Compound synthesis
BfC was synthetically produced as previously described in Hansen et al.20.
Cell culture and cytokine-induced cell death
Rat beta cells RIN-M5F (purchased from ATCC, Manassas, VA, USA, product code ATCC-CRL-11605, passage number below 40 for all experiments presented in this manuscript) were cultured in RPMI-1640 (cat# R5886, Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 10% FBS (Biowest, Nauillé, France), 2 mM L-glutamine (Biowest), 1 mM sodium pyruvate (Merck, Darmstadt, Germany) and 1x Penicillin-Streptomycin (Sigma-Aldrich) according to manufacturer’s instructions. Cells were seeded 3 days before the start of each experiment to allow full attachment. In experiments analysing the effects of cytokines, cells were treated with 20 ng/ml human IL-1β and 20 ng/ml rat IFNγ (Peprotech, Rocky Hill, NJ, USA) for a given time as indicated. Mycoplasma was tested as negative using MycoAlert® Mycoplasma Kit (Lonza, Basel, Switzerland).
Analysis of cell viability
RIN-M5F cells were cultured in 96-well plates, 2 × 104 cells/well. BfC at a concentration of 10 µM was added to the cells. In dose-response experiments, BfC was two-fold serially diluted between 40 µM and 1.25 µM and added to the cells. Control wells with RPMI-1640 alone or supplemented with DMSO (Sigma-Aldrich, final concentration 10%) were used to estimate absorbance at 100% or 0% viability, respectively. 24 h after addition of substances, cytokines (IL1-β and IFNγ) were added to the experiment. The cells were thereafter incubated for an additional 72 h, and cell viability was measured by CellTiter 96®AQueous One Solution Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Absorbance at 490 nm was measured using a Spark® multimode microplate reader (Tecan, Männedorf, Switzerland). Each experiment was performed in triplicates.
Analysis of caspase 3/7 activity
RIN-M5F cells were cultured in 96-well plates, 2 × 104 cells/well. Caspase 3/7 activity was analysed in cells treated for 48 h with 10 or 20 µM of BfC, with or without cytokines. After 24 h of BfC treatment, cytokines were added, and the cells were co-stimulated for an additional period of 24 h. Caspase 3/7 activity was measured by Caspase-Glo (Promega) according to the manufacturer’s instructions. Each experiment was performed in triplicates.
EdU incorporation
RIN-M5F cells were seeded into 6 well plates, 6 × 105 cells per well. Staining with 5-ethynyl 2′-deoxyuridine (EdU) was performed on cells treated with 10 or 20 µM of BfC, with or without cytokines. After 24 h of BfC treatment, cytokines were added, and the cells were stimulated for a total of 48 h. Subsequently, the cells were stained with 10 µM Click-iT EdU (Invitrogen, Waltham, MA, USA) for 2 h, and analysed by flow cytometry (CytoFLEX, Beckman Coulter, Brea, CA, USA) according to the manufacturer’s instructions. EdU incorporation was analysed using the CytExpert software.
Cell cycle
RIN-M5F cells were seeded in 6-well plates, 6 × 105 cells/well. Cell cycle distribution was analysed in cells treated for 48 h with 10 or 20 µM of BfC, with or without cytokines. After 24 h of BfC treatment, cytokines were added, and the cells were co-stimulated for an additional period of 24 h. After stimulation, the cells were washed with phosphate buffered saline (PBS) and trypsinated for 5 min at 37 °C, and fixed in ice-cold ethanol (70%) at 4 °C until analysis. The cells were stained by FxCycle™ PI/RNase Staining Solution kit (Invitrogen) according to manufacturer’s instructions and analysed by flow cytometry (CytoFLEX, Beckman Coulter). The percentages of cells in G1, S1 and G2/M was determined using the CytExpert software.
Kinase activity assay
The IC50 values of BfC against the DYRK1A, DYRK2 and DYRK3 kinases were measured using 33P-ATP radioactive filter binding assays22 at the International Centre for Kinase Profiling. Each kinase was assayed using a duplicate 10-point concentration curve.
