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Title:
ACTIVATORS OF SIRTUINS AND USES THEREOF
Document Type and Number:
WIPO Patent Application WO/2021/018965
Kind Code:
A1
Abstract:
The present invention concerns compounds useful as modulators of sirtuins, in particular as SIRT1 activators. Moreover, the invention refers to the medical use of these compounds, in particular in the prevention and/or treatment of SIRT1 related conditions, such as cardiovascular diseases and of metabolic diseases, to pharmaceutical compositions that comprise them and to an in vitro method to identify a SIRT1 modulator.

Inventors:
ALTUCCI LUCIA (IT)
NEBBIOSO ANGELA (IT)
SCISCIOLA LUCIA (IT)
Application Number:
PCT/EP2020/071401
Publication Date:
February 04, 2021
Filing Date:
July 29, 2020
Export Citation:
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Assignee:
EPI C S R L (IT)
International Classes:
A61K31/00; C07D311/00; C07D311/16
Domestic Patent References:
WO2015009883A12015-01-22
Other References:
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Attorney, Agent or Firm:
CAPASSO, Olga et al. (IT)
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Claims:
CLAIMS

1. A compound of formula I

wherein R, R’ and R” are independently H, linear or branched C1-C6 alkyl, OH or halogen, Y and Y’ are independently H, halogen, C1-C6 alkoxy, C1-C6 haloalkoxy or a salt, tautomer, solvate, stereoisomer or analogue thereof for use as a SIRTl activator medicament.

2. The compound, salt, tautomer, solvate, stereoisomer or analogue for use according to claim 1, wherein R is H or linear or branched C1-C6 alkyl, R’ is OH or linear or branched C1-C6 alkyl, R” is H, halogen or linear or branched C1-C6 alkyl, Y is H or halogen.

3. The compound, salt, tautomer, solvate, stereoisomer or analogue for use according to any one of the previous claims, wherein said linear or branched C1-C6 alkyl is C¾ or CH2CH3.

4. The compound, salt, tautomer, solvate, stereoisomer or analogue for use according to any one of the previous claims, wherein said halogen is F or Cl.

5. The compound, salt, tautomer, solvate, stereoisomer or analogue for use according to any one of the previous claims, wherein said C1-C6 alkoxy is OCH2CH3.

6. The compound, salt, tautomer, solvate, stereoisomer or analogue for use according to any one of the previous claims, wherein said C1-C6 haloalkoxy is OCH(F)2.

7. The compound, salt, tautomer, solvate, stereoisomer or analogue for use according to any one of the previous claims, wherein Y and/or Y’ is not H.

8. The compound, salt, tautomer, solvate, stereoisomer or analogue for use according to any one of the previous claims, wherein at least one of R, R’ and/or R” is OH.

9. The compound, salt, tautomer, solvate, stereoisomer or analogue for use according to any one of the previous claims, wherein R, R’, R”, Y and Y’ are:

or a salt, tautomer, solvate, stereoisomer or analogue thereo:

10. The compound, salt, tautomer, solvate, stereoisomer or analogue for use according to any one of the previous claims, wherein said solvate is a hydrate or a solvate with DMSO.

11. The compound, salt, tautomer, solvate, stereoisomer or analogue for use according to any one of the previous claims, wherein said use is in the prevention, treatment and/or amelioration of a SIRT1 related condition or disorder.

12. The compound, salt, tautomer, solvate, stereoisomer or analogue for use according to any one of the previous claims, wherein said use is in the prevention, treatment and/or amelioration of a condition and/or a disease selected from the group consisting of: a cardiovascular disease, a metabolic disease, aging, 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, coronary atherosclerosis, an age-related cardiological condition, diabetes, dyslipidemia, hyperlipidemia, an immune disease, a complication of diabetes and/or an inflammation, preferably said neurodegenerative disease is Alzheimer’s disease, Parkinson’s disease, Huntington's chorea, or amyotrophic lateral sclerosis, preferably said diabetes is type 2 diabetes.

13. A pharmaceutical or cosmetic composition comprising the compound, salt, tautomer, solvate, stereoisomer or analogue as defined in any one of claims 1 to 10, a vehicle and optionally a further therapeutic agent, said pharmaceutical or cosmetic composition being for use as a SIRT1 activator medicament.

14. The composition for use according to claim 13, wherein said use is in the prevention, treatment and/or amelioration of a condition or disorder as defined in claim 11 or 12.

15. The composition for use according to claim 13 or 14, wherein said further therapeutic agent is selected from the group consisting of: folic acid, vitamin B6, vitamin B12, metformin, incretin-mimetics, DPP4 inhibitors and insulin.

16. An in vitro method to identify a SIRT1 modulator comprising activating SIRT1 with the compound, salt, tautomer, solvate, stereoisomer or analogue as defined in any one of claims 1 to 10 in the presence or absence of said modulator.

Description:
ACTIVATORS OF SIRTUINS AND USES THEREOF

TECHNICAL FIELD

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

STATE OF THE ART

Sir2 proteins (Silent information regulator 2), also known as Sirtuins (SIRTs), form class III of the histone deacetylase (HD AC) family. Their catalytic activity requires nicotinamide adenine dinucleotide (NAD + ) as a cofactor [1]

Following its discovery in Saccharomyces cerevisiae yeast as mating-type regulator 1 (MARI), Sir2 was found to play an important role in suppressing ribosomal DNA recombination and gene silencing [2-4] Moreover, Hstl-4p, its yeast homologs, are involved in silencing of the mating- type loci and telomeres, cell cycle progression, chromosomal stability, and longevity [5, 6] Sir2 homologs were identified in a variety of organisms, from prokaryotes to eukaryotes [7]

Eukaryotes have seven SIRTs, SIRT1-7, which differ in the sequence of the catalytic domain, length of the N/C-terminal domains, cellular localization, and catalytic activity [8] Table 1: The human Sirtuins [26]

The biochemical activity of Sirtuins is not limited to deacetylation, but includes other reactions: ADP-ribosylation (SIRT4 and 6), deacylation (SIRT6), desuccinylation (SIRT5 and 7), deglutarylation (SIRT5), demalonylation (SIRT5), demyristoylation, and depalmitoylation (SIRT6) [10, 11] Although Sirtuins are regulated at different levels, from DNA to proteins, they are mainly controlled by dynamic changes in NAD + levels and the [NAD]/[NADH] ratio [7, 12] Besides histones, a multitude of non-histone proteins such as transcription factors (p53, NF-kB, PGCla) and DNA repair proteins (Ku70, PARP1) [13] are recognized as SIRT targets.

Through their numerous substrates, Sirtuins control different molecular pathways including energy metabolism, cell survival, DNA repair, tissue regeneration, inflammation, neuronal signaling, and even circadian rhythms [14, 15]

Depending on the availability of its cofactor NAD + , SIRT1 is involved in different metabolic pathways regulating the energetic state of the cell. A growing body of evidence indicates that overexpression of NAD + or SIRTl simulates a state of caloric restriction leading to beneficial effects, including enhanced mitochondrial biogenesis, insulin sensitivity, and a lower incidence of age-related diseases [16]

Therefore, the acetylation state of SIRTl substrates is associated to pathogenesis of several pathological conditions such as cancer, metabolic diseases, inflammation, cardiovascular diseases, diabetes and aging [17]

In light of its involvement in different diseases as well as its beneficial effects, SIRTl has emerged as a good therapeutic target for the treatment of different human disorders [14]

In the last years, great attention was focused on the identification of new SIRTl -activating compounds (STACs), able to modulate the activity of Sirtuins with a bigger efficiency than resveratrol.

The effects due to SIRTl activation are noteworthy: SRT1720 [18-20] and oxazole [4,5 -b] pyri dines [21, 22] show anti diabetic action, pyrrolo [3,2-b] quinoxalines [23, 24] and SRT2104 [25] exhibit anti-inflammatory activity, and SRT2104 is used in 14 clinical trials (see http s : //clini caltri al s . gov/ ct2/ results? cond= SRT2104).

On the other hand, Sirtuins activity can be inhibited by several inhibitors (SIRTi), which currently are mainly SIRTl inhibitors [26] The most potent and selective SIRTl inhibitor is EX- 527 (Selisistat or SEN0014196) [27], which displays robust neuroprotective activity in in vitro and in vivo models of Huntington’s disease (HD) [28] In particular, three clinical trials that use EX-527 show that the compound is well-tolerated in HD patients [29] (https://clinicaltrials.gov/ct2/results?cond=&term=SEN00 14196).

Despite the high number of identified SIRTi, none has been approved for clinical use [30] Conversely to SIRTi, few STACs have been identified although they show very promising potential therapeutic activities. So, it is necessary to increase the list of STACs, with the aim to characterize molecules useful in the clinical field.

Actually, STACs could revolutionize the human lifespan improving health, thus showing major therapeutic implications [15] By binding the catalytic domain or an important region for catalytic activity outside of the protein, SIRT activators present more target specificity. However, the activators mimicking the natural mechanism of SIRTs may cause some side effects [15] Hence, there is a pressing need to intensify the discovery of new molecules that can be more potent and selective.

