Identification of an imidazopyridine scaffold to generate potent and selective TYK2 inhibitors that demonstrate activity in an in vivo psoriasis model

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.     

Kinase inhibitors for the treatment of inflammatory and autoimmune disorders

Drugs targeting inhibition of kinases for the treatment of inflammation and autoimmune disorders have become a major focus in the pharmaceutical and biotech industry. Multiple kinases from different pathways have been the targets of interest in this endeavor. This review describes some of the recent developments in the search for inhibitors of IKK2, Syk, Lck, and JAK3 kinases. It is anticipated that some of these compounds or newer inhibitors of these kinases will be approved for the treatment of rheumatoid arthritis, psoriasis, organ transplantation, and other autoimmune diseases.     

Pyrrolo[1,2-f]triazines as JAK2 inhibitors: achieving potency and selectivity for JAK2 over JAK3

SAR studies of pyrrolo[1,2-f]triazines as JAK2 inhibitors is presented. Achieving JAK2 inhibition selectively over JAK3 is discussed.     

Design,synthesis and preliminary biological evaluation of 4-aminopyrazole derivatives as novel and potent JAKs inhibitors

JAKs inhibitors were widely applied in the treatment of immunodeficiency diseases, inflammation and cancers. We designed and synthesized a novel series of 4-aminopyrazole derivatives, which showed inhibitory potency against various JAKs. The in vitro protein kinase inhibition experiment indicated that compounds 17k, 17l, 17m and 17n could inhibit JAKs effectively. Among them, compound 17m possessed the highest protein kinase inhibitory rates (%) at 10 μM, which were 97, 96 and 100 to JAK1, JAK2 and JAK3, respectively. Further evaluation revealed that the IC₅₀ values of 17m against JAK1, JAK2 and JAK3 were 0.67 μM, 0.098 μM and 0.039 μM, respectively. Moreover, western blotting results showed compound 17m could inhibit the phosphorylation of JAK2 in Hela cells effectively. The data supports the further investigation of these compounds as novel JAKs inhibitors.     

Development of a high-throughput cell-based reporter assay for screening of JAK3 inhibitors

JAK3 is an ideal target for the treatment of immune-related diseases and the prevention of organ allograft rejection. Several JAK3 inhibitors have been identified by biochemical enzymatic assays, but the majority display significant off-target effects on JAK2. Therefore, there is a need to develop new experimental approaches to identify compounds that specifically inhibit JAK3. Here, we show that in 32D/IL-2Rβ cells, STAT5 becomes phosphorylated by an IL-3/JAK2- or IL-2/JAK3-dependent pathway. Importantly, the selective JAK3 inhibitor CP-690,550 blocked the phosphorylation and the nuclear translocation of STAT5 following treatment of cells with IL-2 but not with IL-3. In an attempt to use the cells for large-scale chemical screens to identify JAK3 inhibitors, we established a cell line, 32D/IL-2Rβ/6xSTAT5, stably expressing a STAT5 reporter gene. Treatment of this cell line with IL-2 or IL-3 dramatically increased the reporter activity in a high-throughput format. As expected, CP-690,550 selectively inhibited the activity of the 6xSTAT5 reporter following treatment with IL-2. By contrast, the pan-JAK inhibitor curcumin inhibited the activity of this reporter following treatment with either IL-2 or IL-3. Thus, this study indicates that the STAT5 reporter cell line can be used as an efficacious cellular model for chemical screens to identify selective JAK3 inhibitors.     

Janus kinase 3 down-regulates lipopolysaccharide-induced IL-1 beta-converting enzyme activation by autocrine IL-10

