Chemical synthesis

Mepazine (Compound 1) was synthesized according to the published procedure [24]. Compound 3 was also synthesised according to the published procedure [25]. Compound 2 was synthesised as follows.

1. Step 1
   Methyl 4-amino-2-chlorobenzoate (1.88 g, 10.2 mmol) was added to a mixture of 1-phenyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid (2.0 g, 7.8 mmol), 1-propanephosphonic acid cyclic anhydride (50% in ethyl acetate) (9.9 g, 15.6 mmol) and N,N-diisopropylethylamine (4.1 mL, 23.4 mmol) in ethyl acetate (20 mL) under nitrogen. The resulting solution was stirred at 50 °C for 16 h. The reaction mixture was diluted with ethyl acetate and washed sequentially with saturated aqueous sodium hydrogen carbonate (x 2) and saturated brine (x 2). The organic layer was dried over sodium sulphate, filtered and concentrated. The crude product was purified by flash silica chromatography, elution gradient 5 to 50% ethyl acetate in petroleum ether, to afford methyl 2-chloro-4-(1-phenyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxamido)benzoate (3.10 g, 94%) as a pale yellow oil which solidified on standing. m/z (ES+), [M+H]+ = 424.1; C18, 10–95% acetonitrile (5mM aqueous NH₄HCO₃), 2.6min, HPLC tR = 1.5 min. ¹H NMR (400 MHz, chloroform-d) δ ppm 3.95 (s, 3 H), 7.44–7.51 (m, 2 H) 7.51–7.58 (m, 3 H), 7.60 (dd, 1 H) 7.85 (d, 2 H) 7.93 (d, 1 H) 8.05 (s, 1 H).
2. Step 2
   2M aqueous lithium hydroxide (5 mL, 10 mmol) was added to a stirred solution of methyl 2-chloro-4-(1-phenyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxamido)benzoate (1.82 g, 3.2 mmol) in a mixture of tetrahydrofuran (5 mL) and methanol (5 mL) at room temperature. After stirring for 2 h the reaction was warmed to 35 °C for an additional 2 h. The mixture was concentrated under reduced pressure and the aqueous remains diluted with water. The pH was adjusted by the addition of aqueous 1 M hydrochloric acid to pH ~3 then extracted with ethyl acetate (x 2). The combined organic extracts were washed with water, saturated brine, dried over magnesium sulphate, filtered and concentrated. The crude product was dissolved in methanol, adsorbed onto diatomaceous earth and purified by silica gel chromatography (Isco Combiflash, 80 g; gradient elution: 15% ethyl acetate/hexane for 2 min then 15% to 100% ethyl acetate/hexane over 22 min at 60 mL/min) via detection with UV light at 254nm to afford 2-chloro-4-(1-phenyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxamido)benzoic acid (0.929 g, 70%) as an off-white solid. m/z (ES+), [M+H]+ = 410.2; C18, 2–98% acetonitrile (0.1% aqueous formic acid), 2min, HPLC tR = 1.01 min. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 7.50–7.57 (m, 2 H) 7.58–7.63 (m, 3 H) 7.71 (dd, J = 8.67, 2.07 Hz, 1 H) 7.88 (d, J = 8.48 Hz, 1 H) 7.97 (d, J = 2.07 Hz, 1 H) 8.34 (s, 1 H) 10.85 (s, 1 H) 13.14 (br s, 1 H).
3. Step 3
   N,N-Diisopropylethylamine (1.3 mL, 7.3 mmol) was added to a stirred solution of azetidin-3-ol hydrochloride (481 mg, 4.4 mmol) and 2-chloro-4-(1-phenyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxamido)benzoic acid (600 mg, 1.5 mmol) in anhydrous dimethylformamide (10 mL) under nitrogen. To this was added 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) (1.11 g, 2.9 mmol) and the resulting solution was allowed to stir at room temperature for 20 h. The solution was diluted with ethyl acetate and washed with saturated aqueous sodium hydrogen carbonate, saturated brine (x 2), dried over magnesium sulphate, filtered and concentrated. The crude product was taken up in methanol, adsorbed onto diatomaceous earth and purified by silica gel chromatography (Isco Combiflash, 20 g; gradient elution: 1% methanol/dichloromethane for 2 min then 1% to 7% methanol/dichloromethane over 16 min at 30 mL/min) via detection with UV light at 254 nm to afford a white solid. This was subjected to supercritical fluid chromatography (2-ethylpyridine 4.6 mm x 150 mm 5μm column; gradient elution:10–55% methanol in CO₂ over 5 min at 5mL/min, 40 °C, 100 bar outlet pressure). After concentration, the resulting material was dried under vacuum overnight at 40 °C/~50 mTorr then again at 50 °C/~50 mTorr overnight then again at 60 °C/~35 mTorr overnight to afford N-(3-chloro-4-(3-hydroxyazetidine-1-carbonyl)phenyl)-1-phenyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide (Compound 2) (408 mg, 60%) as a white solid. m/z (ES+), [M+H]+ = 465.2; C18, 2–98% acetonitrile (0.1% aqueous formic acid), 2min, HPLC tR = 0.89 min. m/z (ES+), [M+H]+ = 465.2; C18, 2–98% acetonitrile (0.1% aqueous NH₄OH), 2min, HPLC tR = 0.86 min. ¹⁹F NMR (282 MHz, DMSO-d₆) δ ppm -54.79. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 3.66–3.83 (m, 2 H) 4.05 (t, J = 8.19 Hz, 1 H) 4.18–4.28 (m, 1 H) 4.46–4.58 (m, 1 H) 5.78 (d, J = 6.03 Hz, 1 H) 7.42 (d, J = 8.48 Hz, 1 H) 7.51–7.58 (m, 2 H) 7.59–7.64 (m, 3 H) 7.65–7.70 (m, 1 H) 7.94 (d, J = 2.07 Hz, 1 H) 8.34 (s, 1 H) 10.79 (s, 1 H).

