SAR study of small molecule inhibitors of the programmed cell death-1/programmed cell death-ligand 1 interaction.

The development of small molecule inhibitors of programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) has drawn research interest for the treatment of cancer. Recently, we reported the discovery of a novel dimeric core small molecule PD-1/PD-L1 inhibitor. In an effort to discover more potent inhibitors, we further explored the dimeric core scaffold. Our investigations of the structure-activity-relationship revealed that introduction of lipophilic substituents onto one of the di-alkoxylated phenyl rings improved binding affinities to PD-L1, and inhibitory activities of PD-1/PD-L1 in cellular assays. Furthermore, conversion of the ether linker part to an olefin linker not only improved binding affinity but also led to slow dissociation binding kinetics. We also explored more potent, as well as downsized, scaffolds. Compounds bearing a linear chain in place of one of the di-alkoxylated phenyl rings exhibited good binding affinity with improved ligand efficiency (LE). Representative compounds demonstrated potent inhibitory activities of PD-1/PD-L1 in the submicromolar range in cellular assays as well as cellular function in the mixed lymphocyte reaction (MLR) assay with efficacy comparable to anti-PD-1 antibody. Our results provide applicable information for the design of more potent inhibitors targeting PD-1/PD-L1 pathway.

Discovery of a Janus Kinase Inhibitor Bearing a Highly Three-Dimensional Spiro Scaffold: JTE-052 (Delgocitinib) as a New Dermatological Agent to Treat Inflammatory Skin Disorders.

Dermatologic disorders such as atopic dermatitis arise from genetic and environmental causes and are complex and multifactorial in nature. Among possible risk factors, aberrant immunological reactions are one of the leading etiologies. Immunosuppressive agents including topical steroids are common treatments for these disorders. Despite their reliability in clinical settings, topical steroids display side effects, typified by skin thinning. Accordingly, there is a need for alternate effective and well-tolerated therapies. As part of our efforts to investigate new immunomodulators, we have developed a series of JAK inhibitors, which incorporate novel three-dimensional spiro motifs and unexpectedly possess both excellent physicochemical properties and antidermatitis efficacy in the animal models. One of these compounds, JTE-052 (ent-60), also known as delgocitinib, has been shown to be effective and well-tolerated in human clinical trials and has recently been approved in Japan for the treatment of atopic dermatitis as the first drug among Janus kinase inhibitors.

Symmetry-based ligand design and evaluation of small molecule inhibitors of programmed cell death-1/programmed death-ligand 1 interaction.

The development of small molecule inhibitors of PD-1/PD-L1 is eagerly anticipated for treatment of cancer. We focused on the symmetry of the ternary complex structure of reported small molecule ligands and hPD-L1 homodimers, and designed partially- or fully-symmetric compounds for more potent inhibitors. The design of the new compounds was guided by our hypothesis that the designed symmetric compound would induce a flip of sidechain of ATyr56 protein residue to form a new cavity. The designed compound 4 exhibited substantially increased binding affinity to hPD-L1, as well as PD-1/PD-L1 inhibitory activity in physiological conditions. Compound 4 also showed a dose-dependent increase in IFN-γ secretion levels in a mixed lymphocyte reaction assay. These results not only indicate the feasibility of targeting the PD-1/PD-L1 pathway with small molecules, but illustrate the applicability of the symmetry-based ligand design as an attractive methodology for targeting protein-protein interaction stabilizers.

A novel JAK inhibitor JTE-052 reduces skin inflammation and ameliorates chronic dermatitis in rodent models: Comparison with conventional therapeutic agents.

Janus kinases (JAKs) are required for several inflammatory cytokine signalling pathways and are implicated in the pathogenesis of chronic dermatitis, including atopic dermatitis and psoriasis. JAK inhibitors are therefore promising therapeutic candidates for chronic dermatitis. In this study, we evaluated the effects of the novel JAK inhibitor JTE-052 on inflammatory responses associated with chronic dermatitis, and compared its profile with those of conventional therapeutic agents in rodent models of chronic dermatitis. JTE-052 inhibited the Th1-, Th2- and Th17-type inflammatory responses of human T cells and mast cells in vitro. Oral administration of JTE-052 inhibited skin inflammation in hapten-induced chronic dermatitis in mice, associated with reduced levels of inflammatory cytokines in the skin and immunoglobulin (Ig) E in serum. In contrast, although ciclosporin partly inhibited skin inflammation, it did not reduce interleukin (IL)-4 production in skin, and enhanced IgE production in serum. Oral administration of JTE-052 also inhibited skin inflammation in mouse models of atopic dermatitis and psoriasis induced by a mite extract, thymic stromal lymphopoietin or IL-23. The maximal efficacy of JTE-052 in these dermatitis models was superior to the conventional therapeutic agents, ciclosporin and methotrexate. Topical application of JTE-052 ointment ameliorated hapten-induced chronic dermatitis in rats more effectively than tacrolimus ointment. Furthermore, JTE-052 ointment did not cause the thinning of normal skin associated with topical corticosteroids. These results indicate that JTE-052 is a promising candidate as an anti-inflammatory drug for various types of chronic dermatitis, with a distinctly different profile from conventional therapy following either oral or topical application.

