| Description |
SP2509 is a potent and selective antagonist of lysine specific demethylase 1 (LSD1) with IC50 of 13 nM.
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| Related Catalog |
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| Target |
IC50: 13 nM (LSD1)[1]
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| In Vitro |
SP2509 (250, 500, 1000 nM) inhibits LSD1 activity, depletes colony growth and induces apoptosis and cell death of cultured human acute myeloid leukemia cells, and increases H3K4Me3 on the promoters of p57 Kip, KLF4, and p21 and induces mRNA expression of p57Kip, KLF4 and p21 in AML cells. SP2509 (250, 1000 nM) induces features of morphologic differentiation of cultured and primary AML cells. Besides, SP2509 in combination with PS exerts synergistic lethal activity against cultured and primary AML cells[1]. SP2509 does not destabilize the CoREST-LSD1 interaction, and has no major destabilizing effect on the CRC. SP2509 (1 or 10 µM) induces cell death, but there are no morphological changes at a low concertation of 0.1 µM. SP2509 likewise interferes with the viability of medulloblastoma cells[2].
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| In Vivo |
Treatment with SP2509 (25 mg/kg) and/or PS (5 mg/kg) significantly enhances PS-mediated loss of viability of CD34+ primary AML cells and improves the survival of mice bearing AML xenografts and primagrafts[2].
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| Kinase Assay |
To measure the activity of LSD1, a luminol-based assay is used. A total of 5 µL of eluate is diluted in LSD1 reaction buffer (50 mM Tris, pH 8.5, 50 mM KCl, 5 mM MgCl2, 5 nmol luminol, 20 μg/mL horseradish peroxidase, 1% Triton X-100), and 10 µM H3K4me2 (1-21 aa) peptide is added as the substrate. DMSO and inhibitors are added. The total volume per reaction is 100 µL. The samples are measured in a white 96-well plate using an EnSpire multimode plate reader at an emission wavelength of 428 nm. Three individual purifications (n = 3) are used for the assay, with three technical replicates for each.
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| Cell Assay |
Daoy and D283 Med cells are used in the assay. ONS-76 cells are cultured in RPMI medium 1640 supplemented with 10% FBS, 50 U/mL penicillin, and 50 µg/mL streptomycin. All medulloblastoma cell lines are kept in an incubator at 37°C in a 5% CO2/5% O2/90% N2 atmosphere with maximum humidity. XTT assays are performed in triplicates (n = 3) with three replicates for each using 1×103 Daoy cells/well, 4×103 D283 Med cells/well, and 1×103 ONS-76 cells/well in 100 µL of medium at initial seeding in 96-well plates.
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| Animal Admin |
Female NOD/SCID mice are exposed to 2.5 Gy of radiation. The following day, 5 million OCI-AmL3 cells are injected into the lateral tail vein of the mice and the mice are monitored for 7 days. Following treatments are administered in cohorts of 8 mice for each treatment: vehicle alone, 25 mg/kg SP2509, 5 mg/kg panobinostat and SP2509 plus panobinostat. Treatments are initiated on day 7 for OCI-AmL3 cells. SP2509 (formulated in solubilization buffer [20% Cremaphor, 20% DMSO, 60% sterile water]) is administered twice per week (Tues and Thurs) intraperitoneally (IP) for 3 weeks, and then discontinued. Panobinostat (formulated in 5% DMSO/ 95% normal saline) is administered by IP injection 3 days per week (M-W-F) for 3 weeks and discontinued. The doses of PS utilized in these studies are determined to be safe and effective. A separate in vivo experiment is conducted utilizing NSG mice and primary AmL cells. Following engraftment of the AmL cells (presence of greater than 1% CD45+ cells in the peripheral blood), mice are treated with SP2509 and/or PS, for three weeks.
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| References |
[1]. Fiskus W, et al. Highly effective combination of LSD1 (KDM1A) antagonist and pan-histone deacetylase inhibitor against human AML cells. Leukemia. 2014 Nov;28(11):2155-64. [2]. Inui K, et al. Stepwise assembly of functional C-terminal REST/NRSF transcriptional repressor complexes as a drug target. Protein Sci. 2017 Feb 20.
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