Biochemical mechanism and biological effects of the inhibition of silent information regulator 1 (SIRT1) by EX-527 (SEN0014196 or selisistat)

Figures & data

Figure 1. Structures of SIRT1 inhibitors EX-527 and its analogue Compound 35, indicating their absolute stereochemistry and the corresponding names used in the literature¹⁵. EX-527 and CHIC-35 are now commercially available from suppliers.

Scheme 1. Chemical synthesis of EX-527¹⁵.

Table 1. In vitro assays of EX-527 and its analogue 35 on isolated recombinant sirtuins expressed in bacteria.

Compd	SIRT1	SIRT2	Other sirtuins	References
EX-527	0.098^a	19.6^a	SIRT3: 48.7^a	Napper et al.^15^,^c
	1.29^b
	0.038^a^,^d		SIRT5: > 50 µM^e	Solomon et al.^16
	3 [1–5]^f	79 [45–140]^g		Huhtiniemi et al.^17
	0.165 ± 0.050^h			Liu et al.^18
	0.125 ± 0.021^i
	0.74 ± 0.25^j			Smith et al.^19
	1.18 ± 0.24^k
	0.38^a	32.6^l		Peck et al.^20
	0.16 ± 0.01^a	> 10 (∼35% at 0.5 µM)^l		Pasco et al.^21
	0.16 ± 0.01^a	48.5 ± 15.2^l		Rotili et al.^22
	83.6 ± 4.2% at 50 µM^a	45.5 ± 2.8% at 50 µM^l		Mellini et al.^23
	0.26^m	2.9^m	SIRT3: > 50^m	Disch et al.^24
	0.09 ± 0.03^n		SIRT3: 22.4 ± 2.7^o	Gertz et al.^25
			Sir2Tm: 0.90 ± 0.30^n^,^p
			SIRT5: > 25 µM^q
			SIRT6: 56 ± 8% at 200 µM^r	Kokkonen et al.^26
	0.33 ± 0.03^s			Yang et al.^27
	0.5^t	6.5^t		Therrien et al.^28
	0.10 [0.05–0.19]^u	3.0 [2.1–4.4]^u	SIRT3: 165 [63–430]^u	Ekblad et al.^29
			SIRT6: 107 [48–240]^u
	0.1 ± 0.06^a	20.1 ± 4.2^a		Schnekenburger et al.^30
EX-243	0.123^a			Napper et al.^15
EX-242	> 100^a
Rac-35	0.124^a	2.77^a	SIRT3: > 100^a
	0.652^b
(S)-35	0.063^a
(R)-35	23.0^a

IC₅₀ values are given in µM (with errors as published) and/or %inhibition is indicated at the given concentration. This table constitutes an overview of representative data in the literature. It is important to note that only IC₅₀ values from assays performed under the same experimental conditions are comparable.

^a Fluorimetric assay using a peptide substrate derived from the sequence of p53 (K382): Ac-RHKK(Ac)-AMC (AMC = 7-amino-4-methyl-coumarin).

^b Radioactive nicotinamide release assay using unlabelled 19-aminoacid peptide substrate.

^c SEM (standard error of the mean) < 30% for all data in this article.

^d SIRT1 expressed and purified from mammalian cells.

^e Release of [³H]acetate from acetylated cytochrome c.

^f Radioactive nicotinamide release assay using a peptide substrate derived from the sequence of p53 (K382): Ac-RHKK(Ac)-AMC.

^g Radioactive nicotinamide release assay using a peptide substrate derived from the sequence of p53 (K330): Ac-QPKK(Ac)-AMC.

^h Microfluidic mobility shift assay using a labelled peptide substrate derived from the sequence of p53 (K382): fluorescein-SKKGQSTSRHKK(Ac)LMFKTEGPDS.

ⁱ NAD⁺ bioluminescence assay using a peptide substrate derived from the sequence of p53 (K382): HLKSKKGQSTSRHKK(Ac)LMFK.

^j Enzyme-coupled system detecting nicotinamide formation, using a peptide substrate derived from the sequence of histone H3 (K14) named AcH3: KSTGGK(Ac)APRKQ.

