A Novel highly selective allosteric inhibitor of TYK2 can block inflammation/autoimmune pathways

A Novel highly selective allosteric inhibitor of TYK2 can block inflammation/autoimmune pathways | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A Novel highly selective allosteric inhibitor of TYK2 can block inflammation/autoimmune pathways Celia X-J Chen, Wei Zhang, Fucan Xia, Shulan Qu, Bo Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2914960/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Oct, 2023 Read the published version in Cell Communication and Signaling → Version 1 posted 8 You are reading this latest preprint version Abstract Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family, which plays an important part in signal transduction and regulation of the immune system. To minimize the safety concerns and improve the therapeutic effect against autoimmune diseases, we developed a small molecule inhibitor (QL-1200186) targeting the pseudokinase domain of TYK2 protein (JH2). The binding sites of QL-1200186 were predicted and screened by molecular docking. The inhibitory effects of the downstream signaling pathways and transcriptional activators of TYK2 were reflected in cell lines and human peripheral-blood cells. Pharmacokinetics and pharmacodynamics were verified in mice. QL-1200186 showed highly affinity to TYK2 JH2 and had no apparent selectivity for the TYK2 and JAK homologous kinase domains (JH1); these effects were manifested in assays based on biochemical binding, signaling pathway transduction (JAK1/2/3) and off-target effects. We revealed that currently available drugs, such as BMS-986165 and NDI-034858, were the most likely candidates for TYK2 inhibitors, and found that QL-1200186 was functionally comparable to and selectively superior to both agents in vitro . QL-1200186 showed excellent exposure, high bioavailability and afforded low clearance rates in mice. Oral administration of QL-1200186 dose-dependently inhibited interferon-γ production in interleukin-12-driven responses and ameliorated skin lesions significantly in a mouse model of psoriasis, respectively. These findings suggest that QL-1200186 is a highly selective and potent inhibitor of TYK2. QL-1200186 could be developed as a drug for the treatment of psoriasis or other autoimmune diseases. QL-1200186 TYK2 inhibitor psoriasis autoimmune diseases inflammation JAKs BMS-986165 NDI-034858 Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Background Janus kinases (JAKs) are multi-domain tyrosine kinases that mediate cytokine transduction and regulation of the immune system. The JAK family consists of four members (JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2)), which bind selectively to different receptor chains ( 1 , 2 ). JAKs transduction is mediated by a range of interleukin (IL) receptors, interferon (IFN) receptors, colony-stimulating factors (CSFs) as well as hormones. For instance, IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 can activate the JAK1/3 signaling pathway through their receptors, whereas IL-3, IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF) and thrombopoietin can activate the JAK2 signaling pathway, all of which have been shown to be pathogenic pathways for different autoimmune diseases ( 3 ). When cytokines bind to their receptors to activate the JAK signaling pathway, the signal transducer and activator of transcription (STAT), as the substrate of the JAK family, is phosphorylated by JAKs to form dimers, which then pass through the nuclear envelope into the nucleus for transcriptional regulation. This pathway is known as the JAK-STAT signaling pathway, and has an important role in regulation of the immune system ( 4 ). As a member of the JAK family of non-receptor tyrosine kinases, TYK2 can be activated by IL12, IL-23 and type-I IFNs. This action is required for the immune response and for the development of autoimmune diseases ( 5 , 6 ). For example, TYK2-deficient mice have been reported to be viable (but sensitive) to viral infections. Also, TYK2-mediated signaling can be involved in innate and acquired immune responses due to increases in the number and function of T helper type-1 (Th1) and Th17 cells ( 7 – 9 ). Moreover, mice harboring TYK2 polymorphisms show different susceptibility to collagen-induced arthritis, thereby demonstrating that TYK2 deficiency leads to clinical rheumatoid arthritis ( 10 , 11 ). In addition, TYK2 deficiency has been shown to reduce disease scores and lymphocyte infiltration in an inflamed central nervous system in an experimental model of autoimmune encephalomyelitis ( 12 ). Those results indicate that inactivation of the TYK2 mutation could provide protection against various autoimmune diseases, including psoriasis and inflammatory bowel disease (IBD) ( 10 , 12 – 14 ). Therefore, targeted intervention of TYK2 via small molecule inhibitors have been shown to be a viable option for the treatment of autoimmune diseases. Unlike other kinase domains, the pseudokinase domain of TYK2 (JH2) can play a key part in receptor-mediated activation of adjacent catalytic domains through auto-inhibitory interactions ( 15 ). Previous studies have shown that encoding variants of TYK2 that catalyse substitution of proline residues at position 1104 by alanine block receptor-mediated TYK2 activation and have thus been shown to have protective effects against a variety of autoimmune diseases ( 14 , 16 – 18 ). This principle has been applied similarly to inhibition of TYK2 JH2 expression. Studies have shown that selective inhibitors of TYK2 can be used to treat various autoimmune and inflammation-related models of disease in animals (e.g., systemic lupus erythematosus (SLE), psoriasis, IBD and rheumatoid arthritis) by inhibiting the signal transduction cascade of IL-12, IL-23 and type-I IFN receptors (19, 20). TYK2-selective inhibitors are potential drugs for treatment of SLE, IBD and psoriasis. Only deucravacitinib (Bristol Myers Squib, New York, NY, USA) has been approved (in 2022) by the US Food and Drug Administration for the treatment of psoriasis. Therefore, the development of novel, selective inhibitors of TYK2 is needed. We developed a highly selective and potent small molecule inhibitor of TYK2 (QL-1200186) to be used against autoimmune diseases. Its profile suggested that further clinical development could be possible. 2 Methods 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, MgCl 2 (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), MgCl 2 (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. 3 Results 3.1 QL-1200186 can bind the TYK2 pseudokinase domain highly selectively and shows good safety in vitro QL-1200186 (5 6 -methoxy-N-methyl-5 5 -(1-methyl-1H-pyrazol-3-yl)-8-oxa-2,4-diaza-1( 2 , 6 ),3( 2 , 4 )-dipyridina-5( 1 , 3 )-benzenacyclononaphane-3 5 -carboxamide, i.e., SMILES as O = C(NC)C1 = CN = C(N2)C = C1NC3 = CC(CCOCC4 = NC2 = CC = C4) = CC(C5 = NN(C)C = C5) = C3OC), was discovered through a project initiated by Shanghai Qilu Pharmaceutical R&D Center Limited (Shanghai, China). The project was outsourced to our contract research partner, Bioduro-Sundia (San Diego, CA, USA), where all medicinal, computational and chemistry work took place (patent number: WO 2022/213980 A1). Data on the structure–activity relationship to reach compound QL-1200186 will be disclosed in due course. The binding mode of QL-1200186 is shown in Fig. 1 B. QL-1200186 interacts with the TYK2 JH2 domain mainly through two hydrogen-bond networks. One occurs at the hinge region and involves the backbone carbonyl and NH groups of Val690. The other includes the backbone carbonyl group of Glu688 and side-chain of Lys642. QL-1200186 also shows additional aromatic hydrogen bonds to the backbone carbonyl groups of Glu688 and Glu691, which reinforces the interaction network. Furthermore, we verified the binding of QL-1200186 to TYK2 in vitro . QL-1200186 could bind to the recombinant TYK2 JH2 domain with a half-maximal inhibitory concentration (IC 50 ) of 0.06 nM, and to the JAK1 JH2 domain at an IC 50 of 9.85 nM. The selectivity of QL-120086 for the TYK2 JH2 domain was 164-fold greater than that for JAK1 JH2 (Fig. 1 C). QL-1200186 did not show inhibitory activity against TYK2 JAK1/2/3 JH1 kinases (Fig. 1 D, Table 1 ). Compared with a panel of 207 kinases, QL-1200186 was highly selective for TYK2 (Fig. 1 E). Surprisingly, QL-1200186 had no off-target toxicity (Fig. 1 F). These results suggested that QL-1200186 was highly selective for TYK2 pseudokinase. Table 1 A Biochemical binding affinity of TYK2 inhibitors Biochemical Assay Compound Kinase assay (JH1) Binding assay (JH2) Binding assay (JH2) Selectivity fold (TYK2/JAK1) JAK1 IC 50 (nM) JAK2 IC 50 (nM) JAK3 IC 50 (nM) TYK2 IC 50 (nM) TYK2 IC 50 (nM) JAK1 IC 50 (nM) BMS-986165 4416.26 4115.53 2602.45 > 9901.7 0.17 0.9 5 QL-1200186 > 9901.7 > 9901.7 > 9901.7 > 9901.7 0.11 15.57 142 Table 1 B Potency of TYK2 inhibitors in signaling and