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Pursuing on our efforts regarding development of novel multikinase inhibitors, herein we report the design and synthesis of novel 2-indolinone-based ureides 6a-u and amides 10a-j. In this work we adopt a hybridization strategy between type IIA PTK inhibitor (sorafenib) and type IIB PTK inhibitors (sunitinib and nintedanib). This was implemented via linking the indolinone core, in both sunitinib and nintedanib, which is well-fitted in the hinge region in the kinase domain front cleft and the biaryl urea extension, in sorafenib, which is accommodated in the gate area and the hydrophobic back pocket. Molecular docking of the designed hybrid compounds in VEGFR-2 and FGFR-1 active sites revealed, as planned, their ability to establish the binding interactions achieved by both original type IIA and type IIB inhibitors. The designed compounds were evaluated for their multikinase inhibitory activity towards VEGFR-2, PDGFR-b and FGFR-1 and anti-proliferative activity towards HepG2, MCF-7, A549 and A498 cancer cell lines. The ureido analogue 6u emerged as the most potent multikinase inhibitor in the ureido series with VEGFR-2, FGFR-1 and PDGFR-b IC 50 of 0.18, 0.23 and 0.10 μM, respectively. Whereas, the amido congener 10j emerged as the most potent multikinase inhibitor in the amide series with VEGFR-2, FGFR-1 and PDGFR-b IC 50 of 0.28, 0.46 and 0.09 μM, respectively. While, indolinone 6u was the most potent derivative towards HepG2 cells (IC 50  = 2.67 ± 0.14 μM), 6r stood out as the most potent indolinone against A498 cells (IC 50  = 0.78 ± 0.02 μM). Additionally, the target indolinones displayed non-significant cytotoxic impact towards human normal melanocyte (HFB4). ADME prediction study of the designed compounds showed that they are not only with promising multikinase inhibitory activity but also with favorable pharmacokinetic and drug-likeness properties. Compounds 6r and 10j are revealed to be the best compounds in terms of multikinase activity and pharmacokinetics., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Introduction: Tyrosine kinase 2 (Tyk2) is a non-receptor tyrosine-protein kinase, an enzyme that in humans is encoded by the TYK2 gene. Tyk2, together with three other family subtypes, namely, Jak1, Jak2, and Jak3, belong to the JAK family. Before 2014, far more publications and patents appeared in public domain attributing to the development of selective Jak2 and Jak3 inhibitors than those for selective Tyk2 and Jak1 inhibitors., Areas Covered: This review sought to give an overview of patents related to small molecule selective Tyk2 inhibitors published from 2015 to 2018. The article also covers clinical activities of small molecule selective Tyk2 inhibitors in recent years., Expert Opinion: As a key component of the JAK-STAT signaling pathway, Tyk2 regulates INFα, IL12, and IL23. Selective inhibition of Tyk2 can provide pharmacological benefits in the treatment of many diseases such as psoriasis, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), rheumatoid arthritis (RA), cancer, and diabetes. The selectivity against other Jak family subtypes (such as Jak2) is crucial in order to minimize the potential side effects and to maximize the desired pharmacological effects. In this context, this review of recent selective Tyk2 inhibitor patents may prove valid, interesting, and promising within the therapeutic paradigm.

Cytokine signaling is an important characteristic of autoimmune diseases. Many pro-inflammatory cytokines signal through the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) pathway. JAK1 is important for the γ-common chain cytokines, interleukin (IL)-6, and type-I interferon (IFN) family, while TYK2 in addition to type-I IFN signaling also plays a role in IL-23 and IL-12 signaling. Intervention with monoclonal antibodies (mAbs) or JAK1 inhibitors has demonstrated efficacy in Phase III psoriasis, psoriatic arthritis, inflammatory bowel disease, and rheumatoid arthritis studies, leading to multiple drug approvals. We hypothesized that a dual JAK1/TYK2 inhibitor will provide additional efficacy, while managing risk by optimizing selectivity against JAK2 driven hematopoietic changes. Our program began with a conformationally constrained piperazinyl-pyrimidine Type 1 ATP site inhibitor, subsequent work led to the discovery of PF-06700841 (compound 23), which is in Phase II clinical development (NCT02969018, NCT02958865, NCT03395184, and NCT02974868).

Background: Tyrosine kinase 2 (TYK2) signaling pathways, which mediate cytokine signaling, are implicated in the pathophysiology of psoriasis. Selective inhibitors of TYK2 may be effective in treating psoriasis., Methods: We conducted a phase 2, double-blind trial of a TYK2 inhibitor, BMS-986165, in adults with moderate-to-severe psoriasis, excluding patients with a previous lack of response to agents targeting cytokine signaling through the same tyrosine kinase pathway. Patients were randomly assigned to receive the drug orally at a dose of 3 mg every other day, 3 mg daily, 3 mg twice daily, 6 mg twice daily, or 12 mg daily or to receive placebo. The primary end point was a 75% or greater reduction from baseline in the Psoriasis Area and Severity Index (PASI) score at week 12 (higher scores indicate greater severity of psoriasis)., Results: A total of 267 patients received at least one dose in an intervention group of the trial. At week 12, the percentage of patients with a 75% or greater reduction in the PASI score was 7% (3 of 45 patients) with placebo, 9% (4 of 44 patients) with 3 mg of BMS-986165 every other day (P=0.49 vs. placebo), 39% (17 of 44 patients) with 3 mg daily (P<0.001 vs. placebo), 69% (31 of 45 patients) with 3 mg twice daily (P<0.001 vs. placebo), 67% (30 of 45 patients) with 6 mg twice daily (P<0.001 vs. placebo), and 75% (33 of 44 patients) with 12 mg daily (P<0.001 vs. placebo). There were three serious adverse events in patients receiving the active drug, as well as one case of malignant melanoma 96 days after the start of treatment., Conclusions: Selective inhibition of TYK2 with the oral agent BMS-986165 at doses of 3 mg daily and higher resulted in greater clearing of psoriasis than did placebo over a period of 12 weeks. Larger and longer-duration trials of this drug are required to determine its safety and durability of effect in patients with psoriasis. (Funded by Bristol-Myers Squibb; ClinicalTrials.gov number, NCT02931838 .).

