UNC0638 is an inhibitor of β-protein lysine methyltransferases G9a(IC50<15 nM) and GLP(IC50=19 nM) with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets.

GLP:19 nM, G9a:<15 nM

UNC0638是一种针对G9a和GLP具有高选择性、高渗透性的化学探针，其毒性/功能比率大于100，相较于BIX01294的小于6。它是针对G9a和GLP的选择性抑制剂，在广泛的表观遗传及非表观遗传靶点上展现出优越的选择性。UNC0638对SET7/9（一种H3K4 HMTase）、SET8（一种H4K20 HMTase）、PRMT3和SUV39H2的选择性超过10,000倍。在MDA-MB-231细胞中，48小时的UNC0638处理以浓度依赖的方式降低H3K9me2的水平，IC50为81 nM。UNC0638对多种细胞系的处理降低了全球H3K9me2水平，相当于对G9a和GLP进行小发夹RNA敲减的水平，同时UNC0638的功能效力与其毒性之间有着明显的分离。此外，UNC0638显著降低了MCF7细胞的克隆形成能力，在已知G9a调控的内源基因的启动子处降低H3K9me2的丰度，并不成比例地影响了编码微RNA的几个基因组位点。在小鼠胚胎干细胞中，UNC0638以浓度依赖的方式重新激活G9a沉默的基因和一种逆转录病毒报告基因，而不促进分化。[1]

The enzymatic reactions are conducted in duplicate at room temperature for 1 hour in a 50 μL mixture containing PKMT assay buffer, substrate coated plate, 10 M SAM, a HMT enzyme (EZH2 (800 ng/reaction), MLL (300 ng/reaction), PRMT1 (0.5 ng/reaction), SUV39H1 (75 ng/reaction) and UNC0638 (0-1.25 μM). After enzymatic reactions, 100 μL of first antibody is added to each well and the plate is incubated at room temperature for an additional 1 h. 100 μL of secondary antibody is added to each well and the plate is incubated at room temperature for an additional 30 min. 100 μL of developer reagents are added to wells and luminescence is measured using a BioTek SynergyTM 2 microplate reader. Enzyme activity assays are performed in duplicates at each concentration. The luminescence data are analyzed using the computer software, Graphpad Prism[1].

UNC0638 is dissolved in deuterated DMSO (10 mM) and deuterated Water (90:10 ratio)[1]. MDA-MB-231, PC3, HCT116 cells are cultured in RPMI with 10% FBS, 22RV1 cells in alphaMEM and 10% FBS, MCF7 and IMR90 cells in DMEM with 10% FBS. Cells are grown in the presence or absence of UNC0638 (10 nM, 100 nM, 1 μM, 10 μM, and 100 μM ) for stated amount of time. The media is removed and replaced with DMEM 10% FBS without phenol red supplemented with 1 mg/mL of MTT and incubated for 1-2 h. Live cells reduce yellow MTT to purple formazan. The resulting formazan is solubilized in acidified isopropanol and 1% Triton and absorbance measured at 570 nm, corrected for 650 nm background[1].