ProIL-1 beta processing by IL-1 beta-converting enzyme (ICE) and the subsequent release of mature IL-1 beta are highly regulated events in the monocyte/macrophage response to pathogens. This process occurs in a controlled way through the activation of the constitutively expressed 45-kDa ICE precursor (proICE). To characterize the signaling pathways involved in ICE regulation in human monocytes/macrophages, we analyzed ICE activation in the presence of specific inhibitors of classic signaling pathways. Although LPS-induced ICE activity was not significantly affected by interruption of extracellular signal-regulated kinase, p38 kinase, or phosphoinositol 3-kinase, Janus kinase 3 (JAK3) inhibition produced a significant dose-dependent enhancement of LPS-induced ICE activity. Support for the inhibitory role of JAK3 was shown by the fact that IL-4 (which uses JAK1 and JAK3 signaling) suppressed LPS-induced ICE activity and by the finding that JAK3 knockout macrophages have increased LPS-induced ICE activation. To understand how JAK3 down-regulates LPS-induced ICE activity in monocytes, we hypothesized that JAK3 signaling enhances IL-10 production. In support of this model we show that LPS-induced IL-10 expression was synchronous with ICE deactivation, IL-4 induced the release of IL-10, exogenous IL-10 suppressed LPS-induced ICE activity, a neutralizing IL-10 Ab increased LPS-induced ICE activity, and, finally, JAK3 knockout macrophages displayed significantly reduced LPS-induced IL-10 production. These findings support a model in which JAK3 signaling enhances IL-10 production leading to down-regulation of ICE activation and suppression of IL-1 beta processing and release.     

Janus kinase inhibitors role in bone remodeling

Janus kinases (JAKs) play a pleiotropic role in several important physiological processes, such as cell maturation, cell proliferation, and cell death, via providing transmission signals from several molecules, such as cytokines, interferons, hormones, and growth factors, to the nucleus. Bone physiology and remodeling are markedly influenced by proinflammatory cytokines. Among them, interleukin-1 (IL-1) and IL-6 are considered potent stimulator of bone resorption. Several cytokine receptors, such as IL-6 receptors, are characterized by tyrosine kinases of the JAK family associated with their intracellular domains. There is an emerging interest in the effects of JAKs inhibition on the cells involved in bone remodeling. JAK inhibitors represent a new class of molecules involved in the therapy of numerous immune-mediated inflammatory diseases. In this review, we want to focus on the role of JAKs inhibitors on bone remodeling and on RANKL-RANK-OPG signal and inflammatory cytokines which are involved in the regulation of bone cells, such as osteoblasts and osteoclasts.     

Kinome-wide selectivity profiling of ATP-competitive mammalian target of rapamycin (mTOR) inhibitors and characterization of their binding kinetics

An intensive recent effort to develop ATP-competitive mTOR inhibitors has resulted in several potent and selective molecules such as Torin1, PP242, KU63794, and WYE354. These inhibitors are being widely used as pharmacological probes of mTOR-dependent biology. To determine the potency and specificity of these agents, we have undertaken a systematic kinome-wide effort to profile their selectivity and potency using chemical proteomics and assays for enzymatic activity, protein binding, and disruption of cellular signaling. Enzymatic and cellular assays revealed that all four compounds are potent inhibitors of mTORC1 and mTORC2, with Torin1 exhibiting ～20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC(50) = 2 nM) relative to other inhibitors. In vitro biochemical profiling at 10 μM revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 μM but did show that PP242 efficiently inhibited the RET receptor (EC(50), 42 nM) and JAK1/2/3 kinases (EC(50), 780 nM). In addition, Torin1 displayed unusually slow kinetics for inhibition of the mTORC1/2 complex, a property likely to contribute to the pharmacology of this inhibitor. Our results demonstrated that, with the exception of PP242, available ATP-competitive compounds are highly selective mTOR inhibitors when applied to cells at concentrations below 1 μM and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.     

Interleukin-10 activation of the interleukin-10E1 pathway and tissue inhibitor of metalloproteinase-1 expression is enhanced by proteasome inhibitors in primary prostate tumor lines

