2.1 Molecular docking
The structure of TYK2 JH2 (Protein Data Base ID: 6NZP) and QL-1200186 were prepared using Protein Preparation Wizard and LigPrep modules, respectively. Then, the binding site was defined as a 20 × 20 × 20 Å3 cubic box centered to the ligand in TYK2 JH2 protein. After that, molecular docking was undertaken using Glide standard precision, and flexible macrocycle sampling was adopted. Post-docking minimization was undertaken to further refine docking results. All the modules mentioned were implemented in Schrödinger 2021-3 (https://www.schrodinger.com/).
2.2 Assay to measure binding of the TYK2 pseudokinase domain
An assay that measures the ability of a compound to bind to the TYK2 pseudokinase domain through competition with a tracer was employed. First, human polyhistidine-tag (his-tagged) TYK2 pseudokinase was added to different concentrations of QL-1200186. Then, his-terbium-labeled antibody in assay buffer (HEPES (20 mM), pH 7.5, MgCl2 (10 mM), 0.015% Brij-35, dithiothreitol (2 mM) and bovine serum albumin (50 µg/mL)) was added to the recombinant TYK2 pseudokinase domain. Next, fluorescent-labeled tracers were added, and centrifugation for 30 s and incubation for 60 min at room temperature (RT) undertaken. After 1 h at RT, the homogeneous time-resolved fluorescence (HTRF) signal was measured on a plate reader (Envision™; PerkinElmer, Waltham, MA, USA).
2.3 Assay to measure the activity of JAK1/2/3 and TYK2 kinase
We measured the ability of a compound to bind to JAK1/2/3 and the TYK2 JH1 domain through HTRF® KinEASE™-TK kits (Millipore, Bedford, MA, USA). JAK1/2/3 and the TYK2 JH1 domain in assay buffer were added to various concentrations of QL-1200186 and incubation for 15 min at RT undertaken. Then, the plate was incubated with the biotin-labeled TK substrate and adenosine triphosphate for 45 min at RT. Next, SA-XL665 and TK antibody-Eu3 were added to the detection buffer and incubation for 1 h at RT undertaken. The HTRF signal was measured on a plate reader (Envision).
2.4 Kinase panel assay
The selectivity of QL-1200186 was measured using a panel of 207 kinase proteins. QL-1200186 (final concentration = 1 µM) was placed in a 384-well plate.Different kinase solutions were configured in kinase buffer (HEPES (50 mM), MgCl2 (10 mM), 0.01% Brij, dithiothreitol (2 mM) and then transferred to 384-well plates. After incubation for 10 min at RT, the substrate and adenosine triphosphate were added, and the reaction allowed to continue for 60 min. Kinase selectivity was screened by the ADP-Glo™ Kinase Assay (V9103; Promega, Fitchburg, WI, USA). Finally, a microplate reader (BMG Labtech, Ortenberg, Germany) was used to measure the luminescence signal. After data analysis, heatmaps and kinase selection trees were drawn using Prism 8.0.2 (GraphPad, La Jolla, CA, USA) and KinMap (www.kinmap.org/), respectively.
2.5 Assay to measure off-target effects
Forty-four safety targets were tested through evaluation of pharmacologic safety in vitro. The off-target effects of QL-1200186 on 24 G protein-coupled receptors (β1, β2, D1, H2, A2A, CB1, CB2, 5-HT1A, 5-HT1B and D2S) were evaluated by measuring the content changes in cyclic adenosine monophosphate and IP-1. The effects of QL-1200186 on current changes in eight ion-channel targets were detected by methods based on fluorescent imaging plate reading and patch clamps. Using stable transfer or transient plasmid transfer, the excitatory and inhibitory effects of QL-1200186 on glucocorticoid receptors and androgen receptors in the nucleus were detected by the change in luciferase expression. The inhibitory effects of QL-1200186 on seven enzymes (including cyclo-oxygenase-2, phosphodiesterase (PDE)3A and PDE4D2) were detected by biochemical methods based on luminescence and fluorescence. After data analyses, radar charts were used to display results.
2.6 Human type-I IFN reporter cells
After 24-h long incubation with QL-1200186, HEK-Blue™ IFN-α/β cells (Invivogen; NKB-IFNAB) were stimulated with human IFNα (200 U/mL; catalog number: 11200-2; PBL Assay Science; Piscataway, NJ, USA) for 6 h. Stimulation of HEK-Blue IFN-α/β cells with human IFNα activates the JAK/STAT/ISGF3 pathway and subsequently induces the production of SEAP. Levels of SEAP in the supernatant were readily assessable using QUANTI-Blue™ Solution (InvivoGen, San Diego, CA, USA).
