Scintillation Proximity Assay

Methyltransferase activity assays are performed by monitoring the incorporation of tritiumlabeled methyl group from S-adenosylmethionine (3H-SAM) to biotinylated peptide substrates using Scintillation Proximity Assay (SPA) for PRC2-EZH2 trimeric complex (EZH2:EED:SUZ12), PRC2-EZH1 pentameric complex (EZH1:EED:SUZ12:RBBP4:AEBP2), SETD7, G9a, GLP, SETDB1, SETD8, SUV420H1, SUV420H2, SUV39H2, MLL1 tetrameric complex (MLL:WDR5:RbBP5:ASH2L), PRMT1, PRMT3, PRMT5-MEP50 complex and SMYD2. The reaction buffer for SMYD2 and SMYD3 is 50 mM Tris pH 9.0, 5 mM DTT, 0.01% TritonX-100; for G9a, GLP and SUV39H2 is 25 mM potassium phosphate pH 8.0, 1 mM EDTA, 2 mM MgCl2 and 0.01% Triton X-100; and for other HMTs 20 mM Tris pH 8.0, 5 mM DTT, 0.01% TritonX-100. To stop the enzymatic reactions, 10 μL of 7.5 M guanidine hydrochloride is added, followed by 180 μL of buffer, mixed and transferred to a 384-well FlashPlate. After mixing, the reaction mixtures are incubated and the CPM counts are measured using Topcount plate reader. The CPM counts in the absence of compound for each data set are defined as 100% activity. In the absence of the enzyme, the CPM counts in each data set are defined as background (0%). IC50 values are determined using compound concentrations ranging from 100 nM to 100 μM. The IC50 values are determined using SigmaPlot software. EZH2-Y641F assays are performed using 30 nM of enzyme in 20 mM Tris pH 8, 5 mM DTT, 0.01% Triton X-100, 5 μM SAM and 1 μM of H3 (1-24) peptide (same as for the wild-type PRC2-EZH2 complex). For DNMT1, the assay is performed using hemimethylated dsDNA as a substrate. The dsDNA substrate is prepared by annealing two complementary strands (biotintlated forward strand: BGAGCCCGTAAGCCCGTTCAGGTCG and reverse strand: CGACCTGAACGGGCTTACGGGCTC), synthesized by Eurofins MWG Operon. Reaction buffer is 20 mM Tris-HCl, pH 8.0, 5mM DTT, 0.01% Triton X-100. Methyltransferase activity assays for DOT1L is performed using Filter-plates. Reaction mixtures in 20 mM Tris-HCl, pH 8.0, 5 mM DTT, 2 mM MgCl2 and 0.01% Triton X-100 are incubated at room temperature for 1h, 100 μL 10% TCA is added, mixed and transferred to filter-plate. Plates are centrifuged at 2000 rpm for 2 min followed by 2 additional 10% TCA wash and one ethanol wash (180 μL) followed by centrifugation. Plates are dried and 100 μL MicroO is added and centrifuged. 70 μL MicroO is added and CPM are measured using Topcount plate reader.

Cell lines

MCF10A cells, Diffused large B cell lymphoma cell lines

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

100 nM-5 μM for 2-8 days

Applications

UNC1999 concentration-dependently reduced H3K27me3 levels in MCF10A cells and selectively killed diffused large B cell lymphoma cell lines harboring the EZH2Y641N mutant.

Animal models

Male Swiss albino mice model

Dosage form

15, 50, or 150 mg/kg, intraperitoneal injection or oral administration for 24 h

Applications

UNC1999 is orally bioavailable and is an useful tool for the biomedical research community to assess long-term therapeutic benefit(s) and potential toxicity in mice. Moreover, UNC1999 (150 mg/kg, intraperitoneal injection) was well tolerated by all test mice.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

UNC1999 is an orally bioavailable inhibitor of EZH2 and EZH1 with IC50 values of [1].

UNC1999 binds to the EZH2 SAM binding site of EZH2 and competes with the cofactor SAM with a Ki of 4.6nM. It does not compete with the H3 peptide substrate. UNC1999 also has potency for mutant EZH2, including Y641N and Y641F mutants. Besides EZH2, UNC1999 is effective for EZH1 with IC50 value of 45nM. UNC1999 is shown to be more than 200-fold selective for EZH2 over a broad range of non-epigenetic targets, including kinases, GPCRs, transporters and ion channels [1].

EZH2 catalyzes methylation of H3K27, producing H3K27me3. H3K27me3 is a transcriptionally repressive post-translational modification. As an inhibitor of EZH2, UNC1999 reduces H3K27me3 with IC50 value of 124nM in the In-Cell Western assay. Additionally, UNC1999 can inhibit cell proliferation in a DLBCL cell line harboring the Y641N mutant with EC50 value of 633nM. Moreover, UNC1999 is reported to be orally bioavailable in mice [1].

References:
[1] Konze KD, Ma A, Li F, Barsyte-Lovejoy D, Parton T, Macnevin CJ, Liu F, Gao C, Huang XP, Kuznetsova E, Rougie M, Jiang A, Pattenden SG, Norris JL, James LI, Roth BL, Brown PJ, Frye SV, Arrowsmith CH, Hahn KM, Wang GG, Vedadi M, Jin J. An orally bioavailable chemical probe of the Lysine Methyltransferases EZH2 and EZH1. ACS Chem Biol. 2013;8(6):1324-34.