cell-function assays Cellular Assay Compound pSTAT5/IFNα PBMC-TYK2/JAK1 IFNa HEK293 Reporter TYK2/JAK1 IL-23 HEK293 Reporter TYK2/JAK1 IFNa reporter IL23 reporter max pSTAT3/IL-23 TYK2/JAK1 pSTAT5/GM-CSF in U937 JAK2/JAK2 PSTAT5/IL-2 in PBMCs JAK1/JAK3 IFNγ/IL-12 in NK-92 TYK2/JAK2 Selectivity fold (TYK2/JAK2) pSTAT1/IFNα (CD3 + T cells in PBMCs-TYK2/JAK1 pSTAT1/IFNα (CD19 + B cells in PBMC-TYK2/JAK1 pSTAT3&5/TPO whole blood-JAK2/JAK2 IC 50 (nM) Maximum inhibition at 10 µM (%) IC 50 (nM) BMS-986165 19 6.33 6.41 100 100 6 10% inhibition @10 mM ~ 40% inhibition @10 mM 38.83 > 258 7.8 6.29 > 10000 QL-1200186 7.26 13.95 ~ 0.6122 99 108 1.026 no inhibition @10 mM ~ 18% inhibition @10 mM 32.48 >>312 1.61 1.43 > 10000 3.2 QL-1200186 blocks the TYK2 signaling pathway TYK2 can mediate signal transduction through the type-I IFN receptor, IL-12 receptor and IL-23 receptor. Hence, to further verify the function of QL-1200186 in the TYK2-mediated signaling pathway, we first examined the potency of QL-1200186 on human HEK-Blue IFN-α/β reporter cells and TYK2-mediate phosphorylation of STAT proteins in PBMCs. QL-1200186 showed dose-dependent inhibition of the JAK/STAT/ISGF3 pathway (Fig. 2 A) and IFNα-stimulated pSTAT5 levels in CD3 + T cells (Fig. 2 B). Considering the role of IL-23 in the TYK2-STAT signaling pathway, we further examined the effect of QL-1200186 on IL-23-induced phosphorylation of STAT3 (pSTAT3) in human TH17 cells. QL-1200186 inhibited the IL-23-induced pSTAT3 level in human Th17 cells in a dose-dependent manner (Fig. 2 C), demonstrating that QL-1200816 had high potency on TYK2-STAT3 signaling mediated by the IL-23 receptor. QL-1200186 had higher potency in IL-12-induced IFNγ production in NK92 cells (TYK2-dependent cytokines) compared with GM-CSF-stimulated JAK2 activation in U937 cells or IL-2-stimulated JAK1/3 activation in human PBMCs (TYK2-independent pSTAT5) (Fig. 2 D–F). These data indicated that QL-1200186 mediated TYK2 inhibition in a highly selective fashion compared with the JAK1/2/3 signaling pathway. IC 50 values are shown in Table 1 . 3.3 Functional and selectivity profile of QL-1200186 compared with other inhibitors of TYK2 and JAK1/3 Deucravacitinib (BMS-986165) is a novel drug given via the oral route. It is being marketed and has been reported to block key molecules of TYK2 in psoriasis pathogenesis ( 21 ). NDI-034858 is an allosteric inhibitor of TYK2 developed by Nimbus (Cambridge, MA, USA). It is in phase-II clinical trials for the treatment of plaque psoriasis and psoriatic arthritis. Thus, to further explore the inhibitory capacity of QL-1200186, we compared the function of two existing TYK2 inhibitors: BMS-986165 and NDI-034858. First, we attempted to understand the direct effects of three TYK2 inhibitors on TYK2 activation (Fig. 3 A). QL-1200186 blocked IFNα-stimulated phosphorylation of TYK2 in Jurkat cells in a concentration-dependent manner. The blockade elicited by QL-1200186 was significantly greater than that of NDI-034858 and no less than that of BMS-986165. Consistent with receptor-mediated TYK2 activation, QL-1200186 inhibited IFNα-induced STAT1 phosphorylation in CD3 + T cells and CD19 + B cells significantly in human PBMCs. QL-1200186 showed a superior inhibitory effect compared with that of the other two TYK2 inhibitors (Fig. 3 B, C). Type-I IFN plays an important part in inducing monocytes to differentiate into antigen-presenting cells and is closely related to autoimmune diseases ( 22 ). Hence, we further investigated the role of the inhibitors QL-120086 and TYK2 in the differentiation of monocytes into mature dendritic cells induced by GM-CSF and IFNα. According to expression of CD80, CD83 and CD86 (Fig. 3 D–F), QL-1200186 could inhibit the differentiation and maturation of monocytes into dendritic cells significantly. Use of X-VIVO 15 medium verified these results (data not shown). Besides, in accordance with the previous result (Fig. 2 D), IFNγ production was reduced significantly in human whole blood after treatment with QL-1200186, and the inhibition was comparable with that observed with BMS-986165 (Fig. 3 G). We wished to further evaluate the selectivity of QL-1200186. We compared the selectivity of QL-1200186, BMS-986165, NDI-034858 and an approved JAK1/3 inhibitor (tofacitinib) for TYK2 and JAK1/2/3 in assays of human whole blood. QL-1200186 and NDI-034858 showed no selectivity in IL-2-mediated JAK1/3 signaling or thrombopoietin-induced JAK2/2 signaling (Fig. 3 H–J). BMS-986165 showed some selectivity for IL-2-induced JAK1/3 expression at high concentrations. Tofacitinib inhibited expression of JAK1/3 and JAK 2 to varying degrees, but did not inhibit TYK2 expression. Overall, we found that QL-1200186 had excellent inhibitory function and selectivity against TYK2 compared with TYK2 inhibitors (BMS-986165 and NDI-034858) and JAK1/3 inhibitor (tofacitinib). IC 50 values are shown in Table 1 . 3.4 PK study In vitro studies showed that QL-1200186 had high potency and functional selectivity in biochemical binding to TYK2 and inhibition of dependent signaling pathways. Therefore, we further examined the PK properties of QL-1200186 in mice. In mice, oral administration of QL-1200186 (10 mg/kg) could reach 20320 h*ng/mL of the area under the curve and the bioavailability was 137%, whereas intravenous administration (1 mg/kg) showed a lower clearance rate (11.6 mL/min/kg) and a low distribution volume of 0.842 L/kg (Table 2 ). Hence, QL-1200186 exhibited good PK properties, with excellent exposure and high bioavailability in vivo , which could promote its development as a drug. Table 2 PK profiles for QL-1200186 in mice Oral QL-1200186 Intravenous QL-1200186 Dose (mg/kg) 10 Dose (mg/kg) 1 C max (ng/mL) 3543 CL (mL/min/kg) 11.6 AUC (h*ng/mL) 20320 Vss (L/kg) 0.842 T1/2 (h) 2.73 T1/2 (h) 1.45 MRTInf (h) 4.34 MRTInf (h) 1.22 F (%) 137 AUC (h*ng/mL) 1482 Cmax: maximum concentration; AUC: Area Under the Curve; MRTInf: mean residence time F(%): The percentage (or the fraction F) of an administered dose of a xenobiotic that reaches the systemic circulation 3.5 QL-1200186 reduces IL-12-driven IFNγ production in vivo On the basis of the results stated above, QL-1200186 could block TYK2-related functional pathways potently and selectively, and showed encouraging PK characteristics. Therefore, the compound was functionally validated further in a mouse model relying on the TYK2-mediated pathway. In this model, mice were injected (i.p.) with ILs associated with a TYK2-dependent receptor pathway (IL-12 and IL-18) to drive IFNγ production. Oral administration of QL-1200186 dose-dependently inhibited IFNγ production by 77.1%, 86.9% and 97.8% at doses of 0.1, 1 and 10 mg/kg, respectively (Fig. 4 A), which was consistent with in vitro results (Fig. 2 D, Fig. 3 G). BMS-986165 had an inhibitory effect on IFNγ production, but much less so than QL-1200186, indicating that QL-1200186 was a powerful inhibitor of TYK2 signaling. 3.6 QL-1200186 can improve imiquimod-induced psoriasis-like skin inflammation in mice To evaluate whether QL-1200186 could improve psoriasis, we used a mouse model of psoriasis-like skin inflammation induced by imiquimod and treated it with QL-1200186. The PASI scores of mice in each group were evaluated every day, and the differences among groups were significant (n = 8). Compared with the imiquimod group, QL-1200186 treatment improved psoriasis-like inflammatory responses (erythema, scaling and thickening) significantly (Fig. 4 B–D). QL-1200186 alleviated psoriasis severity significantly according to the thickness score on day-7 (Fig. 4 E). In general, QL-1200186 exerted excellent efficacy in improving psoriatic dermatosis. 