MM-102

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 °C. 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.

References:

[1]H. Karatas, E.C. Townsend, F. Cao, Y. Chen, D. Bernard, L. Liu, M. Lei, Y. Dou, S. Wang, High-affinity, small-molecule peptidomimetic inhibitors of MLL1/WDR5 protein-protein interaction. J Am Chem Soc 135 (2013) 669-682.