Table 1.
MALT1 potency and selectivity profile across 156 common pharmacological targets (human unless otherwise stated) of mepazine, and tool Compounds 2 and 3, (data from Eurofins [26]), n.t. = not tested, pIC50 = negative logarithm of the concentration of compound that produce 50% inhibition/antagonism of the named target, pEC50 = negative logarithm of the concentration of the named compound that produces half the maximal agonistic effect of the named target protein.
Fig 1.
Structures of mepazine, compounds 2, 3.
Fig 2.
Characterization of allosteric MALT1 inhibitor Compound 2.
Purified human primary CD4+ T cells were stimulated with PMA and ionomycin in the presence of MALT1 inhibitors. Compound 2 and mepazine were used at 10 μM and z-VRPR-fmk at 300 μM (A-B) or 100 μM (C-D). (A) Western Blot analysis of RELB and CYLD, their cleaved product (N-RELB and N-CYLD). (B) Quantification of cleaved RELB and CYLD. All quantitative data were normalised to actin signal and calculated as a percentage relative to DMSO signal set at 100%. Data are presented as mean+/SEM with n = 4. *:p<0.05; **:p<0.005; ***:p<0.001. (donor-matched one-way ANOVA with Dunnett’s multiple comparison test) (C) Representative FACS plots of phosphorylated IκBα in CD4+ T cells following stimulation with PMA and ionomycin for 10minutes. (D) Quantification of FACS analysis of pIκBα and pERK positive cells. Data are presented as mean+/SEM with n = 3. *p<0.05; **:p<0.005. (donor-matched one-way ANOVA with Dunnett’s multiple comparison test) (E) Inhibition of IFN-γ secretion (black circles) by Compound 2 in human CD4+ cells stimulated with anti-CD3 antibody plus anti-CD28 antibody for 3 days. Proliferation (open triangles) and cytotoxicity (open squares) were measured using CFSE and 7-AAD by flow cytometry 3 days post-stimulation. Data are shown as % inhibition from 6 donors (mean ± 95% CI). EC50 values are annotated.
Fig 3.
MALT1 inhibition in mDCs decreases cytokine production in zymosan-treated cells.
(A) Unstimulated PBMCs, 5-hour LPS- or zymosan-stimulated PBMCs (pre-treated for 30 minutes with DMSO, 10 μM mepazine, 100 μM z-VRPR-fmk or 10 μM Compound 2) were analyzed by flow cytometry for intracellular expression of TNF-α or IL-6 in mDCs. Data is representative for 1 out of 3 independent experiments. Coloured dots indicate data from 3 individual donors. * p<0.05. (donor-matched one-way ANOVA with Dunnett’s multiple comparison test) (B) Expression of cytokines in the supernatant of ex vivo purified zymosan-treated mDCs after MALT1 inhibition as measured by MSD. Coloured dots indicate data from 5 individual donors. *:p<0.05; **:p<0.005; ***:p<0.001; ****:p<0.0001. (donor-matched one-way ANOVA with Dunnett’s multiple comparison test) (C) Flow cytometric analysis of the effect of MALT1 inhibition on the expression CD80, CD83, CD86 and CD40 on mDCs upon 5-hour zymosan stimulation of PBMCs. Bars represent mean ± SD and dots show individual donors (n = 3). In all experiments, values are normalized to zymosan-stimulated cells without MALT1 inhibition (set to 100%).
Fig 4.
Effect of allosteric MALT1 inhibitors on activation, proliferation and cytokine production of human CD4+ T cells.
The top panel (A-C) shows the effect of MALT1 inhibitors on CD4+CD45RA+ human T cells stimulated for 3 days with anti-CD3 + anti-CD28 antibody stimulated. Data is presented as the percentage of (A) cells expressing the activation marker CD25, (B) the percentage of cells that have divided, measured by Cell Trace Violet fluorescence intensity dilution and (C) the percentage of CD4+ cells producing IL-2 and IFN-ɣ analysed by multi-color flow cytometric analysis. Data are presented as mean ± SEM with n = 8. Coloured dots indicate data from individual donors across different stimulations.*:p<0.05; **:p<0.005; ***:p<0.001 (donor-matched one-way ANOVA with Dunnet’s multiple comparison test compared to DMSO control). The lower Panel shows the effect of the allosteric MALT1 inhibitor Compound 2 on human CD4+ CD45RO+ T cells co-cultured for 6 days with tetanus toxoid pulsed monocyte-derived dendritic cells in presence or absence of MALT1 inhibitors. Data is presented as the percentage of (D) cells expressing CD25, (E)the percentage of cells that have divided and (F) the percentage of cells producing IFN-ɣ as measured by flow cytometry. Experiments were considered positive when the mean proliferation of tetanus toxoid stimulated T-cells was greater than the mean+2SD of T-cells stimulated with unpulsed DCs. Data are presented as mean ± SEM with n = 7. Coloured dots indicate data from individual donors across different stimulations.*:p<0.05; **:p<0.001 (donor-matched one-way ANOVA with Dunnett’s multiple comparison test compared to DMSO control). In all experiments, 10 μM Compound 2, 5 μM mepazine or 100 μM z-VRPR-fmk were used.
Fig 5.
Effect of allosteric MALT1 inhibitors on activation, proliferation and cytokine production of human memory CD4+ CD45RO+ T cells.