^k Charcoal-binding assay using [³H]AcH3.

^l Fluorimetric assay using a peptide substrate derived from the sequence of p53 (K330): Ac-QPKK(Ac)-AMC.

^m Mass spectrometry assay using the peptide substrate derived from the sequence of p53 (K382): Ac-RHKK(Ac)W-NH₂.

ⁿ Enzyme-coupled system detecting nicotinamide formation, using a peptide substrate derived from the sequence of p53 (K382): RHKK(Ac)LMFK.

^o Enzyme-coupled system detecting nicotinamide formation, using a peptide substrate derived from the sequence of acetyl-CoA synthetase 2 (ACS2, K642): TRSGK(Ac)VMRRL.

^p Sir2Tm: sirtuin from Thermotoga maritima.

^q Enzyme-coupled system detecting nicotinamide formation, using a peptide substrate derived from the sequence of carbamoyl phosphate synthetase 1 (CPS1, K527): FKRGVLK(Ac)EYGVKV.

^r Fluorimetric assay using a peptide substrate derived from the sequence of histone H3 (K56): Ac-RYQK(Ac)-AMC.

^s Luminescence assay using a peptide substrate derived from the sequence of p53 (K330): Z-QPK(Me)₂K(Ac)-aminoluciferin.

^t Fluorometric assay using the substrate Cbz-K(Ac)-AMC.

^u Fluorometric assay kits, undisclosed substrates.

Scheme 2. Spontaneous hydrolysis of the DMF adduct of EX-527.

Figure 2. (A) Mechanism of sirtuin-catalysed deacetylation of a peptide (or protein) substrate Ac-Pep (acetylated peptide). For simplicity, acidic and basic general catalysis is not represented in this mechanism. (B) Proposed simplified mechanism of sirtuin inhibition by EX-243, adapted from Gertz et al.²⁵. E: enzyme. Note that former studies of SIRT1 inhibition by substrate analogues suggested (i) a random addition of substrates (therefore, Ac-Pep could be added first to the enzyme, not represented here for simplification) and (ii) a departure of the peptide product from the enzyme in the last step (which would disagree here with the existence of the crystallised complex E/2′-O-AcADPr/EX-243)³⁹.

Table 2. Binding parameters of EX-527 with sirtuins.

Sirtuin	Ki (Ac-Pep)	Ki (NAD^+)	Kd (apo)	Kd [Ac-Pep]	Kd [NAD^+]	Kd (Ac-Pep + NAD^+)	References
SIRT1	0.408^a Mixed	0.287^a Mixed/uncompetitive	Not binding^b		1.3^b [1 mM]		Napper et al.^15 Zhao et al.^31
Sir2Tm	1.8 ± 0.4^c Non-competitive	3.3 ± 0.4^c Uncompetitive	>180^e	>170^e [1 mM]	6.0 ± 0.4^e [5 mM]	4.9 ± 0.5^e	Gertz et al.^25
SIRT3	33.4 ± 4.4^d Non-competitive	31.3 ± 2.1^d Uncompetitive	>330^e	>180^e [1 mM]	16.5 ± 2.9^e [5 mM]	10.0 ± 1.4^e

K_i and K_d values are given in µM (Ac-pep: acetylated peptide).

^a Fluorimetric assay using a peptide substrate derived from the sequence of p53 (K382): Ac-RHKK(Ac)-AMC (AMC = 7-amino-4-methyl-coumarin).

^b Determined by SPR.

^c Enzyme-coupled system detecting nicotinamide formation, using a peptide substrate derived from the sequence of p53 (K382): RHKK(Ac)LMFK.

^d Enzyme-coupled system detecting nicotinamide formation, using a peptide substrate derived from the sequence of acetyl-CoA synthetase 2 (ACS2, K642): TRSGK(Ac)VMRRL.

^e K_d values determined using microscale thermophoresis.

