Protein kinases are a large group of intracellular and transmembrane signalling proteins in eukaryotic cells (Manning G. et al, (2002) Science, 298: 1912-1934). Phosphorylation of specific amino acid residues in target proteins by protein kinases can modulate their activity leading to profound changes in cellular signalling and metabolism. Kinases play key roles in the regulation of cellular proliferation, survival, differentiation and function. Many kinases have been implicated in disease and, as such, are attractive therapeutic targets.
The Tec-kinase-family of kinases consists of Tyrosine kinase expressed in hepatocellular carcinoma (TEC), Interleukin-2 inducible T-cell kinase (ITK, also known as Tsk and Emt), Resting lymphocyte kinase (RLK, also known as TXK for Tyrosine Protein Kinase), Bruton's tyrosine kinase (BTK), Bone marrow kinase on the X-chromosome (BMX, also known as Etk) (Bradshaw J M Cell Signal. 2010; 22(8):1175-84). These intracellular kinases play important roles in the development and function of lymphocytes and myeloid cells (Horwood et al. Int. Rev. Immunol. 2012; 31(2):87-103, Felices Metal. Adv. Immunol. 2007; 93:145-84). Additionally, selected Tec-family members such as ITK, TEC and BMX are expressed in cancerous cells where they may play a role in cancer cell survival and malignancy (Carson C C et al. Clin Cancer Res. 2015; 21(9):2167-76, Mano H. et al. Oncogene. 1990; 5(12):1781-6, Cenni B et al. Int. Rev. Immunol. 2012; 31(2):166-73).
ITK is an important component of T-cell signaling function and differentiation. ITK is activated upon stimulation of T-cell receptors and initiates a signaling cascade that results in cellular activation, cytokine release and rapid proliferation. ITK is important in T-helper (Th) cell development and function including Th1, Th2, Th9, Th17 and T-regulatory cell development (Fowell D J et al. 1999 Immunity 11:399-409; Gomez-Rodriguez J. et al. 2014 J. Exp. Med 211:529-543, Gomez-Rodriguez J. et al 2016 Nat Commun. 2016; 7: 10857). For example, ITK −/− CD4+ T cells show significant reduction in the production of Th1 and Th17 cytokines and exhibit skewed T effector/Treg cell ratios with a bias towards FoxP3+ Treg (Kannan A et al 2015. J Neurosci. 35:221-233, Gomez-Rodriguez J. et al. 2014 J. Exp. Med. 211:529-543). Furthermore, specific inhibition of an allele-sensitive ITK mutant shows that ITK is important in Th1, Th2, Th17, and iNKT-cell cytokine production (Kannan A et al Eur. J. Immunol. 2015. 45: 2276-2285). Consequently, ITK is a promising target for prevention or treatment of diseases involving Th cytokines or where modulation of immunosuppressive Treg cells is desired. Furthermore, polymorphisms in the ITK promoter that increase ITK expression in humans have been linked to increased asthma incidence (Lee, S. H. et al. 2011 Ann Hum Genet 75:359-369) and ITK preferentially regulates the secretion of the Th2 cytokines IL-5 and IL-13 in models of allergic asthma suggesting that ITK inhibitors may be useful in the treatment of asthma (Muller C et al. 2003 J Immunol. 170:5056-63). Also, ITK is upregulated in lesioned skin from patients with allergic contact dermatitis, atopic dermatitis and psoriasis (von Bonin A et al. 2010. Exp. Derm; 20, 41-47).
TXK (RLK) is another Tec-kinase-family member that is expressed in T-cells (Hu Q et al. 1995 J. Biol Chem. 270:1928-1934). TXK (RLK) and ITK regulate Th cell-mediated responses via their differential expression in Th1 and Th2 cells, respectively (Sahu N et al. J. Immunol. 2008, 181:6125-6131). Furthermore, while ITK −/− mice have impaired in NKT cell generation this defect is exacerbated in the absence of both TXK and ITK (Felices M. et al. 2008, J Immunol. 180:3007-3018). Increased expression of TXK has been reported in patients with Behcet's disease, an inflammatory disorder associated with increased inflammation and Th1 cytokine production (Suzuki N et al. 2006 Clin. Med. Res. 4:147-151). Knockout of both ITK and TXK produces stronger effects on T-cell function than knockout of either kinase alone (Schaeffer et al. 1999 Science 284:638-641; Felices et al. 2008 J. Immunol. 180:3007-3018).
TEC kinase, after which the family of related kinases is known, was first shown to be expressed in hepatocellular carcinoma (Mano et al. 1990 Oncogene. 5:1781-6). TEC kinase is also expressed in normal B and T-cells and is up-regulated upon T-cell activation in Th1 and Th2 cells (Tomlinson M G et al 2004 Mol. Cell. Biol., 24:2455-2466). TEC may have different roles from either ITK or TXK. TEC has a unique subcellular distribution differential protein interactions compared with ITK and TXK (Tomlinson M G et al 2004 Mol. Cell. Biol., 24:2455-2466) and TEC, but not TXK or ITK, is a tyrosine kinase of c-Maf leading to enhancement of c-Maf-dependent IL-4 promoter activity (Liu C C et al. 2015 PLoS One. 10:e0127617). Lastly, TEC controls assembly of the non-canonical caspase 8 inflammasome involved in fungal sepsis and TEC-deficient mice are highly resistant to candidiasis (Zwolanek F et al. 2014 PLoS Pathog 10, e1004525).
