| In Vitro | In vitro activity: MI-2 inhibits MALT1 functions in ABC-DLBCL cell lines with excellent cell penetration. MI-2 produces selective growth inhibition for MALT1-dependent cell lines with GI50 of 0.2, 0.5, 0.4, and 0.4 礛 in HBL-1, TMD8, OCI-Ly3, and OCI-Ly10 cells, whereas the ABC-DLBCL MALT1-independent cell lines, U2932 and HLY-1, and the two GCB-DLBCL cell lines were resistant
Kinase Assay: 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 Assay: Cell proliferation (MALT1-independent cell lines: U2932 and HLY-1, and the two GCB-DLBCL cell lines; MALT1-dependent cell lines: HBL-1, TMD8, OCI-Ly3, and OCI-Ly10 cells) 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. |
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