M344

Catalog No.S2779 Batch:S277901

Technical Data

Formula	C₁₆H₂₅N₃O₃
Molecular Weight	307.39	CAS No.	251456-60-7
Solubility (25°C)*	In vitro	DMSO	62 mg/mL (201.69 mM)
		Ethanol	4 mg/mL (13.01 mM)
		Water	Insoluble
* <1 mg/ml means slightly soluble or insoluble. * Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations. * Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

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Biological Activity

Description

M344 is a potent HDAC inhibitor with IC50 of 100 nM and able to induce cell differentiation.

Targets

HDAC ^[1]

100 nM

In vitro

M344 produces a more significant effect on cell proliferation than on cell differentiation in MEL DS19 cells. M344 is toxic at concentrations above 10 μM, while a maximum of only 20% of the surviving cell population are induced to differentiate. ^[1] In vitro, M344 shows the significant anti-proliferative activities against the endometrial cancer cell line Ishikawa and the ovarian cancer cell line SK-OV-3 with EC50 of 2.3 μM and 5.1 μM, respectively. While the normal human endometrial epithelial cells shows little sensitivity to M344. In addition, M344 also leads to decreased proportion of cells in the S-phase and increased proportion in the G0/G1 phases of the cell cycle, induces apoptosis and decreases the transmembrane potential of mitochondria. ^[2] M344 potently inhibits proliferation of embryonal nervous system tumor cells including medulloblastoma cells (D341 Med, Daoy) and neuroblastoma cells (CH-LA 90,SHSY-5Y ) with GI50 of 0.65 μM, 0.63 μM, 0.63 μM and 0.67 μM, respectively. ^[3]

Features

An inducer of terminal cell differentiation and a potent HDAC inhibitor.

Protocol (from reference)

Kinase Assay:^{^[1]}	Enzyme Inhibition Radioactively labeled chicken core histones are used as the enzyme substrate. The enzyme liberated tritiated acetic acid from the substrate which is quantitated by scintillation counting. IC50 values are results of triple determinations. 50 μL of maize enzyme (at 30 °C) is incubated (30 minutes) with 10 μL of total [³H]acetate-prelabeled chicken reticulocyte histones (1 mg/mL). Reaction is stopped by addition of 36 μL of 1 M HCl/0.4 M acetate and 800 μL of ethyl acetate. After centrifugation (10000g, 5 minutes) an aliquot of 600 μL of the upper phase is counted for radioactivity in 3 mL of liquid scintillation cocktail. M344 is tested in a starting concentration of 40 μM, and active substances are diluted further.
Cell Assay:^{^[1]}	Cell lines MEL DS19 cells Concentrations 0 to 50 μM Incubation Time 72 hours Method MEL DS19 cells (murine erythroleukemia cells) are maintained in D-MEM containing 100 units/mL penicillin G sodium and 100 μg/mL streptomycin sulfate supplemented with 10% fetal bovine serum at 37 °C in a 5% CO₂ atmosphere. To test M344 for potential to induce cell differentiation, log-phase cells with a population doubling time of 11−13 hours are used. Serial dilutions of M344 are prepared in 24-well plates using 1 mL of D-MEM/well. If M344 are dissolved in DMSO, control wells contains the same amount of solvent (generally 2 μL/mL of medium). Subsequently, the cell suspension is added to the wells. After 72 hours the experiment is evaluated. Cell numbers are counted using a Casy 1 TTC flow cytometer. The proliferation of treated cells is expressed as percent proliferation in comparison with the solvent control. Differentiated MEL cells accumulate hemoglobin. Therefore, the induction of cell differentiation is determined by benzidine staining according to the literature. To 100 μL of cell suspension is added 10 μL of a 0.4% solution of benzidine in 12% acetic acid containing 2% H₂O₂. Within 5 minutes hemoglobin-containing cells stains blue. Benzidine-positive and -negative cells are counted under the microscope in a hemocytometer, and the percentage of positive cells is calculated. M344 is first tested at 10 μM and 50 μM final concentration. According to activity/toxicity profile, a range of concentrations is chosen for a dose−response analysis.