Lantha assay
Kinase binding activity was performed as described in23. Briefly, a 12-points dilution series of the compounds was prepared in DMSO and further diluted 1:33 in assay buffer A (50 mM HEPES (Sigma-Aldrich) pH 7.5, 10 mM MgCl2 (Sigma-Aldrich) 1 mM EGTA (Sigma-Aldrich) and 0.01% BRIJ-35 (ThermoFisher Scientific, Waltham, MA, USA). The GST tagged kinases, antibodies and tracer were purchased from ThermoFisher Scientific. DYRK1A and DYRK1B were each mixed with rabbit anti GST Europium tagged antibody in assay buffer A at concentrations of 15 nM kinase and 6 nM antibodies. Tracer 236 was diluted to 90 nM (DYRK1A) or 30 nM (DYRK1B) in assay buffer A. Finally, the kinase-antibody solution, compound solution and tracer solution were mixed together 1:1:1 (5µL each) in white, non-binding, round bottom 384 well microplates (Corning Inc, Kennebunk, ME, USA) mixed at 1000 rpm for 1 min and incubated in the dark at RT in humid atmosphere for 1 h. Fluorescence measurements were taken using a EnVision 2104 Multilabel reader (PerkinElmer, Shelton, CT, USA), excitation wavelength was set to 340 nm and emission was read at both 615 nm (8.5 nm bandwidth) and 665 nm (7.5 nm bandwidth) over 200ms with a 100ms delay between excitation and emission measurements. Emission ratio signals (EM) were calculated as the ratio between the 665 nm signal and the 615 nm signal. Mean EM values of technical triplicate were plotted against compound concentration and used to calculate an IC50 first using the default “three parameter log (inhibition) vs. response” function from Prism 10.1. for Windows (GraphPad Software, Boston, Massachusetts USA). Because BfC autofluorescence was interfering with the fluorescent readings, the fitting formula was also adjusted as described in24 and the parameter “bot” was fixed to values obtained from control compound 5IT to ensure a correct range.
Docking
BfC was docked into the ATP-binding pocket of DYRK1A using Protein Data Bank (PDB) model 6S1725. BfC and control compounds harmine and 5IT were prepared using LigPrep (Schrödinger, Inc., New York, NY, USA, release 2022-2) and the DYRK1A model was prepared (addition of missing hydrogen, fixing different conformers, adding missing residues and energy minimizing the structure) with the Maestro protein preparation wizard (Schrödinger, Inc.)26. Docking was performed using Glide extra precision algorithm27,28.
Western blot
RIN-M5F cells were seeded in 6-well plates, 6 × 105 cells/well. After treatment with 10 and 20 µM of BfC for 48 h, the cells were washed with ice-cold PBS and lysed in Pierce® RIPA buffer (Thermo Fischer Scientific, Waltham, MA, USA) supplemented with cOmplete™ Protease Inhibitor Cocktail (Roche, Basel, Switzerland) and PhosSTOP™ (Roche). After centrifugation (16.9 RCF, 15 min at 4 °C) the samples were directly diluted in NuPAGE™ LDS Sample Buffer (4X, Invitrogen) supplemented with NuPAGE™ Sample Reducing Agent (10X, Invitrogen), boiled at 95 °C for 5min and loaded on gel (NuPAGE™ 4–12%, Bis-Tris, Invitrogen). Protein transfer to PVDF membrane was performed by the iBlot™ dry blotting system (Invitrogen) according to manufacturer’s instructions. The membrane was blocked using SuperBlock™ Blocking Buffer (Thermo Fisher Scientific) and subsequently cut in half around 60 kDA, the top half was incubated with primary antibodies for phospho-dyrk1a/b (Tyr321, Tyr273) (1:500, Invitrogen, ref: PA5-64574), while the bottom half was incubated with phospho-p27kip1 (Ser10) (1:200, Invitrogen, ref:34-6300) over night at 4oC. After washing, the membrane was incubated with Alexa488-conjugated secondary antibody (1:5000, goat anti-rabbit, Invitrogen) for 2 h and imaged with a PharoxFX imager (Bio-Rad, Hercules, CA, USA). Consequently, the bottom half of the gel was washed and re-stained with primary beta-actin antibodies (1:5000, Invitrogen), and then prepared as described for p-p27kip, and reimaged. Protein quantification was performed with ImageJ (National Institutes of Health, Bethesda MD, USA), using the gel analysis tool to get the integrated pixel intensity for each band.
Statistical analysis
Data are expressed as mean ± SEM or SD. All statistical analyses were performed using GraphPad Prism v.7.0 (GraphPad Software, San Diego, CA, USA). Statistical significance was defined as p < 0.05 and determined by unpaired Student’s t test, one-way ANOVA with Bonferroni’s post hoc test, RM one-way ANOVA with Bonferroni’s post hoc test or two-way ANOVA with Dunnett’s post hoc test, as appropriate.
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