BTK is important in B-cell receptor signaling and regulation of B-cell development and activation (W. N. Khan et al. Immunity, 1995, 3:283-299 and Satterthwaite A B et al. Immunol. Rev. 2000, 175: 120-127). Mutation of the gene encoding BTK in humans leads to X-linked agammaglobulinemia which is characterized by reduced immune function, including impaired maturation of B-cells, decreased levels of immunoglobulin and peripheral B cells, diminished T-cell independent immune response (Rosen F S et al., N Engl. J. Med., 1995, 333:431-440; and Lindvall J M et al. Immunol. Rev. 2005, 203:200-215). BTK is activated by Src-family kinases and phosphorylates PLC gamma leading to effects on B-cell function and survival. Additionally, BTK is important for cellular function of mast cells, macrophage and neutrophils suggesting that BTK inhibition would be effective in treatment of diseases mediated by these and related cells including inflammation, bone disorders, and allergic disease (Kawakami Y. et al., J Leukoc Biol. 1999; 65(3):286-90). BTK inhibition is also important in survival of lymphoma cells (Herman S E M. Blood, 2011, 117:6287-6289) suggesting that inhibition of BTK may be useful in the treatment of lymphomas and other cancers (Uckun F M, Int Rev Immunol. 2008; 27(1-2):43-69). As such, inhibitors of BTK and related kinases are of great interest as anti-inflammatory as well as anti-cancer agents. BTK is also important for platelet function and thrombus formation suggesting that BTK-selective inhibitors may prove to be useful antithrombotic agents (Liu J. Blood, 2006, 108:2596-603). Furthermore, BTK is required for inflammasome activation (Ito M. et al. Nat Commun. 2015 Jun. 10; 6:7360) and inhibition of BTK may be useful in treatment of inflammasome-related disorders including; stroke, gout, type 2 diabetes, obesity-induced insulin resistance, atherosclerosis amyotrophic Lateral sclerosis, Parkinson's disease, and Muckle-Wells syndrome. BTK is expressed in CNS microglia and activated in infiltrating macrophages and neutrophils in models of stroke and inhibition of BTK suppresses neurological damage in a mouse model of stroke (Ito M. et al. Nat Commun. 2015 Jun. 10; 6:7360). In addition BTK is expressed in HIV infected T-cells and treatment with BTK inhibitors sensitizes infected cells to apoptotic death and results in decreased virus production (Guendel I et al. J Neurovirol. 2015; 21:257-75). Accordingly, BTK inhibitors may be useful in the treatment of HIV-AIDS and other viral infections.
Experimental data using Tec-kinase-family null animals supports the therapeutic benefit of kinase inhibition in human disease. ITK modulates neuroinflammation due to experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). ITK−/− mice exhibit reduced disease severity, and transfer of ITK−/−CD4+T cells into T cell-deficient mice results in lower EAE disease severity (Kannan A k et al. J. Neurosci, 2015; 35:221-233). ITK −/− mice exhibit decreased inflammatory response in contact hypersensitivity models (Von Bonin et al. Experimental Dermatology, 2010; 20, 41-47) and secretion of the Th2 cytokines IL-5 and IL-13 is decreased in models of allergic asthma in ITK −/− mice (Mueller C et al. J Immunol. 2003; 170(10):5056-63).
Data obtained with inhibitors of selected Tec-kinase-family members suggests that inhibitors of these kinases may be useful in the treatment of disease. Inhibitors of ITK, TXK and other Tec-kinase-family members may be useful in the prevention or treatment of T-cell related diseases such as multiple sclerosis, asthma, atopic dermatitis, psoriasis and inflammatory bowel diseases well as viral infections. For example, a small molecule inhibitor of ITK and TXK has shown efficacy in the mouse adoptive T-cell transfer model of colitis (Cho H-S et al. 2015; J. Immunol. 195: 4822-31).
Also, a selective ITK inhibitor blocked leukocyte lung infiltration following ovalbumin challenge in a rat model of asthma (Lin T A et al. 2004 Biochemistry. 43:11056-11062). Additionally, an ITK inhibitor was effective in mouse models of skin contact hypersensitivity (von Bonin A et al. 2010. Exp. Derm; 20, 41-47). Furthermore, ITK inhibitors can alter the HIV replication at various stages of viral life cycle including viral entry, gp120-induced actin reorganization, transcription from viral long terminal repeats (LTR) and virion assembly release from T cells (Readinger J A et al. Proc Natl Acad Sci USA. 2008; 105(18):6684-9). Similarly ITK inhibition alleviates T-cell activation and murine myocardial inflammation associated with Coxsackie virus CVB3 infection (He F et al. Mol Immunol. 2014; 59(1):30-8) and ITK is required for efficient replication of influenza virus in infected T-cells (Fan K et al. J Gen Virol. 2012; 93(Pt 5):987-97). These data suggest that inhibitors of the Tec-kinase-family may be useful in the treatment of a variety of human and animal diseases.
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