2.7 Phosphorylation of STAT5 in human cells
With regard to IFNα-stimulated phosphorylation of STAT5 in human peripheral-blood mononuclear cells (PBMCs), after incubation with QL-1200186 for 1 h, human PBMCs were stimulated with recombinant human IFNα (1000 U/mL; 11200-2; PBL Assay Science) for 30 min.
With respect to GM-CSF-stimulated JAK2 activation in U937 cells (Cobioer Biosciences, CBP60277), after 1-h treatment with QL-1200186, U937 cells were stimulated with GM-CSF (20 ng/mL; C003; Novoprotein, Suzhou, China) for 15 min.
With regard to IL-2-stimulated JAK1/3 activation in human PBMCs, after 1-h incubation with QL-1200186, human PBMCs were stimulated with human IL-2 (20 ng/mL; C013; Novoprotein) for 20 min.
With respect to IL-2-stimulated JAK1/3 activation in human whole blood cells, whole blood (50 µL) was pre-incubated with compounds for 30 min and then stimulated with IL-2 (20 ng/mL) for 15 min.
The stimulations stated above were terminated with prewarmed Lyse/Fix Buffer (558049; BD Biosciences, San Jose, CA, USA) for 10 min. Cells were stained with an anti-cluster of differentiation (CD)3 fluorescein isothiocyanate (FITC) antibody (required for PBMCs but not for the other cell types), washed, and permeabilized on ice using Perm III Buffer (558050; BD Biosciences) prior to staining with an Alexa-Fluor 647 anti-phosphorylated (p)STAT5 (pY694) antibody for 30 min and analyzed by flow cytometry. Phosphorylation of STAT5 was quantified by the median fluorescence intensity (MFI) after gating on the CD3-positive population.
2.8 Separation and differentiation of human naïve CD4 + T cells
Human naïve CD4+ T cells were separated by the human naïve CD4+ T cells isolation kit (StemCell, #17852), then cells were stimulated with anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody for 7–10 days under the following Th17-skewing conditions: IL-6, IL-23, IL-1β and transforming growth factor-β. Fresh medium containing a stimulant-inducing differentiation was half-renewed every other day. Th17 cells were incubated overnight at 37℃ for deactivation. Cells were re-inoculated on 96-well plates and co-cultured with gradient-diluted QL-1200186 for 1 h, and then stimulated with recombinant human IL-23(ACRO: #IL-B-H52WS) for 0.5 h. Cells were collected and phosphorylation of STAT3 was quantified according to the MFI.
2.9 IL-12/IL-18-induced IFNγ production in NK-92 cells and human whole blood cells
We wished to ascertain the effect of IFNγ production of NK cells. We stimulated NK92 cells with recombinant human IL-12 (2 ng/mL; 200 − 12; PeproTech, Cranbury, NJ, USA) and recombinant human IL-18 (5 ng/mL; 9124-IL-050/CF; R&D Systems, Minneapolis, MN, USA) with or without QL-1200186 for 24 h at 37℃. For human whole blood cells, whole blood (200 µL) was pre-incubated with the compound for 1 h, then stimulated with recombinant human IL-12 (2 ng/mL) or recombinant human IL-18 (10 ng/mL) for 24 h. After centrifugation, the supernatant was collected and IFNγ production in the supernatant was detected by enzyme-linked immunosorbent assay kits according to manufacturer (Biolegend,430104) instructions.
2.10 TYK2 activation in Jurkat cells
Cells were co-cultured with QL-1200186, BMS-986165 or NDI-034858 for 1 h and stimulated with recombinant human IFNα (1000 U/mL) for 15 min. Then, the phosphorylation of Tyr-1054 and Tyr-1055 in Jurkat cells (Cobioer Biosciences: CBP61444) was detected by western blotting to determine the inhibitory effect of QL-1200186 on TYK2 receptor-mediated activation.
2.11 Phosphorylation of STAT1 in human PBMCs
Human PBMCs were pre-incubated with QL-1200186, BMS-986165 or NDI-034858 for 30 min, then stimulated with recombinant human IFNα (1000 U/mL) for 15 min. The reaction was terminated by collecting cells for surface staining. Cells were stained with anti-CD3 FITC (300406; Biolegend, San Diego, CA, USA), anti-CD19 APC (555415; BD Biosciences) or aqua fluorescent reactive dye BV421 (L34966A; Invitrogen, Carlsbad, CA, USA) antibodies for 20 min. After washing, fixation buffer (557870; BD Biosciences) was added and incubation allowed for 10 min. Cells were washed and permeabilized on ice using Perm III Buffer (558050; BD Biosciences). Cells were stained with PE anti-PSTAT1 antibody (562069; BD Biosciences) for 30 min and analyzed by flow cytometry.