A therapeutic rationale is proposed by selectively targeting tyrosine kinase 2 (TYK 2) to obtain potent TYK 2 inhibitors by molecular modeling studies. In the present study, we have taken tyrosine kinase (TYK 2) inhibitors and carried out molecular docking, 3 D quantitative structure-activity relationship (3D-QSAR) analysis and molecular dynamics (MD). Based on the 3D-QSAR results thirteen new compounds (R-1 to R-13) were designed and synthesized in good yields. The synthesized molecules were evaluated for their in vitro anticancer activity against LnCap and A549 cell lines. The molecules R-1, R-3, R-5, R-7, and R-10 exhibited considerable anti cancer activity.

The safety, tolerability, pharmacokinetics, and pharmacodynamics of PF-06700841 were assessed in a randomized, double-blind, placebo-controlled, single- and multiple-dose escalation, parallel-group study in healthy subjects and patients with plaque psoriasis. The single ascending dose (1, 3, 10, 30, 100, or 200 mg) and multiple ascending dose (MAD; PF-06700841; up to 175 mg once daily or 50 mg twice daily for 10 days) periods included 54 healthy participants. In addition, 30 patients with psoriasis received PF-06700841 30 or 100 mg or placebo once daily for 28 days. Single PF-06700841 doses were rapidly absorbed, with peak plasma concentrations ≤ 1 hour, proportional exposure up to 100 mg, and mean half-life 3.8-7.5 hours. On day 10 of MAD, plasma concentrations peaked at ≤1.5 hours postdose (10-175 mg once daily). Elimination half-life was 4.9-10.7 hours; steady state was reached by day 8. In psoriasis patients on day 28, peak plasma concentrations occurred at 1-2 hours. Biomarkers IP-10 and high-sensitivity C-reactive protein were reduced and returned to near baseline levels after dosing. Maximal mean percent change from baseline in the Psoriasis Area and Severity Index scores for PF-06700841 30 mg once daily and 100 mg once daily were -67.92% and -96.31%, respectively, in week 4. All adverse events were mild/moderate. PF-06700841 was safe and well tolerated up to 200 mg once daily in healthy subjects and 100 mg once daily in patients with psoriasis, suggesting potential therapeutic utility in plaque psoriasis and other inflammatory diseases., (© 2017, The American College of Clinical Pharmacology.)

Herein we report identification of an imidazopyridine class of potent and selective TYK2 inhibitors, exemplified by prototype 6, through constraint of the rotatable amide bond connecting the pyridine and aryl rings of compound 1. Further optimization led to generation of compound 30 that potently inhibits the TYK2 enzyme and the IL-23 pathway in cells, exhibits selectivity against cellular JAK2 activity, and has good pharmacokinetic properties. In mice, compound 30 demonstrated dose-dependent reduction of IL-17 production in a PK/PD model as well as in an imiquimod-induced psoriasis model. In this efficacy model, the IL-17 decrease was accompanied by a reduction of ear thickness indicating the potential of TYK2 inhibition as a therapeutic approach for psoriasis patients., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A series of potent dual JAK1/3 inhibitors have been developed from a moderately selective JAK3 inhibitor. Substitution at the C6 position of the pyrrolopyridazine core with aryl groups provided exceptional biochemical potency against JAK1 and JAK3 while maintaining good selectivity against JAK2 and Tyk2. Translation to in vivo efficacy was observed in a murine model of chronic inflammation. X-ray co-crystal structure determination confirmed the presumed inhibitor binding orientation in JAK3. Efforts to reduce hERG channel inhibition will be described., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The severity and lethality of influenza A virus (IAV) infections is frequently aggravated by secondary bacterial pneumonia. However, the mechanisms in human lung tissue that provoke this increase in fatality are unknown and therapeutic immune modulatory options are lacking.We established a human lung ex vivo co-infection model to investigate innate immune related mechanisms contributing to the susceptibility of secondary pneumococcal pneumonia.We revealed that type I and III interferon (IFN) inhibits Streptococcus pneumoniae -induced interleukin (IL)-1β release. The lack of IL-1β resulted in the repression of bacterially induced granulocyte-macrophage colony-stimulating factor (GM-CSF) liberation. Specific inhibition of IFN receptor I and III-associated tyrosine kinase 2 (Tyk2) completely restored the S. pneumoniae -induced IL-1β-GM-CSF axis, leading to a reduction of bacterial growth. A preceding IAV infection of the human alveolus leads to a type I and III IFN-dependent blockade of the early cytokines IL-1β and GM-CSF, which are key for orchestrating an adequate innate immune response against bacteria. Their virally induced suppression may result in impaired bacterial clearance and alveolar repair.Pharmacological inhibition of Tyk2 might be a new treatment option to sustain beneficial endogenous GM-CSF levels in IAV-associated secondary bacterial pneumonia., Competing Interests: Conflict of interest: Disclosures can be found alongside this article at erj.ersjournals.com, (Copyright ©ERS 2017.)

