In Vitro Histone Methyltransferase (HMT) Assay

The HMT assay is performed in 50 mM HEPES pH 7.8, 100 mM NaCl, 1.0 mM EDTA, and 5% glycerol at 22 ℃. Each reaction contains 1.5 μCi of the co-factor, 3H-S-adenosylmethionine. H3 10-residue peptide is used as the substrate at 50 μM. Compounds are added at concentrations ranging from 0.125 to 128 μM and incubated with the pre-assembled WDR5/RbBP5/ASH2L complex at a final concentration of 0.5 μM for each protein for 2–5 min. Reactions are initiated by addition of the MLL1 protein at a final concentration of 0.5 μM and allowed to proceed for 30 min before preparing scintillation counting. To count samples, reactions are spotted on separate squares of P81 filter paper and precipitated by submerging in freshly prepared 50 mM sodium bicarbonate buffer with pH 9.0. After washing and drying, samples are vortexed in Ultima Gold scintillation fluid and counted. As a negative control, assays are performed using 0.5 μM MLL1/WDR5/RbBP5/ASH2L complex assembled with the non-interacting mutant, WDR5D107A.

Cell lines

MV4-11 cells, KOPN8, and K562 cells

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

6.25, 25, 50 μM for 4-7 days;

Applications

MM-102 significantly inhibited the expression of HoxA9 and Meis-1, two critical MLL1 target genes in MLL1 fusion protein mediated leukemogenesis. MM-102 (6.25, 25, 50 μM) also specifically suppressed cell growth and induced apoptosis in leukemia cells harboring MLL1 fusion proteins.

MM-102 is an antagonist of MLL1 with IC50 value of 2.4nM [1].

Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase. The interaction of MLL1 and WDR5 is essential for MLL1 enzymatic activity and is a target for the treatment of acute leukemia. MM-102 is a peptidomimetic of MLL1. It has a high binding affinity to WDR5 with IC50 value of 2.4nM [1].

MM-102 is reported to reduce the expression of MLL1 targeted genes HoxA9 and Meis-1 in MLL1-AF9 transduced murine cells. Since these two genes are required for MLL1 mediated leukemogenesis, MM-102 is found to inhibit cell growth of leukemia cells harboring MLL1 fusion proteins. It dose-dependently inhibits the cell growth in the MV4;11 and KOPN8 cell lines with IC50 values both of 25μM[1].

References:
[1] Karatas H, Townsend EC, Cao F, Chen Y, Bernard D, Liu L, Lei M, Dou Y, Wang S. High-affinity, small-molecule peptidomimetic inhibitors of MLL1/WDR5 protein-protein interaction. J Am Chem Soc. 2013 Jan 16;135(2):669-82.