We thank Sucharity Mistry for her assistance with the initial draft. serum IL-10 levels exquisitely correlates with the drug pharmacokinetics and degree of MALT1 inhibition in vitro and in vivo and could constitute a useful pharmacodynamic biomarker to evaluate these compounds in clinical trials. Compound 3 revealed insights into the biology of MALT1 Mouse monoclonal antibody to CDC2/CDK1. The protein encoded by this gene is a member of the Ser/Thr protein kinase family. This proteinis a catalytic subunit of the highly conserved protein kinase complex known as M-phasepromoting factor (MPF), which is essential for G1/S and G2/M phase transitions of eukaryotic cellcycle. Mitotic cyclins stably associate with this protein and function as regulatory subunits. Thekinase activity of this protein is controlled by cyclin accumulation and destruction through the cellcycle. The phosphorylation and dephosphorylation of this protein also play important regulatoryroles in cell cycle control. Alternatively spliced transcript variants encoding different isoformshave been found for this gene in ABC DLBCL, such as the role of MALT1 in driving JAK/STAT signaling and suppressing the type I IFN response and MHC class II expression, suggesting that MALT1 inhibition could prime lymphomas for immune recognition by cytotoxic immune cells. control. Cells were stimulated with vehicle or 200 ng/ml PMA and 1 M IO for 2 hours. FC relative to the nontargeting shRNA (shNT). Results are representative of 2 independent experiments performed in triplicate. **** 0.0001, by ANOVA with Tukeys multiple comparisons adjustment. (D) MALT1 expression in MALT1-knockdown Raji MALT1-GloSensor reporter cells assayed in C. Numbers below the blot indicate MALT1 expression FC versus shNT (MALT1/actin). (E) Dose-dependent inhibition of MALT1 reporter activity in response to Z-VRPR-fmk. Cells were pretreated for 30 minutes with the inhibitor before PMA and IO stimulation, as in B. EPZ031686 RLU, relative luciferase units. Data represent the mean SD of 1 1 representative experiment. Next, we generated a stable Raji MALT1-GloSensor reporter cell line and observed that luciferase activity was induced 10-fold following PMA and IO treatment (Figure 1C) (ANOVA followed by Tukeys multiple comparisons test; 0.0001). To verify MALT1 specificity, Raji cells expressing the MALT1-GloSensor reporter were infected by lentiviruses expressing either MALT1 shRNAs or a nontargeting control (shNT). We found that MALT1 knockdown caused a significant reduction in luciferase activity (by 58% and 66% for shMALT1_1 and shMALT_2; ANOVA followed by Tukeys multiple comparisons test; 0.0001 for both shRNAs), which was proportional to the knockdown efficiency of the shRNAs (Figure 1D), demonstrating that the GloSensor reporter activity was MALT1 specific. As EPZ031686 an additional control, we tested whether the specific and irreversible MALT1 inhibitor peptide Z-VRPR-fmk could EPZ031686 extinguish GloSensor activation by PMA and IO. Raji MALT1-GloSensor cells were pretreated with various doses of Z-VRPR-fmk for 30 minutes and then induced with PMA and IO for 1 hour. We observed that increasing concentrations of Z-VRPR-fmk resulted in a dose-dependent decrease in luciferase activity (Figure 1E). To rule out artifact due to interference of peptides with the luminescence signal, we tested activity in parallel, which indeed was not affected by Z-VRPR-fmk (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/JCI99436DS1). Development of a selective substrate-mimetic inhibitor of MALT1. In order to develop superior MALT1 catalytic activity inhibitors, we used the following 3 different assays to support structure-activity relationship (SAR) studies: (a) an in vitro biochemical assay using a recombinant form of MALT1 (aa 340C789) fused to a leucine zipper dimerization motif (LZ-MALT1) that promotes MALT1 dimerization and activation (23); (b) an assay using the above-described cell-based GloSensor reporter that measures MALT1 protease activity in live cells (Figure 1, ACE); and (c) a differential growth inhibition assay of ABC versus GCB EPZ031686 DLBCL cell lines. Equipped with these tools, we used Z-VRPR-fmk as a starting point to develop substrate-mimetic MALT1 inhibitors. Z-VRPR-fmk was derived from the optimal tetrapeptide substrate for the metacaspase AtmC9 (29) and incorporates an electrophilic fluoromethyl ketone warhead, which forms a covalent bond with the active site cysteine residue (Figure 2A). Although Z-VRPR-fmk has detectable activity in cell-based assays (22, 30), its efficacy is highly limited because of poor cell penetration, probably due to the 2 arginine residues. Previous studies of MALT1 substrate specificity based on positional scanning libraries EPZ031686 (31, 32) and co-crystal structures with Z-VRPR-fmk (31, 33) had suggested that the P1 arginine might be critical, given the multiple interactions with acidic residues in the P1 pocket, but that the P3 arginine could be replaced (Supplemental Figure.
We thank Sucharity Mistry for her assistance with the initial draft