4 Discussion QL-1200186 is one of the TYK2 pseudokinase (JH2) inhibitors invented by Qilu Pharmaceuticals. First, molecular docking was conducted to find structurally diverse inhibitors of TYK2 JH2 binding. Surprisingly, the interaction of QL-1200186 with TYK2 JH2 protein revealed a potentially new binding pocket. The latter enabled QL-1200186 to achieve higher potency, selectivity and PK profiles. Therefore, QL-1200186 was shown to be non-selective for kinases other than TYK2, including members of the JAK family ( 1 ). Also, we demonstrated that QL-1200186 had a reduced effect on TYK2-mediated signaling, including type-I IFN signaling and IL-12/IFNγ signaling in NK92 cells and IL-23/pSTAT3 in Th17 cells. Conversely, QL-1200186 exhibited virtually no inhibition of GM-CSF/pSTAT5 expression in U937 cells and IL-2-driven pSTAT5 expression in PBMCs. Hence, the dependence of QL-1200186 on TYK2 regulation was demonstrated again. QL-1200186 inhibited IL-12/IL-18-induced IFNγ production significantly and improved imiquimod-induced psoriasis-like skin inflammation in mice, which is consistent with the importance of these pathways in psoriasis ( 23 , 24 ). Psoriasis is a chronic autoimmune inflammatory skin disease characterized by epidermal hyperplasia overlying an inflammatory infiltrate ( 25 – 27 ). Studies have demonstrated that several JAK1/3 inhibitors used to treat inflammatory diseases are closely associated with adverse effects at therapeutic doses due to the high conservation of the JAK domain, including the risk of infection, lymphocytopenia, thromboembolism, dyslipidemia, altered liver function and metabolism, and even an increased risk of malignancy ( 3 , 28 – 33 ). IL-23 is essential for driving the TH17 pathway, which is crucial in chronic autoimmune inflammatory skin diseases such as psoriasis and other autoimmune conditions ( 23 , 34 – 36 ). In addition, type-I IFNs are important upstream initiators of psoriasis development and SLE pathogenesis ( 37 ). Therefore, targeting the JH2 domain rather than the catalytic domain of TYK2 provides a unique approach with potential advantages. Deucravacitinib is a novel inhibitor of TYK2 developed by Bristol Myers Squibb that binds to the pseudokinase domain (19). A phase-III clinical trial showed that deucravacitinib was effective in the treatment of psoriasis in adults. Although deucravacitinib showed greater selective inhibition for TYK2 than JAK1/JAK2/JAK3, and has been reported to improve SLE, colitis and psoriasis by blocking IL-23 and type-I IFN signaling pathways ( 38 ); binding to the JH2 domain of JAK1 and other subtypes of the JH1 domain (JAKs) to varying degrees has been reported (19, 39, 40). Conversely, QL-1200186 binds very weakly to the JH2 domain of JAK1 at different concentrations, and has no effect on the JH1 domain of other homologous isomers, so QL-1200186 has more advantages in terms of selectivity. Phase-2b clinical studies to evaluate another TYK2 inhibitor, NDI-034858 (Nimbus), in moderate-to-severe plaque psoriasis (NCT04999839) and active psoriatic arthritis (NCT05153148) are ongoing, and have shown superior functionality and an encouraging clinical-activity profile. QL-1200186 outperformed deucravacitinib with regard to TYK2 selectivity, but also combined with NDI-034858 (Nimbus) for powerful efficacy. When the minimum dose of QL-1200186 was 0.1 mg/kg and 1 mg/kg, respectively, it could reduce the IFNγ production induced by IL-12/IL-18 and improve the disease severity of our imiquimod-induced psoriasis model. Hence, QL-1200186 is a strong and efficacious small molecule inhibitor of TYK2, which may also have good efficacy for other indications. Besides the compelling evidence that QL-1200186 can alleviate psoriasis, there remains an urgent need for the development of safe drugs against autoimmune diseases. Off-target drug reactions are the main cause of adverse drug reactions. A considerable proportion of projects on the research and development of drugs stall and terminate at a late stage due to a failure of early detection of off-target effects, and clinical trials fail due to adverse drug reactions. To design lead drugs accurately, improve the success rate of research and development and reduce the failure rate of clinical trials, we tested the in vitro off-target effects of QL-1200186 based on 44 early drug-safety targets jointly proposed by AstraZeneca, GlaxoSmithKline, Novartis and Pfizer to detect off-target effects. QL-1200186 10 µM showed good safety for these 44 targets. QL-1200186 showed good safety in toxicological tests on dogs and rats (data not shown). Therefore, QL-1200186 appears to be more beneficial than current therapies against autoimmune diseases. 5 Conclusions There is an unmet need for safe, rapid-acting and efficacious drugs against psoriasis and other autoimmune diseases. There is a strong rationale for targeting the TYK2 JH2 domain in these diseases, and QL-1200186 may be the best option. QL-1200186 blocks the cytokine-mediated inflammatory response potently through TYK2. Preclinical pharmacologic and safety assessments support clinical development to evaluate its efficacy against autoimmune diseases. Declarations Data availability statement Data may be provided upon reasonable request to the corresponding author. Ethics statement This study was conducted in accordance with the Animal Care and Use Committee of Qilu Pharmaceuticals. Samples of peripheral blood were taken from healthy volunteers who provided written informed consent in accordance with the guidelines and regulations set by Qilu Pharmaceuticals Author contributions Bo Chen and Celia X.-J. Chen developed the concept and supervised the project. Wei Zhang and Fucan Xia carried out the main experiments. Shulan Qu and Bo Chen prepared the manuscript and analyzed the data. All authors were involved in drafting the manuscript or revising it critically for important intellectual content. All authors approved the final version of the manuscript. Funding This study was sponsored by Qilu Pharmaceuticals. Acknowledgements We extend our thanks to the donors and investigators who participated in this study. Conflict of interest Bo Chen, Celia X.-J. Chen, Wei Zhang, Fucan Xia and Shulan Qu are employees of Qilu Pharmaceuticals. References Gadina M, Le MT, Schwartz DM, Silvennoinen O, Nakayamada S, Yamaoka K, et al. 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Supplementary Files PTYK2WBrawdata.pptx Cite Share Download PDF Status: Published Journal Publication published 16 Oct, 2023 Read the published version in Cell Communication and Signaling → Version 1 posted Editorial decision: Major revision 16 Jul, 2023 Reviews received at journal 16 Jul, 2023 Reviews received at journal 03 Jul, 2023 Reviewers agreed at journal 27 Jun, 2023 Reviewers invited by journal 26 Jun, 2023 Editor assigned by journal 26 Jun, 2023 Submission checks completed at journal 26 Jun, 2023 First submitted to journal 10 May, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2914960","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":213129974,"identity":"f4c1fdd3-eead-40ba-8b40-8295fcad0e09","order_by":0,"name":"Celia X-J Chen","email":"","orcid":"","institution":"Shanghai Qilu Pharmaceutical R\u0026D Center Limited","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Celia","middleName":"X-J","lastName":"Chen","suffix":""},{"id":213129975,"identity":"063c16b6-4abc-4c4c-ab08-907dd669b8b7","order_by":1,"name":"Wei Zhang","email":"","orcid":"","institution":"Shanghai Qilu Pharmaceutical R\u0026D Center Limited","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Zhang","suffix":""},{"id":213129976,"identity":"7c5c7a67-5a49-4152-bb50-4a01c3e2d3a3","order_by":2,"name":"Fucan Xia","email":"","orcid":"","institution":"Shanghai Qilu Pharmaceutical R\u0026D Center Limited","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fucan","middleName":"","lastName":"Xia","suffix":""},{"id":213129977,"identity":"184f47af-9ff0-4d07-bf66-00d5b970407a","order_by":3,"name":"Shulan Qu","email":"","orcid":"","institution":"Shanghai Qilu Pharmaceutical R\u0026D Center Limited","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shulan","middleName":"","lastName":"Qu","suffix":""},{"id":213129978,"identity":"0efb7997-545f-48e0-a3da-f8e27d4d8d5f","order_by":4,"name":"Bo Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYDACCQYGZgYGGzl+9uYDJGlJM5bsOZZAkpbDiRtu5BgQp4N/dvOxxwUVIFvOfN34cweDPL8YAQdK3DmWbjzjDMgvvdtu855hMJw5m4ADDSRyzKR520C2nN12m7GNIcHgNkEt+d+AWsB+eXbzJ3FacthgWthu8BKjReJGmpn0jDPgQDa7zdsmQdgv/DOSn0kXVICjEuQwG3l+aQJaMGwlTfkoGAWjYBSMAuwAAH/2RSeP3IyJAAAAAElFTkSuQmCC","orcid":"","institution":"Shanghai Qilu Pharmaceutical R\u0026D Center Limited","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2023-05-10 07:59:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2914960/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2914960/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12964-023-01299-7","type":"published","date":"2023-10-16T15:01:07+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":39322090,"identity":"2f677e1a-f366-4b7a-be64-cac24d81952f","added_by":"auto","created_at":"2023-06-29 18:22:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1086349,"visible":true,"origin":"","legend":"\u003cp\u003eQL-1200186 can bind TYK2 JH2 with high selectively and has good safety \u003cem\u003ein vitro\u003c/em\u003e. (A) Domain structure of TYK2. (B) Docking model of QL1200186 with TYK2 JH2. TYK2 JH2 ribbon and carbons are shown in green and QL-1200186 carbons are shown in pink. TYK2 JH2 surface is shown in transparent gray. (C) The binding of QL-1200186 to JH2 of TYK2 and JAK1. (D) HTRF was used to detect the effects of QL-1200186 on JAK1/2/3 and TYK2 JH1 kinase activities. (E) A kinase selectivity panel was screened by the ADP-Glo™ kinase assay. (F) Evaluation of the pharmacological safety of QL-1200186 \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2914960/v1/f86d8250310fa19a532c7b7f.png"},{"id":39320825,"identity":"b6dae809-42cd-4aa5-8756-44cea706abbd","added_by":"auto","created_at":"2023-06-29 18:14:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":369858,"visible":true,"origin":"","legend":"\u003cp\u003eQL-1200186 blocks the TYK2 signaling pathway. (A) Effect of QL-1200186 on the JAK/STAT/ISGF3 pathway. (B) Human PBMCs were stimulated with human IFNα to detect pSTAT5. (C) Th17 cells were stimulated with IL-23 to detect pSTAT3. (D) IL-12 induced IFNγ production in NK92 cells. (E) GM-CSF-induced pSTAT5 production in U937 cells. (F) Human PBMCs were stimulated with human IL-2 to detect pSTAT5. Data are the mean ± SD. At least three independent experiments were carried out.