High throughput screening

FITC-MBM1 at 15 nM and Menin at 150 nM in the FP buffer were mixed and incubated for 1hr in the dark at room temperature. For point screening, the 0.2 μL of each compound (20 μM final concentration, 1% DMSO) was added to 20 μL of the aliquot of the protein-peptide mixture and incubated on 384-well plates in the dark at room temperature for 1hr. In confirmation screening, the serial dilution plates with compounds in DMSO were prepared and used to titrate the Menin-FITC-MBM1 complex. Change in fluorescence polarization was monitored at 525 nm after excitations at 495 nm using the PHERAstar microplate reader (BMG) and applied to determine IC50 values with the Origin 7.0 program.

Cell lines

MLL-AF9 and MLL-ENL transduced murine BMCs

Preparation method

The solubility of this compound in DMSO is >15.1 mg/mL. 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

3 ~ 25 μM

Applications

In MLL-AF9 and MLL-ENL transduced murine BMCs, MI-2 inhibited the proliferation of BMCs with a GI50 value of about 5 μM. Moreover, MI-2 reduced colony formation in BMCs transduced with MLL-AF9. In a series of human MLL leukemia cell lines with different MLL translocations, MI-2 significantly inhibited cell proliferation and induced cell apoptosis.

MI-2 is a Menin-MLL inhibitor with IC50 value of 0.45 μM [1].

Mixed-lineage leukemia (MLL) is a common target of chromosomal translocations in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). The leukemogenic activity of MLL fusion proteins is dependent on their direct interaction with menin, which is a tumor suppressor that directly controls cell growth in endocrine organs [1].

MI-2 is a Menin-MLL inhibitor. MI-2 bound to the wild-type menin but didn’t bind M278K and Y323K menin mutants. In HEK293 cells transfected with Flag-MLL-AF9, MI-2 effectively inhibited the menin-MLL-AF9 interaction without affecting the expression level of menin and MLL-AF9. In mouse bone marrow cells (BMC) transduced with MLL-AF9 and MLL-ENL, MI-2 blocked proliferation of BMC with GI50 value of about 5 μM. Also, MI-2 inhibited colony formation in BMC transduced with MLL-AF9. In a series of human MLL leukemia cell lines, MI-2 significantly inhibited translocation in a dose dependant way [1].

Reference:
[1].  Grembecka J, He S, Shi A, et al. Menin-MLL inhibitors reverse oncogenic activity of MLL fusion proteins in leukemia. Nat Chem Biol, 2012, 8(3): 277-284.