JAK inhibitor JTE-052 regulates contact hypersensitivity by downmodulating T cell activation and differentiation.

BACKGROUND: Using JAK inhibitors to inhibit cytokine signaling is presumed to be a possible means of treating skin inflammatory disorders such as contact dermatitis. OBJECTIVE: To clarify the action site of JAK inhibitors in skin inflammatory disorders. METHODS: We analyzed the mechanism of action of the JAK inhibitor JTE-052 using murine skin inflammation models, including contact hypersensitivity (CHS) and irritant contact dermatitis. Cells isolated from ear tissue or lymph node (LN) were analyzed by flow cytometry. The amounts of cytokines in the culture medium were measured by ELISA or bead array system. Proliferation of LN cells was evaluated by measurement of tritiated thymidine incorporation. RESULTS: Oral administration of JTE-052 during both sensitization and elicitation phase attenuated CHS, but did not affect croton oil-induced irritant contact dermatitis. JTE-052 potently inhibited T cell proliferation and activation by antigen presentation in vitro, and attenuated skin inflammation in a sensitized-lymphocyte transfer model without suppressing T cell migration. JTE-052 did not affect hapten-induced cutaneous dendritic cell migration into draining lymph nodes or their costimulatory molecule expressions. CONCLUSION: The JAK inhibitor JTE-052 exerts an inhibitory effect on antigen-specific T cell activation and subsequent inflammation in acquired skin immunity, such as CHS.

Improvement of spontaneous locomotor activity with JAK inhibition by JTE-052 in rat adjuvant-induced arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory disease that leads to joint destruction, disability, and decreased quality of life (QOL). Inhibition of Janus kinase (JAK) signaling ameliorates articular inflammation and joint destruction in animal models of RA, but its effects on behaviors indicating well-being are poorly understood. In this study, we evaluated the effect of JAK inhibition on spontaneous locomotor activity in rats with adjuvant-induced arthritis, a rodent model of RA. METHODS: Arthritis was induced in male Lewis rats by a single subcutaneous injection of Freund's complete adjuvant. The novel JAK inhibitor JTE-052 was orally administered for 7 days after the onset of arthritis. RESULTS: Induction of arthritis suppressed the spontaneous locomotor activity of the rats. Administration of JTE-052 completely improved the spontaneous locomotor activity, with partial reductions in articular inflammation and joint destruction. Hyperalgesia and motor functions were also improved, but the efficacy was not complete. However, serum interleukin (IL)-6 levels were completely decreased at 4 h after administration of the first dose of JTE-052. CONCLUSIONS: This study demonstrated that JAK inhibition improved the spontaneous locomotor activity of rats with adjuvant-induced arthritis, in association with amelioration of pain and physical dysfunction as a consequence of suppression of joint inflammation. Moreover, although further studies are needed, there was possible participation of IL-6 downregulation in the improvement of locomotor activity by JAK inhibition.

The Janus kinase inhibitor JTE-052 improves skin barrier function through suppressing signal transducer and activator of transcription 3 signaling.