Figure 3. Crystal structures of sirtuins in complex with indole inhibitors EX-243 and its analogue (S)-35. Left: SIRT1/NAD⁺/(S)-35 (4I5I)³¹; middle: SIRT3/ADPr/EX-243 (4BVB); right: Sir2Tm/2′-O-AcADPr/deacetyl p53 peptide/EX-243 (4BV2)²⁵. Active site close-up representations are displayed below the full structures. Pep: deacetyl p53 peptide.

Table 3. Representative examples of cellular effects of EX-527.

Cell lines^a	Added agent	Effect of EX-527 on cells	Effect of EX-527 at the protein level	Comments	References
NCI-H460 U-2 OS MCF-7 HMEC	Etoposide, adriamycin, hydroxyurea, or hydrogen peroxide	No effect at 1 µM	Increases p53 acetylation (K382) at 1 µM (but no effect on two specific p53 target genes)	No effect on p53 without the genotoxic agent − 1 µM is non-toxic to all cell lines	Solomon et al.^16
HCT-116	5-FU or camptothecin	Decreases cell proliferation and increases apoptosis at 2 µM	–	Increases cell proliferation at 2 µM, without the chemotherapy agent (and under growth factor deprivation)	Kabra et al.^40
MCF-7	None	Decreases proliferation at 50–100 µM	No apparent increase in p53 acetylation, but global increase in lysine acetylation of proteins	Causes cell cycle arrest at G1 phase at 50 µM	Peck et al.^20
U937	None	No cytotoxicity up to 50 µM ∼10 % apoptosis induction at 50 µM	–	No effect on granulocytic differentiation at 50 µM	Rotili at al. ^22
Primary AML Primary B-CLL U937 697 Jurkat	Valproic acid (VA): HDAC class I/II/IV inhibitor	Synergistic effect with VA (100 µg/mL): ∼60% leukaemia cell death at 75 µM	Effect through Bax: in Jurkat with increased Bax expression, ∼70% leukaemia cell death at 75 µM (even without VA)	Low cytotoxic activity in leukaemia cells without VA	Cea et al.^41
SGC transfected with ATF4 (induces MDR effects)	5-FU or cisplatin	Increases the cytotoxicity of 5-FU and cisplatin at 10 µM (synergistic effect)	Downregulates MDR1 expression	Slightly increases the viability at 10 µM without the cytotoxic agent	Zhu et al.^42
MCF-7 U937	None	Cell cycle arrest in the G1 phase (no apoptosis) at 50 µM	At 10 µM, increases p53 and α-tubulin acetylation	No effect on granulocytic differentiation at 50 µM	Mellini et al.^23
CSC: CRC (CRO and 1.1) GBM (30P and 30PT)	None	Weak inhibition of cell viability at 50 µM (up to 20%)	–	In combination with SIRT2 inhibitor AGK2, slight synergic effect proposed	Rotili et al.^43
HCT-116	None	–	At 10 µM, increases p53 acetylation	Ratio (Ac-p53 / total p53) = 0.27 vs control = 0.03	Suzuki et al.^44
BMDMs	LPS-induced production of cytokines	At 4 µM, no effect on cytokine production by macrophages	–	No effect at 120 µM or in combination with SIRT2-selective inhibitors	Lugrin et al.^45
HCC (HepG2)	Trichostatin (TSA): HDAC inhibitor	–	At 20 µM: increases p53 acetylation decreases NAMPT enzymatic activity and increases its extracellular levels	–	Schuster et al.^46
PC-12 expressing mHtt	None	Rescues ∼35% mHtt mediated toxicity at 1 µM (but only ∼25% at 10 µM)	Increases mHtt acetylation and clearance	Protective effect in primary cultures of rat striatal neurons infected with viral vectors expressing a mHtt fragment	Smith et al.^47
SH-SY5Y	None	At 3 µM, restores viability in neuronal cells carrying a G93A SOD1 mutant (ALS-linked mutation)	No increase in p53 acetylation	The authors propose that the observed effects do not come from SIRT1 inhibition	Valle et al.^48
HUVEC	H2O2	At 15 µM, protects against H2O2: Increases cell viability, adhesion, migratory ability Decreases the apoptotic index and ROS production	Reverses H2O2 effects: Decreases SIRT1, p-JNK, p-p38MAPK and increases p-ERK expression	No effect on HUVEC untreated by H2O2	Li et al.^49
PANC-1 BXPC-3 ASPC-1	Gemcitabine or cisplatin	At 1 µM, increases the cytotoxic and pro-apoptotic effects of gemcitabine and cisplatin	At 2 µM, increases p53 acetylation and FOXO3a expression	Pro-apoptotic and anti-proliferative effects also without the cytotoxic agent (IC50 values 5 to 9 µM)	Zhang et al.^50
TNBC MDA-MB-231 BT-549	None	Decreases viability by 20% at 50 µM	At 25 µM, increases p53 acetylation (K382)	Additional complex interplay with AMPK and metadherin studied	Gollavilli et al.^51
CSCs: CD44^high CML K562 CD44^+ HCT-15	Hsp90 inhibitors: 17-AAG and AUY922	At 10 nM, increases the cytotoxicity of Hsp90 inhibitors	Involvement of HSF1 and MDR related molecules proposed	–	Kim et al.^52
CEM/VLB100 MCF7-MDR (MDR variants)	Hsp90 inhibitors: 17-AAG and AUY922	At 10 nM, increases the cytotoxicity of Hsp90 inhibitors (synergistic effect demonstrated)	At 50 nM: Decreases 17-AAG induced expression of Hsp70/Hsp27 Increases 17-AAG induced downregulation of mut p53 and P-gp Decreases P-gp efflux activity with AUY922	Decreases P-gp efflux activity also without AUY922	Kim et al.^53