(A) Representative FACS plots showing IFN-ɣ expression and cell trace violet (CTV) dilution in human memory CD4+ CD45RO+ T cells co-cultured with autologous monocytes and LPS and stimulated with anti-CD3 + anti-CD28 antibodies for 5 days in the absence or presence of 10 μM Compound 2, 5 μM mepazine or 100 μM z-VRPR-fmk. (B) Quantification of the percentage of cells expressing CD25 (top graph) and cells that have divided as defined by CTV dilution (lower graph), (C) and quantification of cells producing either IFN-ɣ, IL-17A, both IFN-ɣ+IL-17A or IL-2. Data are presented as mean ± SEM with n = 6. Coloured dots indicate data from individual donors across different stimulations. (D) Data representing the ratio of CD4+ T cells that have divided and express IFN-ɣ to total IFN-ɣ expressing CD4+ T cells. (E) IFN-ɣ mRNA expression in CD4+ T cells after 3 days culture with 1 μg/mL plate-bound anti-CD3 antibody + 1 μg/mL soluble anti-CD28 antibody was analysed by RT-qPCR. Data is presented relative to DMSO control set at 1 with n = 3 and shown as mean ± SEM. The significance of the data was evaluated by donor-matched one-way ANOVA with Dunnett’s multiple comparison test compared to DMSO control. *:p<0.05; **:p<0.005; ***:p<0.001.
Fig 6.
Allosteric MALT1 inhibitors affect de novo induction of human Tregs in vitro but not the function of in vitro expanded blood Tregs.
(A) Naïve human Tregs expanded in vitro for 14 days in the presence of rapamycin and then treated for 2 days with DMSO (n = 9), Compound 2 (n = 5), mepazine (n = 5), z-VRPR-fmk (n = 5) or rapamycin (n = 9). Expanded and treated Tregs were then co-cultured with CTV labeled CD4+ T cells and stimulated with anti-CD3 at the indicated ratios for 3 days. Suppressive activity was assessed as the reduction in CD4+ responder T cell proliferation normalised to responder cell proliferation in the absence of Tregs. Data is presented as mean ± SEM. (B) Number of CD4+CD25+FoxP3+ human Tregs after 14-days expansion in vitro in the presence of Compound 2, mepazine or z-VRPR-fmk or rapamycin (n = 6). Data are presented as Box and whiskers of number of live Tregs. (C) Percentage of de novo induced Tregs from naïve human CD4+ T cells stimulated for 7 days with anti-CD3 antibody plus anti-CD28 antibody in the absence or the presence of TGFβ + IL-2 + rapamycin in the presence of Compound 2, mepazine or z-VRPR-fmk (n = 5). Media indicates the basal induction of FoxP3 in activated CD4+ T cells in the absence of TGFβ + IL-2 + rapamycin. Coloured dots indicate data from individual donors across different stimulations. Data are presented as Box and whiskers plot. For all experiments, Compound 2 was used at 10 μM, mepazine at 5 μM, z-VRPR-fmk at 100 μM and rapamycin at 100 nM. One-way ANOVA with Dunnett’s for multiple comparison test was used for statistical analyses.**:p<0.01.
Fig 7.
Allosteric MALT1 inhibitor Compound 2 reduces BCR-induced and T-cell induced B-cell immune responses.
(A) and (B) CFSE stained CD19+ cells were stimulated for 5 days with anti-IgM+anti-CD40 + IL-21 in the presence or absence of 10 μM Compound 2, 5 μM mepazine or 100 μM z-VRPR-fmk. (n = 8). Proliferation (A) was assessed as CFSE dilution of CD19+ B cells, presence of CD19+CD27hi CD38hi plasmablasts was measured using flow cytometry and (B) levels of IgG, IgA and IgM in the supernatant culture of unstimulated or stimulated B cells was measured using human MSD multiplex isotyping panel. (C) and (D) CFSE stained human CD19+ cells were co-cultured with FACS sorted CD4+CXCR5+CD45RO+ T-cells and stimulated for 5 days with SEB 100 ng/ml (n = 6) in the presence or absence of 10 μM Compound 2, 5 μM mepazine or 100 μM z-VRPR-fmk. Proliferation (A) was assessed as CFSE dilution of CD19+ B cells, presence of CD19+CD27hi CD38hi plasmablasts was measured using flow cytometry and (B) levels of IgG, IgA and IgM in the supernatant culture of unstimulated or stimulated B cells was measured using human MSD multiplex isotyping panel. Data are presented as mean ± SEM. The significance of the data was evaluated by donor-matched one-way ANOVA with Dunnett’s multiple comparison test compared to DMSO control. *:p<0.05; **:p<0.005;****:p<0.0001.
Fig 8.
Effect of the allosteric MALT1 inhibitors on anti-CD3 antibody induced cytokines and on IFN-γ producing-CD4+ T cells and FoxP3+Tregs in vivo.
Blood of C57BL/6 females was collected 4h after anti-CD3 or IgG isotype control antibody injection (i.p) following 30 min pre-treatment with Compound 2 (i.p) at 30 and 90 mg/kg. (A) plasma cytokine levels in C57BL/6 mice 4 h after intraperitoneal anti-CD3 antibody injection following 30 min pre-treatment with 2 at 30 and 90 mg/kg (n = 7–8). (B) C57BL/6 mice treated orally with Compound 3 at 10 mg/kg twice daily for 4 weeks (n = 8 per group). Quantification of numbers of IFN-ɣ producing CD4+ T cells and FoxP3+ CD4+CD25high regulatory T-cells in spleen and lymph nodes (LN) as assessed by flow cytometry. (C) Quantification of plasma IFN-ɣ and IL-2 levels following 4-week treatment with Cpd 3. Cytokines levels were analysed by multiplex MSD analysis. Dots represent individual mice and data is presented as a Box and whiskers plot. (D) Representative H&E-stained colon (top) and stomach fundus (bottom) from 4-week Cpd 3 treated mice. Data were evaluated by unpaired two-sided t test with Welch’s correction against the vehicle group *:p<0.05; **:p<0.01;***:p<0.001.