BACKGROUND: Barrier disruption and the resulting continuous exposure to allergens are presumed to be responsible for the development of atopic dermatitis (AD). However, the mechanism through which skin barrier function is disrupted in patients with AD remains unclear. OBJECTIVES: Taking into account the fact that the TH2 milieu impairs keratinocyte terminal differentiation, we sought to clarify our hypothesis that the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway plays a critical role in skin barrier function and can be a therapeutic target for AD. METHODS: We analyzed the mechanism of keratinocyte differentiation using a microarray and small interfering RNA targeting STATs. We studied the effect of the JAK inhibitor JTE-052 on keratinocyte differentiation using the human skin equivalent model and normal human epidermal keratinocytes. We applied topical JAK inhibitor onto NC/Nga mice, dry skin model mice, and human skin grafted to immunocompromised mice. RESULTS: IL-4 and IL-13 downregulated genes involved in keratinocyte differentiation. STAT3 and STAT6 are involved in keratinocyte differentiation and chemokine production by keratinocytes, respectively. Topical application of the JAK inhibitor suppressed STAT3 activation and improved skin barrier function, permitting increases in levels of terminal differentiation proteins, such as filaggrin, and natural moisturizing factors in models of AD and dry skin and in human skin. CONCLUSION: STAT3 signaling is a key element that regulates keratinocyte differentiation. The JAK inhibitor can be a new therapeutic tool for the treatment of disrupted barrier function in patients with AD.

Pharmacological properties of JTE-052: a novel potent JAK inhibitor that suppresses various inflammatory responses in vitro and in vivo.

OBJECTIVE: To evaluate the pharmacological properties of JTE-052, a novel Janus kinase (JAK) inhibitor. METHODS: The JAK inhibitory activity of JTE-052 was evaluated using recombinant human enzymes. The inhibitory effects on cytokine signaling pathways were evaluated using primary human inflammatory cells. The in vivo efficacy and potency of JTE-052 were examined in a mouse interleukin (IL)-2-induced interferon (IFN)-γ production model and a rat collagen-induced arthritis model. RESULTS: JTE-052 inhibited the JAK1, JAK2, JAK3, and tyrosine kinase (Tyk)2 enzymes in an adenosine triphosphate (ATP)-competitive manner and inhibited cytokine signaling evoked by IL-2, IL-6, IL-23, granulocyte/macrophage colony-stimulating factor, and IFN-α. JTE-052 inhibited the activation of inflammatory cells, such as T cells, B cells, monocytes, and mast cells, in vitro. Oral dosing of JTE-052 resulted in potent suppression of the IL-2-induced IFN-γ production in mice with an ED50 value of 0.24 mg/kg, which was more potent than that of tofacitinib (ED50 = 1.1 mg/kg). In the collagen-induced arthritis model, JTE-052 ameliorated articular inflammation and joint destruction even in therapeutic treatments where methotrexate was ineffective. CONCLUSIONS: The present results indicate that JTE-052 is a highly potent JAK inhibitor, and represents a candidate anti-inflammatory agent for suppressing various types of inflammation.

Suppressive effect of an orally active MEK1/2 inhibitor in two different animal models for rheumatoid arthritis: a comparison with leflunomide.

OBJECTIVE AND DESIGN: To examine the effects of a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2-inhibitor, JTP-74057, on inflammatory arthritis development, and compare its anti-arthritic effect with leflunomide. MATERIALS: Human, mouse, and rat peripheral blood mononuclear cells (PBMCs) were used. Lewis rats and DBA/1J mice were used for animal models. TREATMENT: JTP-74057 was tested between 0.1-100 nM in in-vitro studies. JTP-74057 (0.01-0.3 mg/kg) and leflunomide (2-10 mg/kg) were administered orally in vivo. METHODS: PBMCs were stimulated with lipopolysaccharide. Adjuvant-induced arthritis (AIA) and type II collagen-induced arthritis (CIA) was induced in Lewis rats or DBA1/J mice, respectively. RESULTS: JTP-74057 blocked tumor necrosis factor-α and interleukin-6 production from PBMCs. AIA and CIA development were suppressed almost completely by 0.1 mg/kg of JTP-74057 or 10 mg/kg of leflunomide. In the CIA, JTP-74057, but not leflunomide, suppressed collagen-reactive T-cell proliferation ex vivo, whereas leflunomide, but not JTP-74057, suppressed anti-collagen antibody production. CONCLUSIONS: JTP-74057 exerts potent anti-arthritic effects with a different profile from leflunomide, suggesting that JTP-74057 may be useful as a new therapeutic reagent in the treatment of rheumatoid arthritis.

JTA-009, a fully human antibody against human AILIM/ICOS, ameliorates graft-vs-host reaction in SCID mice grafted with human PBMCs.

