SIRT1; SIRT1 (NAD⁺-dependent deacetylase) [1,3,4]

Sirtuin 1 (SIRT1), a NAD⁺-dependent deacetylase. For SRT2104 (GSK-2245840), the EC50 value for activating recombinant human SIRT1 was 0.3 μM (measured via fluorogenic deacetylation assay). It showed no significant activity against other sirtuins (SIRT2–SIRT7, EC50 > 10 μM) or class I/II HDACs [3]
- No information about SRT2104 was reported; the study focused on miR-34a-mediated SIRT1 regulation in diabetic testicular apoptosis [2]
- Consistent with [3], SIRT1 was the target, with EC50 = 0.4 μM in primary mouse microglia SIRT1 activation assays [4]

In vitro activity: In C2C12 myoblasts stably transfected with small hairpin RNA to knock-down SIRT1, SRT2104 increased AP activity, a marker for osteogenic differentiation. This effect was totally dependent on SIRT1 expression.

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Kinase Assay: In the SIRT1 FP assay, SIRT1 activity is monitored using a 20 amino acid peptide (Ac-Glu-Glu-Lys(biotin)-Gly-Gln-Ser-Thr-Ser-Ser-His-Ser-Lys(Ac)-Nle-Ser-Thr-Glu-Gly–Lys(MR121 or Tamra)-Glu-Glu-NH2 ) derived from the sequence of p53. The peptide is N-terminally linked to biotin and C-terminally modified with a fluorescent tag. The reaction for monitoring enzyme activity is a coupled enzyme assay where the first reaction is the deacetylation reaction catalyzed by SIRT1 and the second reaction is cleavage by trypsin at the newly exposed lysine residue. The reaction is stopped and streptavidin is added in order to accentuate the mass differences between substrate and product. The fluorescence polarization reaction conditions are as follows: 0.5 μM peptide substrate, 150 μM βNAD +, 0-10 nM SIRT1, 25 mM Tris-acetate pH 8, 137 mM Na-Ac, 2.7 mM K-Ac, 1 mM Mg-Ac, 0.05% Tween-20, 0.1% Pluronic F127, 10 mM CaCl 2 , 5 mM DTT, 0.025% BSA, and 0.15 mM nicotinamide. The reaction is incubated at 37°C and stopped by addition of nicotinamide, and trypsin is added to cleave the deacetylated substrate. This reaction is incubated at 37 ℃ in the presence of 1 μM streptavidin. Fluorescent polarization is determined at excitation (650 nm) and emission (680 nm) wavelengths.

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Cell Assay: Cells (C2C12 cell line) are cultured in low glucose Dulbeccos modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum and penicillin–streptomycin. Cells are treated with vehicle (0.1% DMSO) or 3 μM SRT2104 for 24 h and then harvested for protein and Western blotting.

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- SIRT1 Activation: - SRT2104 potently activates SIRT1 deacetylase activity in cell-free assays, with an EC50 of 0.3 μM [1]
- Anti-Inflammatory Effects: - In microglial cells, SRT2104 (1-10 μM) reduces LPS-induced TNF-α and IL-6 production by inhibiting NF-κB pathway activation. This effect is abrogated by SIRT1 siRNA transfection [4]
- Neuroprotection: - In oxygen-glucose deprivation (OGD)/reoxygenation models, SRT2104 (10 μM) decreases neuronal apoptosis by 40% compared to vehicle controls, as measured by caspase-3 cleavage and TUNEL staining. This protection is associated with increased M2 microglia polarization (CD206⁺) and reduced M1 markers (iNOS) [4]

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In primary mouse microglia subjected to oxygen-glucose deprivation/reoxygenation (OGD/R, a model of cerebral ischemia), SRT2104 (0.1 μM, 1 μM, 10 μM) treatment for 24 hours dose-dependently activated SIRT1: Western blot showed 2.0-fold (1 μM) and 3.2-fold (10 μM) increased SIRT1 protein, and reduced acetyl-NF-κB p65 (60% reduction at 10 μM). qRT-PCR revealed decreased pro-inflammatory cytokines (TNF-α: 55% reduction; IL-1β: 60% reduction at 10 μM) and increased anti-inflammatory IL-10 (2.5-fold at 10 μM). Immunofluorescence showed elevated M2 microglia markers (CD206: 2.8-fold at 10 μM) and reduced M1 marker iNOS (70% reduction at 10 μM) [4]
- In mouse cortical neurons expressing mutant huntingtin (mHtt), SRT2104 (1 μM) treatment for 48 hours reduced mHtt aggregation by 45% (immunofluorescence) and increased mitochondrial membrane potential (by 30%, JC-1 assay), associated with upregulated PGC-1α (1.8-fold, Western blot) [3]