Kinase Assay:^{^[1]}

Enzyme Inhibition
Radioactively labeled chicken core histones are used as the enzyme substrate. The enzyme liberated tritiated acetic acid from the substrate which is quantitated by scintillation counting. IC50 values are results of triple determinations. 50 μL of maize enzyme (at 30 °C) is incubated (30 minutes) with 10 μL of total [³H]acetate-prelabeled chicken reticulocyte histones (1 mg/mL). Reaction is stopped by addition of 36 μL of 1 M HCl/0.4 M acetate and 800 μL of ethyl acetate. After centrifugation (10000g, 5 minutes) an aliquot of 600 μL of the upper phase is counted for radioactivity in 3 mL of liquid scintillation cocktail. M344 is tested in a starting concentration of 40 μM, and active substances are diluted further.

Cell Assay:^{^[1]}

Cell lines
MEL DS19 cells
Concentrations
0 to 50 μM
Incubation Time
72 hours
Method
MEL DS19 cells (murine erythroleukemia cells) are maintained in D-MEM containing 100 units/mL penicillin G sodium and 100 μg/mL streptomycin sulfate supplemented with 10% fetal bovine serum at 37 °C in a 5% CO₂ atmosphere. To test M344 for potential to induce cell differentiation, log-phase cells with a population doubling time of 11−13 hours are used. Serial dilutions of M344 are prepared in 24-well plates using 1 mL of D-MEM/well. If M344 are dissolved in DMSO, control wells contains the same amount of solvent (generally 2 μL/mL of medium). Subsequently, the cell suspension is added to the wells. After 72 hours the experiment is evaluated. Cell numbers are counted using a Casy 1 TTC flow cytometer. The proliferation of treated cells is expressed as percent proliferation in comparison with the solvent control. Differentiated MEL cells accumulate hemoglobin. Therefore, the induction of cell differentiation is determined by benzidine staining according to the literature. To 100 μL of cell suspension is added 10 μL of a 0.4% solution of benzidine in 12% acetic acid containing 2% H₂O₂. Within 5 minutes hemoglobin-containing cells stains blue. Benzidine-positive and -negative cells are counted under the microscope in a hemocytometer, and the percentage of positive cells is calculated. M344 is first tested at 10 μM and 50 μM final concentration. According to activity/toxicity profile, a range of concentrations is chosen for a dose−response analysis.

References

[4] St Germain C, et al. Cancer Cell Int. 2010,10, 32.

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Customer Product Validation

: Data from [Biochem Biophys Res Commun, 2014, 445(2), 320-6]

Selleck's M344 has been cited by 11 publications

Targeting codon 158 p53-mutant cancers via the induction of p53 acetylation. [ Nat Commun, 2020, 29;11(1):2086]	PubMed: 32350249
Quantifying Drug Combination Synergy along Potency and Efficacy Axes. [ Cell Syst, 2019, 8(2):97-108]	PubMed: 30797775
Inhibition of HDAC4 Attenuated JNK/c-Jun-Dependent Neuronal Apoptosis and Early Brain Injury Following Subarachnoid Hemorrhage by Transcriptionally Suppressing MKK7. [ Front Cell Neurosci, 2019, 13:468]	PubMed: 31708743
DRUGPATH - a novel bioinformatic approach identifies DNA-damage pathway as a regulator of size maintenance in human ESCs and iPSCs [ Sci Rep, 2019, 9(1):1897]	PubMed: 30760778
Neutralizing negative epigenetic regulation by HDAC5 enhances human haematopoietic stem cell homing and engraftment. [ Nat Commun, 2018, 9(1):2741]	PubMed: 30013077
Epigenetic Reprogramming with Antisense Oligonucleotides Enhances the Effectiveness of Androgen Receptor Inhibition in Castration-Resistant Prostate Cancer [ Cancer Res, 2018, 78(20):5731-5740]	PubMed: 30135193
Evaluating biological activity of compounds by transcription factor activity profiling. [ Sci Adv, 2018, 4(9):eaar4666]	PubMed: 30263952
Histone deacetylase inhibitor M344 significantly improves nuclear reprogramming, blastocyst quality, and in vitro developmental capacity of cloned pig embryos [ J Anim Sci, 2017, 95(3):1388-1395]	PubMed: 28380503
Histone deacetylase inhibitor M344 significantly improves nuclear reprogramming, blastocyst quality, and in vitro developmental capacity of cloned pig embryos [ J Anim Sci, 2017, 95(3):1388-1395]	PubMed: 28380503
Target Engagement and Drug Residence Time Can Be Observed in Living Cells With BRET [ Nat Commun, 2015, 3;6:10091]	PubMed: 26631872

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