Aminopyrazole Carboxamide Bruton's Tyrosine Kinase Inhibitors. Irreversible to Reversible Covalent Reactive Group Tuning.

Potent covalent inhibitors of Bruton's tyrosine kinase (BTK) based on an aminopyrazole carboxamide scaffold have been identified. Compared to acrylamide-based covalent reactive groups leading to irreversible protein adducts, cyanamide-based reversible-covalent inhibitors provided the highest combined BTK potency and EGFR selectivity. The cyanamide covalent mechanism with BTK was confirmed through enzyme kinetic, NMR, MS, and X-ray crystallographic studies. The lead cyanamide-based inhibitors demonstrated excellent kinome selectivity and rat pharmacokinetic properties.

Selective inhibition of BTK prevents murine lupus and antibody-mediated glomerulonephritis.

Autoantibody production and immune complex deposition within the kidney promote renal disease in patients with lupus nephritis. Thus, therapeutics that inhibit these pathways may be efficacious in the treatment of systemic lupus erythematosus. Bruton's tyrosine kinase (BTK) is a critical signaling component of both BCR and FcR signaling. We sought to assess the efficacy of inhibiting BTK in the development of lupus-like disease, and in this article describe (R)-5-amino-1-(1-cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxy]phenyl)-1H-pyrazole-4-carboxamide (PF-06250112), a novel highly selective and potent BTK inhibitor. We demonstrate in vitro that PF-06250112 inhibits both BCR-mediated signaling and proliferation, as well as FcR-mediated activation. To assess the therapeutic impact of BTK inhibition, we treated aged NZBxW_F1 mice with PF-06250112 and demonstrate that PF-06250112 significantly limits the spontaneous accumulation of splenic germinal center B cells and plasma cells. Correspondingly, anti-dsDNA and autoantibody levels were reduced in a dose-dependent manner. Moreover, administration of PF-06250112 prevented the development of proteinuria and improved glomerular pathology scores in all treatment groups. Strikingly, this therapeutic effect could occur with only a modest reduction observed in anti-dsDNA titers, implying a critical role for BTK signaling in disease pathogenesis beyond inhibition of autoantibody production. We subsequently demonstrate that PF-06250112 prevents proteinuria in an FcR-dependent, Ab-mediated model of glomerulonephritis. Importantly, these results highlight that BTK inhibition potently limits the development of glomerulonephritis by impacting both cell- and effector molecule-mediated pathways. These data provide support for evaluating the efficacy of BTK inhibition in systemic lupus erythematosus patients.

Enhanced GITR/GITRL interactions augment IL-27 expression and induce IL-10-producing Tr-1 like cells.

The glucocorticoid-induced TNFR-related (GITR) protein is a coactivating receptor that is constitutively expressed on Treg cells and induced on activated T cells. To better under-stand the role of long-term GITR signaling, we generated a mouse that constitutively expresses GITR ligand (GITRL) on APCs that mimics the physiological distribution of GITRL in vivo. Despite a five-fold expansion of the Treg-cell pool, there is increased activation and depletion of naive T cells in the transgenic (Tg) mice, suggesting that the increased number of Treg cells cannot fully suppress T-cell activation. Interestingly, GITRL Tg mice have multiorgan lymphocytic infiltrates yet display no overt autoimmunity, indicating the existence of a compensatory immunoregulatory mechanism(s). In the spleens and tissue infiltrates ofGITRL Tg mice, we found increased numbers of Foxp3(-) IL-10-producing type 1 regulatory T (Tr-1)-like cells that suppress naïve T-cell proliferation in an IL-10-dependent fashion. Increased IL-27 production from Tg APCs and activation of c-Maf in the Tr1-like cells suggest a possible mechanism for their induction. Our results demonstrate that enhanced GITR/GITRL interactions have a pleiotropic role on the regulation of T-cell responses, which includes promoting the differentiation of Tr-1-like cells, which contribute to the maintenance of peripheral T-cell tolerance.

A FLIPR-based assay to assess potency and selectivity of inhibitors of the TEC family kinases Btk and Itk.