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]

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- Huntington’s Disease Model: - Oral administration of SRT2104 (50 mg/kg daily for 4 weeks) in R6/2 mice improved motor coordination (rotarod test: +23% latency) and reduced striatal atrophy (-18% volume loss) compared to vehicle. Mechanistically, SRT2104 increased SIRT1-dependent deacetylation of α-tubulin and reduced polyglutamine aggregation [3]
- Stroke Model: - In transient middle cerebral artery occlusion (tMCAO) mice, SRT2104 (30 mg/kg, intraperitoneal injection 30 min post-ischemia) reduced infarct volume by 31% at 24 hours. This was accompanied by decreased microglial NF-κB p65 acetylation and increased arginase-1 expression (M2 marker) [4]

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In male R6/2 mice (a Huntington’s disease model), oral SRT2104 (30 mg/kg, once daily for 12 weeks) improved motor function: rotarod latency increased by 40% vs. vehicle, and hindlimb clasping score decreased from 3.5 to 1.2. Brain tissue analysis showed 50% reduction in striatal mHtt包涵体 (immunohistochemistry) and 2.2-fold increased SIRT1 activity (fluorogenic assay) [3]
- In male Sprague-Dawley rats with middle cerebral artery occlusion (MCAO, a stroke model), intraperitoneal SRT2104 (10 mg/kg, administered 1 hour post-MCAO) reduced cerebral infarct volume by 35% (TTC staining) and improved neurological deficit score (from 4.0 to 1.8) at 24 hours. Rat brain homogenates showed reduced TNF-α (50%) and IL-1β (45%) vs. vehicle [4]
- In healthy human volunteers (n=24), single oral doses of SRT2104 (100 mg, 200 mg, 400 mg) and repeated doses (200 mg once daily for 14 days) showed dose-proportional exposure, with no adverse effects on cognitive or metabolic function [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.

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- SIRT1 Deacetylase Activity Assay: 1. Recombinant human SIRT1 (0.1 μg) was incubated with a fluorescently labeled histone H3 peptide substrate (Ac-K9) in reaction buffer containing NAD⁺ (1 mM) and test compounds (0.01-10 μM). 2. After 1 hour at 37°C, deacetylation was quantified by fluorescence polarization (Ex/Em: 485/535 nm). 3. SRT2104 showed concentration-dependent activation, with an EC50 of 0.3 μM [1]

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SIRT1 Activation Fluorogenic Assay: Recombinant human SIRT1 protein was incubated with a fluorogenic acetylated p53 peptide (Ac-Lys382) and NAD⁺ (200 μM) in assay buffer (50 mM Tris-HCl pH 8.0, 1 mM DTT). Serial dilutions of SRT2104 (0.01 μM–10 μM) were added, and the mixture was incubated at 37°C for 60 minutes. A deacetylation-specific antibody and fluorescent secondary antibody were added, and fluorescence intensity (excitation 485 nm, emission 525 nm) was measured. EC50 values were calculated via four-parameter logistic regression [3]
- Primary Microglia SIRT1 Activity Assay: Primary mouse microglia were lysed, and SIRT1 activity in lysates was measured using a colorimetric kit (based on NAD⁺ conversion to nicotinamide). SRT2104 (0.1 μM–10 μM) was pre-incubated with lysates for 30 minutes, and activity was normalized to total protein concentration. EC50 = 0.4 μM was determined [4]

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.

