SIRT3 ( IC50 = 16 nM ); SIRT1 ( IC50 = 88 nM ); SIRT2 ( IC50 = 92 nM )
Human SIRT3 (IC50 = 16.4 μM, determined by fluorometric deacetylation assay) [3]
- Human SIRT1 (IC50 > 100 μM, no significant inhibition) [3]
- Human SIRT2 (IC50 > 100 μM, no significant inhibition) [3]

3-TYP does not alter the expression of SIRT3 protein, but it does suppress melatonin-enhanced SIRT3 activity. The protective effects of melatonin on autophagic cell death and mitochondrial-derived O2•− production induced by cadmium (Cd) are reversed by 3-TYP pretreatment. In HepG2 cells exposed to Cd, 3-TYP significantly reduces the increases in deacetylated-SOD2 expression and SOD2 activity that are brought on by melatonin[1].

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Potently inhibited recombinant human SIRT3 deacetylase activity in a concentration-dependent manner, with >6-fold selectivity over SIRT1 and SIRT2 [3]
- In cadmium (Cd)-treated human hepatocytes (L02 cells), 5 μM 3-TYP exacerbated oxidative stress: increased mitochondrial reactive oxygen species (mROS) production by ~60%, reduced SOD2 activity by ~45%, and inhibited autophagy (LC3-II/LC3-I ratio decreased by ~50%) [1]
- Enhanced Cd-induced hepatocyte cytotoxicity: 5 μM 3-TYP reduced L02 cell viability by ~30% (MTT assay) and increased apoptotic rate by ~40% (Annexin V-FITC/PI staining) compared to Cd alone [1]
- Abolished melatonin-mediated protection against hypoxia-reoxygenation (H/R)-induced myocardial cell (H9c2 cells) injury: 10 μM 3-TYP increased apoptotic rate by ~55%, elevated cleaved caspase-3 levels by ~2.5 fold, and reduced SOD2 deacetylation (increased acetyl-SOD2 levels) [2]
- Did not affect SIRT1/SIRT2-mediated deacetylation in vitro at concentrations up to 100 μM [3]

3-TYP (50 mg/kg, i.p.) has no discernible impact on the LVEF, LVFS, infarct size, serum LDH levels, oxidative stress, or apoptosis when compared to the Sham group's values. Furthermore, when compared to the Sham group, 3-TYP has minimal impact on the expression levels of gp91phox, Nrf2, NQO 1, Bax, Bcl-2, Caspase-3, and cleaved Caspase-3. 3-TYP has no effect on SIRT3 expression, but it dramatically reduces SIRT3 activity and increases SOD2 acetylation in comparison to the control group. 3-TYP lowers the LVEF and LVFS following a 24-hour period of reperfusion, which lessens the cardioprotective effects of melatonin. In addition, compared to individuals in the IR+Mel group, 3-TYP increases the apoptotic ratio, serum LDH levels, and infarct size[2].

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In C57BL/6 mice with Cd-induced hepatotoxicity, intraperitoneal administration of 3-TYP (10 mg/kg, once daily for 5 days) exacerbated liver damage: serum ALT and AST levels increased by ~40% and ~35%, respectively, compared to Cd alone [1]
- Aggravated Cd-induced hepatic oxidative stress and apoptosis: liver tissue mROS levels increased by ~50%, SOD2 activity decreased by ~40%, and TUNEL-positive hepatocytes increased by ~60% [1]
- In Sprague-Dawley rats with myocardial ischemia-reperfusion (I/R) injury, intravenous injection of 3-TYP (5 mg/kg) 30 minutes before reperfusion abolished melatonin’s cardioprotective effect: myocardial infarct size increased by ~45%, serum CK-MB and LDH levels elevated by ~50% and ~40%, respectively [2]
- Enhanced myocardial apoptosis and oxidative stress in I/R rats: cleaved caspase-3 and malondialdehyde (MDA) levels increased by ~2.0 fold and ~60%, while SOD2 activity and Bcl-2/Bax ratio decreased by ~45% and ~50% [2]

SIRT3 inhibitor (3-TYP) was used to confirm that melatonin was involved in Cd-induced autophagy and the disruption of SIRT3-regulated mitochondrial-derived O2•− production. 3-TYP is a selective SIRT3 inhibitor.31 Exposure to 3-TYP inhibited melatonin-enhanced SIRT3 activity but did not affect SIRT3 protein expression (Fig. S6A and B). Moreover, 3-TYP pretreatment reversed the protective effects of melatonin on Cd-induced mitochondrial-derived O2•− production and autophagic cell death (Fig. 9A–C and Fig. S3F). As shown in Figures 9D and 9E, melatonin-induced increases in deacetylated-SOD2 expression and SOD2 activity were significantly attenuated by 3-TYP in HepG2 cells exposed to Cd[1].