Activation of tyrosine kinase 2 (TYK2) contributes to the aberrant survival of T-cell acute lymphoblastic leukaemia (T-ALL) cells. Here we demonstrate the anti-leukaemic activity of a novel TYK2 inhibitor, NDI-031301. NDI-031301 is a potent and selective inhibitor of TYK2 that induced robust growth inhibition of human T-ALL cell lines. NDI-031301 treatment of human T-ALL cell lines resulted in induction of apoptosis that was not observed with the JAK inhibitors tofacitinib and baricitinib. Further investigation revealed that NDI-031301 treatment uniquely leads to activation of three mitogen-activated protein kinases (MAPKs), resulting in phosphorylation of ERK, SAPK/JNK and p38 MAPK coincident with PARP cleavage. Activation of p38 MAPK occurred within 1 h of NDI-031301 treatment and was responsible for NDI-031301-induced T-ALL cell death, as pharmacological inhibition of p38 MAPK partially rescued apoptosis induced by TYK2 inhibitor. Finally, daily oral administration of NDI-031301 at 100 mg/kg bid to immunodeficient mice engrafted with KOPT-K1 T-ALL cells was well tolerated, and led to decreased tumour burden and a significant survival benefit. These results support selective inhibition of TYK2 as a promising potential therapeutic strategy for T-ALL., (© 2017 John Wiley & Sons Ltd.)

Modeling protein-ligand interactions has been a central goal of computational chemistry for many years. We here review recent progress toward this goal, and highlight the role free energy calculation methods and computational solvent analysis techniques are now having in drug discovery. We further describe recent use of these methodologies to advance two separate drug discovery programs targeting acetyl-CoA carboxylase and tyrosine kinase 2. These examples suggest that tight integration of sophisticated chemistry teams with state-of-the-art computational methods can dramatically improve the efficiency of small molecule drug discovery., (Copyright © 2016. Published by Elsevier Ltd.)

We report herein the discovery and optimization of 3-amino-1,5-dihydro-4H-pyrazolopyridin-4-one TYK2 inhibitors. High-throughput screening against TYK2 and JAK1-3 provided aminoindazole derivative 1 as a hit compound. Scaffold hopping of the aminoindazole core led to the discovery of 3-amino-1,5-dihydro-4H-pyrazolopyridin-4-one derivative 3 as a novel chemotype of TYK2 inhibitors. Interestingly, initial SAR study suggested that this scaffold could have a vertically flipped binding mode, which prompted us to introduce a substituent at the 7-position as a moiety directed toward the solvent-exposed region. Introduction of a 1-methyl-3-pyrazolyl moiety at the 7-position resulted in a dramatic increase in TYK2 inhibitory activity, and further optimization led to the discovery of 20. Compound 20 inhibited IL-23-induced IL-22 production in a rat PD assay, as well as inhibited IL-23 signaling in human PBMC. Furthermore, 20 showed selectivity for IL-23 signaling inhibition against GM-CSF, demonstrating the unique cytokine selectivity of the novel TYK2 inhibitor.

Introduction: ErbB2 overexpression and/or gene amplification is present in 20% of all breast cancers and characterizes an aggressive form of this disease. Despite the availability of several active drugs that have yielded substantial survival improvements, most patients with ErbB2-positive metastatic disease will develop tumor progression, either because of primary or acquired resistance. Therefore, research has focused on drugs that can more efficiently interfere with ErbB2 and with other members of the epidermal growth factor receptor family., Areas Covered: This review focuses on those investigational drugs that inhibit ErbB2 tyrosine kinase activity (TKIs) for treating breast cancer., Expert Opinion: ErbB-targeting TKIs show encouraging activity in patients with ErbB-positive tumors that are resistant to conventional ErbB-therapies (mostly trastuzumab), confirming pre-clinical observations. Efficient interference with the ErbB-network signaling implies also a potential use in ErbB2-normal tumors, where the phenotype is sustained by ErbB-aberrant signaling. Finally, early data suggests that ErbB-targeting TKIs could be active in treating patients with activating ErbB2 mutations. Ongoing and future research efforts should elucidate what is, according to the peculiarities of these compounds, their positioning in the treatment of women with breast cancer.

Aims and Background: The introduction of agents targeting vascular endothelial growth factor has radically changed the approach to metastatic renal cell carcinoma (mRCC): sunitinib and pazopanib are now the standard first-line therapy in mRCC. At sunitinib failure, second-line axitinib or everolimus or sorafenib should be considered to improve the clinical outcome. No data are available for a third-line tyrosine kinase inhibitor (TKI) after 2 previous lines of therapy with TKIs. At pazopanib failure, no prospective data are available., Study Design: The TOKIO study was designed to evaluate progression-free survival, safety, and efficacy of third-line therapy with TKI in 44 patients already treated with 2 previous lines of TKIs in 10 Italian centers, and relapsed from sunitinib-axitinib (group A) or pazopanib-sorafenib (group B). Standard treatment is sorafenib in group A and sunitinib in group B, administered until disease progression or unacceptable toxicity. Secondary endpoints include the evaluation of overall survival, safety, and quality of life.