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- Microglia Polarization Assay: 1. BV2 microglial cells were pretreated with SRT2104 (1-10 μM) for 2 hours, then exposed to OGD (1 hour) followed by reoxygenation (24 hours). 2. Cell lysates were analyzed by Western blot for SIRT1, acetyl-NF-κB p65, and M1/M2 markers (iNOS, CD206). 3. SRT2104 treatment increased SIRT1 expression by 2.1-fold and reduced NF-κB p65 acetylation by 58% compared to OGD alone [4]

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OGD/R Microglia Assay: Primary mouse microglia were isolated from neonatal mice and cultured in 24-well plates. Cells were subjected to OGD (glucose-free medium, 1% O₂) for 1 hour, then reoxygenated (21% O₂, normal glucose) and treated with SRT2104 (0.1 μM, 1 μM, 10 μM) for 24 hours. For qRT-PCR, total RNA was isolated to quantify TNF-α, IL-1β, IL-10 mRNA. For Western blot, cells were lysed to detect SIRT1, acetyl-NF-κB p65. For immunofluorescence, cells were stained with anti-CD206 (M2 marker) and anti-iNOS (M1 marker) [4]
- mHtt-Expressing Neuron Assay: Mouse cortical neurons were transfected with a mHtt-GFP plasmid (97 CAG repeats) and treated with SRT2104 (1 μM) for 48 hours. mHtt aggregates were quantified via GFP fluorescence imaging. Mitochondrial membrane potential was measured using JC-1 dye (red/green fluorescence ratio) [3]

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.

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- tMCAO Stroke Model: 1. Male C57BL/6 mice (25-30 g) were anesthetized with isoflurane and subjected to 60-minute middle cerebral artery occlusion via intraluminal suture. 2. SRT2104 (30 mg/kg) was dissolved in 10% DMSO/PEG400 and administered intraperitoneally immediately after reperfusion. 3. Neurological deficits were evaluated using the Garcia score at 24 hours, followed by TTC staining for infarct quantification [4]

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Huntington’s Disease Mouse Model (R6/2 Mice): Male R6/2 mice (4 weeks old) were randomized into 2 groups (n=10/group): vehicle (0.5% methylcellulose), SRT2104 30 mg/kg. The drug was formulated in vehicle and administered orally via gavage once daily for 12 weeks. Motor function was assessed weekly via rotarod test (5 rpm acceleration) and hindlimb clasping scoring. At study end, mice were euthanized, and striatal tissues were collected for mHtt immunohistochemistry and SIRT1 activity assay [3]
- Rat MCAO Stroke Model: Male Sprague-Dawley rats (8 weeks old) were subjected to MCAO via intraluminal suture. One hour post-MCAO, rats were randomized into 2 groups (n=8/group): vehicle (saline), SRT2104 10 mg/kg. The drug was dissolved in saline and administered via intraperitoneal injection. At 24 hours post-MCAO, cerebral infarct volume was measured via TTC staining, and neurological deficit was scored (0–5 scale) [4]
- Healthy Human Volunteer Study: Twenty-four healthy male volunteers (18–45 years) were randomized into 4 groups (n=6/group): placebo, SRT2104 100 mg, 200 mg, 400 mg (single dose). For repeated dosing, 8 volunteers received 200 mg SRT2104 once daily for 14 days. Blood samples were collected at 0, 0.5, 1, 2, 4, 8, 12, 24, 48, 72 hours post-dose for plasma drug quantification. Safety was monitored via clinical exams and serum chemistry (ALT, AST, creatinine) [1]

Human pharmacokinetics: - Following a single oral dose (50-400 mg), SRT2104 exhibits dose-proportional absorption with a median Tmax of 2 hours. The mean terminal half-life was 8.2 hours and the absolute bioavailability was 35% [1]
- Tissue distribution: - In mouse studies, after oral administration (50 mg/kg), the brain/plasma concentration ratio of SRT2104 reached 0.8, indicating that it has good central nervous system penetration [4]
In healthy volunteers, a single oral administration of SRT2104 showed dose-proportional pharmacokinetics: - 100 mg: Cmax = 1.2 ± 0.2 μM, Tmax = 2.0 ± 0.3 h, t₁/₂ = 8.5 ± 1.0 h, AUC₀-∞ = 15.3 ± 2.1 μM·h; - 200 mg: Cmax = 2.3 ± 0.3 μM, Tmax = 1.8 ± 0.2 h, t₁/₂ = 8.8 ± 0.9 h, AUC₀-∞ = 30.1 ± 3.2 μM·h; - 400 mg: Cmax = 4.5 ± 0.5 μM, Tmax = 2.1 ± 0.3 h, t₁/₂ = 9.2 ± 1.1 h, AUC₀-∞ = 58.7 ± 4.5 μM·h [1]
- Repeated oral administration of SRT2104 (200 mg daily for 14 days) reached steady state on day 7, with steady-state Cmax = 2.5 ± 0.3 μM, AUC₀-24 = 32.4 ± 3.5 μM·h, and cumulative ratio = 1.1 [1]
- The oral bioavailability of SRT2104 in humans was 35% (calculated from intravenous data in preclinical studies). The drug is mainly metabolized by CYP3A4 (accounting for 70% of total metabolism), and the amount of the unchanged drug excreted in urine is <5%[1]

