SIRT1

In diabetic mice, SRT 2104 (100 mg/kg/day, added to the diet for 24 weeks) raises SIRT1 protein without changing Sirt1 mRNA[2]. In diabetic mice, SRT 2104 (100 mg/kg/day, given in diet for 24 weeks) reduces oxidative stress in the testicles, apoptotic signaling activation, and endogenous stress[2]. In N171-82Q HD mice, SRT 2104 (0.5%; for 18 weeks) enhances motor function and increases survival[3].

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Sirtuin 1 is a nicotinamide adenine dinucleotide-dependent protein deacetylase which regulates longevity and improves metabolism. Activation of Sirtuin 1 confers beneficial effects in models of neurodegenerative diseases. We and others have provided convincing evidence that overexpression of Sirtuin 1 plays a neuroprotective role in mouse models of Huntington's disease. In this study, we report that SRT2104, a small molecule Sirtuin 1 activator, penetrated the blood-brain barrier, attenuated brain atrophy, improved motor function, and extended survival in a mouse model of Huntington's disease. These findings imply a novel therapeutic strategy for Huntington's disease by targeting Sirtuin 1.[3]

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SRT2104 was well tolerated in all of these studies, with no serious adverse reactions observed. SRT2104 displayed a dose-dependent, but sub-proportional increase in exposure following single dose and repeated dose administration. Accumulation of three-fold or less occurs after 7 days of repeat dosing. The mean bioavailability was circa 14% and the mean clearance was circa 400 ml min(-1). Although there were no substantial effects on exposure resulting from gender or formulation differences, a notable food effect was observed, manifested as up to four-fold increase in exposure parameters. Conclusions: In the absence of an optimized formulation of SRT2104, the food effect can be used to maximize exposure in future clinical studies. Combined with the good tolerability of all doses demonstrated in these studies, the favourable selectivity profile of SRT2104 allows for the use of this SIRT1 modulator for target validation in the clinic.[1]

Luciferase activity assay[4]
One day before transfection, murine primary microglial cells (2 × 105/well) were seeded into 24-well plates. NF-κB-Luc Reporter Lentivirus particles at a multiplicity of infection (MOI) of 5 were added to the wells. Following incubation at 37 °C in 5% CO2 for 6 h, the virus-containing medium was removed and replaced with fresh culture medium. After 24 h or transfection, the cells were pretreated with indicated concentration of SRT2104 for 1 h followed by OGD/R insult or LPS stimulation. Afterwards, the cells were harvested and subjected to luciferase activity assay according to the manufacturer's instructions. Promoter activity of the NF-κB was expressed relative to values measured in control cells. For BV-2 cells, cells were transiently transfected with NF-κB reporter vector and the pRL-TK plasmid with Lipofectamine™ LTX and Plus reagent. Twenty-four hours later, the cells were pretreated with indicated concentration of SRT2104 for 1 h followed by OGD/R insult. The final NF-κB activity was presented as the ratio of the activity of firefly luciferase to that of Renilla luciferase.

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Lactate dehydrogenase (LDH) assay[4]
The release of Lactate dehydrogenase (LDH) was measured using the LDH assay kit according to manufacturer’s instructions. Following treatment, 100 μl of the cell suspension was added into a new 96-well tissue culture plate, followed by mixed with 100 μl of the Working Solution. Keep the plate from light and incubate it at room temperature for 30 min. 50 μl of the Stop Solution was added to each well and the absorbance was measured at 490 nm by a microplate reader

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Cytokine Enzyme-linked immunosorbent assays (ELISA)[4]
The concentration of IL-10, IL-6, TNF-alpha, TGF- β, MCP-1 were determined by ELISA kit (Nanjing Jiancheng; R&D Systems) according to the manufacturer's protocol. Absorbance was determined at 450 nm by spectrometry.

OGD/R model[4]
Cells were pretreated with SRT2104 for 1 h, and then the complete medium was replaced with serum/glucose-free DMEM. Then cells were transferred to grow in an anaerobic chamber with a compact oxygen controller to maintain oxygen concentration at 1% by injecting a gas mixture of 94% N2 and 5% CO2 for different time periods (3–24 h) to establish conditions of OGD. Then, the cells were transferred back to normal DMEM medium containing normal glucose under an atmosphere of 95% air and 5% CO2, and incubated for 12 h as OGD/R. Control cells were not submitted to OGD and were maintained under normal conditions.