2.12 IFNα-induced differentiation of human monocytes to dendritic cells
First, monocytes in human PBMCs were isolated using EasySep™ Human CD14 Positive Selection Kit II (17858; Stemcell Technologies, Vancouver, Canada). Then, cells were seeded at 300,000 in 96-well plates and stimulated with GM-CSF (100 ng/mL; C003; Novoprotein), recombinant human IL-4 (50 ng/mL; 6507-IL-010/CF; R&D Technologies) plus IFNα (1000 U/mL; 11200-2; PBL Assay Science) for 6 days with or without QL-1200186, BMS-986165 or NDI-034858 at 5 nM, 20 nM or 100 nM. Media were half-changed on days 3 and 5. On day-6, cells were collected and stained with BV421 aqua fluorescent reactive dye (L34966A; Invitrogen), FITC mouse anti-human CD80 (555683; BD Biosciences), PE Mouse Anti-Human CD86 (560957; BD Biosciences) or BUV737 mouse anti-human CD83 (612823; BD Biosciences) antibodies for 20 min. After staining, cells were washed twice and analyzed by flow cytometry.
2.13 Thrombopoietin-stimulated phosphorylation of STAT3 and STAT5 in platelets
After 30 min of preincubation of QL-1200186, BMS-986165, NDI-034858 or tofacitinib with whole blood cells, stimulation was undertaken using recombinant human thrombopoietin (50 ng/mL; 300 − 18; PeproTech) for 15 min. Stimulation was terminated by adding Lyse/Fix buffer. Staining with BUV786 mouse anti-human CD61 antibody (744384; BD Biosciences) was carried out, followed by washing, permeabilization and staining for PE mouse anti-human STAT3 antibody (651004; BD Biosciences) and APC mouse anti-human STAT5 antibody (562076; BD Biosciences), as described above. Expression of p-STAT3 and STAT5 was quantified by the MFI after CD61-positive platelets were controlled.
2.14 Pharmacokinetic (PK) study
The PK study was conducted in healthy male C57BL/6 mice (18–22 g, n = 6). Mice were fed and given free access to water prior to dosing. The drug (QL-1200186) was administered via oral or intravenous routes at doses of 10 mg/kg and 1 mg/kg, respectively. Blood samples were collected at 0.0833, 0.25, 0.5, 1, 2, 4, 8 and 24 h from the saphenous vein. Plasma was separated within 1 h of sampling by centrifugation at 1000g. Liquid chromatography–tandem mass spectrometry of the sample was carried out on an mass spectrometer (API 5500; AB Sciex, Framingham, MA, USA). Chromatographic separation was achieved using a C18 column (2.6 µm, 50 × 3.0 mm; Kinetex; Phenomenex, Torrance, CA, USA). The main PK parameters were calculated by WinNonlin 8.0 (Certara, Princeton, NJ, USA).
2.15 Mouse model of a IL-12- and IL-18-induced IFNγ production
Healthy female, specific pathogen-free C57BL/6 mice (6–8 weeks,) were challenged with injection (i.p.) of IL-12 and IL-18 to drive IFNγ production. Mice were numbered, weighed and divided randomly into seven groups of five according to bodyweight using Excel™ (Office™; Microsoft, Redmond, WA, USA). One hour after drug administration, IL-12 (0.01 µg) was injected (i.p.). One-hour later, recombinant IL-18 (0.1 µg) was injected. Whole blood was collected 3-h later, centrifuged (300g, 10 min, 4°C) and serum collected. IFNγ expression in serum was detected by CBA Flex kit (BD Biosciences).
2.16 Imiquimod-induced model of psoriasis in mice
Healthy male BALB/c mice (18–20 g) were divided randomly into three groups of eight according to bodyweight using Excel™ (Office™; Microsoft). After 1 week of adaptive feeding, mice received a daily topical dose of 62.5 mg of 5% imiquimod cream (20 mg; Aldara®; 3M Pharmaceuticals, Maplewood, MN, USA) on the shaved back and left ear for 7 consecutive days. Control mice were treated similarly with vehicle cream (Vaseline lanette creme; Fagron, Amsterdam, the Netherlands), Mice in the treatment group were given QL-1200186 (5 mg/kg or 1 mg/kg, p.o., b.i.d.).
The Psoriasis Area and Severity Index (PASI) consists of measurements of the erythema, scale and thickness of skin. Mice were evaluated from day-1 that imiquimod was given for 7 consecutive days. The thickness of mouse skin was measured using a micrometer. PK parameters were measured.
2.17 Statistical analyses
Data are presented using Prism 8.0.2 (GraphPad). One-way ANOVA was used for measuring statistical differences in data between multiple groups if a single factor was used. Two-way ANOVA with Bonferroni post-tests was used if two factors were employed. P < 0.05 was considered significant.