SUMMARY OF THE INVENTION

In the present invention, a novel small molecule able to activate SIRTl (herein indicated as SCIC2) was identified. SCIC2 is able to activate SIRTl, with enzymatic activity of 135.8% and an AC50 value of 50 mM. It directly binds the enzyme with a K D of 26.4 ± 0.6 pM. Its optimization led to the identification of a more potent derivative (herein indicated as SCIC2.1) with enzymatic activity for SIRTl of 175% and a stronger binding with the enzyme.

Neither compound is cytotoxic nor alters cell growth. In cancer and normal cell systems, SCIC2 and SCIC2.1 are involved in the increase of the SIRTl -mediated effect in genotoxic response, making them promising agents for various therapeutic applications.

Therefore, it is an object of the present invention a compound of formula I

wherein R, R’ and R” are independently H, linear or branched C1-C6 alkyl, OH or halogen, Y and Y’ are independently H, halogen, C1-C6 alkoxy, C1-C6 haloalkoxy or a salt, tautomer, solvate, stereoisomer or analogue thereof for use as a SIRT1 activator medicament. In particular, said use as a SIRTl activator medicament is in preventing, treating or ameliorating a SIRTl related condition or disorder.

It is a further object of the present invention a SIRTl activator of formula I Wherein R, R’ e R” are independently H, linear or branched C 1 -C 6 alkyl, OH or halogen, Y and Y’ are independently H, halogen, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy or a salt, tautomer, solvate, stereoisomer or analogue thereof.

Thus, the present invention refers to a compound of formula I, or a salt, tautomer, solvate, stereoisomer or analogue thereof, as defined above, wherein said compound, salt, tautomer, solvate, stereoisomer or analogue thereof is a SIRT1 activator or is a SIRT1 activating agent or a SIRTl activator medicament or is an active ingredient having SIRT 1 activating activity.

It is to be understood that the following preferred embodiments may be combined among each other in any way that would give rise to a stable molecule.

In a preferred embodiment, R is H or linear or branched C 1 -C 6 alkyl. Additionally or alternatively, preferably R’ is OH or linear or branched C 1 -C 6 alkyl. Additionally or alternatively, preferably R” is H, halogen or linear or branched C 1 -C 6 alkyl. Additionally or alternatively, preferably Y is H or halogen. Preferably, R is H or linear or branched C 1 -C 6 alkyl, R’ is OH or linear or branched C 1 -C 6 alkyl, R” is H, halogen or linear or branched C 1 -C 6 alkyl, Y is H or halogen.

In a preferred embodiment, said linear or branched C 1 -C 6 alkyl is C¾ or CH 2 CH 3. Additionally or alternatively, preferably said halogen is F or Cl. Additionally or alternatively, preferably said C 1 -C 6 alkoxy is OCH 2 CH 3. Additionally or alternatively, preferably said C 1 -C 6 haloalkoxy is OCH(F) 2. Preferably, said linear or branched C 1 -C 6 alkyl is C¾ or CH 2 CH 3 , said halogen is F or Cl, said C1-C 6 alkoxy is OCH2CH 3 , said C1-C 6 haloalkoxy is OCH(F)2.

In any of the above-defined objects and embodiments, preferably, Y and/or Y’ is not H.

In any of the above-defined objects and embodiments, preferably, at least one of R, R’ and/or R” is OH.

In a preferred form, R, R’, R”, Y and Y’ are:

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

Preferably, said solvate is a hydrate or a solvate with DMSO.

In a particularly preferred form, the compound, salt, tautomer, solvate, stereoisomer, analogue or SIRT1 activator as defined above is for use in the prevention, treatment and/or amelioration of a SIRT1 related condition or disorder.

Additionally or alternatively, preferably, the compound, salt, tautomer, solvate, stereoisomer, analogue or SIRT1 activator as defined above is for use in the prevention, treatment and/or amelioration of a condition and/or a disease selected from the group consisting of: a cardiovascular disease, a metabolic disease, aging, 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, coronary atherosclerosis, an age-related cardiological condition, diabetes, dyslipidemia, hyperlipidemia, an immune disease, a complication of diabetes and/or an inflammation, preferably said neurodegenerative disease is Alzheimer’s disease, Parkinson’s disease, Huntington's chorea, or amyotrophic lateral sclerosis, preferably said diabetes is type 2 diabetes.

Senescence is a form of cell cycle arrest. Non-proliferating cells release pro-inflammatory cytokines that play an important role in the progression of aging and degenerative diseases. So, senescence-related diseases fall into the two categories listed above.

The present invention additionally 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 medicament.

In a particularly preferred form, the pharmaceutical or cosmetic composition is for use in the prevention, treatment and/or amelioration of a SIRT1 related condition or disorder.

Additionally or alternatively, preferably, the composition as defined above is for use in the prevention, treatment and/or amelioration of a condition and/or a disease selected from the group consisting of: a cardiovascular disease, a metabolic disease, aging, 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, coronary atherosclerosis, an age-related cardiological condition, diabetes, dyslipidemia, hyperlipidemia, an immune disease, a complication of diabetes and/or an inflammation, preferably said neurodegenerative disease is Alzheimer’s disease, Parkinson’s disease, Huntington's chorea, or amyotrophic lateral sclerosis, preferably said diabetes is type 2 diabetes.

In a preferred form, said further therapeutic agent is selected from the group consisting of: folic acid, vitamin B6, vitamin B 12, metformin, incretin-mimetics, DPP4 inhibitors and insulin.

The present invention further provides an in vitro method to identify a SIRT1 modulator comprising activating SIRT1 with the compound, salt, tautomer, solvate, stereoisomer, analogue or SIRT1 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 SIRTl activator as defined above, e.g. by contacting SIRTl or a host (such as a cell) comprising SIRTl with the compound, salt, tautomer, solvate, stereoisomer, analogue or SIRTl activator as defined above.

It is to be understood that in the present invention any reference to“the compound”,“the compound of the invention”,“SIRTl activator” or grammatical variants thereof also includes the corresponding salt, tautomer, solvate, stereoisomer or analogue thereof as defined above.

In the invention,“activator of SIRTl”,“SIRTl activator”,“SIRTl activator medicament” and grammatical variants thereof mean a molecule capable of increasing SIRTl’ s 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 SIRTl activation include: in vitro enzymatic assays, in which the level of substrate acetylation due to SIRTl activity is measured in the presence of potential modulators, and Western Blotting by analyzing the acetylation of at least one target of SIRTl. Proper control against which the increase of SIRTl’ s 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 SIRTl include but are not limited to: histones HI, H3 and H4, p53, NFKB, p300, proteins of the FOXO family (such as FOXO 1, 3a and 4), HIVTat, PGC-Ia, PCAF, MyoD, peroxisome proliferator-activated receptor g, Ku70, Hif-la, Hif-2a, MYC, STAT3, Rb, DNMT1, CRTC2, LXRs, AceCSl. In particular, a SIRTl activator can be defined as a molecule that decreases the acetylation level of any one or more of SIRTl’ 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.

SIRT1 is expressed in a wide range of tissues and organs and has been detected in the liver, pancreas heart, muscle, brain and adipose tissue. SIRT1 is activated by high NAD+ levels, a condition caused by low cellular energy status, which could be e.g. caused by calorie restriction or exercise. Activation of SIRTl leads to the deacetylation of target proteins which are important for apoptosis, the cell cycle, circadian rhythms, mitochondrial function, and activated metabolism, including glucose management, lipid metabolism, and energy homeostasis as well as to positive effects on cell protection. Several mouse models have been used in order to investigate the metabolic function of SIRTl. It could be demonstrated that over-expression of SIRTl show decrease in adiposity, serum cholesterol, and insulin, while displaying increased resistance to obesity-generated glucose intolerance and insulin resistance.

SIRTl has the following proven effects linked to the beneficial effects described in this invention:

Glucose or insulin management: SIRTl has beneficial effects on glucose or insulin management in the liver, in the pancreatic beta cells and in the skeletal muscle cells. In the liver SIRTl is upregulated during negative energy balance, like calorie restriction, and inhibits glycolyses and stimulates gluconeogeneses by deacetylation of the transcriptional coactivator PGC1 alpha. In addition, glucose metabolism is regulated by interaction between SIRTl and FOXO transcription factors. SIRTl has cell protective effects on pancreatic beta cells, preventing the hyperglycemia- induced damage of these cells which produce insulin, which is necessary to regulate the glucose uptake into the cell and its metabolism. Insulin secretion itself is also stimulated by SIRTl leading to a higher glucose tolerance of the cells. Within the muscles cells SIRTl improves insulin sensitivity.

Lipid metabolism: SIRTl has beneficial effects on lipid metabolism in the liver, the skeletal muscle and in adipocytes. In the liver SIRTl regulates hepatic fatty acid metabolism by activating the AMPK/LKB1 signaling pathway. Furthermore, it plays an important role in cholesterol homeostasis by deacetylation of the liver X receptor (LXR) and the critical regulator SREBP. In the skeletal muscle SIRTl deacetylates PGC-la to induce mitochondrial fatty acid oxidation in a glucose-scarce environment. Furthermore, SIRTl deacetylates and activates acetyl-CoA synthetase (AceCS), which can induce substantial fatty acid synthesis. In the adipocytes SIRTl favors lipolysis and fatty acid mobilization in response to fasting by repressing PPARg which is essential for adipogenesis. Another pathway involves the deacetylation of FOXOl and stimulation of ATGL gene transcription.