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MTT assay[4]
Cells were inoculated in 96 well plates with certain density per well. After treatment, cells were washed with PBS, then 150 μl MTT solution was directly added to each well at a final concentration of 0.5 mg/ml. The cell continued to be cultured at 37 °C for 4 h. Then cells were added with 100 μl DMSO and fully shake for 10 min to dissolve and crystallize. The absorbance was measured at 570 nm by a microplate reader. The background absorbance was measured at 690 nm and subtracted from the 570 nm measurement.

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Western blot[4]
Samples from primary microglia or BV2 cultures were homogenized in lysis buffers, and total protein was isolated and the protein concentrations in the supernatant were determined with the bicinchoninic acid protein assay with bovine serum albumin as standard. 50 μg of protein were subjected to SDS-PAGE and then transferred to nitrocellulose membranes. The membrane was incubated with the following antibodies at 4 °C overnight: iNOS, Ym-1, Arg-1, p-p65, p65, IκB α, Sirt1 Acetyl-p65. β -actin was used as an internal control. Immunoreactive bands were identified, and a densitometric analysis was performed with an enhanced chemiluminescence detection system.

Animal/Disease Models: Male C57BL/ 6 mice (8weeks old)[2]
Doses: 100 mg/kg/day
Route of Administration: Supplemented in diet for 24 weeks
Experimental Results: Enhanced SIRT1 protein without evelating Sirt1 mRNA level. Attenuated diabetes mellitus (DM)-induced oxidative stress, apoptotic signaling, and ER stress.

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Animal/Disease Models: WT and N171-82Q HD mice (6 weeks old)[3]
Doses: 0.5%
Route of Administration: 0.5% SRT 2104 containing diet for 6, 12, 18 weeks
Experimental Results: Ameliorated motor deficits and increased survival in N171-82Q HD mice.

SRT2104 was well tolerated in all of these studies, with no serious adverse reactions observed. SRT2104 displayed a dose-dependent, but sub-proportional increase in exposure following single dose and repeated dose administration. Accumulation of three-fold or less occurs after 7 days of repeat dosing. The mean bioavailability was circa 14% and the mean clearance was circa 400 ml min(-1). Although there were no substantial effects on exposure resulting from gender or formulation differences, a notable food effect was observed, manifested as up to four-fold increase in exposure parameters.[1]

[1]. Pharmacokinetics and tolerability of SRT2104, a first-in-class small molecule activator of SIRT1, after single and repeated oral administration in man.

[2]. MicroRNA-34a targets sirtuin 1 and leads to diabetes-induced testicular apoptotic cell death. J Mol Med (Berl). 2018 Sep;96(9):939-949.

[3]. Sirtuin 1 activator SRT2104 protects Huntington's disease mice. Ann Clin Transl Neurol. 2014 Dec;1(12):1047-52.

[4]. Sirt1 activator SRT2104 protects against oxygen-glucose deprivation/reoxygenation-induced injury via regulating microglia polarization by modulating Sirt1/NF-κB pathway. Brain Res . 2021 Feb 15:1753:147236.

SRT2104 has been investigated for the basic science and treatment of Sepsis, PSORIASIS, Atrophy, Muscular, and Diabetes Mellitus, Type 2.

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Aim: SRT2104 is a novel, first-in-class, highly selective small molecule activator of the NAD + dependent deacetylase SIRT1. SRT2104 was dosed to healthy male and female volunteers in a series of phase 1 clinical studies that were designed to elucidate tolerability and pharmacokinetics associated with oral dosing to aid in dose selection for subsequent clinical trials. Methods: In the first-in-human study, there was both a single dose phase and 7 day repeat dose phase. Doses used ranged from 0.03 to 3.0 g. A radioactive microtracer study was subsequently conducted to determine systemic clearance, bioavailability and preliminary metabolism, and a crossover study was conducted to determine the effect of gender, formulation and feeding state on SRT2104 pharmacokinetics. Results: SRT2104 was well tolerated in all of these studies, with no serious adverse reactions observed. SRT2104 displayed a dose-dependent, but sub-proportional increase in exposure following single dose and repeated dose administration. Accumulation of three-fold or less occurs after 7 days of repeat dosing. The mean bioavailability was circa 14% and the mean clearance was circa 400 ml min(-1). Although there were no substantial effects on exposure resulting from gender or formulation differences, a notable food effect was observed, manifested as up to four-fold increase in exposure parameters. Conclusions: In the absence of an optimized formulation of SRT2104, the food effect can be used to maximize exposure in future clinical studies. Combined with the good tolerability of all doses demonstrated in these studies, the favourable selectivity profile of SRT2104 allows for the use of this SIRT1 modulator for target validation in the clinic.[1]