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2914960/v1/e8799ebb28e292444eb4821f.png"},{"id":39320829,"identity":"d49fb705-74bd-44b2-92a2-1fda5a192464","added_by":"auto","created_at":"2023-06-29 18:14:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":561866,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional and selectivity profile of QL-1200186 compared with other TYK2 inhibitors. (A) Effect of TYK2 inhibitors on IFNα-induced pTYK2 expression. (B, C) Effects of TYK2 inhibitors on pSTAT1 expression in CD3+ T and CD19+ B cells (n = 3). (D–F) Effects of TYK2 inhibitors on the differentiation of monocytes into dendritic cells (n = 2). (G) Effects of TYK2 inhibitors on IL-12-induced IFNγ production in human whole blood cells (n = 3). (H) Effect of a TYK2 inhibitor and tofacitinib on the activity of JAK 1/3 was detected by IL-2-induced pSTAT5 expression (n = 3). (I, J) Effect of compounds on JAK 2/2 activity was determined by thrombopoietin-induced pSTAT3/5 expression (n = 3).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-2914960/v1/0ec021f70172965197c3623f.png"},{"id":39320828,"identity":"3a3af700-bde1-4223-8006-3b3f897b31a5","added_by":"auto","created_at":"2023-06-29 18:14:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1081333,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of QL-1200186 against IL-12-driven responses and imiquimod-induced psoriasis-like skin inflammation. (A) Effects of QL-1200186 on serum IFNγ production in a mouse model induced by IL-12 and IL-18. (B) Total PASI scores and area under the PASI curve (AUC) on day-7 in an imiquimod-induced psoriasis-like model (n = 8). (C) Back-skin photographs of mice were taken at day-7 in the imiquimod-induced psoriasis-like model. (D) Profiles for QL-1200186 in imiquimod-induced psoriasis-like skin inflammation\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-2914960/v1/83537ea03ef5b56cb73c4a37.png"},{"id":45091353,"identity":"5abf87f7-8963-43c6-9d65-042551630717","added_by":"auto","created_at":"2023-10-23 15:09:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1513299,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2914960/v1/787fc6b4-71dd-41a7-a99b-3cd2be0683d8.pdf"},{"id":39320826,"identity":"5747ddff-4de3-4221-88bc-dfcbc35c6bc5","added_by":"auto","created_at":"2023-06-29 18:14:30","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":501783,"visible":true,"origin":"","legend":"","description":"","filename":"PTYK2WBrawdata.pptx","url":"https://assets-eu.researchsquare.com/files/rs-2914960/v1/b3706f41ad45c486893cd56d.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Novel highly selective allosteric inhibitor of TYK2 can block inflammation/autoimmune pathways","fulltext":[{"header":"1 Background","content":"\u003cp\u003eJanus kinases (JAKs) are multi-domain tyrosine kinases that mediate cytokine transduction and regulation of the immune system. The JAK family consists of four members (JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2)), which bind selectively to different receptor chains (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eJAKs transduction is mediated by a range of interleukin (IL) receptors, interferon (IFN) receptors, colony-stimulating factors (CSFs) as well as hormones. For instance, IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 can activate the JAK1/3 signaling pathway through their receptors, whereas IL-3, IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF) and thrombopoietin can activate the JAK2 signaling pathway, all of which have been shown to be pathogenic pathways for different autoimmune diseases (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). When cytokines bind to their receptors to activate the JAK signaling pathway, the signal transducer and activator of transcription (STAT), as the substrate of the JAK family, is phosphorylated by JAKs to form dimers, which then pass through the nuclear envelope into the nucleus for transcriptional regulation. This pathway is known as the JAK-STAT signaling pathway, and has an important role in regulation of the immune system (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs a member of the JAK family of non-receptor tyrosine kinases, TYK2 can be activated by IL12, IL-23 and type-I IFNs. This action is required for the immune response and for the development of autoimmune diseases (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). For example, TYK2-deficient mice have been reported to be viable (but sensitive) to viral infections. Also, TYK2-mediated signaling can be involved in innate and acquired immune responses due to increases in the number and function of T helper type-1 (Th1) and Th17 cells (\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Moreover, mice harboring TYK2 polymorphisms show different susceptibility to collagen-induced arthritis, thereby demonstrating that TYK2 deficiency leads to clinical rheumatoid arthritis (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). In addition, TYK2 deficiency has been shown to reduce disease scores and lymphocyte infiltration in an inflamed central nervous system in an experimental model of autoimmune encephalomyelitis (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Those results indicate that inactivation of the \u003cem\u003eTYK2\u003c/em\u003e mutation could provide protection against various autoimmune diseases, including psoriasis and inflammatory bowel disease (IBD) (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Therefore, targeted intervention of TYK2 \u003cem\u003evia\u003c/em\u003e small molecule inhibitors have been shown to be a viable option for the treatment of autoimmune diseases.\u003c/p\u003e \u003cp\u003eUnlike other kinase domains, the pseudokinase domain of TYK2 (JH2) can play a key part in receptor-mediated activation of adjacent catalytic domains through auto-inhibitory interactions (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Previous studies have shown that encoding variants of TYK2 that catalyse substitution of proline residues at position 1104 by alanine block receptor-mediated TYK2 activation and have thus been shown to have protective effects against a variety of autoimmune diseases (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e18\u003c/span\u003e). This principle has been applied similarly to inhibition of TYK2 JH2 expression. Studies have shown that selective inhibitors of TYK2 can be used to treat various autoimmune and inflammation-related models of disease in animals (e.g., systemic lupus erythematosus (SLE), psoriasis, IBD and rheumatoid arthritis) by inhibiting the signal transduction cascade of IL-12, IL-23 and type-I IFN receptors (19, 20). TYK2-selective inhibitors are potential drugs for treatment of SLE, IBD and psoriasis. Only deucravacitinib (Bristol Myers Squib, New York, NY, USA) has been approved (in 2022) by the US Food and Drug Administration for the treatment of psoriasis.\u003c/p\u003e \u003cp\u003eTherefore, the development of novel, selective inhibitors of TYK2 is needed. We developed a highly selective and potent small molecule inhibitor of TYK2 (QL-1200186) to be used against autoimmune diseases. Its profile suggested that further clinical development could be possible.\u003c/p\u003e"},{"header":"2 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Molecular docking\u003c/h2\u003e \u003cp\u003eThe 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 \u0026times; 20 \u0026times; 20 \u0026Aring;3 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 Schr\u0026ouml;dinger 2021-3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.schrodinger.com/\u003c/span\u003e\u003cspan address=\"https://www.schrodinger.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Assay to measure binding of the TYK2 pseudokinase domain\u003c/h2\u003e \u003cp\u003eAn 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, MgCl\u003csub\u003e2\u003c/sub\u003e (10 mM), 0.015% Brij-35, dithiothreitol (2 mM) and bovine serum albumin (50 \u0026micro;g/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\u0026trade;; PerkinElmer, Waltham, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Assay to measure the activity of JAK1/2/3 and TYK2 kinase\u003c/h2\u003e \u003cp\u003eWe measured the ability of a compound to bind to JAK1/2/3 and the TYK2 JH1 domain through HTRF\u0026reg; KinEASE\u0026trade;-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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Kinase panel assay\u003c/h2\u003e \u003cp\u003eThe selectivity of QL-1200186 was measured using a panel of 207 kinase proteins. QL-1200186 (final concentration\u0026thinsp;=\u0026thinsp;1 \u0026micro;M) was placed in a 384-well plate.Different kinase solutions were configured in kinase buffer (HEPES (50 mM), MgCl\u003csub\u003e2\u003c/sub\u003e (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\u0026trade; 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 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://www.schrodinger.com/\" target=\"_blank\"\u003ewww.kinmap.org/\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.kinmap.