Bruton's tyrosine kinase (Btk) and interleukin-2-inducible T cell kinase (Itk) are members of the TEC family of nonreceptor tyrosine kinases and are expressed primarily in B and T cells, respectively. Both kinases are critically involved in lymphocyte development and signal transduction. In particular, Btk and Itk regulate calcium mobilization subsequent to antigen receptor stimulation. Small molecule antagonists that specifically inhibit either Btk or Itk may allow for selective modulation of B cell or T cell activity and may be useful in treating inflammatory and autoimmune conditions. We have developed a medium-throughput fluorescent imaging plate reader (FLIPR)- based calcium flux assay that can be used to assay potential Btk and Itk inhibitors. This assay takes advantage of Btk-deficient DT40 (DT40-Btk-/-) chicken B cells, which are unable to mobilize calcium in response to cross-linking of their B cell receptor (BCR). Ectopic expression of TEC family kinases can restore antigen receptor signaling in these cells. We have generated stable DT40-Btk-/- lines expressing either wild-type human Btk (huBtk) or a chimeric Btk-Itk kinase (huBtk-Itk) molecule-a Btk protein whose kinase domain has been replaced by the kinase domain of Itk. Expression of either huBtk or huBtk-Itk in DT40-Btk-/- cells restores calcium flux in response to BCR engagement. Using Btk- and Itk-selective inhibitors, we show that inhibition of calcium responses in huBtk-Itk-DT40-Btk-/- cells and huBtk-DT40-Btk-/- cells is dependent on the Itk or Btk kinase domain, respectively. Thus, the FLIPR assay described here can be used to assess, compare, and rank the potency and selectivity of inhibitors of Itk and Btk kinases.

Exploiting genotypic differences to identify genes important for EAE development.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of the human autoimmune disease multiple sclerosis (MS) and is primarily driven by T helper type 1 (Th1) cells. Interleukin (IL)-12 and interferon (IFN)-gamma are important cytokines involved in the differentiation and amplification of Th1 cells, however mice deficient in either IFN-gamma or IL-12 still develop EAE. We have used microarray analysis of EAE-affected CNS tissues in wild-type, IFN-gamma -/- and IL-12 -/- animals to identify genes critical for development of EAE. Over 500 genes were regulated in at least one genotype and over 94 genes were regulated in all three. Of those, 17 were also upregulated in spleen during the disease. We show that a majority of the genes regulated in EAE are also regulated in diseased regions of human MS tissues. The genes in the pool of 94 are more likely to be found regulated in MS patients than the genes regulated in only one or two of the mouse strains suggesting that analyzing gene expression under these multiple genetic conditions may lead to better identification of the genes critical for disease development.

Cytosolic phospholipase A2 alpha-deficient mice are resistant to experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE), a Th1-mediated inflammatory disease of the central nervous system (CNS), is a model of human multiple sclerosis. Cytosolic phospholipase A2alpha (cPLA2alpha), which initiates production of prostaglandins, leukotrienes, and platelet-activating factor, is present in EAE lesions. Using myelin oligodendrocyte glycoprotein (MOG) immunization, as well as an adoptive transfer model, we showed that cPLA2alpha-/- mice are resistant to EAE. Histologic examination of the CNS from MOG-immunized mice revealed extensive inflammatory lesions in the cPLA2alpha+/- mice, whereas the lesions in cPLA2alpha-/- mice were reduced greatly or completely absent. MOG-specific T cells generated from WT mice induced less severe EAE in cPLA2alpha-/- mice compared with cPLA2alpha+/- mice, which indicates that cPLA2alpha plays a role in the effector phase of EAE. Additionally, MOG-specific T cells from cPLA2alpha-/- mice, transferred into WT mice, induced EAE with delayed onset and lower severity compared with EAE that was induced by control cells; this indicates that cPLA2alpha also plays a role in the induction phase of EAE. MOG-specific T cells from cPLA2alpha-/- mice were deficient in production of Th1-type cytokines. Consistent with this deficiency, in vivo administration of IL-12 rendered cPLA2alpha-/- mice susceptible to EAE. Our data indicate that cPLA2alpha plays an important role in EAE development and facilitates differentiation of T cells toward the Th1 phenotype.

