SP2509 is a specific histone demethylase LSD1 inhibitor(IC50 =13 nM).

LSD1:13 nM

在负荷OCI-AML3异种移植物的小鼠中,SP2509（25 mg/kg i.p. ）,能够抑制肿瘤生长,延迟动物的存活.

在OCI-AML3中,SP250能够抑制集落形成,诱导细胞凋亡。在AML细胞中,SP2509能够抑制LSD1与CoREST作用,增加启动子特异性H3K4Me3,同时诱导p53,p21和C/EBPα。在原代AML细胞中,SP2509诱导细胞的增殖。

SP2509 activity assays: Test compounds are diluted to 20 × the desired test concentration in 100% DMSO and 2.5?μL of the diluted drug sampleis added to a black 384-well plate. The LSD1 enzyme stock is diluted 17-fold with assay buffer and 40?μL of the diluted LSD1 enzyme is added to the appropriate wells. Substrate, consisting of horseradish peroxidase, dimethyl K4 peptide corresponding to the first 21 amino acids of the N-terminal tail of histone H3, and 10-acetyl-3,7-dihydroxyphenoxazine is then added to wells. Resorufin is analyzed on an Envision plate reader with an excitation wavelength of 530?nm and an emission wavelength of 595?nm. The activity of SP2509 on the other oxidases is determined by using commercially available kits. The glucose oxidase activity (which also noncovalently binds FAD in an elongate conformation), is determined using the glucose oxidase kit. The MAO assays are performed using the MAO-glo kit with MAO-A and MAO-B.

Cultured AML cells are treated with SP2509 and/or PS for 96?h. At the end of treatment, cells are washed free of the drugs and 500 cells per condition are plated in methylcellulose and incubated at 37?°C. Colony formation is measured 7–10 days after plating.(Only for Reference)