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G9a (EHMT2) and GLP (EHMT1) catalyze the mono and dimethylation of lysine 9 of histone 3 (H3K9) and other non-histone substrates such as p53 and WIZ.
Here we present UNC0642, a G9a/GLP chemical probe with improved PK properties relative to UNC0638. UNC0642 exhibits an in vitro IC50 <15 nM with selectivity > 100-fold over 13 other HMTs and selected representatives of kinases, ion channels, 7TMs, and other epigenetic proteins.
In cells, UNC0642 results in a potent reduction of H3K9me2 in MDA MB231 cells with IC50 = 106 nM.
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Below we show UNC0642 is equipotent to UNC0638 in the G9a in vitro assay:
G9a: Ki = 4 ± 2 (nM)
Competitive with peptide substrate
Non-competitive with SAM cofactor
Similar potency as UNC0638 (Ki = 3 nM)
In addition, UNC0642 has been shown to be inactive versus 50 kinases at 10 uM and has a similar GPCR selectivity as UNC0638.
a i.p. administration of a single 5 mg/kg dose in male Swiss Albino mice. 8 time points and 3 animals per time point
MDA-MB231 cells were cultured in RPMI with 10% FBS and MCF7 cells cultured in DMEM with 10% FBS.
Cells were grown in the presence or absence of inhibitors for stated amount of time. The media was removed and replaced with DMEM 10% FBS without phenol red supplemented with 1mg/ml of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and incubated for 1-2h. Live cells reduce yellow MTT to purple formazan. The resulting formazan was solubilized in acidified isopropanol and 1% Triton and absorbance measured at 570nm, corrected for 650nm background.
Cells were grown in 96-well plates in the presence of inhibitors as stated in figures. Media was removed by flicking and 2% formaldehyde in PBS added for 15min. After five washes with 0.1% Triton X100 in PBS, cells were blocked for 1h with 1% BSA in PBS. Three out of four replicates were exposed to primary H3K9m2 antibody, Abcam #1220 at 1/800 dilution in 1% BSA, PBS for 2h. One replicate was reserved for background control. The wells were washed five times with 0.1% Tween 20 in PBS, then secondary IR800 conjugated antibody (LiCor) and DNA-intercalating dye, DRAQ5 (LiCor) added for 1h. After 5 washes with 0.1% Tween 20 in PBS, the plates were read on Odyssey (LiCor) scanner at 800nm (H3K9m2 signal; 764nm excitation) and 700nm (DRAQ5 signal; 683nm excitation). Fluorescence intensity was quantified, normalized to background and DRAQ5 signal expressed as percentage of control.
Discovery of an in Vivo Chemical Probe of the Lysine Methyltransferases G9a and GLP; Feng Liu, Dalia Barsyte-Lovejoy, Fengling Li, Yan Xiong, Victoria Korboukh, Xi-Ping Huang, Abdellah Allali-Hassani, William P. Janzen, Bryan L. Roth, Stephen V. Frye, Cheryl H. Arrowsmith, Peter J. Brown, Masoud Vedadi, and Jian Jin; J. Med. Chem., 2013, 56 (21), pp 8931–8942.