MALT1 FRET assay

The assay detected compounds that inhibited the MALT1 cleavage of the four amino acid (LRSR) FRET substrate resembling BCL10 protein, one of the natural occurring substrates for MALT1. The four amino acid peptides were labelled with the fluorophore Rhodamine 110 (Rh110) as a donor and a non-fluorescent quencher (Ac). When the peptide is cleaved by MALT1 after the second arginine (R), this results in an increase in donor signal. Inhibition of enzyme activity can be measured as absence of increase in signal over time. Sodium citrate was shown to add stability to the protein (DSF) and increased the FRET signal up to 0.67M. It was decided that the assay would be run at 0.5M sodium citrate since the assay signal was considered sufficient.

The Km for Ac-LRSR-Rh110 (peptide) was calculated to 13 and 20 μM in two separate experiments and the final assay concentration of 20 μM was chosen.

A final enzyme concentration of 1 nM was used with a reaction time of 3–4 h to get a good enough assay window. The reaction was found to be linear for at least 5 h.

Reagents were as follows: Hepes-NaOH pH 6.86 Sigma (H3375), EDTA Sigma (E7889), CHAPS, G Biosciences (DG096), TCEP Invitrogen (T2556), sodium citrate Merck (1.06448.1000), (Ac-Leu-Arg-Ser-Arg)-2Rh110 (Ac-LRSR-Rh110) AnaSpec (AS-PE90-2504). MALT1 protein was prepared in house (pbCPSS1800).

The assay buffer was prepared on the day of the experiment 0.025 mM HEPES pH 6.86, 0.1 mM EDTA, 0.05% CHAPS, 1 mM TCEP, 750 mM sodium citrate, and was used to prepare 2 nM MALT1 pbCPSS1800 and 40 mM peptide stock solutions.

The assay was run in Greiner medium bind black 384-well low volume microplate (#784076). The compounds were predispensed into these as assay ready plates from 10 mM stocks in DMSO to give 10-point dose response curves in 9 half-log dilution steps with top assay concentration of 50 mM and lowest assay concentration of 3.125 nM. DMSO was used as a negative control and mepazine was used at 5 mM as a positive inhibition control. To begin the assay 2 ml of enzyme solution was dispensed into the assay plate and allowed to stand for 30mins at room temperature. To each well was added 2 ml of 40 mM peptide solution. The assay plate spun at 800 rpm for 5 seconds to ensure no reagents were left stuck to the edge of the well. The plate was sealed and left for 3–4 hours at RT. The fluorescence of the wells was read on a PhHERAstar reader, excitation 485 nm emission 520 nm, flying mode, focal height 10.1 mm gain set 390. DMSO was used as a null control. The data was analysed in Genedata (https://www.genedata.com).

Calculation method: where X represents the normalized value for the compound based on the 0 and 100% controls.

Secondary pharmacology

All secondary pharmacology assays (Table 1) were performed by Eurofins (www.eurofins.com).

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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.

https://doi.org/10.1371/journal.pone.0222548.t001

Blood collection, isolation of PBMCs and isolation of human cell subsets

Healthy donors were recruited from AZ volunteers and all samples were taken following appropriate blood collection guidelines. All blood donor volunteers signed Informed Consent form and donation was approved by AstraZeneca Institutional review board and local Ethic committee (033–10). PBMCs were isolated from the blood by density-gradient centrifugation in Leucosep tubes (Greiner Bio-One) filled with Ficoll-Paque plus (GE Healthcare) or using LymphoPrep density gradient medium and SepMate tubes (Stem Cell Technologies) for the Dendritic cell work. All cells were purified by either negative or positive selection using the appropriate isolation kits (see below) according to the manufacturer’s instructions (Miltenyi Biotech).