HCC (HepG2)	H2O2	–	At 10 µM, aggravates H2O2 induced: Decrease in MnSOD and Bcl-xL Increase in cleaved caspase 3	–	Hu et al.^54
HHUA, HHUA-SIRT1, HEC151 and HEC1B	Cisplatin	At 1 µM, inhibits the proliferation with a synergic effect with cisplatin	Independent of p53 mutation status	Inhibits the proliferation at 1 µM also without cisplatin	Asaka et al.^55
Human platelets	None	At 10 µM, induces apoptosis-like changes: enhances annexin V binding, ROS production and drop in mitochondrial transmembrane potential	Increases p53 acetylation and the level of conformationally active Bax	–	Kumari et al.^56
Naïve CD4 T cells	None	At 12.5 µM, decreases Th17 effector cells differentiation from CD4 T cells	SIRT1 deacetylates RORγt and increases its transcriptional activity	–	Lim et al.^57
HeLa	None	At 10 µM, decreases colony formation (> 50 %) and migration At 50 µM, causes cell cycle arrest in the G1 phase (no apoptosis)	Increases HSF1 acetylation, ubiquitination, and degradation Causes G1 phase arrest mediated by inhibition of Cdk4, Cdk6 and cyclin D1	–	Kim et al.^58
Pluripotent P19 cells (mouse embryonic carcinoma)	None	At 100 µM, accelerates the differentiation of P19 cells into functionally active neurons	Identification of neuron-specific proteins and glutamate receptor in differentiated neurons	–	Kim et al.^59
A549	MK-1775: WEE1 kinase inhibitor (induces DNA damage)	At 5 µM, enhances the anti-proliferative and pro-apoptotic effects of MK-1775.	Reduces homologous recombination (HR) repair activity by acetylation of machinery proteins NBS1 and Rad51	Several other lung cancer cells lines tested give similar results	Chen et al.^60
THP-1 macrophages	Ox-LDL induced inhibition of autophagy	At 2 µM, increases the inhibition of autophagy	Exacerbates acetylation of Atg5	Macrophage accumulation is linked to atherosclerosis	Yang et al.^61
AML12 RAW264.7 macrophages	[Ru(CO)3Cl2]2 (Carbon monoxide releasing molecule)	At 10 µM, decreases the protective effect of [Ru(CO)3Cl2]2 after hypoxia/reoxygenation injury	Decreases the inhibition of acetylation, translocation to the cytoplasm, and release of HMGB1 by [Ru(CO)3Cl2]2	A direct deacetylation of HMGB1 by SIRT1 was also demonstrated with isolated enzymes	Sun et al.^62
U373 Hs683	None	Inhibits cell growth with IC50 = 157.4 ± 23.0 (U373) and 115.9 ± 23.3 µM (Hs683)	–	–	Schnekenburger et al.^30
HCC (HepG2 and Huh7)	None	Decreases cell survival with IC50 = 195 ± 12 (HepG2) and 33 ± 6 µM (Huh7) and increases early apoptosis at 1 µM	Increases p53 and FoxO1 acetylation at 1 µM Decreases ABC transporters P-gp and MRP3 protein levels at 40 µM in HepG2	3D cultures: decreases spheroid growth and viability with IC50 = 567 ± 41 (HepG2) and 67 ± 16 µM (Huh7)	Ceballos et al.^63
T cells	None	At 50 µM, increases the number and the suppressive function of Tregs	Increases both the acetylation and the expression levels of FOXP3	T cells isolated from patients suffering from abdominal aortic aneurysm	Jiang et al.^64
HCC (HepG2)	Hesperetin	At 10 µM, no effect on cell viability	Inhibits the increase of SIRT1 activity and AMPK phosphorylation caused by hesperetin	–	Shokri Afra et al.^65
BMMs	RANKL-induced Osteoclastogenesis	Promotes RANKL-stimulated osteoclastogenesis	Increases TNF-α mRNA and protein levels and ROS production	Dose of EX-527 not found	Yan et al.^66
HUVEC	High glucose conditions Resveratrol	At 10 µM, abolishes resveratrol-mediated anti-apoptosis and pro-proliferation effects	Involvement of the transcription factors Foxo1 and c-Myc	–	Huang et al.^67
HL-7702	Isoniazid (antituberculosis drug)	At 1 µM, aggravates the cell damages caused by isoniazid	In combination with isoniazid, increases further the expression of inflammatory regulators and cytokines, and the level of H3K9 acetylation in the promoter region of the IL-6 gene	No effects on cells and proteins when used alone	Zhang et al.^68
T cells stimulated with allogenic APC (co-cultures)	None	At 10 µg/mL, reduces T cell proliferation	Increases p53 acetylation and total protein acetylation	–	Daenthanasanmak et al.^69
MDA-MB-231 (high NNMT expression)	Adriamycin or paclitaxel	Increases the cytotoxicity, the inhibition of colony formation, and the apoptosis caused by the cytotoxic agents	Decreases the protection against cytotoxic agents given by the high NNMT expression	No effect without a cytotoxic agent Dose of EX-527 not found	Wang et al.^70