Energy homeostasis: SIRT1 activates PGC-la (peroxysome proliferator-activated receptor gamma coactivator-la) which directly coactivates factors relating to mitochondrial biogenesis and respiration rates as well as the uptake and utilization of substrates for energy production in different tissues (e.g. in the liver where SIRT1 controls the neogeneration of glucose by modulating PGC-la and CREB (cAMP response element-binding protein) regulated transcription coactivator 2. Furthermore, activated SIRTl results in deacetylation of PGC-la in muscle and brown fat tissue and leads to an increase in its transcriptional activity which would then allow the cell to increase mitochondrial respiration and meet energy requirements when exposed to energy stress (Carles Canto and Johan Auwerx. PGC-1 alpha, SIRTl and AMPK, an energy sensing network that controls energy expenditure. Curr Opin Lipidol 2009, 20(2): 98-105; Carles Canto and Johan Auwerx. PGC-1 alpha, SIRTl and AMPK, an energy sensing network that controls energy expenditure. Curr Opin Lipidol 2009, 20(2): 98-105).

Muscle mass (Body composition): SIRTl modulates muscle differentiation (Nogueiras R et al. Sirtuin 1 and Sirtuin 3: Physiological modulators of metabolism. Physiol Rev 2012, 92(3): 1479- 1514). Furthermore, SIRTl plays a role in deacetylation and of PGC-la and improved mitochondrial content and fatty acid utilization (Gurd BJ et al. Does SIRTl determine exercise- induced skeletal muscle mitochondrial biogenesis: differences between in vitro and in vivo experiments? J Appl Physiol 2012, 112(5):926-8).

Cell protection and DNA repair: Substrates which are deacetylated by SIRTl include also proteins such as p53 and transcription factors which are involved in DNA repair, e.g. Ku70, FOXLB or NBSl (Nijmegen breakage syndrome protein, STAT3, Rb, PCGlalpha). Deacetylation of these targets by SIRTl results in reduction of stress induced apoptosis, increased DNA repair in cells after radiation exposures and cell survival by delayed cell cycle progression. By activating FOXO proteins, e.g. FOX03a (forkhead box group) resistance to oxidative stress is increased. A further mechanism for cell survival is autophagy, which is a cellular housekeeping process for cleansing aberrant and dysfunctional molecules and organelles. SIRTl regulates the autophagy process via several factors, e.g. the FoxO and p53 pathways (Salminen A and Kaamiranta K, Cellular Signalling, 2009, 21(9): 1356-60).

"Prevention, treatment and/or amelioration of a SIRTl related disorder or condition" comprises according to the invention e.g. maintaining well aging, maintaining healthy body composition, maintaining healthy glucose or insulin metabolism, treating and/or preventing overweight and/or obesity, reducing risk to develop diabetes type II, maintaining a healthy fat (or lipid) metabolism, reducing risk to develop elevated blood lipid levels, reducing risk to develop atherosclerosis and/or cardiovascular diseases, protecting cells, repairing DNA, maintaining physical power and/or muscle mass during aging.

As used herein,“C 1 -C 6 alkyl” refers to any linear or branched aliphatic hydrocarbon chain comprising from 1 to 6 carbon atoms. Preferably, said C 1 -C 6 alkyl is a C 1 -C 3 alkyl or a C 1 -C 4 alkyl. Examples of C 1 -C 6 alkyl include but are not limited to: methyl, ethyl propyl, /propyl, butyl, sec-butyl, /f/V-butyl, pentyl, hexyl, etc. Preferably, said C 1 -C 6 alkyl is methyl or ethyl.

As used herein,“C 1 -C 6 alkoxy” refers to an -O-alkyl group, wherein O is an oxygen atom and alkyl is a C 1 -C 6 alkyl as defined above. Preferably, said C 1 -C 6 alkoxy is a C 1 -C 3 alkoxy or a Ci- C 4 alkoxy. Examples of C 1 -C 6 alkoxy include but are not limited to: methoxy, ethoxy propoxy, /propoxy, butoxy, etc. Preferably, said C 1 -C 6 alkoxy is methoxy or ethoxy.

As used herein,“C 1 -C 6 haloalkoxy” refers to a C 1 -C 6 alkoxy as defined above wherein the alkoxy is substituted at any one or more positions with a halogen atom. Preferably, said C 1 -C 6 haloalkoxy is a C1-C 3 haloalkoxy or a C1-C4 haloalkoxy. Preferably, said C1-C 6 haloalkoxy is OCF3 or OCH(F) 2.

As used herein, the terms "treat", "prevent" or "ameliorate" and grammatical variants thereof refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development of a disease, e.g. a gut disease or a cardiovascular disease. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Those in need of treatment include those already with the condition or disorder as well as those prone to develop the condition or disorder or those in which the manifestation of the condition or disorder is to be prevented.

The compounds of the invention may be obtained by chemical synthesis according to standard procedures and/or may be purchased by commercial suppliers as described in the Materials and Methods below.

In the invention, a cardiovascular disease comprises acute myocardial infarction, angina pectoris, ischemic and hemorrhagic stroke. As used herein, a tumor comprises solid or hematopoietic tumor, such as leukemia, breast, hepatic, pancreatic, colorectal and lung cancer. Sai tumor may be primary or metastatic.

A metabolic disease includes diabetes mellitus, phenylketonuria, citrullinemia, dyslipidemia. Cellular senescence includes Alzheimer’s disease and Parkinson’s disease. A neurodegenerative disease comprises Alzheimer’s disease, Parkinson’s disease and Huntington’s chorea and amyotrophic lateral sclerosis.

An endothelial dysfunction includes hypertension. DNA damage comprises cancer. Oxidative stress includes early or premature aging and chronic degenerative diseases. Brain damage or cerebral damage comprises an ischemia or a stroke. Cardiovascular damage includes thrombosis. Coronary artery disease (or more simply coronary disease) comprises cardiovascular diseases such as ischemic heart disease and myocardial infarction.

An age-related cardiological condition includes a heart attack.

An immune disease comprises rheumatoid arthritis, systemic erythematous lupus, Hashimoto's thyroiditis, psoriasis.

A complication of diabetes includes retinopathy, nephropathy, diabetic foot, coronaropathy and cerebral vasculopathy.

Inflammation includes tuberculosis and hepatitis.

DDP4 inhibitors include Saxagliptin, Sitagliptin, Linagliptin and Alogliptin.

Incretin-mimetics include Exenatide, Lixisenatide, Dulaglutide and Liraglutide.

The compounds of the invention could be in the form of a salt, in particular of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts comprise conventional non toxic salts obtained by salification with inorganic acids (such as hydrochloric, hydrobromic, sulfuric or phosphoric acid), or with organic acids (such as acetic, propionic, succinic, benzoic, sulfanilic, 2-acetoxy-benzoic, cinnamic, mandelic, salicylic, glycolic, lactic, oxalic, malic, maleic, fumaric, tartaric, citric, p-toluenesulfonic, methansulfonic and ethanesulfonic acid or naphthalene sulfonic acids). For information about pharmaceutically suitable salts please refer to Berge S. M. et ah, 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.

Moreover, 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 a person skilled 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 those with alkali metals or alkaline earth metals and pharmaceutically acceptable salts such as sodium, potassium, calcium or magnesium salts; in particular pharmaceutically acceptable salts of one or more carboxylic acid moieties which may be present in the compounds of the invention. Salts may also be internal salts such as zwitterions.

Furthermore, the compounds of the invention can be administered in non-solvated as well as solvated forms with pharmaceutically acceptable solvents such as water, EtOH, DMSO and others like these.

The 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 in accordance to the present invention. The present invention covers individual isomers as well as isomers mixtures in which one or more chiral centers are reversed.

Analogously, it is to be understood that compounds of the invention may exist in tautomeric forms different from the other depicted in the formulas and these are also within the scope of the present invention.

The invention also includes all isotopic variants of the compounds of the invention. An isotopic variation is defined as a variation in which at least one atom of the molecule is substituted with 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 into the compounds 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 C1, 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, could lead to therapeutic advantages deriving from greater metabolic stability. Isotopic variants of the compounds could be generally prepared by conventional procedures by using appropriate 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 pure or as pharmaceutical formulations, i.e. suitable for parenteral, oral, vaginal or rectal administrations. Each of these formulations can contain excipients and/or fillers and/or additives and/or binders and/or coatings and/or suspending agents and/or emulsifiers and/or preservatives and/or controlled-release agents suitable for the selected pharmaceutical form. Pharmaceutical compositions can be chosen according to the treatment needs. These compositions are prepared by mixing and can be administered in tablets, capsules, oral preparations, powders, granules, pills, liquid solutions for injection or infusion, suspensions, suppositories or preparations for inhalation. Tablets and capsules for oral administration are normally formulated in unit-dose 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 starch sodium glycolate), coloring agents, flavoring agents, and wetting agents (e.g. sodium lauryl sulfate).