In this study, we synthesized compound 12 with potent Tyk2 inhibitory activity from FBDD study and carried out a cell-based assay for Tyk2/STAT3 signaling activation upon IFNα5 stimulation. Compound 12 completely suppressed the IFNα5-mediated Tyk2/STAT3 signaling pathway as well as the basal levels of pSTAT3. Stimulation with IFNα/β leads to the tyrosine phosphorylation of the JAK1 and Tyk2 receptor-associated kinases with subsequent STATs activation, transmitting signals from the cell surface receptor to the nucleus. In conclusion, the potency of compound 12 to interrupt the signal transmission of Tyk2/STAT3 appeared to be equivalent or superior to that of the reference compound., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Structure- and property-based drug design is an integral part of modern drug discovery, enabling the design of compounds aimed at improving potency and selectivity. However, building molecules using desktop modeling tools can easily lead to poor designs that appear to form many favorable interactions with the protein's active site. Although a proposed molecule looks good on screen and appears to fit into the protein site X-ray crystal structure or pharmacophore model, doing so might require a high-energy small molecule conformation, which would likely be inactive. To help scientists make better design decisions, we have built integrated, easy-to-use, interactive software tools to perform docking experiments, de novo design, shape and pharmacophore based database searches, small molecule conformational analysis and molecular property calculations. Using a combination of these tools helps scientists in assessing the likelihood that a designed molecule will be active and have desirable drug metabolism and pharmacokinetic properties. Small molecule discovery success requires project teams to rapidly design and synthesize potent molecules with good ADME properties. Empowering scientists to evaluate ideas quickly and make better design decisions with easy-to-access and easy-to-understand software on their desktop is now a key part of our discovery process.

Pancreatic adenocarcinoma upregulated factor (PAUF), a novel oncogene, plays a crucial role in the development of pancreatic cancer, including its metastasis and proliferation. Therefore, PAUF-expressing pancreatic cancer cells could be important targets for oncolytic virus-mediated treatment. Panc-1 cells expressing PAUF (Panc-PAUF) showed relative resistance to parvovirus H-1 infection compared with Panc-1 cells expressing an empty vector (Panc-Vec). Of interest, expression of type I IFN-α receptor (IFNAR) was higher in Panc-PAUF cells than in Panc-Vec cells. Increased expression of IFNAR in turn increased the activation of Stat1 and Tyk2 in Panc-PAUF cells compared with that in Panc-Vec cells. Suppression of Tyk2 and Stat1, which are important downstream molecules for IFN-α signaling, sensitized pancreatic cancer cells to parvovirus H-1-mediated apoptosis. Further, constitutive suppression of PAUF sensitized Bxpc3 pancreatic cancer cells to parvovirus H-1 infection. Taken together, these results suggested that PAUF conferred resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNAR-mediated signaling., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Psoriasis is a chronic autoimmune disease affecting the skin and characterized by aberrant keratinocyte proliferation and function. Immune cells infiltrate the skin and release proinflammatory cytokines that play important roles in psoriasis. The Th17 network, including IL-23 and IL-22, has recently emerged as a critical component in the pathogenesis of psoriasis. IL-22 and IL-23 signaling is dependent on the JAK family of protein tyrosine kinases, making JAK inhibition an appealing strategy for the treatment of psoriasis. In this study, we report the activity of SAR-20347, a small molecule inhibitor with specificity for JAK1 and tyrosine kinase 2 (TYK2) over other JAK family members. In cellular assays, SAR-20347 dose dependently (1 nM-10 μM) inhibited JAK1- and/or TYK2-dependent signaling from the IL-12/IL-23, IL-22, and IFN-α receptors. In vivo, TYK2 mutant mice or treatment of wild-type mice with SAR-20347 significantly reduced IL-12-induced IFN-γ production and IL-22-dependent serum amyloid A to similar extents, indicating that, in these models, SAR-20347 is probably acting through inhibition of TYK2. In an imiquimod-induced psoriasis model, the administration of SAR-20347 led to a striking decrease in disease pathology, including reduced activation of keratinocytes and proinflammatory cytokine levels compared with both TYK2 mutant mice and wild-type controls. Taken together, these data indicate that targeting both JAK1- and TYK2-mediated cytokine signaling is more effective than TYK2 inhibition alone in reducing psoriasis pathogenesis., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The family of JAK comprises four members and has received significant attention in recent years from the pharmaceutical industry as a therapeutic target. The role of JAK is central to cytokine signaling pathways. It is believed that selective modulation of one specific JAK can lead to the inhibition of a restricted set of cytokines, which should avoid undesired side effects and get closer to the profile of biologic therapies. Consequently, selective JAK inhibition has become a major focus area of drug discovery research. A review of the TYK2 patents indicates that industry attention has recently turned toward the development of specific inhibitors. Importantly, despite the increasing number of published patents, none of these drugs have yet made it to the clinical trials.