^a Cell lines: 697: B cell precursor leukaemia; A549: adenocarcinomic human alveolar basal epithelial cells (lung cancer); AML12: alpha mouse liver 12 (from hepatocytes); ASPC-1: pancreatic cancer; B-CLL: B cell chronic lymphocytic leukaemia; BM(D)Ms: bone-marrow derived macrophages; BXPC-3: pancreatic cancer; CEM/VLB₁₀₀: MDR variant of acute lymphoblastic leukaemia cells (overexpressing P-gp); CML: human chronic leukaemia; CRC: colorectal cancer; CSCs: cancer stem-like cells; GBM: glioblastoma multiforme; HCC: hepatocellular carcinoma; HCT-116/HCT-15: human colon cancer; Hela: cervical cancer; HHUA, HEC151, and HEC1B: human endometrial carcinoma; HMEC: primary human mammary epithelial cells; HL-7702: human normal liver cells; Hs683: glioblastoma; HUVEC: human umbilical vein endothelial cells; Jurkat: acute T cell leukaemia; MCF-7: human breast cancer; MDA-MB-231: breast cancer; NCI-H460: human non-small cell lung cancer; PANC-1: pancreatic cancer; PC-12: rat pheochromocytoma cells; SGC7901: human gastric adenocarcinoma; SH-SY5Y: subclone from bone marrow cells from neuroblastoma; Th17: T helper 17 cells (not naïve CD4 T cells); THP-1: human leukaemia monocyte; TNBC: triple negative breast cancer; Tregs: T regulatory cells; U373: glioblastoma; U937: human myeloid leukaemia (AML: acute myelogenous leukaemia); U-2 OS: human bone osteosarcoma epithelial cells.