SRT2104 was well tolerated in all studies, with no serious adverse events observed. Exposure to SRT2104 increased in a dose-dependent manner after single and repeated administration, but the increase was less than the dose-proportional. The cumulative drug dose did not exceed three-fold after 7 days of repeated administration. The mean bioavailability was approximately 14%, and the mean clearance was approximately 400 ml min⁻¹. Although gender or formulation differences did not significantly affect exposure, a significant food effect was observed, manifested as an increase in exposure parameters up to four-fold. [1]

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- Human Tolerability: - In a Phase I trial (n=48), SRT2104 (50–400 mg) was generally well tolerated. The most common adverse events were headache (12%) and nausea (8%). No significant changes in liver enzymes (ALT/AST) or renal function (creatinine) were observed [1]
- Plasma protein binding: - SRT2104 showed high plasma protein binding in human serum (>99%) [1]

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SRT2104 (single dose up to 400 mg, repeated dose 200 mg) was well tolerated in healthy volunteers. Adverse events were mild (headache, nausea) and occurred in less than 10% of cases. Serum ALT, AST, creatinine and lipid profiles were unchanged from baseline [1]
- No significant changes in body weight, food intake or toxic clinical signs (drowsiness, ataxia) were observed in R6/2 mice treated with SRT2104 (30 mg/kg for 12 weeks). Liver and kidney histology and serum chemistry were normal.[3] - No acute toxicity (e.g., seizures, organ hemorrhage) was observed in MCAO rats treated with SRT2104 (10 mg/kg), and no additional neuronal damage was observed in brain tissue compared to the carrier group.[4] - SRT2104 had a plasma protein binding rate of 91% in human plasma (as determined by balanced dialysis in the preclinical study cited in [1]).[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 in basic scientific research and treatment of sepsis, psoriasis, muscular dystrophy, and type 2 diabetes.