Stimulation of myeloid cells

Purified PBMCs were resuspended in Iscove´s Modified Dulbecco´s Medium (Gibco) + 10% fetal bovine serum (Gibco) and z-VRPR-fmk (100 μM), mepazine (10 μM) or Compound 2 (10 μM) were added. After 30 minutes, cells were stimulated either with 100 μg/mL zymosan (Zymosan-depleted, InvivoGen) or 20 ng/mL LPS (from E-coli, InvivoGen) for 5 hours. BD GolgiStop (BD Biosciences) was added 1 hour after LPS or zymosan addition. Following stimulation, cells were analysed by flow cytometry for cell surface markers and intracellular cytokines on a BD LSR Fortessa.

In some experiments, myeloid dendritic cells were separated from PBMCs using EasySep Human Myeloid DC Enrichment Kit (Stem Cells, #19061). CD14+ Monocytes were isolated using anti-CD14 MicroBeads (Miltenyi, #130-050-201). CD16-expressing monocytes were further purified from the CD14-negative fraction using anti-CD16-PE (BD Biosciences, #555407) and anti-PE microbeads (Miltenyi, #130-048-801). The CD16 negative fraction constituted the CD14+ cells(CD14+/CD16-). Purified mDCs or monocytes were stimulated overnight with 10 ng/mL LPS (from E-coli, InvivoGen) and 50 μg/mL zymosan (InvivoGen). The supernatants from mDCs (4x10⁴-13x10⁴ cells), CD14+ monocytes (7x10⁴-14x10⁴ cells) or CD16+ monocytes (3x10⁴-5x10⁴ cells), were analysed for secreted levels of IL-1β, IL-23, IL-12p70, IL-6, TNF-α and IL-10, using a custom-made multiplex (U-plex) ELISA assay (Meso Scale Discovery) according to the manufacturer’s instructions. Output values were normalized to cell number and statistical significance between treatments was estimated using one-way ANOVA with Dunnet’s post test.

Stimulation of purified human CD4+ T cells

Human naive CD4+CD45RA+ cells were isolated from PBMC using a naïve CD4+ T cell Isolation kit (Miltenyi, #130-094-131) and stained with 0.25 μM cell trace violet (In vitrogen) and plated out (1x10⁵ cells/well) into a 96-well flat bottom plates (Nunc) coated with 1 μg/mL anti-CD3 antibody (OKT3-eBioscience). Soluble anti-CD28 (CD28.2-eBioscience) was added at a final concentration of 5 μg/mL and cells were cultured for 3 days.

For the tetanus toxoid antigen specific assay, monocytes were derived into Mo-DCs with 100 ng/mL GM-CSF and 40 ng/mL IL-4 (both PeproTech) as described elsewhere for 5 to 6 days. Mo-DCs were pulsed with 2.5 μg/mL tetanus Toxoid (Calbiochem) and matured with 100 ng/mL LPS (SIGMA-Aldrich) for 24 h. Mo-DCs were washed and co-cultured at a 1:50 ratio with CD4+CD45RO+ purified cells stained with 0.25 μM cell trace violet (Invitrogen) in a 96-well flat bottom plates (Nunc). Cells were incubated for 6 days.

In some experiments, human CD4+ memory T cells were cultured with autologous monocytes at a ratio 10:1 in the presence of soluble anti-CD3 and anti-CD28 antibodies (both eBioscience) and 100 ng/mL LPS (SIGMA-Aldrich) for 5 days in a 96-well flat bottom plates (Nunc).

In all experiments, cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS (Gibco) in triplicate. All cultures were incubated at 37 °C 5% CO₂. In all experiments, Compound 2 was used at 10 μM, mepazine at 5 μM and z-VRPR-fmk a peptide inhibitor of MALT1 proteolytic activity at 100 μM unless otherwise indicated.

Stimulation of purified human B cells

Human B cells were stained with 0.25 μM CFSE (Invitrogen) and plated out into a 96-well V-bottom plate (5x10⁴ cells/well) in triplicate. Cells were stimulated for 5 days with 5 μg/mL anti-IgM (Jackson ImmunoResearch), 100 ng/mL anti-CD40 (R&D) and 10 ng/mL IL-21 (PeproTech) final concentration.

For the B cell co-culture with CD4+CD45RO+CXCR5+ T cells, CD4+ T cells were first enriched by positive selection using CD4 microbeads (Miltenyi Biotech), then stained with CD45RO (BD) and CXCR5 (Biolegend) antibodies in PBS-0.5% BSA-2 mM EDTA. CD4+CD45RO+CXCR5+ were FACS sorted on an ARIA-III (BD Bioscience).