The solid oral compositions can be prepared with conventional methods of mixing, filling or tableting. The mixing operation can be repeated to distribute the active ingredient in all the compositions containing large quantities of fillers. These operations are conventional. Oral liquid preparations can be for example in the form of aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or they can be provided as a dry product to be reconstituted with water or with a suitable vehicle before use. These liquid preparations may include conventional additives such as suspending agents, for example, sorbitol, syrup, methylcellulose, gelatin, hydroxyethyl cellulose, 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, flavoring agents or conventional colorants.

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

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

Parenteral solutions are normally prepared by dissolving the compound in a vehicle, sterilizing through filtration, filling suitable containers and sealing.

Advantageously, adjuvants such as local anesthetics, preservatives and buffer agent can be dissolved in the vehicle. To enhance stability, the composition could be frozen after having filled the vials and removed the water under vacuum. Parenteral suspensions are prepared substantially in the same manner, except that the compound could be suspended in the vehicle instead of being dissolved and sterilized by ethylene oxide exposition before its suspension into the sterile vehicle.

Advantageously, a surfactant or a wetting agent can be included in the composition to favor the uniform distribution of the compound of the invention.

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

For buccal or sublingual administration, compositions can be tablets, pastilles, lozenges, or gels. The compounds can be pharmaceutically formulated as suppositories or retention enemas, for instance as suppositories containing conventional bases such as cocoa butter, polyethylene glycol, or other glycerides, for rectal administration.

Another way to administer the compounds of this invention regards topical treatment. Topical formulations can contain for examples 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, semi-solid hydrocarbons, or emulsifiable ointments such as hydroxystearyl sulfate, anhydrous lanolin, hydrophilic petrolatum, cetyl alcohol, glycerol monostearate, stearic acid, or water-soluble ointments containing polyethylene glycols of various molecular weights.

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

Another way to administer compounds of the invention regards transdermal delivery. Topical transdermal formulations include conventional aqueous and non-aqueous vehicles, such as creams, oils, lotions or pastes or they can be in the form of membranes or medicated plasters or patches. Formulations are known in literature (Remington Remington“The Science and Practice of Pharmacy”, Lippincott Williams & Wilkins, 2000). The compounds of the present invention can be used for the treatment and/or prevention of the conditions mentioned above alone as sole therapy, in a combination among themselves or in combination with other therapeutic agents, either through separate administrations or including them in the same formulation. The compounds can be administered simultaneously or sequentially.

The combination can be administered as separate compositions (simultaneous, sequential) of individual components of treatment or as a single dosage form containing all agents. When the compounds 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 therefore provided as combined preparations, which are administered at the same time or at different times, or can be formulated together in preparation with two or more ingredients.

The compounds can be administered to a patient in a total daily dose of, for example, from 0,001 to 1000 mg/kg of body weight. The unit dosage compositions can contain submultiples of these quantities to compensate the daily dose. The compounds could also be administered weekly or once a day. The determination of the optimal dosages for a certain 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.

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. 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.5 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

(FOX01_HUMAN)(https://www.uniprot.org/uniprot/Q12778) - 043524

(FOX03_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 conserved DNA domain binding, FOX or forked box. In human, the family includes more than 100 members, 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 DNA binding motif. This motif is also known as“winged helix” due to the butterfly aspect that its pockets take.

The present invention will now be described in terms of non-limiting examples with reference to the following figures:

Fig. 1 Identification and characterization of two SIRTl activators. A The chemical structures of SCICl and SCIC2. B In vitro enzymatic assay to evaluate SCICl and SCIC2 capability to modulate SIRTl activity. Primary enzymatic assay was performed using library molecules at a fixed concentration of 10 mM, in comparison to the control without any modulator, to the known activator (STAC -2) [31] and to the known inhibitor (EX527). In this assay SCICl and SCIC2 percentage activity is 151.8% and 135.8%, respectively. C Counter-screening performed to evaluate if SCICl and SCIC2 at 10 pM modulate the activity of the nicotinamidase enzyme (NMase), in comparison to the control, where no modulator is present. Neither compound displays activity on NMase enzyme. D/E Dose curves (from 0.5 to 250 pM) for enzyme activity to evaluate AC50 of SCICl and SCIC2. AC50 value is plotted as the percentage of activity over the compound concentration (log), fit to a dose-response equation (variable-slope, this model does not assume a standard slope of -1-0 but fits another slope called variable slope).

Fig. 2 Interaction studies of SCICl and SCIC2 with the SIRTl enzyme by Surface Plasmon Resonance (SPR). Sensorgrams obtained from analysis of SPR interaction of EX-527, SCICl and SCIC2 with immobilized SIRTl. Each compound has been injected at five different concentrations (from 3.12 to 50 pM). Equilibrium dissociation constants (K D ) derive from the ratio between kinetic dissociation (kd) and association (ka) constants, obtained by analyzing data from all injections at different concentrations of each compound using BIAevaluation software. Fig. 3 Molecular modelling of the recognition between the STACs of invention and hSIRTl. A Binding of SCIC2 to the hSIRTl opened structure. The protein is represented with its NTD and CD while the ligand as spheres indicated by L. B Close-up view of the opened SCIC2 binding to the enzyme, as predicted by docking calculations. The protein is represented as stick and ribbons while the ligand as sticks. C Binding mode of SCIC2 to the hSIRTl closed conformation structure. The protein is represented as NTD and CD surface while the ligand as spheres (L). D Close-up view of the closed binding of SCIC2 to the enzyme, as predicted by docking calculations. The protein is represented as stick and ribbons while the ligand as grey sticks. E X- ray binding mode of resveratrol superimposed on the theoretical one of SCIC2.

Fig. 4 Identification and characterization of SCIC2.1, compound analogous to SCIC2. A The chemical structure of SCIC2.1 B In vitro enzymatic assay to evaluate SCIC2.1 capability to modulate SIRT1 activity. The enzymatic assay was performed using the molecules at a concentration of 10 mM, in comparison to the control without any modulator, to the known activator (STAC -2) [31] and to the known inhibitor (EX527). In this assay SCIC2.1 percentage activity is 175.1%. C Counter-screening performed to evaluate if SCIC2.1 at 10 pM modulates nicotinamidase (NMase) enzyme, in comparison to the control, where no modulator is present. The compound does not display activity on NMase. D Dose curve (from 0.5 to 250 pM) for enzyme activity to evaluate AC50 of SCIC2.1. AC50 value is plotted as the percentage of activity over the compound concentration (log), fit to a dose-response equation (variable-slope, this model does not assume a standard slope of -1-0 but fits another slope called variable slope).

Fig. 5 SCIC2 e SCIC2.1 directly bind SIRT1. Western blot for SIRTl and its relative quantization based on Cellular Thermal Shift Assay (CETSA). Cells were treated with SCIC2 at 50 pM and SCIC2.1 at 25 pM and an equal amount of DMSO for 1 h. The samples are divided into aliquots and heated at 4°C, 37°C, 47°C for 3 min. SIRTl primary antibody was used for Western blotting.

Fig. 6 SCIC2 and SCIC2.1 do not affect cell cycle of HepG2 and H9c2 cell lines. A, B FACS analysis showing cell cycle progression of HepG2 (A) and H9c2 (B) cells. C,D Cell death analysis of HepG2 (C) and H9c2 (D) cells. Cell cycle and cell death were acquired using ModFit LT v3 software (Verity) and Cell Quest Pro software (BD Biosciences), respectively.

Fig. 7 Effects of SCIC2 and SCIC2.1 on p53 acetylation. A/B Western blot analyses for p53 and p53K382/381ac from whole protein extracts of HepG2 and H9c2 cells treated with Doxo for 12 h, and for additional 6 h with SCIC2, SCIC2.1, and EX-527 at the indicated doses. ERKs were used as a loading control. Semi -quantitative analysis was performed using ImageJ software. Values are mean ± SD.

Fig. 8 Effect of SCIC2 and SCIC2.1 on the senescence in HepG2 and H9c2 cell systems. A/B HepG2 and H9c2 are treated with Doxo (0.5 pM) for 48 h to induce the senescence and the single compounds are administrated two times. Senescence-associated b-galactosidase (SA-b- gal) activity is then measured according to manufacturer’s protocol. Panels represent the ratio between positive cells and total cells.

Fig. 9 Schematic representation of SIRT1 enzymatic assay.

DETAILED DESCRIPTION OF THE INVENTION

METHODS

Enzyme purification

The SIRT1-GST (glutathione S-transf erase) enzyme was purified by Escherichia coli BL21 bacteria after transfection with pGEX-SIRTl (Addgene) plasmid. One selected bacterial colony was grown in LB broth medium (Lennox) supplemented with antibiotics (100 pg/mL ampicillin) in a shaking incubator overnight. When optical density was in a range between 0.6 and 0.8, protein expression was induced by i sopropyl -b-D- l -thi ogal actopyranosi de (IPTG; AppliChem) at a concentration of 200 pM for 5 h. The bacteria were centrifuged at 3000 rpm (Beckman centrifuge) for 20 minutes and the pellet was then lysed by sonication (Bioruptor; Diagenode) in lysis buffer containing phosphate buffered saline (PBS), 1 mM dithiothreitol (DTT) (Applichem), 0.5 mM phenylmethylsulfonyl fluoride (AppliChem), and 1 tablet of mini protease inhibitor cocktail (PIC) (Roche) for each 10 mL. Sonication was performed for 10 cycles of 45 seconds at 14,000 MHz with 30 seconds intervals between each sonication. Then, Triton X100 0.1% (Acros) was added and the solution was incubated for 15 minutes on ice. The sonicate was then centrifuged at 13,000 rpm for 30 minutes and filtered with a filter of 0.45 pm pore size.

