MI-2 (MALT1 inhibitor) is an irreversible MALT1 inhibitor.

MALT1:5.84 μM

Daily intraperitoneal injection of 25 mg/kg MI-2 effectively inhibits the growth of TMD8 and HBL-1 ABC-DLBCL xenografts, while it has no effect on the growth of OCI-Ly1 tumors.

MI-2 demonstrates excellent cellular permeability and inhibits the function of MALT1 in the ABC-DLBCL cell lines. It selectively inhibits the growth of MALT1-dependent cell lines, with GI50 values of 0.2, 0.5, 0.4, and 0.4 μM in HBL-1, TMD8, OCI-Ly3, and OCI-Ly10 cells, respectively. Conversely, ABC-DLBCL cell lines not dependent on MALT1, such as U2932 and HLY-1, along with two types of GCB-DLBCL cell lines, exhibit resistance to MI-2.

High-Throughput Screening for MALT1 Proteolytic Activity Inhibitors: Ac-LRSR-AMC is used as substrate and reactions are measured with excitation/emission wavelengths of 360/465 nm. Two time points are measured for each reaction to eliminate false positives due to compound autofluorescence. The final percent inhibition is calculated with the formula {[fluorescencetest compound (T2-T1)-fluorescenceneg ctrl (T2-T1)]/[fluorescencepos ctrl (T2-T1)-fluorescenceneg ctrl (T2-T1)]} × 100, where Z-VRPR-FMK (300 nM) is used as positive control and buffer only as negative control. The positive hits are validated in concentration-response experiments within a dose range of 0.122–62.5 μM to determine IC50 of the compounds. Activity is validated using recombinant full-length wild-type MALT1.

Cell proliferation is determined by ATP quantification using a luminescent method and trypan blue dye exclusion. Standard curves for each cell line are calculated by plotting the cell number (determined using trypan blue) against their luminescence values, and cell number is calculated accordingly. Cell viability in drug-treated cells is normalized to their respective controls (fractional viability), and results are given as 1-fractional viability. CompuSyn software is used to determine GI25 and GI50 values.(Only for Reference)