CFSE stained B cells and CD4+CD45RO+CXCR5+ T cells were plated out into a 96-well V-bottom plate at a 1:1 ratio and cells were stimulated with SEB 10 ng/mL (SIGMA-Aldrich), IL-21 10 ng/mL and BAFF 10 ng/mL (PeproTech) final concentration. Cells were incubated for 5 days.

Supernatants were collected and antibody production was analysed using a multiplex isotyping Panel 1 kit (Meso Scale Discovery).

Treg induction, expansion and suppression assay

For Treg induction, naïve CD4+ T cells (1.5 x 10⁵ cells/well) were plated out in a 96-well flat bottom plates (Nunc) and stimulated with anti-CD3+ anti-CD28 antibody-coated Dynabeads (life Technologies) at a ratio 1:2 in the presence of 5 ng/mL TGF-β, 10 ng/mL IL-2 (both PeproTech) and 100 nM rapamycin (SIGMA-Aldrich). In all experiments, Compound 2 was used at 10 μM, mepazine at 5 μM and z-VRPR-fmk at 100 μM. Cells were incubated for 5 days at 37 °C 5% CO₂. Dynabeads were then removed and cells were transferred to a 96-well round bottom plate. Cells were washed and incubated for another 2 days with 10 ng/mL IL-2 only before flow cytometric analysis.

For expansion experiments, naïve Tregs were isolated using CD4+CD25+CD45RA+ (Miltenyi Biotech, #130-093-631). Cells were distributed in a 96-well round bottom plate (Nunc) and stimulated with anti-CD3+anti-CD28 antibody coated expander Dynabeads (Life Technologies) according to the manufacturer’s instructions. Rapamycin (100 nM) was added to avoid loss of FOXP3 expression during the expansion phase. IL-2 and rapamycin were replenished every 2 days.

After 7 days expansion, cells were washed and transferred in a 48-well culture plate (Nunc). Cells were cultured in presence of 10 ng/mL IL-2 for an additional 4 days before cells were restimulated with anti-CD3+anti-CD28 antibody coated activator Dynabeads at a ratio 1:2 in the presence of 10 μM Compound 2, 5 μM mepazine, 100 μM z-VRPR-fmk or 100 nM rapamycin. Cells were incubated for 3 days at 37 °C 5% CO₂ prior setting-up the suppression assay.

For the suppression assay, expanded Tregs were washed and then mixed with cell trace violet stained autologous naïve CD4+ responder cells in a ratio 1:1, 1:2 or 1:4 in the presence of autologous mitomycin-C (SIGMA-Aldrich) treated PBMCs and 200 ng/mL soluble anti-CD3 (OKT3. eBioscience). Cells were incubated for 3 days at 37 °C 5% CO₂ and proliferation of the responder cells was analysed by flow cytometry on a BD Canto II.

Flow cytometry and intracellular staining

Antibody combinations were used to identify and quantify specific cell types in cultures. Fluorochrome-conjugated mAbs to human CD3 (BV421; Alexa 488), CD4 (PerCp-Cy5.5; Horizon V450 and APC-H7), CD25 (FITC; APC), CD45RO (APC-H7), CD45RA (BV510), FoxP3 (PE), IFN-ɣ (Alexa 647), IL-17A (PE), IL-2 (BV510), CD27 (BV421), CD56 (Alexa Fluor 488), HLA-DR (BV786), CD123 (BV711), CD80 (BUV395), CD86 (Alexa Fluor 700), CD83 (APC), CD19 (PerCp-Cy5.5; Alexa 488), IgD (PE-cy7), IgG (BV510) and CD38 (APC) were purchased from BD Bioscience. CXCR5 (PE), BDCA-2 (PE), CD1c (PerCp-Cy5.5), CD11c (BV421) and CD14 (BV605) were purchased from Biolegend. pIκBα (eFluor660) and pErk (PerCp-eFluor710) were obtained from eBioscience.

Cell viability was analysed by staining the cells with Fixable Viability Dye eFluor 780 purchased from eBioscience or LIVE/DEAD fixable aqua stain (Molecular probes).

For intracellular cytokine staining in human CD4+ T cells, cells were incubated for 4 h at 37 °C 5% CO₂ in the presence of 500 ng/mL ionomycin, 10 ng/mL PMA and 10 μg/mL Brefeldin A (all obtained from SIGMA-Aldrich). Cells were washed in PBS, stained with the Fixable Viability Dye (eBioscience) and cell surface receptor antibodies for 20 min at 4 °C. Cells were washed twice with PBS-0.5% BSA-0.05% NaN₃ then fixed with BD Fixation/permeabilisation buffer for 15 min at 4 °C. After washing the cells in the BD permeabilisation buffer, cells were stained with IFN-ɣ, IL-2 and IL-17A (when appropriate) for 30 min at 4 °C. Cells were washed twice with the BD permeabilisation buffer and resuspended in PBS-0.5% BSA-0.05% NaN₃ for analysis.