The enzyme was purified using GSTrap 4B columns (GE Healthcare Life Sciences). The columns were equilibrated with 20 mL lysis buffer. Next, the lysate was loaded onto columns and subsequently they were washed with the lysis buffer.

The elution was carried out with 20 mL of elution buffer (EB) composed of 50 mM Tris-HCl pH 8.0, 1 mM DTT, 20 mM reduced L-glutathione (AppliChem) and ddH20. The SIRT1-GST protein was detected using colorimetric methods - Bradford protein assay (Biorad). The purified human SIRTl recombinant was dialyzed in a buffer solution composed of 50 mM Tris-HCl pH 8.0, 100 mM NaCl (Sigma), 1 mM DTT, 1 PIC tablet (for every lOmL) and H20dd overnight at 4 °C. The following day, the samples were cryopreserved in 20% glycerol (Sigma-Aldrich). Histidine-tagged nicotinamidase (NMase) enzyme was expressed in E. coli BL21 bacteria. Bacteria were grown in LB broth medium supplemented with antibiotics (100 pg/mL ampicillin) in a shaking incubator overnight at 37 °C. When optical density was 0.7, IPTG was added at 1 mM concentration for 5 h. The bacteria were centrifuged at 3000 rpm for 20 minutes and the pellet was then lysed using lysis buffer A (50 mM NaH2P04 pH 8.0, 300 mM NaCl, 10 mM imidazole (Applichem), 100 mg/50 mL lysozyme (Applichem) in H20, and 1 tablet of PIC for each 10 mL). The bacterial lysate was incubated on ice for 30 minutes and then sonicated for 10 cycles of 45 seconds at 14,000 MHz with 30 second intervals between each sonication. The lysate was then filtered with a filter of 0.45 mih pore size and incubated for 3 h at 4 °C with 1 mL nickel-NTA agarose resin (ABT) pre-equilibrated with equilibration buffer (EQ) (50 mM NaH2P04 pH 8.0, 300 mM NaCl, 20 mM imidazole in ddH20). Subsequently, the column was washed with EQ and the enzyme was eluted with a buffer composed of 50 mM NaH2P04 pH 8.0, 300 mM NaCl, 250 mM imidazole, and 1 tablet of PIC for each 10 mL, and the samples were cryopreserved in 20% glycerol.

SIRT1 enzymatic fluorescence assay in HST mode

The assay was performed in 384-well plates (Corning 384 flat bottom black polystyrene) with a reaction volume of 15 pL. Plates were pre-loaded with the reaction controls (positive/negative), STAC2 as activator, EX-527 (Selisistat; Sigma) as inhibitor, in the upper left and bottom right wells with a D300 Digital Dispenser (TEC AN) using HP’s Direct Digital Dispensing technology, and for each plate 160 compounds were tested. All compounds were dissolved in DMSO (Sigma- Aldrich). A control of DMSO with no modulator was also present.

The reaction buffer (PBS, ImM DTT, and 0.6% DMSO), compounds at 10 mM, SIRT1 at a dilution of 1 mg/mL and the Nmase enzyme mix (NMase-purified enzyme, b-NAD intermediate dilution at 1 mM, and acetylated peptide p53K382 intermediate dilution at 250 mM, synthesized by INBIOS), were placed in the automated dispenser.

Firstly, the automated platform generated compound dilutions (from 10 mM to 10 pM) and added SIRT1 -purified enzyme to the whole plate except for the negative control wells. After incubation for 15 minutes at 37 °C, the NMase enzyme mix was added and incubated for 40 minutes at 37 °C, after which the buffer developer (70% PBS, 30% ethanol, 10 mM DTT, and 10 mM OPT [Acros]) was added and incubated for 30 minutes in the dark. Fluorescence reading was performed using an Infinite M1000 microplate reader (TEC AN) at 420/460 nm.

The HTS platform enabled to obtain highly reproducible data in terms of z' and standard deviation. The compound was identified as a SIRTl activator when its activity was > 120% and as a SIRTl inhibitor when its activity was < 70%.

Counter-screening for NMase

The compounds were re-tested by fluorescence assay in the presence of NMase enzyme only to exclude the potential activity of the molecules on this enzyme. The assay was carried out in a 96- well plate with the same positive and negative reaction controls as the SIRTl fluorescence assay. Each compound was always tested at 10 mM and then 5 pL of intermediate compound dilution (50 mM) was added to 5 pL reaction buffer (that simulated SIRTl enzyme volume) and to NMase enzyme mix (NMase-purified enzyme, intermediate nicotinammide dilution (1 mM) and peptide p53 acetylated at K382). After 40 minutes of incubation at 37 °C, the developer buffer was added and the plate was again incubated in the dark for 30 minutes at 37 °C. Fluorescence was read at 420/460 nm using an Infinite Ml 000 reader.

Evaluation of intrinsic fluorescence

The compounds, resuspended in the reaction buffer, were placed in a 96-well black plate and incubated for 40 minutes at 37 °C. Fluorescence was read at 420/460 nm using an Infinite Ml 000 reader.

IC50/ AC50 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 (from 0.01 mM to 100 pM) into a 96-well plate using HP D300 Digital Dispenser technology (TECAN). The assay was conducted following the same conditions as the SIRT1 enzymatic fluorescence assay. AC50 and/or IC50 values were calculated using GraphPad Prism software (GraphPad Software).

Surface Plasmon Resonance (SPR)

SPR analyses were performed on a Biacore 3000 optical biosensor equipped with research-grade CM5 sensor chips (Biacore, cat. no. 29104988 by CYTIVA). Recombinant SIRT1-GST enzyme was immobilized (30 pg/ml enzyme in 10 mM sodium acetate, pH 4.5) at a flow rate of 10 pL/min using standard amine-coupling protocols to obtain densities of 8-9 kRU (kilo Resonance Units). All compounds were dissolved in DMSO (100%) to obtain 50 mM solutions and diluted in HBS-P (10 mM HEPES pH 7.4, 0.15 M NaCl, 0.005% surfactant P20), always maintaining a final DMSO concentration of 0.2%. Binding experiments were performed at 25°C by using a flow rate of 30 pL/min, with 60 s monitoring of association and 200 s monitoring of dissociation. Regeneration of the surfaces was performed, when necessary, by an injection of 10 mM NaOH. The simple 1 :1 Langmuir binding fit model of the BIAevaluation software was used for determining equilibrium dissociation constants (K D ) and kinetic dissociation (K d ) and association (K a ) constants by using the equation:

Where: K d =dissociation constant; K a = association constant; K D = equilibrium dissociation constant = K d /K a ; R = response unit; R eq = response at equilibrium; R max = maximal response; C = concentration of the analyte.

Molecular modelling methods SIRT1 enzyme structures (PDB ID: 4ZZH and 5BTR) were retrieved from the PDB. These structures were prepared for docking calculations with the Protein Preparation Wizard utility in Schrodinger’s Maestro suite. Specifically, using the“preprocess and analyse structure” tool, hydrogen atoms were added, every water molecule was removed, and bond orders and disulfide bonds were calculated. The Epik 2.0 program predicted the ionization of side chain hetero groups and tautomeric states while“H-Bond assignment” was used to optimize the hydrogen bonds. Lastly, minimization of the hydrogen atoms was carried out with the“impref utility”, while restraining the heavy atoms in place.

Subsequently, the structure of SCIC2 was built using the 2D Sketcher tool in Schrodinger’s Maestro suite. For the SCIC2 analogues, the .sdf file of the Mcule database was downloaded from the vendor’s website and then ligands were prepared with“LigPrep”, another Schrodinger utility. Specifically, every hydrogen atom was added and the tautomeric and ionization states were generated. Subsequently, the obtained ligands underwent minimization with the OPLS- 2005 force field. Then, the pairwise Tanimoto similarity between this database and SCIC2 was calculated using the“Canvas Similarity and Clustering” utility in Schrodinger’s Maestro suite. Subsequently, the latest version of the docking software AutoDock 4.2 (AD4) [32] in conjunction with the graphical user interface AutoDockTools (ADT) [32] was used to perform docking calculations.

Afterward, both the ligand and the receptor were converted to the PDBQT format. This format is very similar to a standard PDB file but includes“Q” (partial charges) and“T” (torsional angles) atom types. Each atom has one line, and special keywords are used if some are required to be flexible in the docking calculations. Preparing the structures involves ensuring that their atoms are assigned the correct atom types, adding Gasteiger charges if necessary, merging non-polar hydrogens, detecting aromatic carbons, and setting up the“torsion tree” in the case of ligands. Therefore, the python scripts prepare_receptor4.py and prepare_ligand4.py, part of ADT, were adopted applying the standard settings.