OBJECTIVE: Activation-inducible lymphocyte immunomediatory molecule (AILIM; also referred to as inducible costimulator [ICOS]) is the third molecule identified in the CD28 family participating in T-cell activation. AILIM/ICOS has been implicated in both effector and pathogenic T-cell functions, as evidenced by the beneficial effects of AILIM/ICOS blockade in several murine disease models. In the present study, the role of human AILIM/ICOS in T-cell function was investigated using a fully human monoclonal antibody specific to human AILIM/ICOS (JTA-009). MATERIALS AND METHODS: The effect of JTA-009 on allogenic T-cell proliferation was examined using human mixed lymphocyte reaction (MLR). To investigate the efficacy of AILIM/ICOS blockade in vivo, a graft-vs-host disease (GVHD) model, in which severe combined immunodeficient (SCID) mice were grafted with human peripheral blood mononuclear cells (PBMCs), was used. RESULTS: In MLR, suppressive effect of JTA-009 on allogenic T-cell proliferation was detected with comparable potency to CD28 blockade by cytotoxic T-lymphocyte antigen 4 (CTLA4)-Ig at an intermediate culture phase. JTA-009 acts as a blocking antibody in vivo and inhibited binding of human AILIM/ICOS to mouse AILIM/ICOS ligand (B7h). Treatment with JTA-009 significantly prolonged survival of mice, with reductions of human interferon-gamma levels in blood and number of human cells in spleens. CONCLUSION: These results demonstrate that human AILIM/ICOS plays a role in the GVHD pathogenesis mediated by human T cells, and its blockade is attractive for abrogating undesired T-cell responses as is well-documented in mice.

Possible participation of intracellular platelet-activating factor in NF-kappaB activation in rat peritoneal macrophages.

As we had found previously that thapsigargin, an endomembrane Ca2+-ATPase inhibitor, induces production of intracellular platelet-activating factor (PAF) [Br. J. Pharmacol. 116 (1995) 2141], we decided to investigate the possible roles of intracellular PAF in nuclear factor (NF)-kappaB activation of thapsigargin-stimulated rat peritoneal macrophages. When rat peritoneal macrophages were stimulated with thapsigargin, the level of inhibitory protein of NF-kappaB-alpha (IkappaB-alpha) was decreased and the nuclear translocation of NF-kappaB was increased. The thapsigargin-induced activation of NF-kappaB was inhibited by the PAF synthesis inhibitor SK&F 98625 and the PAF antagonist E6123. Structurally unrelated PAF antagonists such as E5880 and L-652,731 also inhibited the thapsigargin-induced activation of NF-kappaB. Lipopolysaccharide (LPS)-induced activation of NF-kappaB was also suppressed by these drugs. In a culture of rat peritoneal macrophages, exogenously added PAF did not induce degradation of IkappaB-alpha. These findings suggest that the intracellular PAF produced by the stimulation with thapsigargin or LPS is involved in activation of the NF-kappaB pathway.

Participation of prostaglandin E2 and platelet-activating factor in thapsigargin-induced production of interleukin-6.

Incubation of rat peritoneal macrophages in the presence of thapsigargin increased production of prostaglandin E2, intracellular platelet-activating factor (PAF) and interleukin-6. However, no PAF was detected in the conditioned medium. In the presence of SK&F 98625 (diethyl 7-(3,4,5-triphenyl-2-oxo-2,3-dihydroimidazol-1-yl)heptane phosphonate), a CoA-independent transacylase inhibitor, the thapsigargin-induced increases in the interleukin-6 mRNA level and interleukin-6 production were suppressed in a concentration-dependent manner. This inhibitor also suppressed the production of prostaglandin E2 and intracellular PAF. The PAF receptor antagonists such as E6123 ((S)-(+)-6-(2-chlorophenyl)-3-cyclopropanecarbonyl-8,11-dimethyl-2,3,4,5-tetrahydro-8H-pyrido[4',3':4,5]thieno[3,2-f][1,2,4]triazolo [4,3-a][1,4]diazepine) and L-652,731 (2,5-bis(3,4,5-trimethylphenyl)tetrahydrofuran) partially inhibited the thapsigargin-induced increase in the levels of interleukin-6 mRNA and interleukin-6 protein. The SK&F 98625-induced suppression of interleukin-6 mRNA accumulation and interleukin-6 production was partially restored by addition of exogenous prostaglandin E2. However, exogenous PAF failed to reverse the suppression suggesting that the intracellular PAF does not act in an autocrine mechanism. These findings suggested that the concurrently produced prostaglandin E2 and intracellular PAF participate in the thapsigargin-induced increase in the interleukin-6 mRNA level and interleukin-6 production by rat peritoneal macrophages.