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Assay to measure off-target effects\u003c/h2\u003e \u003cp\u003eForty-four safety targets were tested through evaluation of pharmacologic safety \u003cem\u003ein vitro\u003c/em\u003e. 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Human type-I IFN reporter cells\u003c/h2\u003e \u003cp\u003eAfter 24-h long incubation with QL-1200186, HEK-Blue\u0026trade; 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\u0026trade; Solution (InvivoGen, San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Phosphorylation of STAT5 in human cells\u003c/h2\u003e \u003cp\u003eWith 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.\u003c/p\u003e \u003cp\u003eWith 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.\u003c/p\u003e \u003cp\u003eWith 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.\u003c/p\u003e \u003cp\u003eWith respect to IL-2-stimulated JAK1/3 activation in human whole blood cells, whole blood (50 \u0026micro;L) was pre-incubated with compounds for 30 min and then stimulated with IL-2 (20 ng/mL) for 15 min.\u003c/p\u003e \u003cp\u003eThe 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Separation and differentiation of human na\u0026iuml;ve CD4\u0026thinsp;+\u0026thinsp;T cells\u003c/h2\u003e \u003cp\u003eHuman na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells were separated by the human na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells isolation kit (StemCell, #17852), then cells were stimulated with anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody for 7\u0026ndash;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 IL-12/IL-18-induced IFNγ production in NK-92 cells and human whole blood cells\u003c/h2\u003e \u003cp\u003eWe wished to ascertain the effect of IFNγ production of NK cells. We stimulated NK92 cells with recombinant human IL-12 (2 ng/mL; 200\u0026thinsp;\u0026minus;\u0026thinsp;12; PeproTech, Cranbury, NJ, USA) and recombinant human IL-18 (5 ng/mL; 9124-IL-050/CF; R\u0026amp;D Systems, Minneapolis, MN, USA) with or without QL-1200186 for 24 h at 37℃. For human whole blood cells, whole blood (200 \u0026micro;L) 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 TYK2 activation in Jurkat cells\u003c/h2\u003e \u003cp\u003eCells 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Phosphorylation of STAT1 in human PBMCs\u003c/h2\u003e \u003cp\u003eHuman 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 IFNα-induced differentiation of human monocytes to dendritic cells\u003c/h2\u003e \u003cp\u003eFirst, monocytes in human PBMCs were isolated using EasySep\u0026trade; 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\u0026amp;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Thrombopoietin-stimulated phosphorylation of STAT3 and STAT5 in platelets\u003c/h2\u003e \u003cp\u003eAfter 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\u0026thinsp;\u0026minus;\u0026thinsp;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Pharmacokinetic (PK) study\u003c/h2\u003e \u003cp\u003eThe PK study was conducted in healthy male C57BL/6 mice (18\u0026ndash;22 g, n\u0026thinsp;=\u0026thinsp;6). Mice were fed and given free access to water prior to dosing. The drug (QL-1200186) was administered \u003cem\u003evia\u003c/em\u003e 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\u0026ndash;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 \u0026micro;m, 50 \u0026times; 3.0 mm; Kinetex; Phenomenex, Torrance, CA, USA). The main PK parameters were calculated by WinNonlin 8.0 (Certara, Princeton, NJ, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Mouse model of a IL-12- and IL-18-induced IFNγ production\u003c/h2\u003e \u003cp\u003eHealthy female, specific pathogen-free C57BL/6 mice (6\u0026ndash;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\u0026trade; (Office\u0026trade;; Microsoft, Redmond, WA, USA). One hour after drug administration, IL-12 (0.01 \u0026micro;g) was injected (i.p.). One-hour later, recombinant IL-18 (0.1 \u0026micro;g) was injected. Whole blood was collected 3-h later, centrifuged (300g, 10 min, 4\u0026deg;C) and serum collected. IFNγ expression in serum was detected by CBA Flex kit (BD Biosciences).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16 Imiquimod-induced model of psoriasis in mice\u003c/h2\u003e \u003cp\u003eHealthy male BALB/c mice (18\u0026ndash;20 g) were divided randomly into three groups of eight according to bodyweight using Excel\u0026trade; (Office\u0026trade;; Microsoft). After 1 week of adaptive feeding, mice received a daily topical dose of 62.5 mg of 5% imiquimod cream (20 mg; Aldara\u0026reg;; 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.).\u003c/p\u003e \u003cp\u003eThe 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17 Statistical analyses\u003c/h2\u003e \u003cp\u003eData 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\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.1 QL-1200186 can bind the TYK2 pseudokinase domain highly selectively and shows good safety \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eQL-1200186 (5\u003csup\u003e6\u003c/sup\u003e-methoxy-N-methyl-5\u003csup\u003e5\u003c/sup\u003e-(1-methyl-1H-pyrazol-3-yl)-8-oxa-2,4-diaza-1(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e),3(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)-dipyridina-5(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)-benzenacyclononaphane-3\u003csup\u003e5\u003c/sup\u003e-carboxamide, i.e., SMILES as O\u0026thinsp;=\u0026thinsp;C(NC)C1\u0026thinsp;=\u0026thinsp;CN\u0026thinsp;=\u0026thinsp;C(N2)C\u0026thinsp;=\u0026thinsp;C1NC3\u0026thinsp;=\u0026thinsp;CC(CCOCC4\u0026thinsp;=\u0026thinsp;NC2\u0026thinsp;=\u0026thinsp;CC\u0026thinsp;=\u0026thinsp;C4)\u0026thinsp;=\u0026thinsp;CC(C5\u0026thinsp;=\u0026thinsp;NN(C)C\u0026thinsp;=\u0026thinsp;C5)\u0026thinsp;=\u0026thinsp;C3OC), was discovered through a project initiated by Shanghai Qilu Pharmaceutical R\u0026amp;D Center Limited (Shanghai, China). The project was outsourced to our contract research partner, Bioduro-Sundia (San Diego, CA, USA), where all medicinal, computational and chemistry work took place (patent number: WO 2022/213980 A1). Data on the structure\u0026ndash;activity relationship to reach compound QL-1200186 will be disclosed in due course.\u003c/p\u003e \u003cp\u003eThe binding mode of QL-1200186 is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. QL-1200186 interacts with the TYK2 JH2 domain mainly through two hydrogen-bond networks. One occurs at the hinge region and involves the backbone carbonyl and NH groups of Val690. The other includes the backbone carbonyl group of Glu688 and side-chain of Lys642. QL-1200186 also shows additional aromatic hydrogen bonds to the backbone carbonyl groups of Glu688 and Glu691, which reinforces the interaction network.\u003c/p\u003e \u003cp\u003eFurthermore, we verified the binding of QL-1200186 to TYK2 \u003cem\u003ein vitro\u003c/em\u003e. QL-1200186 could bind to the recombinant TYK2 JH2 domain with a half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) of 0.06 nM, and to the JAK1 JH2 domain at an IC\u003csub\u003e50\u003c/sub\u003e of 9.85 nM. The selectivity of QL-120086 for the TYK2 JH2 domain was 164-fold greater than that for JAK1 JH2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). QL-1200186 did not show inhibitory activity against TYK2 JAK1/2/3 JH1 kinases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Compared with a panel of 207 kinases, QL-1200186 was highly selective for TYK2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Surprisingly, QL-1200186 had no off-target toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These results suggested that QL-1200186 was highly selective for TYK2 pseudokinase.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eA\u003c/b\u003e Biochemical binding affinity of TYK2 inhibitors\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e \u003cp\u003eBiochemical Assay\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eKinase assay (JH1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBinding assay (JH2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBinding assay (JH2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSelectivity fold (TYK2/JAK1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJAK1 IC\u003csub\u003e50\u003c/sub\u003e (nM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJAK2 IC\u003csub\u003e50\u003c/sub\u003e (nM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJAK3 IC\u003csub\u003e50\u003c/sub\u003e (nM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTYK2 IC\u003csub\u003e50\u003c/sub\u003e (nM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTYK2 IC\u003csub\u003e50\u003c/sub\u003e (nM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eJAK1 IC\u003csub\u003e50\u003c/sub\u003e (nM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMS-986165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4416.