The interleukin-10 (IL-10) activation of Janus kinase (JAK) family members (JAK1/TYK2) and IL-10E1 is subsequently inactivated by approximately 3-4 h in primary prostate tumor lines. We examined the effect of proteasome inhibition on IL-10 activation of the IL-10E1 pathway following stimulation of HPCA-10a cells. Treatment of HPCA-10a cells with the proteasome inhibitor, N-acetyl-L-leucinyl-L-leucinyl-norleucinal (LLnL), led to stable tyrosine phosphorylation of the IL-10 receptor and IL-10E1 following stimulation. Further investigation showed that these stable phosphorylation events were the result of prolonged activation of JAK1 and TYK2 plus IL-10E1. IL-10E1 signaling normally induced the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and LLnL treatment of the HPCA-10a and HPCA-10c cells significantly enhanced IL-10 induction of TIMP-1 levels to block tumor cell invasion in modified Boyden chamber invasion assays. These observations were confirmed using pharmacologic inhibitors by Western blot and ELISAs. In the presence of LLnL, stable phosphorylation of IL-10E1 and induction of TIMP-1 was abrogated if the tyrosine kinase inhibitor, staurosporine, was added. The effect of staurosporine on IL-10E1 phosphorylation and TIMP-1 could be overcome if the phosphatase inhibitor, vanadate, was also added, suggesting that phosphorylated IL-10E1 could be stabilized by phosphatase, but not by proteasome inhibition. These observations are consistent with the hypothesis that proteasome-mediated protein degradation can modulate the activity of the IL-10E1 pathway and TIMP-1 induction by regulating the deactivation of JAK1/TYK2.     

2,7-Pyrrolo[2,1-f][1,2,4]triazines as JAK2 inhibitors: modification of target structure to minimize reactive metabolite formation

The JAK2/STAT pathway has important roles in hematopoiesis. With the discovery of the JAK2 V617F mutation and its presence in many patients with myeloproliferative neoplasms, research in the JAK2 inhibitor arena has dramatically increased. We report a novel series of potent JAK2 inhibitors containing a 2,7-pyrrolotriazine core. To minimize potential drug-induced toxicity, targets were analyzed for the ability to form a glutathione adduct. Glutathione adduct formation was decreased by modification of the aniline substituent at C2.     

TGF-beta does not inhibit IL-12- and IL-2-induced activation of Janus kinases and STATs

The immune system is an important target for the cytokine TGF-beta1, whose actions on lymphocytes are largely inhibitory. TGF-beta has been reported to inhibit IL-12- and IL-2-induced cell proliferation and IFN-gamma production by T cells and NK cells; however, the mechanisms of inhibition have not been clearly defined. It has been suggested by some studies that TGF-beta blocks cytokine-induced Janus kinase (JAK) and STAT activation, as in the case of IL-2. In contrast, other studies with cytokines like IFN-gamma have not found such an inhibition. The effect of TGF-beta on the IL-12-signaling pathway has not been addressed. We examined this and found that TGF-beta1 did not have any effect on IL-12-induced phosphorylation of JAK2, TYK2, and STAT4 although TGF-beta1 inhibited IL-2- and IL-12-induced IFN-gamma production. Similarly, but in contrast to previous reports, we found that TGF-beta1 did not inhibit IL-2-induced phosphorylation of JAK1, JAK3, and STAT5A. Furthermore, gel shift analysis showed that TGF-beta1 did not prevent activated STAT4 and STAT5A from binding to DNA. Our results demonstrate that the inhibitory effects of TGF-beta on IL-2- and IL-12-induced biological activities are not attributable to inhibition of activation of JAKs and STATs. Rather, our data suggest the existence of alternative mechanisms of inhibition by TGF-beta.     

Combination treatment for myeloproliferative neoplasms using JAK and pan‐class I PI3K inhibitors

Current JAK2 inhibitors used for myeloproliferative neoplasms (MPN) treatment are not specific enough to selectively suppress aberrant JAK2 signalling and preserve physiological JAK2 signalling. We tested whether combining a JAK2 inhibitor with a series of serine threonine kinase inhibitors, targeting nine signalling pathways and already used in clinical trials, synergized in inhibiting growth of haematopoietic cells expressing mutant and wild‐type forms of JAK2 (V617F) or thrombopoietin receptor (W515L). Out of 15 kinase inhibitors, the ZSTK474 phosphatydylinositol‐3′‐kinase (PI3K) inhibitor molecule showed strong synergic inhibition by Chou and Talalay analysis with JAK2 and JAK2/JAK1 inhibitors. Other pan‐class I, but not gamma or delta specific PI3K inhibitors, also synergized with JAK2 inhibitors. Synergy was not observed in Bcr‐Abl transformed cells. The best JAK2/JAK1 and PI3K inhibitor combination pair (ruxolitinib and GDC0941) reduces spleen weight in nude mice inoculated with Ba/F3 cells expressing TpoR and JAK2 V617F. It also exerted strong inhibitory effects on erythropoietin‐independent erythroid colonies from MPN patients and JAK2 V617F knock‐in mice, where at certain doses, a preferential inhibition of JAK2 V617F mutated progenitors was detected. Our data support the use of a combination of JAK2 and pan‐class I PI3K inhibitors in the treatment of MPNs.     