5-FU: 5-fluorouracil; ABC: ATP binding cassette; AMPK: AMP-activated protein kinase; APC: antigen-presenting cells; ATF4: activating transcription factor 4; Atg5: autophagy-related 5; Bcl-xL: B cell lymphoma-extra-large; FoxO: forkhead box O; FOXP3: human forkhead box P3; HMGB1: high-mobility group box 1; HSF1: heat shock factor 1; Hsp: heat shock protein; LPS: lipopolysaccharides; MRP3: multidrug resistance-associated protein 3; mHtt (mHttex1pQ72): mutated Htt (huntingtin) exon 1 fragment with expanded Q repeat, presenting aggregates, and cytotoxicity, model of Huntington’s disease (HD); MnSOD: manganese superoxide dismutase; NNMT: nicotinamide N-methyl transferase; Ox-LDL: oxidised low-density lipoprotein; P-gp/MDR1: P-glycoprotein/multidrug resistance protein 1; RANKL: receptor activator of nuclear factor-κB ligand; RORγt: RAR-related orphan receptor γ-t; TNF-α: tumour necrosis factor-α

Table 4. Selected pharmacokinetics parameters of EX-527 (in plasma).

Organism	Dose	Cmax (µM)	tmax (h)	t1/2 (h)	Css,avg (µM)	References
C57bl/6J mice	10 mg/kg			2.3		Napper et al.^15
R6/2 mice (mean ± SD, n = 3)	5 mg/kg	6.9 ± 6.9	0.3 ± 0.1	2.7 ± 2.3	0.4 ± 0.2	Smith et al.^47
	10 mg/kg	10.5 ± 3.6	0.8 ± 0.4	1.4 ± 0.5	1.5 ± 0.4
	10 mg/kg^a	21.5 ± 3.3^a	1.0 ± 0.0^a	2.8 ± 0.4^a	3.0 ± 0.4^a
	20 mg/kg	29.3 ± 6.4	0.5 ± 0.0	0.9 ± 0.2	3.2 ± 0.4
Healthy human volunteers^b	150 mg	6.7 ± 1.8	3.7	3.9 ± 1.6	1.6 ± 0.6	Westerberg et al.^37
	300 mg	13.1 ± 4.5	3.5	4.9 ± 0.8	3.9 ± 2.2
	600 mg	26.6 ± 10.5	4.0	6.1 ± 1.4	11.8 ± 6.0
HD patients^b	10 mg/d	0.6 ± 0.2	2.0	2.3 ± 0.9	0.11 ± 0.05	Süssmuth et al.^73
	100 mg/d	5.9 ± 1.9	3.0	3.3 ± 1.6	1.8 ± 0.9

R6/2 is a mice model of Huntington’s disease (HD).

C_max: maximal plasma concentration; t_1/2: terminal plasma half-life; C_ss,avg: average plasma concentration over 24 h.

^a Values measured in brain.

^b Data selected for males (larger samples and dose ranges).

Table 5. Representative examples of in vivo assays of EX-527.