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4115.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2602.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;9901.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQL-1200186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;9901.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;9901.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;9901.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;9901.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e142\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eB\u003c/b\u003e Potency of TYK2 inhibitors in signaling and cell-function assays\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"14\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"14\" nameend=\"c14\" namest=\"c1\"\u003e \u003cp\u003eCellular Assay\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epSTAT5/IFNα PBMC-TYK2/JAK1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIFNa HEK293 Reporter TYK2/JAK1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIL-23 HEK293 Reporter TYK2/JAK1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIFNa reporter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIL23 reporter max\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003epSTAT3/IL-23\u003c/p\u003e \u003cp\u003eTYK2/JAK1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003epSTAT5/GM-CSF in U937 JAK2/JAK2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePSTAT5/IL-2 in PBMCs JAK1/JAK3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eIFNγ/IL-12 in NK-92 TYK2/JAK2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eSelectivity fold (TYK2/JAK2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003epSTAT1/IFNα (CD3\u0026thinsp;+\u0026thinsp;T cells in PBMCs-TYK2/JAK1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003epSTAT1/IFNα (CD19\u0026thinsp;+\u0026thinsp;B cells in PBMC-TYK2/JAK1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003epSTAT3\u0026amp;5/TPO whole blood-JAK2/JAK2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (nM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eMaximum inhibition at 10 \u0026micro;M (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"8\" nameend=\"c14\" namest=\"c7\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (nM)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMS-986165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10% inhibition @10 mM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e~\u0026thinsp;40% inhibition @10 mM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e38.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;258\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e6.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;10000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQL-1200186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e~\u0026thinsp;0.6122\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.026\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eno inhibition @10 mM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e~\u0026thinsp;18% inhibition @10 mM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e32.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026gt;312\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e1.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e1.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;10000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.2 QL-1200186 blocks the TYK2 signaling pathway\u003c/h2\u003e \u003cp\u003eTYK2 can mediate signal transduction through the type-I IFN receptor, IL-12 receptor and IL-23 receptor. Hence, to further verify the function of QL-1200186 in the TYK2-mediated signaling pathway, we first examined the potency of QL-1200186 on human HEK-Blue IFN-α/β reporter cells and TYK2-mediate phosphorylation of STAT proteins in PBMCs.\u003c/p\u003e \u003cp\u003eQL-1200186 showed dose-dependent inhibition of the JAK/STAT/ISGF3 pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and IFNα-stimulated pSTAT5 levels in CD3\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Considering the role of IL-23 in the TYK2-STAT signaling pathway, we further examined the effect of QL-1200186 on IL-23-induced phosphorylation of STAT3 (pSTAT3) in human TH17 cells. QL-1200186 inhibited the IL-23-induced pSTAT3 level in human Th17 cells in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), demonstrating that QL-1200816 had high potency on TYK2-STAT3 signaling mediated by the IL-23 receptor. QL-1200186 had higher potency in IL-12-induced IFNγ production in NK92 cells (TYK2-dependent cytokines) compared with GM-CSF-stimulated JAK2 activation in U937 cells or IL-2-stimulated JAK1/3 activation in human PBMCs (TYK2-independent pSTAT5) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u0026ndash;F). These data indicated that QL-1200186 mediated TYK2 inhibition in a highly selective fashion compared with the JAK1/2/3 signaling pathway. IC\u003csub\u003e50\u003c/sub\u003e values are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.3 Functional and selectivity profile of QL-1200186 compared with other inhibitors of TYK2 and JAK1/3\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eDeucravacitinib (BMS-986165) is a novel drug given \u003cem\u003evia\u003c/em\u003e the oral route. It is being marketed and has been reported to block key molecules of TYK2 in psoriasis pathogenesis (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e21\u003c/span\u003e). NDI-034858 is an allosteric inhibitor of TYK2 developed by Nimbus (Cambridge, MA, USA). It is in phase-II clinical trials for the treatment of plaque psoriasis and psoriatic arthritis. Thus, to further explore the inhibitory capacity of QL-1200186, we compared the function of two existing TYK2 inhibitors: BMS-986165 and NDI-034858.\u003c/p\u003e \u003cp\u003eFirst, we attempted to understand the direct effects of three TYK2 inhibitors on TYK2 activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). QL-1200186 blocked IFNα-stimulated phosphorylation of TYK2 in Jurkat cells in a concentration-dependent manner. The blockade elicited by QL-1200186 was significantly greater than that of NDI-034858 and no less than that of BMS-986165. Consistent with receptor-mediated TYK2 activation, QL-1200186 inhibited IFNα-induced STAT1 phosphorylation in CD3\u003csup\u003e+\u003c/sup\u003e T cells and CD19\u003csup\u003e+\u003c/sup\u003e B cells significantly in human PBMCs. QL-1200186 showed a superior inhibitory effect compared with that of the other two TYK2 inhibitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). Type-I IFN plays an important part in inducing monocytes to differentiate into antigen-presenting cells and is closely related to autoimmune diseases (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Hence, we further investigated the role of the inhibitors QL-120086 and TYK2 in the differentiation of monocytes into mature dendritic cells induced by GM-CSF and IFNα. According to expression of CD80, CD83 and CD86 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026ndash;F), QL-1200186 could inhibit the differentiation and maturation of monocytes into dendritic cells significantly. Use of X-VIVO 15 medium verified these results (data not shown). Besides, in accordance with the previous result (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), IFNγ production was reduced significantly in human whole blood after treatment with QL-1200186, and the inhibition was comparable with that observed with BMS-986165 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eWe wished to further evaluate the selectivity of QL-1200186. We compared the selectivity of QL-1200186, BMS-986165, NDI-034858 and an approved JAK1/3 inhibitor (tofacitinib) for TYK2 and JAK1/2/3 in assays of human whole blood. QL-1200186 and NDI-034858 showed no selectivity in IL-2-mediated JAK1/3 signaling or thrombopoietin-induced JAK2/2 signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH\u0026ndash;J). BMS-986165 showed some selectivity for IL-2-induced JAK1/3 expression at high concentrations. Tofacitinib inhibited expression of JAK1/3 and JAK 2 to varying degrees, but did not inhibit TYK2 expression.