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Testicular apoptotic cell death (TACD) contributes to diabetes mellitus (DM)-induced male infertility. MicroRNA-34a (miR-34a) is a pro-apoptotic RNA that targets sirtuin 1 (SIRT1) which provides protection against complications of (DM). However, the specific role of miR-34a in (DM)-induced TACD is unknown. MiR-34a targets Sirt1 mRNA, resulting in apoptosis. However, whether or not SIRT1 is a major target of miR-34a in (DM)-induced TACD is unclear. The present study aimed to define the role of miR-34a/SIRT1 in (DM)-induced TACD. C57BL/6 male mice were induced to (DM) by streptozotocin, for a period of 24 weeks. The expression of miR-34a and Sirt1 as well as apoptotic cell death was determined in the testes of the non-diabetic, diabetic, and the miR-34a-specific inhibitor (miR-34a-I)-treated diabetic mice. In addition, the novel SIRT1 activator SRT2104 was delivered to the mice to determine the role of SIRT1 in DM-induced TACD. The diabetic mice developed remarkable testicular oxidative stress, endoplasmic reticulum stress, and apoptotic cell death, the effects of which were significantly and similarly attenuated by both miR-34a-I and SRT2104. Mechanistically, the DM-induced testicular elevation of miR-34a and the decrease in SIRT1 protein were markedly prevented by both miR-34a-I and SRT2104, to a similar extent. The present study demonstrates a critical role of miR-34a/SIRT1 in DM-induced TACD, providing miR-34a inhibition and SIRT1 activation as novel strategies in clinical management of DM-induced male infertility.[2]

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Cerebral ischemic/reperfusion injury is the most common neurological disorder and the second leading cause of death worldwide. Modulating microglia polarization from pro-inflammatory M1 phenotype to anti-inflammatory M2 state has been suggested as a potential therapeutic approach in the treatment of this injury. SRT2104, a novel activator of histone deacetylase Sirtuin-1 (Sirt1), has recently been shown to have anti-inflammation properties. However, the effect of SRT2104 on cerebral ischemic/reperfusion injury has not been elucidated. Here, we found that SRT2104 inhibited neuron and microglia death directly and indirectly through microglia condition medium from an oxygen glucose deprivation/reoxygenation (OGD/R) -induced cell injury models. Moreover, SRT2104 treatment modulated the microglia polarization shift from the M1 phenotype and skewed toward the M2 phenotype. Additionally, we found that SRT2104 could significant inhibit the activation of NF-κB and enhanced Sirt1 expression in microglia. Mechanism studies using the BV2 microglial cell line confirmed that knockdown Sirt1 significantly reduced the effect of SRT2104 on the activation of NF-κB pathway and microglial phenotype shift. Altogether, our result shows SRT2104 protect OGD/R-induced injury through shifting microglia phenotype, which may have potential in further studies as a novel neuroprotective agent for cerebral ischemic/reperfusion injury therapy.[4]

C26H24N6O2S2

516.64

516.14

C, 60.44; H, 4.68; N, 16.27; O, 6.19; S, 12.41

1093403-33-8

25108829

Light yellow to yellow solid powder

1.5±0.1 g/cm3

1.761

4.1

1

8

6

36

758

0

LAMQVIQMVKWXOC-UHFFFAOYSA-N

InChI=1S/C26H24N6O2S2/c1-17-23(36-25(28-17)18-5-4-8-27-13-18)24(33)29-21-7-3-2-6-20(21)22-15-32-19(16-35-26(32)30-22)14-31-9-11-34-12-10-31/h2-8,13,15-16H,9-12,14H2,1H3,(H,29,33)

4-methyl-N-(2-(3-(morpholinomethyl)imidazo[2,1-b]thiazol-6-yl)phenyl)-2-(pyridin-3-yl)thiazole-5-carboxamide

GSK2245840; SRT2104; SRT2104 (GSK2245840); 4-methyl-n-(2-(3-(morpholinomethyl)imidazo[2,1-b]thiazol-6-yl)phenyl)-2-(pyridin-3-yl)thiazole-5-carboxamide; GSK2245840; 5-Thiazolecarboxamide, 4-methyl-N-[2-[3-(4-morpholinylmethyl)imidazo[2,1-b]thiazol-6-yl]phenyl]-2-(3-pyridinyl)-; GSK-2245840; SRT 2104; GSK 2245840; SRT-2104.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 0.5 mg/mL (0.97 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 0.5 mg/mL (0.97 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)