to 10 mM in DMSO then again at 50 μM 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 IDY-18 column of ONYX 50 x 2 mm, applying a gradient of CH3CN, 0.1% of TFA on H2O, 0.1% of TFA from the 1% to 35% in 4 minutes. Total duration: 9 minutes. The compound was monitored by 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.

Extraction of histone proteins

After treatment, cells were harvested, washed with PBS (Euroclone) and lysed in Triton Extraction Buffer (TEB; PBS containing 0.5% Triton X 100 (v / v), 2 mM PMSF, 0.02% (w / v) NaN3) at a cell density of 10 <7> cells / ml for 10 minutes on ice, with gentle shaking. After centrifugation (2000 rpm at 4 ° C for 10 min), the supernatant was removed and the pellet was washed to half the volume of TEB and centrifuged again. The pellet was suspended in 0.2 N HCl at a cell density of 4 × 107 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 harvested, 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-HCl pH 8.0, 150 mM NaCl, 1% NP40, 10 mM sodium fluoride, 0.1 mM sodium orthvanadate (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). Cell extracts were diluted 1: 1 in Laemmli 2X buffer (0.217 M Tris-HCl pH 8.0, 52.17% SDS, 17.4% glycerol, 0.026% bromo-phenol blue, 8.7% beta-mercaptoethanol ), then boiled for 3 min. Equal amounts of proteins (20 µg) were analyzed and separated by SDS-PAGE gel (acrylamide gel).

Cell viability

MTT assay: Quantification of cell viability was performed by reduction of 3- (4,5-dimethyl-thiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) to 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. Cell viability values were expressed as a percentage of the control (100% cell viability) treated with DMSO. All experiments were performed in triplicate.

Cell count

Cells were incubated with trypan blue (Euroclone) at a ratio of 1: 1. Trypan positive blue cells (dead cells) and the total cell population were counted using a light microscope to calculate the percentage of viable cells.

Western blotting

Western blotting analysis was performed following the recommendations of antibody suppliers and loading 20 μg of protein extracts obtained by total extraction of proteins on 10% polyacrylamide gel and 8 μg of histone extracts obtained by extraction of histone proteins on 15% polyacrylamide gel and subsequently transferred to nitrocellulose membranes (Trans-Blot Turbo Mini Nitrocellulose Transfer No. 1704158, BioRad). Membranes were blocked with 5% milk in 0.5% Tween-20 TrisHCl buffered saline (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 anti-mouse antibodies (Amersham No. NA931) diluted in 3% milk in TBST. The antibodies used included: Anti-SIRT1 (Abcam, ab12193), Anti-p53 (acetyl K382) (Abcam, 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 loading control; anti-H4 antibodies (Abcam, No. ab16483 and anti-H3 (Abcam, No. ab213257) were used as loading control.

RNA extraction 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 RNA degradation 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 ratio of 1:10 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 washed in cold with 75% ethanol. Finally, they were dried at 42 ° C and suspended in DEPC water. For mRNA expression, the total RNA (1 μg) was back-transcribed 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 has been made to minimize the number of animals used and their suffering. The vessels used for the ex vivo experiments were obtained from wild-type and heterozygous Mthfr 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 (10mg / kg) or ISIDE11 (10 mg / kg), alone or after 30 minutes following 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; ISIDE11 was prepared in a solution of DMSO (2%) in sesame oil (Sigma-Aldrich, No. 1612404)

Animals used as controls were treated similarly, but with the drugs replaced with the vehicle alone (2% DMSO in saline). At the end of the in-vivo treatments, the mesenteric arteries were explanted to perform the ex-vivo studies of vascular reactivity. While, the femoral arteries were cryopreserved in OCT (Vector Labs) and then dissected at the cryostat (Leica) in 10 μm sections and analyzed by haematoxylin / 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 isolation of 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 a 37 ° C (chamber size 5 mL DMT, Danish Myosystem). After an equilibrium period of 60 minutes, the arteries were pre-contracted with potassium chloride (KCl 80 mM) until they reached 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 to cumulative concentrations of acetylcholine (10 <-9> M to 10 <-5> M) or nitroglycerin (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 KCl). Vascular relaxation is reported as a negative percentage, considering the basal tension, i.e. that before the stimulus with phenylephrine as -100% of the vascular relaxation and the tension induced by phenylephrine (vasoconstriction) as 0% of the vascular relaxation. For ex vivo studies, vascular reactivity was measured before (baseline) and after 1 hour of incubation in the organ bath with 25 µM of resveratrol or 50/100 µM of ISIS 11 as shown in the figure.