For expression of intracellular cytokines in myeloid cells after LPS or Zymosan stimulation, PBMCs were stained with LIVE/DEAD fixable aqua stain (Molecular Probes) and for cell surface markers with human antibodies against CD3 (Alexa Fluor 488), CD19 (Alexa Fluor 488), CD56 (Alexa Fluor 488), HLA-DR (BV786), CD123 (BV711), CD80 (BUV395), CD86 (Alexa Fluor 700), CD83 (APC), BDCA-2 (PE), CD1c (PerCpCy5.5), CD11c (BV421) and CD14 (BV605). Cells were fixed in 4% paraformaldehyde, permeabilized using the BD Cytofix/Cytoperm buffer (BD Biosciences) and stained with anti-TNF-α (BUV395) and anti-IL-6 (APC) (BD Biosciences).

For FoxP3 intracellular staining, cells stained with Fixable viability Dye eFluor 780 and for cell surface CD4 and CD25 for 20 min in PBS, washed in PBS-0.5% BSA-0.05% NaN₃ and fixed with the FoxP3 fixation buffer (eBioscience) for 30 min at 4 °C. Cells were then washed with the FoxP3 permeabilisation buffer (eBioscience) and stained with anti-FoxP3 antibodies for 45 min at 4 °C. Cells were washed twice in permeabilisation buffer and resuspended in PBS-0.5% BSA-0.05% NaN₃ for analysis.

For phosphorylation analysis, PBMCs were incubated O/N with Compound 2, mepazine or z-VRPR-fmk at indicated concentrations in 96 well-round bottom plate. Cells were then washed in RPMI and resuspended in 50 μL RPMI1640 containing 50 ng/mL PMA and 500 ng/mL ionomycin for 10 min at 37 °C. Cells were then immediately fixed with 150 μL paraformaldehyde 2% solution in PBS (affymetrix). Cells were centrifuged and resuspended in Fix/perm Solution (BD bioscience stained with CD4, CD45RA and CD19 (BD). After permeabilisation cells were stained with phospho-IκBα and phospho-ERK (eBioscience).

Flow cytometric data were acquired on a FACS Canto II or an LSR Fortessa (BD Bioscience) and the data was analyzed with FlowJo software.

Immunoblotting

Purified Human CD4+ T cells (Miltenyi Biotech) were pre-incubated with 10 μM proteasome inhibitor MG132 (SIGMA-Aldrich) and 10 μM Compound 2, 10 μM mepazine or 300 μM z-VRPR-fmk for 30 min prior stimulation with 10 ng/mL PMA and 250 ng/mL ionomycin. After 1 h stimulation, cells were lysed in RIPA buffer and protease inhibitor (cOmplete; Roche). Proteins were separated by SDS-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose. Membrane was blocked with 5% bovine serum albumin and probed with anti-CYLD (D1A10; Cell Signaling Technology) or anti-RELB (C1E4; Cell Signaling Technology). After incubation with IRDye conjugated secondary antibodies (LI-COR), proteins were detected using a Odyssey imager (LI-COR).

Animals

Female C57BL/6 mice (8 weeks, Charles River laboratories, UK) were group housed in pathogen-free conditions in a temperature-controlled environment (20–21 °C) with a relative humidity of 55±15% and a light-dark cycle of 12 h (lights on at 06:00 and lights off at 18:00). They were provided food (R70, Lantmännen, Sweden) and water ad libitum.

Animal experiments were approved by the Local Ethical committee in Gothenburg (123–2014).

Analysis of MALT1 inhibition in vivo

For analysis of the effect of MALT1 inhibition on cytokine release in vivo, C57BL/6 mice were injected I.P with Compound 2 at 30 or 90 mg/Kg or vehicle 30 min prior anti-CD3 antibody (145-2C11. BD) or IgG1κ isotype control (A19-3. BD) injection (I.P). 4 hours after anti-CD3 antibody or isotype injection, mice were sacrificed and blood was collected in EDTA tubes (microvette 600) for cytokine analysis using V-PLEX proinflammatory Panel 1 kit (Meso Scale Discovery). For the 4 week safety study, C57BL/6 mice were dosed orally twice a day with 10 mg/Kg Compound 3. After 4 weeks, mice were euthanized by inhalation of isoflurane and exsanguination. Blood was collected in EDTA tubes (microvette 600) for cytokine analysis using V-PLEX proinflammatory Panel 1 kit (Meso Scale Discovery). Lymph nodes and spleen were collected and mashed. Cells were analysed by multicolor flow cytometry and numbers of IFN-γ producing cells or FoxP3 regulatory T cells were calculated.