TH1-mediated airway hyperresponsiveness independent of neutrophilic inflammation.

BACKGROUND: T(H)2-mediated allergic asthma is characterized by eosinophilia, mucus overproduction, and airway hyperresponsiveness (AHR). Although it is clear that T(H)2 cells and their cytokines play an important role in AHR, the roles of T(H)1 cells and neutrophils in AHR are controversial. OBJECTIVE: We sought to determine the roles of T(H)1 cells and neutrophils in AHR. METHODS: Ovalbumin-specific CD4(+) T cells were purified from DO11.10 mice, differentiated into T(H)1 cells, and injected into naive BALB/c, IL-4RalphaKO, or IL-8RKO mice. After ovalbumin antigen challenge, cytokine mRNA levels in lung samples, as well as inflammatory cell types and numbers in bronchoalveolar lavage fluid (BALF), were determined. AHR was assessed by measuring resistance in tracheostomized mice and enhanced pause in freely moving mice. RESULTS: T(H)1 cells induced AHR as robust as T(H)2 cells. They also induced lung inflammation dominated by neutrophils. Neither AHR nor inflammation were reduced when T(H)1 cells were transferred into IL-4RalphaKO mice. When IL-8RKO mice were used as recipients of T(H)1 cells, neutrophilia was greatly reduced, but the AHR was as strong as that seen in wild-type mice. On the other hand, dexamethasone treatment had no effect on neutrophilia but has significantly reduced AHR. Reduction in AHR was accompanied by a reduction in the numbers of lymphocytes and macrophages in BALF. CONCLUSIONS: T(H)1 cells can induce strong AHR independent of IL-4 and IL-13. The AHR is associated with the presence of lymphocytes and macrophages, but not neutrophils, in BALF. Our results point to a pathway whereby T(H)1 cells mediate AHR independent of neutrophilic inflammation.

Human bronchial epithelial cells express and secrete MMP-12.

Matrix metalloproteinases (MMPs) degrade extracellular matrix proteins, which may be responsible for enlargement of alveoli in chronic obstructive pulmonary disease (COPD) and remodeling of pulmonary tissue associated with chronic asthma. Here, we provide novel evidence that MMP-12 is expressed and secreted by normal human bronchial epithelial cell cultures (NHBECs) and reveal the regulation of MMP-12 gene expression by tumor necrosis factor-alpha (TNF-alpha), epidermal growth factor (EGF), and interferon gamma (IFN-gamma). Reverse transcription-polymerase chain reaction analyses demonstrated MMP-12 mRNA presence in unstimulated differentiated NHBEC cultures. Cultures stimulated independently with EGF or IFN-gamma failed to alter MMP-12 mRNA abundance, while TNF-alpha, TNF-alpha+EGF, or TNF-alpha+IFN-gamma elicited relatively early (6 h) peak increases in MMP-12 mRNA levels. Western blot analyses specifically indicated the presence of MMP-12 in differentiated NHBEC-conditioned media. These findings indicate that the bronchial epithelium may be an important source of elastolytic activity in COPD and tissue remodeling in chronic asthma.

Local delivery of granulocyte macrophage colony-stimulating factor by retrovirally transduced antigen-specific T cells leads to severe, chronic experimental autoimmune encephalomyelitis in mice.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) that can be induced in susceptible mice by the transfer of autoreactive T cells that recognize myelin basic protein (MBP). The onset and subsequent recovery from disease are associated with distinct patterns of cytokine and chemokine expression within the inflammatory lesions of the CNS. Given the likely importance of the local cytokine milieu in regulating the disease process, it would be preferable to administer cytokines locally to the CNS and reduce systemic delivery in order to evaluate their immunoregulatory roles in EAE. For this purpose, we have used retrovirally transduced T cells from MBP-specific T cell receptor transgenic mice in an attempt to target cytokine delivery to the CNS where MBP is primarily expressed. We have found that T cells expressing granulocyte macrophage colony-stimulating factor (GM-CSF) induce severe, chronic EAE from which mice fail to recover. Our results indicate that increased local GM-CSF expression could play an important role in inducing chronic EAE.