Organism	Physiology/pathology	Effect of EX-527	Proposed protein(s) and/or pathway(s) involved	References
Transgenic nematodes Caenorhabditis elegans	Oculopharyngeal muscular dystrophy (OPMD)	Fully rescues motility at 33.3 µM	Sir2^a inhibition modulates the activity of FoxO transcription factor, therefore, decreasing polyalanine expansion in PABPN1	Pasco et al.^21
Transgenic flies Drosophila melanogaster	Model of Huntington’s disease (HD)	At 0.1 and 1 µM, limits the loss of photoreceptor neurons At 10 µM, increases the survival of flies	Sir2^a inhibition increases acetylation of mHtt exon 1 fragment, increasing its rate of clearance. Beneficial effects were eliminated in Sir2 (−/−) flies	Smith et al.^47
C57BL/6 mice	Heart allograft	At 1 mg/kg/d in combination with rapamycin, prolonged heart allograft survival	Involvement of Foxp3 in Tregs cells	Beier et al.^74
Mice	Adoptively transferred Tregs (potential applications in autoimmune diseases and graft rejections)	At 40 mg/kg/d i.p., increases Tregs stability	Promotes Foxp3 expression in Tregs, by increasing acetylation on 3 of its lysine sites	Kwon et al.^75
R6/2 mice	Model of HD	At 20 mg/kg, increases the median survival by 3 weeks and decreases the number of aggregates in brains At 5 mg/kg, reduces the ventricular volume in brains (but not significant at 20 mg/kg)	Increases acetylation of mHtt exon 1 fragment, increasing its rate of clearance Possibly other SIRT1 substrates involved	Smith et al.^47
Mice	Thrombocytopenia	At 20 mg/kg, decreases the platelet count and the number of reticulated platelets	Increases the acetylation of p53 and the level of conformationally active Bax	Kumari et al.^56
C57BL/6J mice	Sepsis induced by caecal ligation and puncture	At 5 mg/kg i.p., abolishes the protective effects of melatonin	FoxO1, p53, NF-κB, and Bax	Zhao et al.^76
Mice	Model of multiple sclerosis	At 10 mg/kg subcutaneous injection, strongly suppresses the number of paralysed mice (from 100 to ∼20%)	Effect on Th17 effector cells through RORγt	Lim et al.^57
Mice	Endometrial cancer model with HHUA and HEC1B cells xenografts	At 10 mg/kg/week i.p.:Decreases the tumour volumes No apparent adverse effects	This study also shows that SIRT1 stimulates the proliferation of endometrial carcinoma cells	Asaka et al.^55
Mice	Pancreatic cancer model with PANC-1 xenograft	At 10 mg/kg i.p. alone, promotes the tumour growth No synergic effect with gemcitabine (however, almost no tumour growth was observed with gemcitabine alone)	–	Oon et al.^77
Mice	Model of depression induced by chronic social defeat stress procedure	Injection in the nucleus accumbens at 0.5 µg/d blocks anxiety-like (open field, elevated maze) and social avoidance behaviours	BDNF signalling	Kim et al.^78
Mice	Model of Parkinson’s disease (PD) induced by MPTP	At 10 mg/kg/d i.p., blocks the protective effects of resveratrol (which ameliorates the motor deficit and physiopathological changes)	Reduces SIRT1-mediated (activated by resveratrol) LC3 deacetylation and subsequent autophagic degradation of α-synuclein	Guo et al.^79
Mice	Lung cancer model with A549 cells xenografts	At 30 mg/kg/d: Synergistically represses lung cancer growth with MK-1775 (WEE1 kinase inhibitor) No apparent toxicity on normal tissues	Reduces homologous recombination (HR) repair activity by acetylation of machinery proteins NBS1 and Rad51	Chen et al.^60
Male Balb/C mice	Acute lung injury associated to endotoxemia, induced by LPS exposition	At 10 mg/kg, suppressed LPS-induced elevation of TNF-α and IL-6, and attenuated histological abnormalities	The beneficial effects were reversed by addition of an mTOR activator	Huang et al.^80
Mice (ApoE^−/−)	Atherosclerosis induced by collar placement around the carotid artery	At 10 mg/kg i.p., increases the atherosclerotic lesion	Decreases the autophagy process and enhances intraplaque macrophage infiltration	Yang et al.^61
Mice (db/db)	Diabetic wound healing on diabetic mice	At 10 µM (topical application), delays diabetic wound healing promoted by resveratrol	Foxo1 and c-Myc transcription factors involved	Huang et al.^67