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

Rat tail bleeding

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

Thrombosis method

Mice were anesthetized and the longitudinal skin incision made on the right paw to expose both common and superficial femoral arteries. Distal portions of the common and superficial femoral arteries were temporarily ligated 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 0.4mm wire was then introduced from the incision through the superficial femoral artery to its proximal portion, 2-3mm below the bifurcation with the common femoral artery. The wire was then retracted and reintroduced 5 times to induce endothelial damage. Subsequently, the wire was withdrawn and the distal portion of the superficial femoral artery was permanently ligated proximal to the incision used to introduce the wire. On the other hand, the surgical ring previously placed in the distal portion of the common femoral artery was removed and blood flow was restored accordingly. 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 leg which was considered the internal control (not thrombotic). In detail, the reduction in flow was calculated by measuring the difference in flow velocity between the femoral artery of the left and right paw, and then calculating the percentage of reduction obtained by making the ratio between the measured difference and the femoral flow. healthy by 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 analyzes were conducted with Prism statistical software (GraphPad, La Jolla, CA).

After ultrasound analysis, the mice were euthanized and the femoral arteries of the two legs were harvested for histological analyzes.

Histological preparation

The isolated femoral arteries were incubated in Krebs solution for 15 minutes, cleaned and then the adventitial layer was mechanically removed to reveal the smooth muscle layer and 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 a cryopreservative OCT solution (Vector Labs) and stored at -20 ° C and then dissected at a thickness of 10 μm to perform the staining with hematoxylin and eosin 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.) onto a ZAISS AXIO-phot microscope. (morphometric not available)

EXAMPLES

Example 1: Identification of ISIDE11 in HTS mode

The SIRT1 fluorescence assay was used to develop a method in HTS in order to examine a small molecule library 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 SIRT1 deacetylase activity with ammonia production (and quantification) by coupling two reactions catalyzed by SIRT1 and nicotinamidase (NMase).

In the first reaction, SIRT1 removes the acetyl group from the lysine at position 382 of the p53 peptide (aa 374-389) (SEQUENCE: GQSTSRHKKacLMFKTEG SEQ ID NO: 5) by reaction with its cofactor NAD <+> 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). Eventually, ammonia is detected as a fluorescent adduct at wavelength 420/460 nm, in the presence of optaldehyde (OPT) present in the reaction stop solution (Fig. 1). Both enzymes were produced through the expression of recombinant SIRT1-GST and NMase-His in E.Coli bacteria by FPLC, as indicated in the Materials and Methods.

Using a TECAN robotic station, the enzyme 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. Specifically, screening was performed in 384-well plates, each plate loaded with 160 compounds (in duplicate), using a 15 μl reaction volume. The optimization of the test for high-throughput screening (HTS) is based on several experimental factors: enzyme kinetic parameters, which include the concentration of the enzyme, the Km and Vmax, the optimal concentration of the substrate, response to known compounds, referents of reaction; concentration of DMSO in which the compounds to be tested were 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 then the fluorescence was detected (420 nm - 460 nm).

This work in HTS mode allowed 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 ≥ 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 μM. The HTS station was used extensively for screening allowing the identification of potential active molecules. From the massive screening of the entire library, 70 active molecules were identified as both activators and inhibitors. (Fig.2A). Once the activity was reconfirmed, each identified compound was subjected to intrinsic fluorescence assessments and a counter-assay for the NMase enzyme. About 20 compounds were selected from these steps, the 50 compounds discarded showed intrinsic fluorescence at the wavelength of the SIRT1 assay. None of the compounds were 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 determining the AC50 / IC50. As shown in Fig.2B: both compounds ISIDE11 and ISIDE20 showed an enzymatic activity on SIRT1 superior to the reference activator, approximately 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 was shown to be dose-dependent. By processing the dose-response curves with the GraphPad Prism6 program, the AC50 values were evaluated, resulting in 20.11 μM for ISIDE11 and 396 μM for ISIDE20 (Fig. 2D-E). The chemical structures of the compounds ISIDE11 and ISIDE20 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 ISIDE11 and the SIRT1 protein in a physiological cellular 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 the “native state”) and ISIDE11 has also been studied by fluorescence spectroscopy. SIRT1 contains several tyrosine residues that emit fluorescence and its spectrum is characterized by the typical emission at 305 nm. The emission spectra of SIRT1 fluorescence were recorded in the absence and in the presence of ISIDE11 at 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 directly interacts with SIRT1 and induces the decrease in the fluorescence emitted by tyrosine. To exclude a collision effect by ISIDE11, the same experiment was also performed on the monomeric tyrosine residue (N-acetyl-L-tyrosine ethyl ester) and the fluorescence variation is shown in Fig. 3B. The F0 / F values recorded for free tyrosine are significantly lower than those recorded for SIRT1 at each molar ratio, indicating the formation of a SIRT1-ISIDE11 complex, as no collisional effect is involved.