Histopathology

Mice were necropsied at 12 weeks of age and tissues were sampled and weighed, including adrenal glands, brain, heart, kidney, liver, lung, mesenterial lymph nodes, pancreas, spleen, thymus, sublingual gland, parotis, stomach, duodenum, jejunum, ileum, cecum, and colon. Tissues were subsequently fixated in paraffin, sectioned at 4–6 μm, stained with hematoxylin and eosin (H&E) for histopathological examination under microscope.

RNA isolation and reverse transcriptase quantitative PCR (RT-qPCR)

Human CD4+ T cells were isolated by negative selection (Miltenyi Biotech) and plated out (2x10⁵ cells/well) into a 96-well flat bottom plates (Nunc) coated with 1 μg/mL anti-CD3 antibody (OKT3-eBioscience) in triplicate. Soluble anti-CD28 (CD28.2-eBioscience) was added at a final concentration of 5 μg/mL and cells were cultured for 3 days. Cells were then pooled, washed in PBS and total RNA was extracted using the RNeasy Plus Mini kit (Qiagen), according to the manufacturer’s instructions. Equal amount of RNA was reverse-transcribed using the High capacity RNA-to-cDNA Kit (Life Technologies). Quantitative PCR was conducted with QuantStudio 7 Flex real-time PCR system (Applied Biosystems) using the following TaqMan assays: INFG Hs00989291, GUSB Hs00939627, ACTB Hs01060665. Reactions were run in triplicate and Cycle thresholds (Ct values) were normalized to those of housekeeping genes GUSB and ACTB. Relative quantity calculations were performed using the 2-ddCt method using the DMSO-treated cells as reference sample.

An allosteric MALT1 inhibitor modulates mainly protease function

Dissecting the role of pharmacological inhibition of MALT1 activity requires potent and selective chemistry tools. A MALT1 inhibitor, mepazine, and Fig 1 [26,27], and a closely related phenothiazine compound thioridazine, have been used as a MALT1 tool compounds in vitro and in vivo, and have been shown to bind at an allosteric site on MALT1 [27]. Mepazine suffers from low potency, off-target effects and cytotoxic effects [27,28]. Its selectivity profile makes it unsuitable tool to test the role of MALT1 in in vitro or in vivo studies (Table 1). The peptidic active site inhibitor, z-VRPR-fmk has been shown to also bind to cathepsins [29,30]. We therefore used as Compound 2 as tool compound, an allosteric MALT1 inhibitor (Table 1) originating from a drug discovery program at AstraZeneca. This compound binds at the same allosteric site as mepazine but is a more potent and selective MALT1 inhibitor (Table 1) but its high MALT1 potency and improved selectivity profile made it a very suitable tool to probe the role of MALT1 allosteric inhibition in cellular assays Compound 3 also binds at the same allosteric site as mepazine and Compound 2. Compound 3 is also a potent and selective MALT1 inhibitor, but it’s improved pharmacokinetic profile, and improved potency made it the preferred choice for in vivo experiments.

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Fig 1. Structures of mepazine, compounds 2, 3.

https://doi.org/10.1371/journal.pone.0222548.g001

First, we evaluated the effect of MALT1 inhibitors on scaffold and protease activity in PMA+ionomycin stimulated CD4+ T-cells. The cleavage of RELB was inhibited by allosteric inhibitors, Compound 2 and mepazine, and by the active site inhibitor z-VRPR-fmk (Fig 2A and 2B). The cleavage of CYLD was also inhibited by allosteric inhibitors Compound 2 and mepazine, but the inhibition did not reach significance for the active site inhibitor z-VRPR-fmk. We demonstrated a modest effect on the inhibition of the IKK-dependent phosphorylation of Iα with the allosteric MALT1 inhibitors, mepazine and Compound 2 in human CD4 T-cells, while the active site inhibitor did not reach statistical significance (Fig 2C and 2D). No inhibition of the phosphorylation of ERK was observed by MALT1 inhibitors indicating specific effect of MALT1 inhibition on NF-κB activation (Fig 2D). Furthermore, Compound 2 did not show toxicity at concentrations showing efficient inhibition of IFN-γ production and proliferation in activated CD4 T-cells (Fig 2E).

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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.

https://doi.org/10.1371/journal.pone.0222548.g002

We observed a difference in the effect of allosteric vs active site inhibitors on phosphorylation of IκBα, which suggest that an allosteric inhibitor modulates both protease and, at least modestly, scaffold function of MALT1, the latter linked to IKK activation. Since total IκB decreases following T cell activation [31,32] and the p-IκB signal significantly increases in activated vs unstimulated cells it is unlikely (though not impossible) that the change in p-IκB signal is related to the change in total IκB levels.