Balb/C and several other mice	Graft-versus-host disease (GVHD) after mismatch grafts, and graft-versus leukaemia (GVL) treatment	At 2 mg/kg/d i.p., improves the clinical scores and prolongs survival in GVHD. Preserves the beneficial effect of graft in GVL treatment	Reduces T cell proliferation Less pathogenic T cells are generated Reduces pro-inflammatory cytokines production	Daenthanasanmak et al.^69
Male Sprague-Dawley rats	Food intake of fasted animals	At 5 µg twice daily i.c.v. injection, decreases food intake and reduces body weight	Involvement of melanocortin receptors through SIRT1 mediated FoxO1 activity regulation	Çakir et al.^81
Male Sprague-Dawley rats	Orexigenic action of ghrelin (food intake)	At 1 µg/rat i.c.v., decreased the orexigenic action of ghrelin	Blocks the activation of hypothalamic AMPK by ghrelin through p53 pathway (does not block the GH release)	Velásquez et al.^82
Male Sprague-Dawley rats	Model of cerebral oxidative stress by intrastriatal infusion of malonate	At 1 µg (cerebrospinal concentration of ∼6 µM) reverses the beneficial effects (neurological improvement and reduction of striatal lesion) of PARP inhibition by 3-aminobenzamide	No effect on the neurological score and lesion when used alone (without 3-aminobenzamide)	Gueguen et al.^36
Male Sprague-Dawley rats	Light-induced retinal damage	At 10 µg intravitreal injection, reduces the retinal protection by hydrogen-rich saline	Targets SIRT1 inhibition of apoptosis (through Bax and Bcl-2) and oxidative stress (through SOD)	Qi et al.^83
Sprague-Dawley rats	Compression-induced skeletal muscle injury	At 1 mg/mg i.p., abolishes the protective effect of unacetylated ghrelin	Increases the levels of apoptosis and necroptosis in compressed muscle tissues despite the presence of unacetylated ghrelin	Ugwu et al.^84
Male Sprague-Dawley rats	Model of partial hepatic warm ischaemia/reperfusion injury (microvascular clamp)	At 5 mg/kg i.v., decreases the beneficial effects on liver injury of a carbon monoxide-releasing molecule [Ru(CO)3Cl2]2	Decreases the inhibition of acetylation, translocation to the cytoplasm, and release of HMGB1 by [Ru(CO)3Cl2]2	Sun et al.^62
Male Wistar rats	MCAO model of cerebral ischaemia	At 10 µg i.c.v., reduces the infarction volume of ischaemic brains and improves the survival (but not the neurological deficits)	Decreases rip3 and mlkl gene expression and protein levels (regulators of necroptosis)	Nikseresht et al.^85
Male Sprague-Dawley rats	Model of myocardial ischaemia/reperfusion injury	At 5 mg/kg/d i.p.: Abolished the beneficial effects of punicalagin (enhanced cardiac function and reduced myocardial infarction) No effect when administered alone on sham-operated rats	Blocks the beneficial effects of punicalagin on oxidative/nitrosative damage and inflammation, and reverses its activation of the NRF-2-HO-1 pathway	Yu et al.^86
HD patients	HD	At doses up to 100 mg/d for 14 d, no observable clinical effects and no change in immune markers	No effect on levels of total circulating mHtt	Süssmuth et al.^73

^a Sir2 is the homologue of mammalian SIRT1.

AMPK: AMP-activated protein kinase; ApoE: apolipoprotein E; BDNF: brain-derived neurotrophic factor; FoxO: forkhead box class O; Foxp3: forkhead box P3; HHUA and HEC1B: human endometrial carcinoma cells; HMGB1: high-mobility group box 1; HO-1: haem oxygenase-1; i.c.v.: intracerebroventricular; i.p.: intraperitoneal; LC3: microtubule-associated protein 1 light chain 3; LPS: lipopolysaccharides; MCAO: middle cerebral artery occlusion; mHtt: mutated Htt (huntingtin) exon 1 fragment with expanded Q repeat, presenting aggregates and cytotoxicity, model of Huntington’s disease; mlkl: mixed lineage kinase domain-like protein; MPTP: 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine; mTOR: mammalian target of rapamycin; NRF-2: nuclear factor erythroid 2-related factor 2; PABPN1: polyadenylate-binding protein, nuclear 1; rip3: receptor-interacting protein kinase 3; Th17: T helper 17 cells (not naïve CD4 T cells); TNF-α: tumour necrosis factor-α; Tregs: T regulatory cells.

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