Furthermore, the cellular permeability of the drug was evaluated. 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 was studied in vivo (Fig.4B). The compound was tested at a concentration of 50 μM and the stability profile was performed in DMEM with incubation at RT for 6 hours. The residual amounts of compound were determined over time by RP-HPLC. ISIDE11 was quantified in chromatograms extracted at 375 nm. The stable residual amount of the compound after 1h was 5%.

In conclusion, these data confirmed that the compound is an excellent activator of SIRT1 and a good candidate for further in vivo studies.

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-type cell systems were evaluated. Several cell lines were untreated and treated with the compounds ISIDE11 and ISIDE20 to evaluate their effects on histone modifications. HepG2 cells were treated and untreated with ISIDE11 and ISIDE20 at two different concentrations (5 and 50 μM) for 24 hours. Western blotting analyzes were then conducted to investigate the main targets of SIRT1, these analyzes showed that ISIDE11 is able to induce a dose-dependent decrease in the level of acetylation 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 haematological tumor cell lines, as revealed by Western blotting analyzes performed on MCF7, HCT1116 and U937 cells (Fig. 5B). These data confirm that ISIDE11 is a SIRT1 activator by acting on the histone targets of the enzyme. This confirms its good enzymatic activity with an AC50 value of 20.11 µM compared to that of ISIDE20 which in fact shows an AC50 value of 396 µM.

Example 4: ISIDE11 enhances SIRT1-mediated effects in the stress response

Many genotoxic stresses induce the acetylation of p53 in the carboxyterminal 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 investigate the role of ISIDE11 and ISIDE20 in modulating SIRT1-mediated functions in such responses, the deacetylation activity of SIRT1 on p53 was evaluated. Western blotting analyzes were performed by pre-treating HepG2 cells with a genotoxic drug, etoposide, for 6 hours, and subsequently with ISIDE11 or ISIDE20 at 50 μM 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 activation of SIRT1, ISIDE11 could strongly attenuate 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 an inhibitor of SIRT1 decreases the level of expression of both of SIRT1 that of p53. This indicates that ISIDE11 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 ISIDE11 compound is capable of increasing the deacetylase activity of SIRT1 and counteracting the genotoxic effects on DNA.

Example 5: Cellular effects of ISIDE11

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

Example 6: Treatment with ISIDE11 recovers endothelial dysfunction in MTHFR / - heterozygous mice

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

A common variant in MTHFR, C677T, is associated with decreased enzymatic activity that leads to an increase in plasma homocysteine levels in humans. Generally, patients with heterozygous MTHFR are predisposed to developing cerebro and cardiovascular events [41]. In fact, although the combined administration of folic acid, vitamin B6 and vitamin B12 represents the only therapeutic approach at low homocysteine levels, it has been shown that these supplements do not reduce the risk of vascular events [42], setting the basis for investigating a possible direct correlation between MTHFR mutation and vascular alteration, independent of homocysteine levels. In this context, down-regulation of SIRT1 has been reported in animal models of heterozygous MTHFR associated with nitric oxidase enzyme dysfunction and thrombotic events [41, 43.44]. Therefore, in order to formulate a new therapeutic strategy capable of reducing endothelial dysfunction and preventing the typical brain and cardiovascular damage 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, ex vivo vascular function was assessed in wild-type and MTHFR <+/-> mesenteric arteries in the presence of ISIDE11 or RSV using the pressure myography system (Fig. 8). These results show the ability of ISIDE11 to induce a significant improvement in endothelial function in vessels from animals MTHFR <+/-> (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]. It is interesting to highlight that 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 the in vivo treatment of MTHFR <+/-> mice and wild type mice for 21 days with ISIDE11 or RSV, alone or in combination with a SIRT1 inhibitor, EX-527.

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

Based on the ability of ISIDE11 to regulate the homeostatic process, we tested its effect on induced thrombosis in the femoral artery using ultrasound and histological approaches.