Introduction: Tyrosine kinase 2 (Tyk2) is a Janus kinase family member that is crucial for signaling transduction in response to a wide variety of cytokines, including type I IFNs, IL-6, IL-10, IL-12 and IL-23. An appropriate expression of Tyk2-mediated signaling might be essential for maintaining normal immune responses., Areas Covered: This review summarizes that Tyk2 is essential for the differentiation and function of a wide variety of immune cells, including natural killer cells, B cells, as well as T helper cells. In addition, Tyk2-mediated signaling promoted the production of autoimmune-associated components, which is implicated in the pathogenesis of autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis. Aberrant expression of Tyk2 was observed in many autoimmune conditions., Expert Opinion: Until recently, no patent filings had claimed selective inhibitors of Tyk2. Both CP-690,500 and CMP6 failed to be used in clinical treatment due to the difficulties of finding suitable selective leads or due to detrimental toxicities. Although the result of Cmpd1 is promising, it remains to be seen how specific the Tyk2 inhibitor is and how they are working. Currently, structure-based drug design (SBDD) technology has provided us with a quite useful window for SBDD of Tyk2 inhibitors.

Tyrosine kinase 2 (Tyk2), a member of the Jak kinase family, mediates signals triggered by various cytokines, which are related to the pathogenesis of psoriasis. In this study, we investigated the role of Tyk2 in IL-23-induced psoriasis-like skin inflammation. Tyk2(-/-) mice when injected with IL-23 showed significantly reduced ear skin swelling with epidermal hyperplasia and inflammatory cell infiltration compared with wild-type mice. In addition, Tyk2 deficiency reduced production of pro-inflammatory cytokines and psoriasis-relevant anti-microbial peptides. More noteworthy is that Tyk2 directly regulated IL-22-dependent inflammation and epidermal hyperplasia. Taken together with the inhibition of IL-23-induced inflammation by treatment with neutralizing antibodies against IL-17 or IL-22, Tyk2 participates in both IL-23 and IL-22 signal transduction to mediate psoriasis-like skin inflammation. On the basis of these findings, we demonstrated for the first time that a small-molecule Tyk2 inhibitor significantly inhibited IL-23-induced inflammation and cytokine production in the skin. These observations demonstrate the important role of Tyk2 in experimental skin inflammation and indicate the therapeutic potential of Tyk2 inhibition in human psoriasis.

Tyrosine kinase inhibitors show great promise as clinical therapies, but small molecule inhibitors that are available in the clinic and under development bind to the adenosine triphosphate binding domain of the kinase, potentially limiting efficacy and selectivity. The development of antisense peptide inhibitors is a relatively unexplored area of research, and here we investigate inhibitory peptides specific for the Janus-associated kinase (JAK) family member, tyrosine kinase 2 (TYK2). We have developed peptides that are 2-3 times more selective for TYK2 than other JAK family members, with a TYK2 IC50 of 1.2 μM. In addition, TYK2 inhibitory peptides show specificity for TYK2-mediated functions over JAK1 functions in cell-based assays. These peptides provide a new tool for the development of specific peptide inhibitors for closely related tyrosine kinases.

Three patent applications, from two different companies, claim structurally novel Tyk2 inhibitors and their uses for the treatment of autoimmune diseases. In EP-2634185 Sareum claims 5-anilino-2-(2-halophenyl)-oxazole-4-carboxamide derivatives which are shown to be nanomolar potency Tyk2 inhibitors with 10 - 100-fold selectivity over JAK1, JAK2 and JAK3. Takeda's WO-2013125543 and WO-2013146963 claim two distinct structural classes of Tyk2 inhibitors. The first application claims inhibitors based on an unusual 1,5-dihydro-4H-pyrazolo[4,3-c]pyridine-4-one scaffold and the second claims 1-(2-arylaminopyrimidin-4-yl)-pyrrolidin-2-one derivatives. One example of the latter was shown to be orally active in an IL-23-induced inflammation model.

A therapeutic rationale is proposed for the treatment of inflammatory diseases, such as psoriasis and inflammatory bowel diseases (IBD), by selective targeting of TYK2. Hit triage, following a high-throughput screen for TYK2 inhibitors, revealed pyridine 1 as a promising starting point for lead identification. Initial expansion of 3 separate regions of the molecule led to eventual identification of cyclopropyl amide 46, a potent lead analog with good kinase selectivity, physicochemical properties, and pharmacokinetic profile. Analysis of the binding modes of the series in TYK2 and JAK2 crystal structures revealed key interactions leading to good TYK2 potency and design options for future optimization of selectivity., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Herein we report our lead optimization effort to identify potent, selective, and orally bioavailable TYK2 inhibitors, starting with lead molecule 3. We used structure-based design to discover 2,6-dichloro-4-cyanophenyl and (1R,2R)-2-fluorocyclopropylamide modifications, each of which exhibited improved TYK2 potency and JAK1 and JAK2 selectivity relative to 3. Further optimization eventually led to compound 37 that showed good TYK2 enzyme and interleukin-12 (IL-12) cell potency, as well as acceptable cellular JAK1 and JAK2 selectivity and excellent oral exposure in mice. When tested in a mouse IL-12 PK/PD model, compound 37 showed statistically significant knockdown of cytokine interferon-γ (IFNγ), suggesting that selective inhibition of TYK2 kinase activity might be sufficient to block the IL-12 pathway in vivo.