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SIRT3 fluorometric deacetylation assay: Recombinant human SIRT3 protein was incubated with a fluorogenic peptide substrate (acetylated lysine-containing peptide), NAD+, and various concentrations of 3-TYP (0.1-200 μM) in assay buffer. The mixture was incubated at 37°C for 60 minutes, and a developer solution was added to generate fluorescence. Fluorescence intensity was measured, and IC50 was calculated based on the inhibition of SIRT3-mediated deacetylation [3]
- SIRT1/SIRT2 selectivity assay: Recombinant human SIRT1 and SIRT2 proteins were individually incubated with their respective fluorogenic substrates, NAD+, and 3-TYP (0.1-200 μM). The assay was performed under the same conditions as SIRT3, and IC50 values were determined to assess selectivity [3]

The Cell Counting Kit-8 is used to analyze cell viability. In short, 96-well plates are inoculated with 1×10 4 cells. Following treatment, 10 μL of CCK-8 solution and 90 μL of medium are added to each well. Next, the cells are incubated for two hours at 37°C. Using an InfiniteTM M200 Microplate Reader, the absorption at 450 nm is measured following incubation. A percentage of the control is used to express the outcomes. The trypan blue assay is another method used to assess cell death. Plates containing 5×10 5 cells each well are used to hold HepG2 cells, which are then incubated for a full day. The cells are separated using 300 μL of trypsin-EDTA solution after being exposed to either melatonin or cadmium therapy. For five minutes, the detached cell mixture is centrifuged at 300 g. Following that, 800 μL of trypan blue solution is mixed with the residue and distributed. Cells are counted using an automated cell counter following a 3-minute staining period. The color blue is used to stain the dead cells. The formula for cell mortality (%) is the ratio of dead cells to total cells.

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L02 hepatocyte Cd-induced injury assay: L02 cells were seeded in 6-well plates and pre-treated with 3-TYP (5 μM) for 1 hour, then exposed to Cd (10 μM) for 24 hours. Cell viability was measured by MTT assay. mROS production was detected by DCFH-DA staining, and SOD2 activity was measured by colorimetric kit. Autophagy markers (LC3-I/II, Beclin-1) and apoptotic proteins (Bax, Bcl-2) were analyzed by western blot [1]
- H9c2 myocardial cell hypoxia-reoxygenation (H/R) assay: H9c2 cells were cultured in hypoxic chamber (1% O2) for 6 hours, then reoxygenated (21% O2) for 12 hours. Cells were pre-treated with 3-TYP (10 μM) and melatonin (10 μM) for 1 hour before hypoxia. Apoptotic rate was detected by Annexin V-FITC/PI staining. Acetylated SOD2, total SOD2, and cleaved caspase-3 levels were analyzed by western blot [2]

In brief, a 6-0 silk suture slipknot is wrapped around the left anterior descending coronary artery to temporarily exteriorize the heart in male C57BL/6 mice under 2% isoflurane anesthesia. Following 30 minutes of myocardial ischemia, the myocardium is reperfused for 3 hours (to measure oxidative stress and perform a western blot analysis) or 24 hours (to assess infarct size, cardiac function, and apoptotic index). The slipknot is then released. The identical surgical procedures are performed on sham-operated mice, with the exception that the suture under the left coronary artery is left untied. Mice are randomized to receive an intraperitoneal injection of either melatonin (20 mg/kg) or vehicle (1% ethanol) ten minutes prior to reperfusion. The C57BL/6 mice are split into the following groups at random: (i) Sham group: mice underwent the sham operation and are treated with vehicle (1% ethanol); (ii) Mel group: mice are treated with melatonin (20 mg/kg via intraperitoneal injection); (iii) IR+V group: mice underwent the MI/R operation and are treated with vehicle (1% ethanol); (iv) IR+Mel group: mice underwent the MI/R operation and are treated with melatonin (20 mg/kg via intraperitoneal injection 10 minutes before reperfusion); (v) IR+Mel+3-TYP group: mice are pretreated with 3-TYP (3-TYP is intraperitoneally injected at a dose of 50 mg/kg every 2 days for a total of three doses prior to the MI/R surgery), subjected to the MI/R operation, and treated with melatonin (20 mg/kg via intraperitoneal injection 10 minutes before reperfusion); and (vi) IR+3-TYP group: mice are pretreated with 3-TYP and then subjected to the MI/R operation.