The docking area was centered on the enzyme active site. A set of grids of 60 A c 60 A c 60 A with 0.375 A spacing was calculated around the docking area for all the ligand atom types using AutoGrid4. For each ligand, 100 independent docking runs were calculated. For any single compounds, each docking run consisted of 10 million energy evaluations employing the Lamarckian genetic algorithm local search (GALS) method. This method creates a random population of feasible docking solutions and keeps the most successful individuals from each generation into the subsequent generation of feasible solutions. A low-frequency local search according to the method of Solis and Wets was applied to docking attempts to guarantee that the final solution represents a local minimum. All dockings were performed with a population size of 250, and 300 rounds of Solis and Wets local search were employed with a probability of 0.06. A mutation rate of 0.02 and a crossover rate of 0.8 were employed to create new docking attempts for following generations, and the best individual from each generation was propagated over the next generation. The docking results from each of the 100 calculations were clustered together on the basis of root-mean-square deviation (rmsd) (solutions differing by less than 2.0 A) between the Cartesian coordinates of the atoms and were ranked on the basis of the calculated free energy of binding (AGAD4).

Cellular and human serum stability

The reaction solutions were prepared by mixing 10 pL water solution (30% DMSO for SCIC2, 10% for SCIC2.1) of each compound (5 mM) and 90 pL cellular extract of HepG2 (at 0.5 mM concentration, 90% HepG2 cellular extract, 3% DMSO for SCIC2, 1% DMSO for SCIC2.1) or 90 pL human serum (at 0.5 mM concentration, 90% serum, 3% DMSO for SCIC2, 1% DMSO for SCIC2.1) and incubated at 37 °C. 10 pL aliquots were collected at different times (0, 15 min, 30 min, 60 min, 90 min, and 180 min), subjected to precipitation by addition of 20 pL acetonitrile (ACN)/0.1% trifluoracetic acid (TFA) solution, and then centrifuged (12,000 rpm, 15 min, 4 °C). The supernatant was recovered and analysed by RP-HPLC-ESI (LCMS 2020; Shimadzu) equipped with a Phenomenex Kinetex column (Cl 8, 150 mm x 4.6 mm, 5 pm, 100 A) with a flow rate of 1 mL/min, using a linear elution gradient from 10% to 90% acetonitrile/0.1% TFA in water in 20 min. The experiments were performed in triplicate.

Cell lines

Human hepatocellular carcinoma cells (HepG2; HB-8065) and rat cardio myoblasts (H9c2; CRL-1446) were purchased from the American Type Culture Collection (ATCC). Cell lines were tested and authenticated following the supplier’s instructions. All cell lines were maintained in an incubator at 37 °C and 5% C02. The cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Euroclone) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich) and antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin, 250 mg/mL amphotericin-B). HepG2 and H9c2 cells were incubated with DMSO or DOXO for 12 h. Then, cells were treated with or without SCIC2, SCIC2.1 and/or EX-527 for 3 h.

Reagents

SCIC2 (Enamine, cat. no. Z44566160) and SCIC2.1 (Enamine, cat. no. Z44459246) were used at 50 pM and 25 pM, respectively. Doxorubicin (Adriblastina; Pfizer) was used at 0.5 pM and EX- 527 (E7034; Sigma-Aldrich) at 10 pM. Propanolol (Sigma P0884 cat.318-98-9) and Furosemide (Sigma 257753 cat.54-31-9) were used at 100 pg/ml. Protein extraction and Western blotting

Cells were dissolved in lysis buffer containing protease inhibitors (10 mM Tris HC1 pH 8.0, 150 mM NaCl, 10 mM NaF, 1% NP40, 1 mM PMSF). The proteins were then subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.22 pm polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk in TBS-T (Tris buffer pH 8.0/0.15% Tween 20) at room temperature for 1 h and then incubated with primary antibodies diluted in TBS-T, including antibodies against p53 (Santa Cruz), p53K382ac (Abeam), SIRT1 (Abeam), p53K381ac (Abeam), and ERK (Santa Cruz), overnight at 4 °C. After three washes in TBS-T, the membranes were incubated with corresponding secondary antibodies (GE Healthcare), horseradish peroxidase-conjugated anti rabbit IgG (GE Healthcare) and horseradish peroxidase-conjugated anti-mouse IgG (GE Healthcare), for 1 h at room temperature. Immunocomplexes were visualized using Amersham EC Western Blotting Detection Reagents (GE Healthcare). The molecular weight of proteins was estimated with pre-stained protein markers (Fermentas). Densitometry analysis was performed using ImageJ software.

Cellular Thermal Shift Assay (CETSA)

HepG2 cells were harvested and washed with PBS after treatment with SCIC2 (50 pM) or SCIC2.1 (25 pM) and an equal amount of DMSO, as control, for 1 hour. The samples were suspended in PBS (1.5 mL), divided into aliquots (100 mL), and heated at different temperatures (4 °C, 37 °C, 47 °C, 54 °C, 57 °C, and 58 °C) for 3 minutes using Thermo Mixer (Eppendorf), followed by cooling for 3 minutes at 4 °C. After incubation, lysis buffer (100 mL) was added to the samples and incubated for 15 min. The samples were then centrifuged at 13,000 rpm for 30 minutes at 4 °C, the supernatant was removed, and protein concentration was determined by a Bradford assay (Bio-Rad). Of the total protein extract (30 pg) was loaded onto 10% SDS-PAGE, and Western blot analysis was performed. SIRTl (abl 10304 Abeam) was used as antibody. Densitometry analysis was performed using ImageJ software.

Permeability assay

Caco-2 cells (ATCC-HTB-37) were cultured in Modified Eagle Medium (DMEM) containing 10% FBS, 1% nonessential amino acids (Euroclone), 100 U/mL penicillin-streptomycin, and 2 mM L-glutamine. Cells were pre-incubated with culture medium for 1 h at 37 °C and then 20,000 cells were resuspended in 100 pL of complete DMEM and placed in the upper chamber (0.33 cm 2 per insert) on Transwell permeable inserts. Then, 600 pL of medium was added to the lower chamber of the Transwell system. The medium was changed only 24 h after seeding and then 3 times per week. Caco-2 monolayers were cultured for 21 days before use. When the monolayer was ready, cells were washed with PBS solution. Compounds SCIC2 and SCIC2.1 at 100 pg/ml, 100 mM Lucifer yellow (Sigma- Aldrich), negative control and propranolol both at 100 pg/ml were solubilized in PBS and 0.1% DMSO. Lucifer yellow is a fluorescent marker used to verify tight junction integrity during the assay. Transport assays were performed using 200 pL of apical donor solution and 600 pL of basolateral PBS acceptor solution. All compounds were tested in three replicate monolayers. Monolayers were incubated with all compounds at room temperature, shaking for 2 h. Concentration of Lucifer yellow was evaluated by Infinite M200 (TECAN) at 480/530 nm.

The other compound concentrations were evaluated by HPLC. Fluorescence was determined without any fluorescence interference. The permeability coefficient (Pa) of the compound after 2 h of incubation was calculated by the following equation:

where“Vd” and“Vr” are the donor (0.2 cm 3 ) and acceptor (0.6 cm 3 ) volume, respectively.“A” is the area of membrane (0.33 cm 2 ),“t” is the time of compound incubation, expressed in seconds (s or sec), and“r” is the ratio between the compound area in donor chamber and the compound area at equilibrium. The permeability percentage (P%) of molecules was calculated as: P% = r x 100

Experiments were performed in triplicate.

Cell cycle

For cell cycle analyses, HepG2 and H9c2 cells were plated (2 c 10 5 cells/mL) and after stimulation with the modulator (SCIC2 at 50 pM, SCI2.1 at 25 pM, EX-527 at 10 pM) harvested, centrifuged at 1200 rpm for 5 minutes and resuspended in 500 pL of a hypotonic solution containing IX PBS, 0.1% sodium citrate, 0.1% NP-40, 0.5 pg/ml RNase A and 50 mg/mL propidium iodide (PI). After 30 minutes at room temperature in the dark, samples were acquired by FACS-Calibur (BD Biosciences, San Jose, CA, USA) using Cell Quest Pro software (BD Biosciences). The percentage in different phases of the cell cycle was determined by ModFit LT v3 software (Verity). Cell death was measured as a percentage of cells in pre-Gl phase. All experiments were performed in triplicate.

Senescence assay

Senescence was assessed by b-gal staining. HepG2 and H9c2 cells were incubated with Doxo for 48 h and single compounds were administered every 24 h (i.e. two administrations) b-gal senescence assay was performed using Senescence Cells Histochemical Staining Kit (Sigma cod. CS0030) according to the manufacturer’s instructions. Staining was visualized and captured using an optical microscope (Carl Zeiss). Statistical analysis

Data are presented as the mean ± SD of biological triplicates. Differences between the treatment groups and controls were compared using one-way analysis of variance (ANOVA) and Dunnetf s multiple-comparison test. Differences between groups were considered significant at a p value < 0.05. Statistical analyses were performed using GraphPad Prism 6.0 software (GraphPad Software, Inc., San Diego,CA).

