Fig 2: M1i-124 and M1i-124d1 inhibit MALT1 protease and scaffolding functions in stimulated Jurkat T cells.(A) Schematic of the CBM complex highlighting the 2 functions of MALT1. MALT1 proteolytic activity cleaves RelB and N4BP1, both of which are analyzed in this study. MALT1 scaffolding activity leads to activation of the IKK complex and phosphorylation of IκB. (B and C) Effect of 1 μM compound pretreatment on CD3/CD28-induced RelB and N4BP1 cleavage in Jurkat T cells. Representative Western blots showing full-length (FL) and cleaved (Cl) proteins are shown. Quantification of the Cl/FL ratio for multiple experiments is plotted. Statistical analyses were performed using 1-way ANOVA and Dunnett’s multiple-comparison test (mean ± SEM; n = 4). (D and E) Effect of 1 μM M1i-124 (D) and 1 μM M1i-124d1 (E) on IKKα/β and ERK phosphorylation following CD3/CD28 stimulation of Jurkat T cells. Representative Western blots are shown along with quantification of the phosphorylated-to-total protein ratios for multiple experiments. Unpaired t test was used to compare each stimulation time point (mean ± SEM; n = 5). (F) IL2 mRNA induction in Jurkat T cells pretreated with 1 μM M1i-124, 1 μM M1i-124d1, or 5 μM mepazine and stimulated with PMA/Iono (mean ± SEM; n = 3). (G) Secreted IL-2 protein measured by ELISA in Jurkat T cells pretreated with M1i-124 or M1i-124d1 and stimulated with PMA/Iono (mean ± SEM; n = 3). For F and G, statistical analyses were performed using 1-way ANOVA and Dunnett’s multiple-comparison test. For all panels, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: In silico drug screen identifies M1i-124 as a BCL10-MALT1 PPI inhibitor.(A) Schematic of in silico drug screen. Three million compounds were screened using LibDock followed by application of Lipinski’s rule of 5 (Ro5) filters, which led to the identification of 9 small-molecule candidates, 7 of which were commercially available. (B) Apoptosis of TMD8 cells, quantified by flow cytometric analysis of annexin V/SYTOX Blue staining, after 6 days of treatment with 1 μM of the 7 candidate molecules from the LibDock screen (mean ± SEM; n = 6). Structure of the only active compound, M1i-124, is shown. (C) Viability of TMD8 cells after 8 days of treatment with 1 μM of the 7 commercially available molecules (mean ± SEM; n = 3). (D) Lead compound, M1i-124, is docked onto the groove between Ig1 and Ig2 near the proposed BCL10-binding site with MALT1(Ig1-2) (PDB ID: 3K0W). (E) SPR analysis of the binding of M1i-124 to immobilized full-length MALT1 confirmed target engagement using a steady-state affinity model. (F) Structures of lead compound, M1i-124, and its analog, M1i-124d1. (G and H) ELISA schematic (G) and analysis (H) showing that M1i-124 and M1i-124d1, but not C741-0547, inhibit the binding of MALT1 to immobilized BCL10 in a dose-dependent manner with indicated IC50 values (mean ± SEM; n = 2–3). For B and C, statistical analyses were performed using a 1-way ANOVA with Dunnett’s multiple-comparison test. ****P < 0.0001.

Fig 4: M1i-124 treatment leads to loss of BCL10 and MALT1 protein content in ABC-DLBCL cells.(A) Representative Western blots showing loss of BCL10 and MALT1 proteins, but not ERK1/2 protein, in TMD8 and OCI-Ly3 cells after 72 hours of treatment with 1 μM M1i-124. BCL10 or MALT1 loss was not observed after treatment with 1 μM of the negative control compound A0070495 or mepazine. (B) Representative Western blots showing time-dependent loss of BCL10 and MALT1 in TMD8 cells treated with 1 μM M1i-124. (C) Representative Western blots showing dose-dependent loss of BCL10 and MALT1 proteins in TMD8 cells treated with 1 μM M1i-124. For A–C, band quantifications, normalized for GAPDH, are indicated below each blot. (D) Time-dependent change in BCL10 and MALT1 mRNA expression levels with 1 μM M1i-124 treatment in TMD8 cells. Statistical analysis was performed using unpaired t test between 0 hours and 72 hours (mean ± SEM; n = 3). *P < 0.05, ****P < 0.0001.

Fig 5: Identification of a BCL10-binding site at the junction of MALT1 Ig1 and Ig2 domains.(A) Schematic of full-length MALT1 protein highlighting the death domain (DD), immunoglobulin-like domains (Ig), and caspase-like proteolytic domain. Specific residues used to create deletion mutants are indicated. (B and C) Co-IP of HA-tagged MALT1 protein fragments with Myc-tagged BCL10. Myc-tagged RICK and MAVS served as negative controls. (D) SPR curves quantifying the binding of MALT1 protein fragments to immobilized BCL10, with calculated KD values. (E) Co-IP of HA-tagged MALT1(Ig1-2) harboring indicated mutations with Myc-tagged BCL10. (F) Co-IP of either HA-tagged full-length (FL) MALT1 or HA-tagged MALT1(Ig1-2) harboring indicated mutations with Myc-tagged BCL10. (G) The location of each mutation that disrupts binding of MALT1(Ig1-2) to BCL10 in co-IP experiments is highlighted in purple in the published MALT1(Ig1-2) crystal structure (PDB ID: 3K0W). Data in B–F are representative of a minimum of 3 independent repeats. All co-IP experiments were performed by expression of proteins in transiently transfected 293T cells.