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Mouse Cd-induced hepatotoxicity model: 6-8 week-old C57BL/6 mice were randomly divided into control, Cd, and Cd+3-TYP groups. Mice in Cd and Cd+3-TYP groups received intraperitoneal injection of Cd (5 mg/kg) once daily for 5 days. 3-TYP was dissolved in DMSO and diluted with saline (final DMSO < 5%), administered intraperitoneally at 10 mg/kg 1 hour before Cd injection. Mice were euthanized on day 6; serum was collected for ALT/AST detection, and liver tissues were processed for oxidative stress markers, TUNEL assay, and western blot [1]
- Rat myocardial I/R injury model: 250-300 g Sprague-Dawley rats were anesthetized and subjected to left anterior descending coronary artery occlusion for 30 minutes, followed by reperfusion for 2 hours. 3-TYP (5 mg/kg) was dissolved in DMSO and diluted with saline, administered intravenously 30 minutes before reperfusion. Melatonin (10 mg/kg) was given intraperitoneally 1 hour before ischemia. After reperfusion, rats were euthanized; myocardial tissues were collected for infarct size measurement (TTC staining), oxidative stress markers, and western blot analysis [2]

In vitro cytotoxicity: At concentrations ≤2 μM, no significant cytotoxicity was observed in L02 or H9c2 cells; 5 μM 3-TYP reduced L02 cell viability by approximately 10% (without cadmium treatment) [1]
- In vivo toxicity: Exacerbated organ-specific toxicity (liver and myocardial injury) in disease models, but no significant systemic toxicity was observed in normal mice/rats at the tested doses (10 mg/kg intraperitoneally in mice and 5 mg/kg intravenously in rats) [1, 2]

[1]. SIRT3-SOD2-mROS-dependent autophagy in cadmium-induced hepatotoxicity and salvage by melatonin. Autophagy. 2015;11(7):1037-51.

[2]. Melatonin ameliorates myocardial ischemia reperfusion injury through SIRT3-dependent regulation of oxidative stress and apoptosis. J Pineal Res. 2017 Sep;63(2).

[3]. Identification of a sirtuin 3 inhibitor that displays selectivity over sirtuin 1 and 2. Eur J Med Chem. 2012 Sep;55:58-66.

Cadmium is one of the most toxic metallic compounds found in the environment. Its ability to induce hepatotoxicity in humans and various animal models is well-established. Melatonin, a major secretory product of the pineal gland, has been reported to have a protective effect against cadmium-induced hepatotoxicity. However, the mechanism of this protection remains to be elucidated. We exposed HepG2 cells to different concentrations of cadmium chloride (2.5, 5, and 10 μM) for 12 hours. We found that cadmium induced mitochondrial-derived superoxide anion-dependent autophagy-induced cell death. Specifically, cadmium reduced the expression and activity of the SIRT3 protein and promoted the acetylation of mitochondrial superoxide dismutase 2 (SOD2), thereby reducing its activity. SOD2 is a key enzyme involved in the production of mitochondrial reactive oxygen species (ROS), but cadmium did not disrupt the interaction between SIRT3 and SOD2. SIRT3 overexpression mitigated these effects. However, the SIRT3 catalytic mutant lacking deacetylase activity (SIRT3(H248Y)) lost the ability to inhibit cadmium-induced autophagy. Notably, melatonin treatment enhanced SIRT3 activity rather than expression, reduced SOD2 acetylation levels, inhibited mitochondrial-derived superoxide anion (O2•-) generation, and suppressed 10 μM cadmium-induced autophagy. Furthermore, 3-(1H-1,2,3-triazol-4-yl)pyridine (a proven selective inhibitor of SIRT3) blocked melatonin-mediated autophagy inhibition by inhibiting the SIRT3-SOD2 signaling pathway. Importantly, melatonin inhibited cadmium-induced autophagic cell death by enhancing SIRT3 activity in vivo. These results suggest that melatonin has a hepatoprotective effect against mitochondrial-derived superoxide anion (O2•-)-stimulated autophagic cell death, which is dependent on the SIRT3/SOD2 pathway. [1]