EXAMPLES

EXAMPLE 1. Identification of new STACs by HTS

To identify novel SIRT1 modulators, a library of 30,000 natural and synthetic compounds was screened. An in vitro fluorescence assay was developed by the inventors and was optimized in HTS mode by using a TEC AN robotic station. This assay correlates SIRT1 deacetylase activity with production (and following quantification) of ammonia by coupling two reactions catalyzed by SIRT1 and nicotinamidase (NMase) (see Material and methods and Fig. 9). The library in question was created using various computational methods such as the study of a 3D pharmacophore and 2D fingerprinting.

All the 30,000 compounds were first tested at 10 mM concentration and about 150 molecules were pre-selected (data not shown). A compound was considered to be a SIRT1 activator when its activity was > 120% and as SIRTl inhibitor when its activity was < 70%. The enzymatic activity expressed as a percentage is calculated as the ratio between the fluorescence value of each compound (420 nm - 460 nm) and that of the control, treated with the DMSO vehicle ([Fluo cmpd]/[Fluo Ctrl]* 100).

Having confirmed its effect, each hit was subjected to intrinsic fluorescence (to exclude false positives) and counter-screening (to exclude NMase modulators) assays. Around 30 compounds were consequently selected as true SIRTl modulators and a concentration-response curve for AC50/IC50 determination was performed for each one. The entire screening process identified SCICl and SCIC2 as promising SIRTl activators (Fig. la).

Both compounds showed higher enzymatic activity on SIRTl than the reference activator with enzymatic activity of 151.8% and 135.8% for SCICl and SCIC2, respectively (Fig. lb). No NMase modulation was observed for either compound (Fig. lc). As reported in Fig. Id and le AC 50 values of SCICl and SCIC2 are 55 ± 0.33 mM and 50 ± 1.8 pM, respectively. Therefore, SCIC2 was further investigated.

EXAMPLE 2. Characterization of SIRTl-molecules interaction

To assess whether SCICl and SCIC2-mediated SIRTl activation was due to a direct enzyme/compound interaction, a SPR (surface plasmon resonance) analysis was performed (Fig. 2). In SPR, SIRT1 was immobilized on a sensor chip with gold surface, which was then exposed to the two molecules in flow phase. Compound binding induced a change in the refractive index on the sensor surface. The ability of the drugs to bind SIRTl was defined by its dissociation constant (K D ) [33]

Fig. 2 shows SPR response data obtained for a series of SCICl and SCIC2 injections from 50 mM to 3.12 mM concentration. The kinetic affinity constants of the molecules were compared to the K D values of EX-527, used as a positive control [34] due to its direct binding properties at the SIRTl catalytic domain. The present findings show that both molecules were able to bind SIRTl, but SCIC2 binding was stronger than that of SCICl, with a K D of 26.4 ± 0.6 pM vs 46 ± 3 pM.

EXAMPLE 3. Molecular modelling calculations for the SIRTl-compounds binding

To gain a greater insight into the binding interactions between SCIC2 and the hSIRTl, molecular modelling calculations were performed. The crystal structure of human SIRTl in complex with a STAC was recently reported [35] Given the intrinsic difficulties in crystallizing hSIRTl, in the above-referenced study the authors developed a less flexible construct containing the minimal structural elements required for maintaining the enzymatic activity of the wild-type enzyme as well as the capability of being inhibited and activated by small molecules. With this protein model, the authors demonstrated [35] that the STAC co-crystal is able to make direct contact with the N-terminal domain (NTD) of the enzyme. In this position, the ligand occupies a shallow pocket where hydrophobic and polar contacts mediate ligand-protein recognition. Of note, the NTD in this structure is distant from the catalytic domain (CD).

In another structural study [36], the three-dimensional conformation (3D) of a hSIRTl construct in complex with the resveratrol STAC ligand and a 7-amino-4-methylcoumarin (AMC)- containing peptide was solved. Here, resveratrol was sandwiched between the AMC moiety and the NTD domain, which is now located close to the rest of the protein. Regardless of the different experimental conformations as well as protein constructs used, both these studies propose a shared mechanism of action for hSIRTl activation in which the ligand induces a conformational transition bringing the NTD and CD close to each other, thereby enhancing binding to the enzyme substrate. In view of these data, the inventors speculated whether the compound SCIC2 might be able to positively interact with the hSIRTl allosteric binding site and induce such a transition. To this end, molecular docking simulations were performed using both the above-described X-ray structures with PDB codes 4ZZH (open and inactive state) and 5BTR (closed and activated state). Analysis of the docking results obtained using the inactive state structure revealed that SCIC2 is able to make several contacts with residues lining the NTD shallow pocket of SIRT1 (Fig. 3a). Specifically, the coumarin core structure is able to engage in H-bond interactions with the N226 side chain as well as with several hydrophobic contacts with 1223 and 1227 (Fig. 3b). Interestingly, the negatively charged oxygen atom in position 7 of the coumarin scaffold points towards the E230 residue. This residue is reported to be critical for SIRT1 allosteric activation [31] mediated by many STACs. Noteworthy, analysis of the X-ray structure reveals that the STAC co-crystal also places an aromatic electron-rich ring (oxazole) in proximity to the negatively charged E230 residue, while the pendant benzyl(methyl)aminom ethyl chain in position 4 of the coumarin core makes favorable contact with LI 06, P211, and L215 chains. When docking was attempted on the activated state structure (5BTR), SCIC2 was predicted to interact at the interface between NTD and CD regions (Fig. 3c). In the activated hSIRTl conformation, the ligand makes contact with the hydrophobic residues of the NTD (L202, L206, and 1223), while the benzyl moiety points towards the E230 side chain (Fig. 3d).

Of note, in this conformation, the latter residue forms an ionic interaction with the R446 side chain belonging to the hSIRTl CD. Importantly, such an interaction was postulated to stabilize the activated conformation of hSIRTl. Comparison of the achieved binding pose with the crystal structure of hSIRTl and resveratrol shows that the two ligands occupy the same enzyme region, supporting the idea that both ligands might activate the enzyme through the same mechanism of action (Fig. 3e).

Taken together, molecular modelling studies suggest that the NTD/SCIC2 recognition event may trigger stabilization of a closed/activated conformation of hSIRTl, which is more conductive to enzymatic catalysis. In this respect, it could be postulated that the unfavorable electrostatic interactions between SCIC2 and the adjacent E230 residue would shift the protein conformational equilibrium to the closed state in which the E230-R446 ionic interaction anchors the NTD and CD in close proximity. Here the ligand, just like resveratrol, should be able to stabilize the enzyme/substrate interactions thereby enhancing the hSIRTl processivity.

EXAMPLE 4. SCIC2 lead optimization

In an attempt to potentiate the promising capacity of SCIC2 to activate hSIRTl, additional molecular modelling studies were carried out. SCIC2 has a coumarin scaffold. Coumarin is a natural chemotype present in several compounds with different pharmacological activities (anticoagulant, CNS-active, anti -HIV, antitumor, and anti-inflammatory) [37] Because of the number of its documented pharmacological activities and its amenability to combinatorial chemistry, the coumarin scaffold is a privileged structure in medicinal chemistry. Given the high synthetic accessibility of coumarins, the inventors first searched for SCIC2 structural analogues in commercial molecular databases. This strategy has the benefit of quickly delivering a database of structural congeners of the lead compound with relative cost-efficiency. Next, the simple pairwise Tanimoto similarity score [38] was computed between SCIC2 and the compounds in the Mcule Purchasable database (In Stock) (https://mcule.com/database/) (~9 million compounds) using a 70% similarity threshold. This resulted in 2,500 compounds that were docked into the SIRT1 closed structure employing the same protocol used to dock SCIC2 binding. The best compounds in terms of predicted binding affinity were subsequently purchased as pure substances and analysed by enzymatic assay in HTS mode. From this screening, SCIC2.1 was identified as an active SCIC2 derivative (Fig. 4a). SCIC2.1 strongly activated SIRT1, inducing higher enzymatic activity than SCIC2 (175% vs 135.8%;) (Fig. 4b). No NMase modulation was observed (Fig. 4c). As reported in Fig. 4d, SCIC2.1 activity occurred, with an AC 50 value of 36.83 mM.

EXAMPLE 5. SCIC2 and SCIC2.1 directly bind SIRT1 in cells

To further support the molecular modelling calculations and to investigate the interaction between the compounds and SIRT1 in a physiological cellular environment, Cellular Thermal Shift Assay (CETSA) was performed in HepG2 cells. Compared to the control (DMSO), both SCIC2 and SCIC2.1 protected SIRT1 from thermal degradation in protein extract of HepG2 cells treated with the drugs. SIRTl signal remained at the highest temperature of 47 °C, while no signal was detected in the DMSO control extract (Fig. 5). Of note, SCIC2.1 protected SIRTl better than SCIC2.

These results confirm a physical interaction between the two molecules and SIRTl.