Unlabelled: Targeted molecular therapy has yielded remarkable outcomes in certain cancers, but specific therapeutic targets remain elusive for many others. As a result of two independent RNA interference (RNAi) screens, we identified pathway dependence on a member of the Janus-activated kinase (JAK) tyrosine kinase family, TYK2, and its downstream effector STAT1, in T-cell acute lymphoblastic leukemia (T-ALL). Gene knockdown experiments consistently showed TYK2 dependence in both T-ALL primary specimens and cell lines, and a small-molecule inhibitor of JAK activity induced T-ALL cell death. Activation of this TYK2-STAT1 pathway in T-ALL cell lines occurs by gain-of-function TYK2 mutations or activation of interleukin (IL)-10 receptor signaling, and this pathway mediates T-ALL cell survival through upregulation of the antiapoptotic protein BCL2. These findings indicate that in many T-ALL cases, the leukemic cells are dependent upon the TYK2-STAT1-BCL2 pathway for continued survival, supporting the development of molecular therapies targeting TYK2 and other components of this pathway., Significance: In recent years, "pathway dependence" has been revealed in specific types of human cancer, which can be important because they pinpoint proteins that are particularly vulnerable to antitumor-targeted inhibition (so-called Achilles’ heel proteins). Here, we use RNAi technology to identify a novel oncogenic pathway that involves aberrant activation of the TYK2 tyrosine kinase and its downstream substrate, STAT1, which ultimately promotes T-ALL cell survival through the upregulation of BCL2 expression

Aberrant regulation in the chemotaxis and migration ability of cancer cells is closely associated with their metastatic activity. The chemokine receptor 7 (CCR7) has recently been shown to play an important role in regional lymph node metastasis of squamous cell carcinoma of the head and neck (SCCHN). In this study, we examined the role of proline-rich tyrosine kinase-2 (Pyk2) in CCR7-induced chemotaxis and migration ability of metastatic SCCHN cells. We showed that Pyk2 is overexpressed in squamous cell carcinoma of the head and neck. We also found that CCR7 induced Pyk2 and cofilin activation. Inhibition of Pyk2 activity using a pharmacological inhibitor, Tyrphostin A9, significantly attenuated CCR7-induced Pyk2 tyrosine phosphorylation, activation of cofilin and sequentially abolished F-actin rearrangment, diminished the chemotaxis and migration ability of SCCHN cells. In summary, our data suggest that CCR7 via Pyk2 and cofilin regulates the chemotaxis and migration ability of metastatic SCCHN cells.

Introduction: The JAK family comprises of the four non-receptor tyrosine kinases JAK1, JAK2, JAK3 and Tyk2, which play key, but differing, roles in cytokine receptor signal transduction. A non-selective JAK inhibitor, ruxolitinib, has recently been approved to treat myelofibrosis whereas tofacitinib is poised for approval to treat rheumatoid arthritis. Selective inhibition of JAK3, JAK1 or Tyk2 provides the opportunity to achieve clinical efficacy in the treatment of inflammatory diseases while reducing the risk of dose-limiting effects attributable to JAK2 inhibition., Areas Covered: This review considers the small number of published patent filings that claim either selective JAK1 or selective Tyk2 inhibitors. These are considered in the context of the considerably larger number of disclosures and patent filings claiming selective JAK2 or JAK3 inhibitors., Expert Opinion: The recent disclosure of the clinical efficacy of a selective JAK1 inhibitor (GLPG-0634) in rheumatoid arthritis and detailed disclosure of the some potent and highly selective JAK1 inhibitors provide a clear stimulus for further activity in this area. The availability of a selective Tyk2 inhibitor will provide the opportunity for better understanding of the physiological role of this kinase. Recent patent applications indicate that Tyk2 selectivity is achievable and Tyk2 inhibitors have potential in the treatment of multiple sclerosis.

Background: Structure-based drug design (SBDD) can accelerate inhibitor lead design and optimization, and efficient methods including protein purification, characterization, crystallization, and high-resolution diffraction are all needed for rapid, iterative structure determination. Janus kinases are important targets that are amenable to structure-based drug design. Here we present the first mouse Tyk2 crystal structures, which are complexed to 3-aminoindazole compounds., Results: A comprehensive construct design effort included N- and C-terminal variations, kinase-inactive mutations, and multiple species orthologs. High-throughput cloning and expression methods were coupled with an abbreviated purification protocol to optimize protein solubility and stability. In total, 50 Tyk2 constructs were generated. Many displayed poor expression, inadequate solubility, or incomplete affinity tag processing. One kinase-inactive murine Tyk2 construct, complexed with an ATP-competitive 3-aminoindazole inhibitor, provided crystals that diffracted to 2.5-2.6 Å resolution. This structure revealed initial "hot-spot" regions for SBDD, and provided a robust platform for ligand soaking experiments. Compared to previously reported human Tyk2 inhibitor crystal structures (Chrencik et al. (2010) J Mol Biol 400:413), our structures revealed a key difference in the glycine-rich loop conformation that is induced by the inhibitor. Ligand binding also conferred resistance to proteolytic degradation by thermolysin. As crystals could not be obtained with the unliganded enzyme, this enhanced stability is likely important for successful crystallization and inhibitor soaking methods., Conclusions: Practical criteria for construct performance and prioritization, the optimization of purification protocols to enhance protein yields and stability, and use of high-throughput construct exploration enable structure determination methods early in the drug discovery process. Additionally, specific ligands stabilize Tyk2 protein and may thereby enable crystallization.