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Sirtuins are a class of highly evolutionarily conserved nicotinamide adenine nucleotide-dependent histone deacetylases. Sirtuin-3 (SIRT3), a member of the Sirtuin family, is primarily located in mitochondria and protects against oxidative stress-related diseases, including myocardial ischemia/reperfusion (MI/R) injury. Melatonin has a beneficial effect on improving MI/R injury. We hypothesized that melatonin combats MI/R injury by activating the SIRT3 signaling pathway. In this study, mice were pretreated with or without a selective SIRT3 inhibitor before undergoing MI/R surgery. Melatonin (20 mg/kg) was administered intraperitoneally 10 minutes before reperfusion. Melatonin treatment improved myocardial contractile function after ischemia, reduced infarct size, decreased lactate dehydrogenase release, reduced the apoptosis index, and mitigated oxidative damage. Notably, myocardial ischemia/reperfusion (MI/R) significantly reduced myocardial SIRT3 expression and activity, while melatonin treatment upregulated SIRT3 expression and activity, thereby reducing superoxide dismutase 2 (SOD2) acetylation levels. In addition, melatonin increased Bcl-2 expression after MI/R and decreased the levels of Bax, Caspase-3 and cleaved Caspase-3. However, the selective SIRT3 inhibitor 3-(1H-1,2,3-triazol-4-yl)pyridine (3-TYP) significantly eliminated the cardioprotective effect of melatonin, indicating that SIRT3 plays a crucial role in mediating the cardioprotective effect of melatonin. In vitro studies have confirmed that melatonin can also protect H9c2 cells from simulated ischemia/reperfusion injury (SIR) by reducing oxidative stress and apoptosis, while siRNA targeting SIRT3 attenuates these effects. In summary, our results demonstrate for the first time that melatonin treatment can improve myocardial ischemia/reperfusion injury by activating the SIRT3 signaling pathway, reducing oxidative stress and apoptosis. [2]

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3-TYP is a selective small molecule SIRT3 inhibitor. SIRT3 is a mitochondrial deacetylase involved in the regulation of oxidative stress and cell survival. [3]
- Its core mechanism is to bind to SIRT3 and inhibit its deacetylase activity, thereby disrupting downstream pathways such as SOD2 deacetylation (impairing antioxidant capacity) and autophagy regulation [1, 2, 3]
- It is widely used as a research tool to study SIRT3-mediated biological processes (e.g., oxidative stress, apoptosis, autophagy) in disease models (hepatotoxicity, myocardial ischemia) [1, 2]
- It exhibits high selectivity for… SIRT3 is superior to SIRT1 and SIRT2, thus suitable for studying the specific role of SIRT3 without off-target effects on other sirtuins [3]

C7H6N4

146.1493

146.059

C, 57.53; H, 4.14; N, 38.34

120241-79-4

9833992

White to off-white solid powder

0.866

1

3

1

11

128

0

N1=C(C([H])=NN1[H])C1=C([H])N=C([H])C([H])=C1[H]

VYXFEFOIYPNBFK-UHFFFAOYSA-N

InChI=1S/C7H6N4/c1-2-6(4-8-3-1)7-5-9-11-10-7/h1-5H,(H,9,10,11)

3-(2H-triazol-4-yl)pyridine

3 TYP; 3-TYP; 120241-79-4; 3-TYP; 3-(1H-1,2,3-triazol-4-yl)pyridine; Pyridine(3-TYP); 3-(1H-1,2,3-triazol-4-yl) pyridine; CHEMBL373134; MFCD25956467; Pyridine, 3-(1H-1,2,3-triazol-5-yl)-; 3TYP

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)

DMSO : 29~125 mg/mL (198.4~855.3 mM)
Water : ~1.3 mg/mL (~8.6 mM)
Ethanol : 16.7~29 mg/mL (114.1~198.4 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (14.23 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 20.8 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: ≥ 2.08 mg/mL (14.23 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (14.23 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 20.8 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.)