Inhibitor‐based affinity probes for the investigation of JAK signaling pathways

The Janus Kinase (JAK) signaling pathway plays a key role for many cellular processes and has recently been correlated with neuronal disorders. In order to understand new links of JAK family members with other signaling pathways, chemical proteomics tools with broad kinase coverage are desirable. A probe that shows outstanding kinase selectivity and allows for the enrichment of up to 133 kinases including many mitogen activated kinase (MAPK) members and JAK kinases has been developed. Furthermore, this probe was applied to establish the selectivity profile of the JAK1/2 inhibitor momelotinib that is currently evaluated in clinical phase 3 studies. These results render this probe a valuable tool for the investigation of JAK and JAK related signaling pathways and the selectivity profiling of kinase inhibitors.     

Anilino-monoindolylmaleimides as potent and selective JAK3 inhibitors

We designed a series of anilino-indoylmaleimides based on structural elements from literature JAK3 inhibitors 3 and 4, and our lead 5. These new compounds were tested as inhibitors of JAKs 1, 2 and 3 and TYK2 for therapeutic intervention in rheumatoid arthritis (RA). Our requirements, based on current scientific rationale for optimum efficacy against RA with reduced side effects, was for potent, mixed JAK1 and 3 inhibition, and selectivity over JAK2. Our efforts yielded a potent JAK3 inhibitor 11d and its eutomer 11e. These compounds were highly selective for inhibition of JAK3 over JAK2 and TYK. The compounds displayed only modest JAK1 inhibition.     

Design,synthesis and anti-inflammatory evaluation of novel pyrrolo[2,3-d]pyrimidin derivatives as potent JAK inhibitors

Aiming to develop potent JAK inhibitors, two series of 4-(1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine derivatives (8a–8p and 11a–11i) were designed and synthesized by coalescing various N-acylpiperidine motifs with baricitinib. The pharmacological results based on enzymatic and cellular assays identified the optimized compound 11e, which exerted over 90% inhibition rates against JAK1 and JAK2, and displayed the most compelling anti-inflammatory efficacy superior to baricitinib by inhibiting NO generation from LPS-induced RAW264.7 macrophages. Importantly, low cytotoxity of 11e was revealed by the IC₅₀ value of 88.2 μM against normal RAW264.7 cells. The binding mode of 11e with JAK1 and JAK2 identified the essential structural bases in accord with SARs analysis. Furthermore, cellular morphology observation and western blot analysis disclosed the ability of 11e to relieve cells inflammatory damage by significantly down-regulating LPS-induced high expression of JAK1, JAK2, as well as pro cytokine IL-1β. Together, 11e was verified as a promising lead for JAK inhibitors for the treatment of inflammatory diseases.     

Discovery of a series of novel 5H-pyrrolo[2,3-b]pyrazine-2-phenyl ethers,as potent JAK3 kinase inhibitors

We report the discovery of a novel series of ATP-competitive Janus kinase 3 (JAK3) inhibitors based on the 5H-pyrrolo[2,3-b]pyrazine scaffold. The initial leads in this series, compounds 1a and 1h, showed promising potencies, but a lack of selectivity against other isoforms in the JAK family. Computational and crystallographic analysis suggested that the phenyl ether moiety possessed a favorable vector to achieve selectivity. Exploration of this vector resulted in the identification of 12b and 12d, as potent JAK3 inhibitors, demonstrating improved JAK family and kinase selectivity.