The Janus kinase (JAK) family consists of four members: JAK-1, -2, -3 and tyrosine kinase 2 (TYK-2). Recent work suggests that cytokine signaling through TYK-2 may play a critical role in a number of inflammatory processes. We recently described the purification and characterization of phosphorylated isoforms of the TYK-2 kinase domain (TYK-2 KD) and its high resolution 3D structure in the presence of inhibitors. We now report the expression and a two-step purification procedure for the doubly tagged full-length construct, H6-FL-TYK-2-FLAG, and examine its properties compared to those of TYK-2 KD. In the presence of ATP and a peptide substrate, H6-FL-TYK-2-FLAG showed a marked lag in phosphopeptide product formation, while TYK-2 KD showed no such lag. This lag could be eliminated by ATP pretreatment, suggesting that the H6-FL-TYK-2-FLAG enzyme was activated by phosphorylation. The potencies of several nanomolar inhibitors were similar for TYK-2 KD and H6-FL-TYK-2-FLAG. However, these same inhibitors were about 1000 times less potent inhibiting the autophosphorylation of H6-FL-TYK-2-FLAG than they were inhibiting the phosphorylation of a peptide substrate modeled after the activation loop sequence of TYK-2. This intriguing result suggests that autophosphorylation and, thus, activation of H6-FL-TYK-2-FLAG is relatively insensitive to inhibition and that present inhibitors act to inhibit TYK-2 subsequent to activation. Inhibition of TYK-2 autophosphorylation may represent a new area of investigation for the JAK family.

Herein, we describe the synthesis and SAR of a series of small molecule macrocycles that selectively inhibit JAK2 kinase within the JAK family and FLT3 kinase. Following a multiparameter optimization of a key aryl ring of the previously described SB1518 (pacritinib), the highly soluble 14l was selected as the optimal compound. Oral efficacy in the murine collagen-induced arthritis (CIA) model for rheumatoid arthritis (RA) supported 14l as a potential treatment for autoimmune diseases and inflammatory disorders such as psoriasis and RA. Compound 14l (SB1578) was progressed into development and is currently undergoing phase 1 clinical trials in healthy volunteers.

Background & Aims: The combination of pegylated interferon (IFN) α and ribavirin (RBV) is the standard therapy for patients with chronic HCV infection. However, it produces a sustained virologic response (SVR) in only half of the treated individuals and is associated with significant side effects. Recently, several single-nucleotide polymorphisms (SNPs) near the IL28B locus, also known as IFNλ3, were identified to be strong predictors of SVR in patients receiving PEG-IFN and RBV. We sought to determine whether IL28B was capable of inhibiting HCV replication and to determine the pathway by which IL28B exhibits anti-HCV activity., Methods: Using the full-length HCV replicon OR6 and the infectious HCV clones JFH1 and Jc1, we assessed the anti-HCV effect of IL28B on HCV and characterized the key steps of the JAK-STAT pathway by real time PCR, luciferase assay, and Western blot. Finally, we evaluated the anti-HCV effect of IL28B in the presence of JAK-STAT pathway inhibitors such as blocking antibodies, a pharmacological inhibitor, and siRNAs., Results: We found that IL28B inhibits HCV replication in a dose- and time-dependent manner. Like IFNα, IL28B induces the phosphorylation of STAT1 and STAT2, ISRE-driven transcription, and expression of known ISGs. The anti-HCV effects of IL28A, IL28B, and IL29 were abrogated by an IL10R2 blocking antibody, a pharmacological inhibitor of JAK1/TYK2, and by siRNA against IL28R1, STAT1, STAT2, and IRF9., Conclusions: Our data demonstrate that IL28A, IL28B, and IL29 signal through the JAK-STAT pathway to inhibit HCV. These data suggest possible applications of new approaches in HCV treatment., (Copyright © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The type I interferons (IFNs) normally suppress tumor growth by phosphorylating and activating the signal transducer and activator of transcription 1 (STAT1), but also briefly activate STAT3, which promotes cell growth. In chronic lymphocytic leukemia (CLL) cells, the duration of IFN-mediated STAT3 phosphorylation was found to exhibit significant interpatient variability and was prolonged in cells with high risk features, such as 11q- and 17p-deletions involving ataxia telangiectasia mutated (ATM) and p53. This aberrant signaling pattern was associated with a paradoxical increase in cell size and number in response to IFN and similar alterations in IFN-signaling and responses were seen in cell lines that developed in the absence of p53 or ATM. However, direct inhibition of p53 or ATM failed to cause these changes, and CLL cells with aggressive clinical features were found to also express high levels of reactive oxygen species (ROS), which decrease tyrosine phosphatase activity. Prolonged IFN-mediated STAT3 phosphorylation and lowered phosphatase activity could be reversed by antioxidants. These findings suggest that increased ROS levels may corrupt IFN-signaling processes in aggressive CLL cells, causing IFN to be used as a growth factor rather than a tumor suppressor. Antioxidants or STAT3 kinase inhibitors might improve the outcome of IFN therapy in CLL by restoring normal signaling., (© 2011 by The American Society of Hematology)

Members of the JAK family of protein kinases mediate signal transduction from cytokine receptors to transcription factor activation. Over-stimulation of these pathways is causative in immune disorders like rheumatoid arthritis, psoriasis, lupus, and Crohn's disease. A search for selective inhibitors of a JAK kinase has led to our characterization of a previously unknown kinase conformation arising from presentation of Tyr962 of TYK2 to an inhibitory small molecule via an H-bonding interaction. A small minority of protein kinase domains has a Tyrosine residue in this position within the αC-β4 loop, and it is the only amino acid commonly seen here with H-bonding potential. These discoveries will aid design of inhibitors that discriminate among the JAK family and more widely among protein kinases., (© 2010 Wiley-Liss, Inc.)

