Selisistat (EX 527)

Biological Activity

Selisistat (EX 527) is a potent and selective inhibitor of the SIRT1 class III histone deacetylase enzyme. EX 527 has been used a powerful tool for studying the relationship between SIRT1 and cell regulation. The deacetylation of cortactin, a protein responsible for rearrangements of the actin cytoskeleton, is associated with cell motility and possibly tumorigenesis, and blocking of this deacetylation by EX 527 correlated to a decrease in cell motility. Blocking of SIRT1 by EX 527 also demonstrated that deacetylation of the important tumor suppressor protein p53 is mediated by SIRT1 as well.

Product Citations

• 

  Mol Neurobiol. 2018 Apr;55(4):3067-3078.

  17β-Estradiol via SIRT1/Acetyl-p53/NF-kB Signaling Pathway Rescued Postnatal Rat Brain Against Acute Ethanol Intoxication
  Selisistat (EX 527) purchased from AbMole

• 

  Free Radic Biol Med. 2018 Oct 17.

  Resveratrol protects photoreceptors by blocking caspase- and PARP-dependent cell death pathways.
  Selisistat (EX 527) purchased from AbMole

Customer Product Validations & Biological Datas

	Source	Free Radical Biology and Medicine (2018). Figure 4. Ex-527 (Abmole Bioscience, Houston, TX, USA)
	Method	western blot
	Cell Lines	661W cells
	Concentrations	180 μM
	Incubation Time
	Results	As shown in Figure 4A–D, there is a lower cell death rate in the presence of RSV in both the GD and the light exposure groups compared to the control group, whereas treatment with Ex-527 reversed this trend (P < 0.01).

	Source	Proc Natl Acad Sci U S A (2013). Figure 3. EX 527
	Method	Continuous Deacetylation Assay
	Cell Lines	Sir2Tm crystals
	Concentrations	1 mM
	Incubation Time	80 min
	Results	Ex-527 showed negligible affinities (Sir2Tm: Kd > 180 μM; Sirt3 Kd > 330 μM) to apo-enzymes and to the Sirtuins saturated with 1 mM peptide substrate (Sir2Tm: Kd > 170 μM; Sirt3: Kd > 180 μM).

	Source	Proc Natl Acad Sci U S A (2013). Figure 1. EX 527
	Method	Activity assay
	Cell Lines	Sir2Tm crystals
	Concentrations	1.5 mM
	Incubation Time	80 min
	Results	Activity assays in presence of varying Ex-527 concentrations showed that inhibition of Sir2Tm and Sirt3 by Ex-527 is noncompetitive with substrate peptide (Fig. 1 E and F; Fig. S1A; Sir2Tm Ki = 1.8 ± 0.4 μM, Sirt3 Ki = 33.4 ± 4.4 μM).

Protocol

Cell Experiment
Cell lines	NCI-H460 cells, MCF-7 cells, U-2 OS cells, or HMEC cell lines
Preparation method	Cell viability assay. NCI-H460 cells, MCF-7 cells, U-2 OS cells, or HMEC were plated at 2,000 cells per well in opaque-walled 96-well plates (Corning) for the viability assay and 800 cells per well in 96-well Cytostar-T scintillating microplates (Amersham) for the proliferation assay. Cells were incubated for 1 day (NCI-H460) or 2 days (MCF-7, U-2 OS, and HMEC) prior to exposure to DNA-damaging agents and deacetylase inhibitors. All experiments were performed in triplicate.For viability assays, cells were treated with the indicated compounds for 48 h. Cell viability was then determined using the Cell Titer-Glo luminescent assay (Promega), which measures total ATP levels as an index of cell number. Luminescence was measured on a Luminoskan Ascent (ThermoLabSystems).
Concentrations	0~100 μM
Incubation time	48 h

Animal Experiment
Animal models	Male Sprague-Dawley rats (8 weeks old, 200–250 g) and C57/B6 mice (8 weeks old) model
Formulation	DMSO
Dosages	5-10 μg in a total volume of 5 μL
Administration	Intracerebroventricular administration

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

Chemical Information

References

Resveratrol attenuates microvascular inflammation in sepsis via SIRT-1-Induced modulation of adhesion molecules in ob/ob mice
Xianfeng Wang, et al. Obesity (Silver Spring). 2015 Jun;23(6):1209-17. PMID: 25959124.

Activating transcription factor 4 confers a multidrug resistance phenotype to gastric cancer cells through transactivation of SIRT1 expression.
Zhu et al. PLoS One. 2012;7(2):e31431. PMID: 22363646.

The central Sirtuin 1/p53 pathway is essential for the orexigenic action of ghrelin.
Velásquez DA, et al. Diabetes. 2011 Apr;60(4):1177-85. PMID: 21386086.

Inhibition of SIRT1 catalytic activity increases p53 acetylation but does not alter cell survival following DNA damage.
Solomon JM, et al. Mol Cell Biol. 2006 Jan;26(1):28-38. PMID: 16354677.