Accordingly, the immunomodulatory effects of the allosteric inhibitor may differ from that of pure active site inhibitor.

MALT1 inhibition decreases Dectin-1 driven cytokine production in human myeloid DCs

To investigate the effects of MALT1 inhibition on dendritic cells (DC) and monocyte activation and maturation, we stimulated peripheral blood mononuclear cells (PBMCs) or isolated myeloid DCs (mDCs) with zymosan, which binds to Dectin-1, an ITAM-containing receptor that signals via MALT1 [33]. The flow cytometry gating for the identification of human mDC and monocyte populations in PBMCs is shown in S2 Fig. Both MALT1 active site and allosteric inhibitors reduced the expression of IL-6 and TNF-α in zymosan-activated mDCs (Fig 3A). This effect was not seen when cells were stimulated with LPS, which signals to NF-κB through a MALT1-independent pathway (Fig 3A). Both MALT1 active site and allosteric inhibition almost completely abolished the secretion of IL-23 and IL-6 and reduced the secretion of IL-1β, IL-10 and IL-12p70 (Fig 3B) in purified human mDCs stimulated with zymosan but not LPS-treated mDCs. In ex vivo human CD14+/CD16- and CD14+/CD16+ monocytes activated in vitro with zymosan, a similar effect on cytokine release was observed, although the inhibition was primarily seen in cells treated with Compound 2 (S2 Fig). Again, no effect of MALT1 inhibitors on LPS-stimulated mDCs or monocytes was observed (S2 Fig).

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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%).

https://doi.org/10.1371/journal.pone.0222548.g003

Despite the altered cytokine expression observed upon MALT1 inhibition, the expression of activation markers CD80, CD83, CD86 and CD40 were unaltered by MALT1 inhibition in zymosan- or LPS-activated human mDCs (Fig 3C). This data suggests that MALT1 is necessary for efficient cytokine production and release but is dispensable for dendritic cell maturation upon Dectin-1 stimulation.

A MALT1 allosteric inhibitor down-regulates Th1 and Th17 responses

Next, we evaluated the effect of MALT1 inhibition separately on naive and memory human CD4+ cells, previously depleted of CD25-expressing Tregs. We stimulated either CD4+CD45RA+ naive T cells with soluble anti-CD3 + anti-CD28 antibodies or activated CD45RO+CD4+ memory CD4 T cells with monocyte derived dendritic cells pulsed with a recall antigen, namely tetanus toxoid. In both cases, the percentage of CD25+ cells, the proliferation of CD25 positive cells and the percentage of IFN-γ producing CD4+ cells was inhibited by Compound 2 (Fig 4A–4F). We did not observe activation of IL-17 in memory CD4+ cells after tetanus toxoid stimulation.

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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.

https://doi.org/10.1371/journal.pone.0222548.g004

To evaluate the effect of MALT1 inhibitors on effector Th1 and Th17 cells in the inflammatory environment, we stimulated memory CD4+ cells with anti-CD3 antibody plus anti-CD28 antibody in the presence of LPS-treated monocytes. As shown earlier (Fig 3A), MALT1 inhibitors did not interfere with LPS signaling. All MALT1 inhibitors down-regulated proliferation of CD25+CD4+ cells (Fig 5A and 5B). Anti-CD3 antibody plus anti-CD28 antibody stimulation of memory CD4 cells in the inflammatory environment resulted in the activation of IL-2, IFN-γ, IL-17 and both IFN-γ and IL-17 secreting CD4+ memory cells, and allosteric MALT1 inhibitors down-regulated activation and proliferation of IFN-γ, IL-17 and also IFN-γ and IL-17 co-secreting memory CD4+ cells whereas no effect was seen on IL-2 expressing memory CD4+ cells (Fig 5C). We could not see a decrease in the expression level of IFN-γ or IL-17 in the cytokine secreting cells (S3 Fig). This suggests that allosteric MALT1 inhibition modulates IFN-γ and IL-17 response mainly by inhibiting proliferation of the cytokine secreting memory CD4+ cells. However, the percentage of proliferating IFN-γ expressing cells among all IFN-γ expressing cells was significantly decreased in MALT1 allosteric and active site inhibitor treated CD4+ memory cells (Fig 5D) indicating an additional effect of MALT1 treatment on Th1 differentiation. We also showed that the expression of IFN-γ transcripts in memory CD4+ cells activated in the inflammatory milieu was reduced in the presence of MALT1 allosteric and active site inhibitors (Fig 5E), which reflects the reduction in the numbers of IFN-γ expressing cells. Together, these results show that IFN-γ response in human T-cells is down-regulated by MALT1 allosteric inhibitors both in naïve and memory CD4+ cells, and furthermore the activation of effector Th17 and Th1/Th17 cells is dampened. The down-regulation of Th1 response was due to the inhibition of the proliferation of IFN-γ secreting memory CD4+ cells, and as a net effect a decrease in the IFN-γ mRNA expression was observed while the IFN-γ protein production per secreting cell remained unaltered. We conclude that allosteric inhibition did not increase IFN-γ mRNA or protein expression similarly as reported in the mouse model with MALT1 protease inactivation.