EXAMPLE 6. SCIC2 and SCIC2.1 are stable compounds and cross cell membrane

Cell permeability and stability can be major concerns for drug candidates interacting with cytoplasmic targets, hampering in many cases their full biological capitalization. In light of this consideration, the inventors investigated the cell permeability in Caco-2 cell line and metabolic stability in both HepG2 cellular lysate and human serum. The cell-based studies allow highlighting potential drawbacks of SCIC2 and SCIC2.1, in terms of low cytoplasmic permeability and/or stability that could lead to misinterpretation of the outcome of subsequent cellular experiments. Moreover, inventors also preliminary assessed the serum stability of the two compounds to gather information that could assist in setting up future in vivo experiments. Specifically, for the cell permeability assay, Caco-2, which is an intestinal cancer cell line that under specific culture conditions becomes differentiated and polarized, resembling the epithelial cells of human intestine and representing a suitable in vitro model to study the permeability of drugs [9] [39], was used. The present results demonstrated that both compounds were able to cross the Caco-2 cell monolayer. Particularly, after 3 h of treatment, SCIC2.1 showed a higher permeability than SCIC2 with values of 25% for SCIC2.1 and 18% for SCIC2 (Table 1). Propanolol and furosemide were used as technical controls.

Table 1. In vitro evaluation of SCIC2 and SCIC2.1 permeability

Next, stability experiments in cell-medium and human serum (HS) were carried out by modifying a previously described protocol [40] Briefly, a 5 mM water solution (3% DMSO) of SCIC2 and SCIC2.1 was diluted with 90% HepG2 cellular medium and at different time intervals, aliquots of the mixture were collected, treated with 0.1% trifluoroacetic acid (TFA) acetonitrile solution to precipitate the proteins, analysed by ESI-RP-HPLC (see Methods for “Cellular and human serum stability”). As reported in table 2, SCIC2 and its analogue SCIC2.1 remained substantially unaltered up to 180 minutes (maximum time of observation) in both HepG2 cell medium and HS. Taken together, these results encourage the employment of the compounds in all the subsequent cell-based experiments, aimed at verifying the effects of the interaction between SCIC2/SCIC2.1 and SIRT1.

Table 2. HepG2 cell medium and Human Serum stability profiles of SCIC2 and 2.1 after different intervals of incubation. Relative concentrations and identity of the compounds were determined by integration of the A220 peaks from ESI-RP-HPLC

in)

SCIC 2 13.1 348.08 347.95 > 99 > 99 > 99 > 99 > 99 > 99

HepG2

SCIC2.1 11.5 314.12 314.10 > 99 > 99 > 99 > 99 > 99 > 99 HS SCIC 2 13.1 348.08 347.95 > 99 > 99 > 99 > 99 > 99 > 99

SCIC2.1 11.5 314.12 314.10 > 99 > 99 > 99 > 99 > 99 > 99 EXAMPLE 7. SCIC2 and SCIC2.1 do not affect cell growth

After molecular characterization, the cytotoxic effects of the two compounds were tested in both normal and cancer cell lines. Cardiomyoblast H9c2 cells and hepatocellular carcinoma cells HepG2 cells were left untreated or treated for 24 h with SCIC2 at 50 mM, SCIC2.1 at 25 pM, or the well-characterized SIRTi EX-527 at 10 pM. After incubation, cell cycle progression was determined by FACS analysis. Compared to untreated cells, compounds did not significantly affect cell distribution over cell cycle phases (Fig. 6a and b). Furthermore, neither molecule was able to induce cell death, expressed as a percentage of cells in pre-Gl phase.

These results show that SCIC2 and SCIC2.1 did not affect cancer and normal cell growth.

EXAMPLE 8. SCIC2 and SCIC2.1 potentiate SIRTI effects in stress response

Many genotoxic stresses are reported to enhance acetylation of p53 in its C-terminal region, increasing its activity and thus leading to cell growth arrest. SIRTI is able to deacetylate p53, attenuating p53 transcription-dependent apoptosis upon DNA damage and oxidative stress. To study the role of SCIC2 and SCIC2.1 in modulating SIRTI -mediated functions in stress responses, the deacetylation activity of SIRTI on p53 was investigated. Western blotting analyses were performed on HepG2 cells pre-treated with the genotoxic drug doxorubicin (DOXO or Doxo) at 0.5 pM for 12 h, and then with SCIC2 or SCIC2.1 at 50 pM and 25 pM and/or with EX-527 at 10 pM for 6 h. The effect of the molecules on SIRTI -deacetylating activity was monitored by following the signal of p53 acetylated at K382 (p53K382ac) (Fig. 7a). The data confirmed that SCIC2 and SCIC2.1 played an opposite role to EX-527 on acetylation status of p53 in regulating DNA damage. Specifically, SCIC2 and SCIC2.1 decreased DOXO- mediated p53K382ac increase, suggesting their potential role in enhancing SIRTI -dependent p53 deacetylation. Interestingly, both drugs, though SCIC2.1 more effectively, competed with EX-527 in binding to SIRTI, as shown in cells treated with all three compounds. Via SIRTI activation, the two drugs strongly attenuated the effects of DNA damage. In the same experimental conditions, p53K381ac resulted decreased in H9c2 cells, corroborating this hypothesis. EX-527-mediated SIRTI inactivation exerted the opposite effect, increasing p53 acetylation. This EX-527-induced effect was again attenuated by the co-presence of SCIC2 and SCIC2.1 in the triple treatment (Fig. 7b).

EXAMPLE 9. SCIC2 and SCIC2.1 attenuate cellular senescence

Increasing evidence correlates senescence and aging with sirtuins. Specifically, down-regulation of sirtuins induces early cell senescence and accelerates aging processes [41]

Therefore, senescence-associated b-galactosidase (SA b-gal) activity was quantified to assess the effect of SCIC2 and SCIC2.1 in HepG2 and H9c2 cell lines. Briefly, the cells were induced to senescence by co-treatment with DOXO at 0.5 mM, and SCIC2/SCIC2.1. After 48 h, the percentage of SA b-gal -positive cells was quantified for each condition. As show in Fig. 8, the presence of DOXO induced about 60% and 35% of SA b-gal -positive in HepG2 and H9c2 cells, respectively. The addition of SCIC2 and SCIC2.1 led to a reduction of ~20% and of 15% in the number of SA b-gal-positive HepG2 and H9c2 cells, respectively. As a good SIRTli, EX-527 did not affect levels of SA b-gal. Taken together, these data show that SCIC2 and SCIC2.1 may attenuate the induction of senescence.

Discussion

A growing body of evidence suggests that SIRTs play a key role in modulating several cellular mechanisms [42]

The acetylation state of numerous SIRT substrates is associated with lifespan-related diseases such as diabetes, metabolic syndrome, cancer, inflammation, and neurodegenerative disorder [43, 44]

Major efforts are currently focusing on SIRTl, the most studied SIRT [45] which is involved in regulating metabolic homeostasis in several tissues [12] in various cell processes such as stress response [46] apoptosis [47] and gene stability [48], and in human diseases including neurodegenerative disorders, cancer, and cardiovascular disease [49, 50]

SIRTl has been suggested as an anti-aging protein and its up-regulation leads to health benefits in different organisms. Scientific interest is therefore increasingly focused on SIRTl as a potential therapeutic target. Several scientific investigations are aimed at increasing SIRTl activity to delay the onset of aging and age-associated diseases. Although resveratrol remains the most well-characterized first-generation STAC, its therapeutic role in degenerative and metabolic diseases is still controversial. Hence, the scientific challenge is to discover and characterize novel STACs to improve quality of life, reduce age-associated diseases, and extend lifespan [14]

Here, by HTS of a large library of compounds, SCIC2 was identified as a potent SIRTl activator. This small molecule showed enzymatic activity of 135.8%, an AC50 value of 50 ± 1.8 mM, and bound SIRTl with a K D of 26.4 ± 0.6 mM. Molecular modelling studies showed that the NTD/SCIC2 recognition event presumably triggers the stabilization of a closed/activated conformation of SIRTl, which is more conducive to enzymatic catalysis. It is also tempting to speculate that the unfavourable electrostatic interactions between SCIC2 and the adjacent E230 residue, detected in the theoretical model, might shift the protein conformational equilibrium to the closed state in which the E230-R446 ionic interaction anchors NTD and CD in close proximity. Here, like resveratrol, the ligand could be able to stabilize enzyme/substrate interactions, thereby enhancing SIRT1 processivity (Fig. 3).

In order to potentiate its SIRT1 -activating capability, SCIC2 was subjected to modelling studies, leading to the identification of a more potent derivative, SCIC2.1. SCIC2.1 displayed higher SIRT1 activity (175%; AC50 = 36.83 ± 2.23 mM) and stronger binding to SIRTl than SCIC2. SCIC2.1 also showed greater cell permeability than SCIC2, probably due to the presence of different groups, as the absence of a chlorine atom makes SCIC2.1 negatively charged and not a zwitterion like SCIC2. At cellular level, both molecules did not alter the cell cycle progression of cancer cells and normal cells (Fig. 6) and potentiated SIRTl -mediated effects in stress response (Fig. 7). Finally, SCIC2 and SCIC2.1 attenuated senescence induction by reducing SA b-gal activity (Fig. 8).

Taken together, the present results identify SCIC2 and SCIC2.1 as promising SIRTl activators, making useful in therapeutic field.

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