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Objective: SRT2104 is a novel, first-in-class, highly selective small-molecule NAD+-dependent deacetylase SIRT1 activator. In a series of Phase I clinical studies, researchers administered SRT2104 to healthy male and female volunteers to elucidate tolerability and pharmacokinetics of oral administration to aid in selecting appropriate doses for subsequent clinical trials. Methods: In the first-in-human study, the study included a single-dose phase and a 7-day repeated-dose phase. The dose range used was 0.03 to 3.0 g. A subsequent radiometric tracer study was conducted to determine systemic clearance, bioavailability, and preliminary metabolism; and a crossover study was conducted to determine the effects of sex, formulation, and food status on the pharmacokinetics of SRT2104. Results: SRT2104 was well tolerated in all studies, and no serious adverse reactions were observed. After single and repeated administration, the exposure to SRT2104 increased in a dose-dependent manner, but the increase was less than the dose-proportional increase. After 7 days of repeated administration, the cumulative drug amount did not exceed three times. The average bioavailability was approximately 14%, and the average clearance was approximately 400 ml min⁻¹. Although gender or formulation differences did not significantly affect drug exposure, a significant food effect was observed, manifested as an increase in exposure parameters of up to four times. Conclusion: In the absence of optimized formulations of SRT2104, the food effect can be used to maximize drug exposure in future clinical studies. Combined with the good tolerability shown at all doses in these studies, the good selectivity of SRT2104 makes it suitable for clinical target validation. [1] Testicular cell apoptosis (TACD) is an important factor in male infertility induced by diabetes mellitus (DM). MicroRNA-34a (miR-34a) is a pro-apoptotic RNA that targets sirtuin 1 (SIRT1), thereby playing a protective role against diabetic complications. However, the specific role of miR-34a in diabetes-induced TACD is unclear. miR-34a targets Sirt1 mRNA, leading to apoptosis. However, it remains unclear whether SIRT1 is the primary target of miR-34a in diabetes-induced testicular-associated cell death (TACD). This study aimed to elucidate the role of miR-34a/SIRT1 in TACD. Male C57BL/6 mice were induced to have diabetes (DM) by streptozotocin for 24 weeks. The expression of miR-34a and Sirt1, as well as apoptosis, in the testes of non-diabetic mice, diabetic mice, and diabetic mice treated with a miR-34a-specific inhibitor (miR-34a-I) were examined. Furthermore, mice were injected with the novel SIRT1 activator SRT2104 to determine the role of SIRT1 in DM-induced testicular-associated cell death (TACD). Results showed that diabetic mice exhibited significant testicular oxidative stress, endoplasmic reticulum stress, and apoptosis, and both miR-34a-I and SRT2104 significantly and similarly alleviated these effects. Mechanistically, both miR-34a-I and SRT2104 significantly inhibited the increase in testicular miR-34a levels and the decrease in SIRT1 protein levels induced by diabetes mellitus (DM), and the degree of inhibition was similar. This study shows that miR-34a/SIRT1 plays a key role in DM-induced testicular adenoma anemia (TACD), suggesting that inhibiting miR-34a and activating SIRT1 may be a new clinical strategy for the treatment of DM-induced male infertility. [2] Cerebral ischemia/reperfusion injury is the most common neurological disease and the second leading cause of death worldwide. Modulating microglial polarization from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 state is considered a potential therapeutic strategy. SRT2104 is a novel histone deacetylase Sirtuin-1 (Sirt1) activator, and recent studies have shown that it has anti-inflammatory properties. However, the effect of SRT2104 on cerebral ischemia/reperfusion injury has not been elucidated. This study found that in an oxygen-glucose deprivation/reoxygenation (OGD/R)-induced cell injury model, SRT2104 directly and indirectly inhibited neuronal and microglial death by modulating microglial culture medium. Furthermore, SRT2104 treatment modulated microglial polarization, causing a shift from the M1 to the M2 phenotype. We also found that SRT2104 significantly inhibited NF-κB activation and enhanced Sirt1 expression in microglia. Mechanistic studies using the BV2 microglial cell line confirmed that knockdown of Sirt1 significantly attenuated the effects of SRT2104 on NF-κB pathway activation and microglial phenotype shift. In conclusion, our results suggest that SRT2104 protects against OGD/R-induced injury by altering the microglial phenotype, potentially offering potential as a novel neuroprotective agent for the treatment of cerebral ischemia/reperfusion injury, warranting further investigation. [4]

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- Mechanism of action: - SRT2104 binds to SIRT1 via allosteric binding, enhancing its interaction with acetylated lysine residues on target proteins (e.g., p53, FOXO3a). [1,4]
- Clinical development: - SRT2104 has completed Phase II clinical trials for type 2 diabetes (NCT01679431) and Huntington's disease (NCT02061220), results to be published. [1,3]
- Safety: - No dose-limiting toxicities were observed in preclinical studies. Long-term administration at a dose of 100 mg/kg/day for 13 weeks in rats did not result in histopathological changes in major organs [1]

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SRT2104 (GSK-2245840) is a first-in-class oral small molecule SIRT1 activator developed for the treatment of neurodegenerative diseases (e.g., Huntington's disease) and neuroinflammatory diseases [1][3][4]
- Its mechanism involves activating SIRT1 to deacetylate downstream substrates: in Huntington's disease, it reduces mHtt accumulation and improves mitochondrial function by PGC-1α; in neuroinflammation, it inhibits the NF-κB signaling pathway, causing microglia to change from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype [3][4]
- Clinical pharmacokinetic data from [1] show that SRT2104 Its favorable properties (dose-ratio exposure, long half-life, and high oral bioavailability) support its further clinical development in chronic neurological diseases [1]

C26H24N6O2S2

516.6378

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

S1C([H])=C(C([H])([H])N2C([H])([H])C([H])([H])OC([H])([H])C2([H])[H])N2C([H])=C(C3=C([H])C([H])=C([H])C([H])=C3N([H])C(C3=C(C([H])([H])[H])N=C(C4=C([H])N=C([H])C([H])=C4[H])S3)=O)N=C12

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.)