\u003c/p\u003e \u003cp\u003eOverall, we found that QL-1200186 had excellent inhibitory function and selectivity against TYK2 compared with TYK2 inhibitors (BMS-986165 and NDI-034858) and JAK1/3 inhibitor (tofacitinib). IC\u003csub\u003e50\u003c/sub\u003e values are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.4 PK study\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e studies showed that QL-1200186 had high potency and functional selectivity in biochemical binding to TYK2 and inhibition of dependent signaling pathways. Therefore, we further examined the PK properties of QL-1200186 in mice.\u003c/p\u003e \u003cp\u003eIn mice, oral administration of QL-1200186 (10 mg/kg) could reach 20320 h*ng/mL of the area under the curve and the bioavailability was 137%, whereas intravenous administration (1 mg/kg) showed a lower clearance rate (11.6 mL/min/kg) and a low distribution volume of 0.842 L/kg (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Hence, QL-1200186 exhibited good PK properties, with excellent exposure and high bioavailability \u003cem\u003ein vivo\u003c/em\u003e, which could promote its development as a drug.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePK profiles for QL-1200186 in mice\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOral\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQL-1200186\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIntravenous\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eQL-1200186\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDose (mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDose (mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e (ng/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3543\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCL (mL/min/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAUC (h*ng/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20320\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVss (L/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.842\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT1/2 (h)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT1/2 (h)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMRTInf (h)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMRTInf (h)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAUC (h*ng/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1482\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eCmax: maximum concentration; AUC: \u003cem\u003eArea Under the Curve;\u003c/em\u003e MRTInf: mean residence time\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eF(%): The percentage (or the fraction F) of an administered dose of a xenobiotic that reaches the systemic circulation\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.5 QL-1200186 reduces IL-12-driven IFNγ production \u003cem\u003ein vivo\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eOn the basis of the results stated above, QL-1200186 could block TYK2-related functional pathways potently and selectively, and showed encouraging PK characteristics. Therefore, the compound was functionally validated further in a mouse model relying on the TYK2-mediated pathway. In this model, mice were injected (i.p.) with ILs associated with a TYK2-dependent receptor pathway (IL-12 and IL-18) to drive IFNγ production. Oral administration of QL-1200186 dose-dependently inhibited IFNγ production by 77.1%, 86.9% and 97.8% at doses of 0.1, 1 and 10 mg/kg, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), which was consistent with \u003cem\u003ein vitro\u003c/em\u003e results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). BMS-986165 had an inhibitory effect on IFNγ production, but much less so than QL-1200186, indicating that QL-1200186 was a powerful inhibitor of TYK2 signaling.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.6 QL-1200186 can improve imiquimod-induced psoriasis-like skin inflammation in mice\u003c/h2\u003e \u003cp\u003eTo evaluate whether QL-1200186 could improve psoriasis, we used a mouse model of psoriasis-like skin inflammation induced by imiquimod and treated it with QL-1200186. The PASI scores of mice in each group were evaluated every day, and the differences among groups were significant (n\u0026thinsp;=\u0026thinsp;8). Compared with the imiquimod group, QL-1200186 treatment improved psoriasis-like inflammatory responses (erythema, scaling and thickening) significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u0026ndash;D). QL-1200186 alleviated psoriasis severity significantly according to the thickness score on day-7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). In general, QL-1200186 exerted excellent efficacy in improving psoriatic dermatosis.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eQL-1200186 is one of the TYK2 pseudokinase (JH2) inhibitors invented by Qilu Pharmaceuticals. First, molecular docking was conducted to find structurally diverse inhibitors of TYK2 JH2 binding. Surprisingly, the interaction of QL-1200186 with TYK2 JH2 protein revealed a potentially new binding pocket. The latter enabled QL-1200186 to achieve higher potency, selectivity and PK profiles. Therefore, QL-1200186 was shown to be non-selective for kinases other than TYK2, including members of the JAK family (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Also, we demonstrated that QL-1200186 had a reduced effect on TYK2-mediated signaling, including type-I IFN signaling and IL-12/IFNγ signaling in NK92 cells and IL-23/pSTAT3 in Th17 cells. Conversely, QL-1200186 exhibited virtually no inhibition of GM-CSF/pSTAT5 expression in U937 cells and IL-2-driven pSTAT5 expression in PBMCs. Hence, the dependence of QL-1200186 on TYK2 regulation was demonstrated again. QL-1200186 inhibited IL-12/IL-18-induced IFNγ production significantly and improved imiquimod-induced psoriasis-like skin inflammation in mice, which is consistent with the importance of these pathways in psoriasis (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePsoriasis is a chronic autoimmune inflammatory skin disease characterized by epidermal hyperplasia overlying an inflammatory infiltrate (\u003cspan additionalcitationids=\"CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Studies have demonstrated that several JAK1/3 inhibitors used to treat inflammatory diseases are closely associated with adverse effects at therapeutic doses due to the high conservation of the JAK domain, including the risk of infection, lymphocytopenia, thromboembolism, dyslipidemia, altered liver function and metabolism, and even an increased risk of malignancy (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR29 CR30 CR31 CR32\" citationid=\"CR26\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e33\u003c/span\u003e). IL-23 is essential for driving the TH17 pathway, which is crucial in chronic autoimmune inflammatory skin diseases such as psoriasis and other autoimmune conditions (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan additionalcitationids=\"CR35\" citationid=\"CR32\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e36\u003c/span\u003e). In addition, type-I IFNs are important upstream initiators of psoriasis development and SLE pathogenesis (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Therefore, targeting the JH2 domain rather than the catalytic domain of TYK2 provides a unique approach with potential advantages.\u003c/p\u003e \u003cp\u003eDeucravacitinib is a novel inhibitor of TYK2 developed by Bristol Myers Squibb that binds to the pseudokinase domain (19). A phase-III clinical trial showed that deucravacitinib was effective in the treatment of psoriasis in adults. Although deucravacitinib showed greater selective inhibition for TYK2 than JAK1/JAK2/JAK3, and has been reported to improve SLE, colitis and psoriasis by blocking IL-23 and type-I IFN signaling pathways (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e38\u003c/span\u003e); binding to the JH2 domain of JAK1 and other subtypes of the JH1 domain (JAKs) to varying degrees has been reported (19, 39, 40). Conversely, QL-1200186 binds very weakly to the JH2 domain of JAK1 at different concentrations, and has no effect on the JH1 domain of other homologous isomers, so QL-1200186 has more advantages in terms of selectivity. Phase-2b clinical studies to evaluate another TYK2 inhibitor, NDI-034858 (Nimbus), in moderate-to-severe plaque psoriasis (NCT04999839) and active psoriatic arthritis (NCT05153148) are ongoing, and have shown superior functionality and an encouraging clinical-activity profile. QL-1200186 outperformed deucravacitinib with regard to TYK2 selectivity, but also combined with NDI-034858 (Nimbus) for powerful efficacy. When the minimum dose of QL-1200186 was 0.1 mg/kg and 1 mg/kg, respectively, it could reduce the IFNγ production induced by IL-12/IL-18 and improve the disease severity of our imiquimod-induced psoriasis model. Hence, QL-1200186 is a strong and efficacious small molecule inhibitor of TYK2, which may also have good efficacy for other indications.