Tyrosine kinase 2 (TYK2) is required for signaling of interleukin-23 (IL-23), which plays a key role in rheumatoid arthritis. Presented is the design and synthesis of 1,2,4-triazoles, and the evaluation of their inhibitory activity against the Janus associated kinases TYK2 and JAKs 1-3., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The Janus kinase family consists of four members: JAK-1, -2, -3 and TYK-2. While JAK-2 and JAK-3 have been well characterized biochemically, there is little data on TYK-2. Recent work suggests that TYK-2 may play a critical role in the development of a number of inflammatory processes. We have carried out a series of biochemical studies to better understand TYK-2 enzymology and its inhibition profile, in particular how the TYK-2 phosphorylated forms differ from each other and from the other JAK family members. We have expressed and purified milligram quantities of the TYK-2 kinase domain (KD) to high purity and developed a method to separate the non-, mono- (pY(1054)) and di-phosphorylated forms of the enzyme. Kinetic studies (k(cat(app))/K(m(app))) indicated that phosphorylation of the TYK-2-KD (pY(1054)) increased the catalytic efficiency 4.4-fold compared to its non-phosphorylated form, while further phosphorylation to generate the di-phosphorylated enzyme imparted no further increase in activity. These results are in contrast to those obtained with the JAK-2-KD and JAK-3-KD, where little or no increase in activity occurred upon mono-phosphorylation, while di-phosphorylation resulted in a 5.1-fold increase in activity for the JAK-2-KD. Moreover, ATP-competitive inhibitors demonstrated 10-30-fold shifts in potency (K(i(app))) as a result of the TYK-2-KD phosphorylation state, while the shifts for JAK-3-KD were only 2-3-fold and showed little or no change for JAK-2-KD. Thus, the phosphorlyation state imparted differential effects on both activity and inhibition within the JAK family of kinases., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Human parainfluenza virus type 3 (HPIV3), one of the paramyxoviruses, uses its accessory C protein as an antagonist against interferon (IFN)-mediated host innate immunity. We have previously shown that the C protein significantly decreased the IFN-induced phosphorylation of signal transducer and activator of transcription (Stat) 1 and the formation of gamma IFN activation factor (GAF) complex, thus abrogating the antiviral activity of the IFNs against vesicular stomatitis virus (VSV) replication. Here, by mutational analyses we demonstrated that the N-terminal truncation of the C protein (CNdelta25 and CNdelta50) substantially (approximately 50%) recovers the IFN-induced responses, suggesting the critical role of the N-terminal region of the C protein in IFN signaling. Furthermore, our results indicate that the charged amino acid residues within the N-terminal region of the C protein regulate the antagonistic effect of the C protein on IFN signaling.

A series of novel pyrrolopyridazine derivatives have been discovered to be HER-2 inhibitors. These compounds selectively inhibited HER-2 kinase activity at low nanomolar concentrations. Compound 7d was identified as a potent HER-2 inhibitor with an IC(50) of 4 nM.

Summary: Cytokines play pivotal roles in immunity and inflammation, and targeting cytokines and their receptors is an effective means of treating such disorders. Type I and II cytokine receptors associate with Janus family kinases (JAKs) to effect intracellular signaling. These structurally unique protein kinases play essential and specific roles in immune cell development and function. One JAK, JAK3, has particularly selective functions. Mutations of this kinase underlie severe combined immunodeficiency, indicative of its critical role in the development and function of lymphocytes. Because JAK3 appears not to have functions outside of hematopoietic cells, this kinase has been viewed as an excellent therapeutic target for the development of a new class of immunosuppressive drugs. In fact, several companies are developing JAK3 inhibitors, and Phase II studies are underway. Mutations of Tyk2 cause autosomal recessive hyperIgE syndrome, and in principle, Tyk2 inhibitors might also be useful as immunosuppressive drugs. JAK2 gain-of-function mutations (V617F) underlie a subset of disorders collectively referred to as myeloproliferative diseases and phase 2 trials using JAK inhibitors are underway in this setting. Thus, we are learning a great deal about the feasibility and effectiveness of targeting Janus kinases, and it appears likely that this will be a fruitful strategy in a variety of settings.

The role of Janus-activated kinase (JAK) signaling in cell cycle transit and maintenance of genomic stability was determined in HL-60 myeloblastic leukemia cells. Inhibition of JAKs, all JAKs (JAK1, JAK2, JAK3, and tyrosine kinase 2), JAK2, or JAK3, caused a significant reduction in cell growth with a major G2-M arrest evident 24 hours after treatment. Targeting all JAKs also caused endoreduplication 48 and 72 hours after treatment. We discovered mitotic cells in both G2 (4N DNA) and G4 (8N DNA) subpopulations of cells treated with an inhibitor of all JAKs as detected by phosphorylated histone H3 expression. Treatment with inhibitors of just JAK2 or JAK3 drastically reduced such mitotic cells. We observed a complete blockage of IFN-gamma and interleukin-6-induced signal transducer and activator of transcription (STAT)-1 and STAT-3 response when all JAKs were inhibited. At the same time, we found baseline phosphorylated extracellular signal-regulated kinase (ERK) 1/2 to be elevated by JAK inhibition, particularly when all JAKs were inhibited. The G2-M arrest and endoreduplication induced by JAK inhibitors were reduced in cells pretreated with PD98059 to inhibit ERK. PD98059 also increased back the expression of the MAD2 cell cycle checkpoint protein that was down-regulated during "all JAKs inhibitor"-mediated endoreduplication. These data suggest that JAK signaling is needed for G2-M transit with inhibition of ERK.