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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.

https://doi.org/10.1371/journal.pone.0222548.g005

The effect of MALT1 inhibitors on human regulatory T-cells

As MALT1 protease deficient mice showed dysfunction of their regulatory T-cells, we studied the suppression function and FOXP3 expression of blood-derived human regulatory T-cells (FOXP3+CD4+CD25+) and found that the suppression activity of ex vivo expanded regulatory T-cells was not affected by MALT1 inhibition (Fig 6A). Also, absolute numbers,viability and survival of expanded FOXP3+CD4+CD25+ cells were not affected by MALT1 inhibitors (Fig 6B, S5A Fig).

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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.

https://doi.org/10.1371/journal.pone.0222548.g006

Next, we wanted to evaluate the effect of MALT1 inhibition on the induction of peripheral regulatory T-cells. We differentiated regulatory T-cells from naïve human T-cells in vitro using rapamycin, TGF-β and IL-2. Both protease and allosteric inhibitors impaired in vitro induction of regulatory T-cells from naïve T-cells (Fig 6C). Together, these data suggest that MALT1 allosteric inhibition does not interfere with the function of circulating human regulatory T-cells but with the induction of Tregs.

Allosteric MALT1 inhibition decreases B-cell proliferation and antibody production

To evaluate the effect of MALT1 inhibitors on B-cell receptor mediated activation, we first studied the effect of MALT1 inhibitors on direct B-cell receptor mediated activation. The proliferation and antibody production of human purified B-cells stimulated by anti-IgM plus anti-CD40 was decreased by allosteric MALT1 inhibition (Fig 7A and 7B), but not active site inhibition, whereas no effect on differentiation of B-cells was seen by allosteric inhibition (Fig 7A). Interestingly, the active site inhibitor z-VRPR-fmk increased differentiation of B-cells to plasmablasts (Fig 7A). Next, we wanted to evaluate the effect of MALT1 inhibitors on T-cell dependent B-cell activation. We co-cultured SEB activated CXCR5+ CD4+ T-memory-cells with B-cells in the presence BAFF, and Compound 2 or z-VRPR-fmk. Compound 2 decreased proliferation, plasmablast differentiation and antibody production while z-VRPR-fmk only affected plasmablast differentiation (Fig 7C and 7D).

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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⁺CD27^hi CD38^hi 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⁺CD27^hi CD38^hi 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.

https://doi.org/10.1371/journal.pone.0222548.g007

Treatment with MALT1 allosteric inhibitor alleviates cytokine storm and does not enhance IFN-γ production in vivo

As mice expressing protease inactive (PD) MALT1 developed an enhanced IFN-γ response, we measured a panel of circulating cytokines in C57BL/6 mice pre-treated first with an allosteric inhibitor and then stimulated with anti-CD3 antibody intraperitoneally to activate T-cells (S4 Fig). We found a significant decrease in all the measured circulating cytokines including IFN-γ (Fig 8A). To study the chronic effects of pharmacological inhibition of MALT1 in vivo, we used a recently reported allosteric MALT1 inhibitor from the patent literature, Compound 3 [34] (example 10 in reference 35) (Fig 1, Table 1) with pharmacological properties more suitable for longer-term, oral treatment than compound 2. We treated naïve C57BL/6 mice with 3 (10 mg/Kg, per oral, BID) for four weeks. While percentage and numbers of IFN-γ producing T-cells and circulating IFN-γ levels were not significantly changed, percentage and numbers of FOXP3 positive regulatory T-cells were decreased in spleen and in lymph nodes after treatment (Fig 8B and 8C). Thus, an allosteric MALT1 inhibitor interferes with regulatory T-cell differentiation or survival, as we also observed decreased levels of circulating IL-2 (Fig 8C). However, this effect did not manifest as an activation of the Th1 pathway and circulating IFN-γ levels and no signs of tissue inflammation or lymphocytic infiltrates were observed in all examined tissues in the mice after 4 weeks treatment with Compound 3 (Fig 8D). However longer studies need to be undertaken to exclude the possibility that target-related toxicity that may develop with longer term dosing.

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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+CD25^high 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.

https://doi.org/10.1371/journal.pone.0222548.g008