\u003c/p\u003e \u003cp\u003eBesides the compelling evidence that QL-1200186 can alleviate psoriasis, there remains an urgent need for the development of safe drugs against autoimmune diseases. Off-target drug reactions are the main cause of adverse drug reactions. A considerable proportion of projects on the research and development of drugs stall and terminate at a late stage due to a failure of early detection of off-target effects, and clinical trials fail due to adverse drug reactions. To design lead drugs accurately, improve the success rate of research and development and reduce the failure rate of clinical trials, we tested the \u003cem\u003ein vitro\u003c/em\u003e off-target effects of QL-1200186 based on 44 early drug-safety targets jointly proposed by AstraZeneca, GlaxoSmithKline, Novartis and Pfizer to detect off-target effects. QL-1200186 10 \u0026micro;M showed good safety for these 44 targets. QL-1200186 showed good safety in toxicological tests on dogs and rats (data not shown). Therefore, QL-1200186 appears to be more beneficial than current therapies against autoimmune diseases.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eThere is an unmet need for safe, rapid-acting and efficacious drugs against psoriasis and other autoimmune diseases. There is a strong \u003cem\u003erationale\u003c/em\u003e for targeting the TYK2 JH2 domain in these diseases, and QL-1200186 may be the best option. QL-1200186 blocks the cytokine-mediated inflammatory response potently through TYK2. Preclinical pharmacologic and safety assessments support clinical development to evaluate its efficacy against autoimmune diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData may be provided upon reasonable request to the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics\u0026nbsp;statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the Animal Care and Use Committee of Qilu Pharmaceuticals. Samples of peripheral blood were taken from healthy volunteers who provided written informed consent in accordance with the guidelines and regulations set by Qilu Pharmaceuticals\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBo Chen and Celia X.-J. Chen developed the concept and supervised the project. Wei Zhang and Fucan Xia carried out the main experiments. Shulan Qu and Bo Chen prepared the manuscript and analyzed the data. All authors were involved in drafting the manuscript or revising it critically for important intellectual content. All authors approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was sponsored by Qilu Pharmaceuticals. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our thanks to the donors and investigators who participated in this study. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBo Chen, Celia X.-J. Chen, Wei Zhang, Fucan Xia and Shulan Qu are employees of Qilu Pharmaceuticals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGadina M, Le MT, Schwartz DM, Silvennoinen O, Nakayamada S, Yamaoka K, et al. Janus kinases to jakinibs: from basic insights to clinical practice. Rheumatology (Oxford) (2019) 58(Suppl 1):i4\u0026ndash;16. doi.org/10.1093/rheumatology/key432 \u003c/li\u003e\n\u003cli\u003eMorris R, Kershaw NJ, Babon JJ. The molecular details of cytokine signaling via the JAK/STAT pathway. Protein Sci (2018) 27(12):1984\u0026ndash;2009. doi.org/10.1002/pro.3519 \u003c/li\u003e\n\u003cli\u003eSchwartz DM, Kanno Y, Villarino A, Ward M, Gadina M, O\u0026apos;Shea JJ. JAK inhibition as a therapeutic strategy for immune and inflammatory diseases. 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PLoS One (2015) 10(4):e0122271. doi.org/10.1371/journal.pone.0122271 \u003c/li\u003e\n\u003cli\u003eL\u0026oacute;pez-Isac E, Campillo-Davo D, Bossini-Castillo L, Guerra SG, Assassi S, Sime\u0026oacute;n CP, et al. Influence of TYK2 in systemic sclerosis susceptibility: a new locus in the IL-12 pathway. Ann Rheum Dis (2016) 75(8):1521\u0026ndash;6. doi.org/10.1136/annrheumdis-2015-208154 \u003c/li\u003e\n\u003cli\u003eBurke JR, Cheng L, Gillooly KM, Strnad J, Zupa-Fernandez A, Catlett IM, et al. Autoimmune pathways in mice and humans are blocked by pharmacological stabilization of the TYK2 pseudokinase domain. Sci Transl Med (2019) 11(502). doi.org/10.1126/scitranslmed.aaw1736 \u003c/li\u003e\n\u003cli\u003eZarrin AA, Bao K, Lupardus P, Vucic D. Kinase inhibition in autoimmunity and inflammation. 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Plasmacytoid predendritic cells initiate psoriasis through interferon-alpha production. J Exp Med (2005) 202(1):135\u0026ndash;43. doi.org/10.1084/jem.20050500 \u003c/li\u003e\n\u003cli\u003eCatlett IM, Aras U, Liu Y, Bei D, Girgis IG, Murthy B, et al. SAT0226 A first-in-human, study of BMS-986165, a selective, potent, allosteric small molecule inhibitor of tyrosine kinase 2. Ann Rheum Dis (2017) 76: 859\u0026ndash;59. \u003c/li\u003e\n\u003cli\u003eGerstenberger BS, Ambler C, Arnold EP, Banker ME, Brown MF, Clark JD, et al. Discovery of tyrosine kinase 2 (TYK2) inhibitor (PF-06826647) for the treatment of autoimmune diseases. J Med Chem (2020) 63(22):13561\u0026ndash;77. doi.org/10.1021/acs.jmedchem.0c00948 \u003c/li\u003e\n\u003cli\u003eLiu C, Lin J, Langevine C, Smith D, Li J, Tokarski JS, et al. Discovery of BMS-986202: a clinical Tyk2 inhibitor that binds to Tyk2 JH2. J Med Chem (2021) 64(1):677\u0026ndash;94. doi.org/10.1021/acs.jmedchem.0c01698 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"QL-1200186, TYK2 inhibitor, psoriasis, autoimmune diseases, inflammation, JAKs, BMS-986165, NDI-034858","lastPublishedDoi":"10.21203/rs.3.rs-2914960/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2914960/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family, which plays an important part in signal transduction and regulation of the immune system. To minimize the safety concerns and improve the therapeutic effect against autoimmune diseases, we developed a small molecule inhibitor (QL-1200186) targeting the pseudokinase domain of TYK2 protein (JH2). The binding sites of QL-1200186 were predicted and screened by molecular docking. The inhibitory effects of the downstream signaling pathways and transcriptional activators of TYK2 were reflected in cell lines and human peripheral-blood cells. Pharmacokinetics and pharmacodynamics were verified in mice. QL-1200186 showed highly affinity to TYK2 JH2 and had no apparent selectivity for the TYK2 and JAK homologous kinase domains (JH1); these effects were manifested in assays based on biochemical binding, signaling pathway transduction (JAK1/2/3) and off-target effects. We revealed that currently available drugs, such as BMS-986165 and NDI-034858, were the most likely candidates for TYK2 inhibitors, and found that QL-1200186 was functionally comparable to and selectively superior to both agents \u003cem\u003ein vitro\u003c/em\u003e. QL-1200186 showed excellent exposure, high bioavailability and afforded low clearance rates in mice. Oral administration of QL-1200186 dose-dependently inhibited interferon-γ production in interleukin-12-driven responses and ameliorated skin lesions significantly in a mouse model of psoriasis, respectively. These findings suggest that QL-1200186 is a highly selective and potent inhibitor of TYK2. QL-1200186 could be developed as a drug for the treatment of psoriasis or other autoimmune diseases.\u003c/p\u003e","manuscriptTitle":"A Novel highly selective allosteric inhibitor of TYK2 can block inflammation/autoimmune pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-06-29 18:14:25","doi":"10.21203/rs.3.rs-2914960/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-07-16T21:10:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-07-16T20:19:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-07-03T20:23:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3cc2ec62-8dba-4f23-b473-d6443102fd09","date":"2023-06-27T06:52:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-06-26T15:59:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-06-26T05:54:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-06-26T05:54:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Communication and Signaling","date":"2023-05-10T07:44:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"33307fb0-76b4-41cc-961a-99c644e5e898","owner":[],"postedDate":"June 29th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-23T15:08:39+00:00","versionOfRecord":{"articleIdentity":"rs-2914960","link":"https://doi.org/10.1186/s12964-023-01299-7","journal":{"identity":"cell-communication-and-signaling","isVorOnly":false,"title":"Cell Communication and Signaling"},"publishedOn":"2023-10-16 15:01:07","publishedOnDateReadable":"October 16th, 2023"},"versionCreatedAt":"2023-06-29 18:14:25","video":"","vorDoi":"10.1186/s12964-023-01299-7","vorDoiUrl":"https://doi.org/10.1186/s12964-023-01299-7","workflowStages":[]},"version":"v1","identity":"rs-2914960","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2914960","identity":"rs-2914960","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}