Interestingly, the assessment of the percentage reduction in the blood flow velocity of the superficial femoral artery of MTHFR <+/-> mice treated with ISIDE11 was significantly reduced compared to heterozygous mice treated with the vehicle alone. In fact, it is possible to observe a reduction in the percentage of flux similar to that observed in wild-type mice or in heterozygous MTHFR animals treated with RSV. Furthermore, the concomitant treatment with the SIRT1 inhibitor is able to block the protective effect of ISIDE11 (Fig.10A).

Subsequently, a histological analysis was performed to visualize the extent of thrombotic injury and, as shown in Fig. 10B, treatment with ISIDE11 counteracts thrombotic injury more efficiently than RSV.

To validate the efficacy of ISIDE11 on endothelial function, the vascular reactivity of the mesenteric arteries of the same groups of animals treated in vivo was evaluated to assess the impact on thrombus formation. 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 ISIDE11 is able to induce a significant improvement in endothelial function compared to to vessels from animals treated with vehicle only, which instead show an important endothelial dysfunction (Fig. 11A) which contributes to the cardiovascular risk associated with the mutation.

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

Example 8: Analogous compounds of ISIDE11

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

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

Claims (11)

CLAIMS 1. An activator of SIRT1 of formula I wherein R = aryl or heteroaryl optionally replaced with a group selected from: alkoxy, phenoxy, alkyl group, amino group and halide or a salt, tautomer, solvate, stereoisomer or analogue thereof, or of formula or a salt, tautomer, solvate, stereoisomer or analogue thereof. 2. The activator of SIRT1 according to claim 1 wherein said aryl is phenyl or said heteroaryl is pyridinyl and / or said alkoxy is methoxy. 3. The activator of SIRT1 according to claim 1 or 2 having the formula: N '- (1,3-dioxo-1,3-dihydro-2H- N'-benzoyl-3,4-dichlorobenzohydrazide inden-2-diene) isonic nohydrazide N '- (1,3-dioxo-1,3-dihydro-2H-inden-2- N' - (1,3-dioxo-1,3-dihydro-2H-diene) -4-methoxybenzohydrazide inden-2- diene) benzohydrazide or a salt, tautomer, solvate, stereoisomer or analogue thereof. The solvate according to any one of the preceding claims wherein said solvate is a hydrate. The activator of SIRT1, salt, tautomer, solvate, stereoisomer or analogue thereof according to any one of the preceding claims for use in a method of therapy and / or prevention of a condition and / or a disease selected from the group consisting of : cardiovascular disease, metabolic disease, aging, cellular senescence, neurodegenerative disease, endothelial dysfunction, DNA damage, oxidative stress, brain damage, cardiovascular damage, coronary artery disease, thrombosis, hyperhomocysteinemia, resistance insulin, obesity, neuronal loss, an age-related cardiological condition, diabetes, preferably type 2 diabetes, dyslipidemia, hyperlipidemia, an immune disease, a complication of diabetes and / or inflammation. 6. The activator of SIRT1, salt, tautomer, solvate, stereoisomer or analogue thereof according to any of the preceding claims for use as an apoptotic drug, vasodilator and / or antithrombotic. 7. The activator of SIRT1, salt, tautomer, solvate, stereoisomer or analogue thereof for use according to claim 5 or 6, characterized in that it is administered to a subject carrying at least one mutation in the methylene tetrahydrofolate reductase enzyme gene (MTHFR), preferably the mutation is the C677T or A1298C mutation. 8. A pharmaceutical or cosmetic composition comprising the activator of SIRT1, salt, tautomer, solvate, stereoisomer or analogue thereof as defined in any one of claims 1 to 4, a vehicle and optionally a further therapeutic agent for use as an activator of SIRT1. The pharmaceutical composition according to claim 8 for use in a method of therapy and / or prevention of a condition and / or a disease selected from the group consisting of: a cardiovascular disease, a metabolic disease, aging, a cellular senescence, neurodegenerative disease, endothelial dysfunction, DNA damage, oxidative stress, brain damage, cardiovascular damage, 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 inflammation. The composition for use according to claim 8 or 9 wherein said further therapeutic agent is selected from the group consisting of: folic acid, vitamin B6 and vitamin B12. 11. An in vitro method for identifying a SIRT1 modulator comprising activating SIRT1 with the SIRT1 activator, salt, tautomer, solvate, stereoisomer or analog thereof as defined in any one of claims 1 to 4 in the presence or absence of said modulator.