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Targeting Interleukin-2-inducible T-cell Kinase (ITK) and Resting Lymphocyte Kinase (RLK) Using a Novel Covalent Inhibitor PRN694

Open AccessPublished:January 15, 2015DOI:https://doi.org/10.1074/jbc.M114.614891
      Interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK or TXK) are essential mediators of intracellular signaling in both normal and neoplastic T-cells and natural killer (NK) cells. Thus, ITK and RLK inhibitors have therapeutic potential in a number of human autoimmune, inflammatory, and malignant diseases. Here we describe a novel ITK/RLK inhibitor, PRN694, which covalently binds to cysteine residues 442 of ITK and 350 of RLK and blocks kinase activity. Molecular modeling was utilized to design molecules that interact with cysteine while binding to the ATP binding site in the kinase domain. PRN694 exhibits extended target residence time on ITK and RLK and is highly selective for a subset of the TEC kinase family. In vitro cellular assays confirm that PRN694 prevents T-cell receptor- and Fc receptor-induced cellular and molecular activation, inhibits T-cell receptor-induced T-cell proliferation, and blocks proinflammatory cytokine release as well as activation of Th17 cells. Ex vivo assays demonstrate inhibitory activity against T-cell prolymphocytic leukemia cells, and in vivo assays demonstrate durable pharmacodynamic effects on ITK, which reduces an oxazolone-induced delayed type hypersensitivity reaction. These data indicate that PRN694 is a highly selective and potent covalent inhibitor of ITK and RLK, and its extended target residence time enables durable attenuation of effector cells in vitro and in vivo. The results from this study highlight potential applications of this dual inhibitor for the treatment of T-cell- or NK cell-mediated inflammatory, autoimmune, and malignant diseases.

      Introduction

      IL-2-inducible T-cell kinase (ITK)
      The abbreviations used are: ITK
      interleukin-2-inducible T-cell kinase
      RLK
      resting lymphocyte kinase
      NK
      natural killer
      TCR
      T cell receptor
      FcR
      Fc receptor
      PLCγ1
      phospholipase Cγ1
      NFAT
      nuclear factor of activated T-cells
      DTH
      delayed type hypersensitivity
      PBMC
      peripheral blood mononuclear cell
      CFSE
      carboxyfluorescein succinimidyl ester
      BisTris
      2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol
      DNP
      dinitrophenyl
      BTK
      Bruton's tyrosine kinase
      T-PLL
      T-cell prolymphocytic leukemia.
      and resting lymphocyte kinase (RLK; also known as TXK) are members of the TEC family of non-receptor tyrosine kinases that play important roles in signal transduction in T-cells and natural killer (NK) cells (
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      The Tec kinases Itk and Rlk regulate NKT cell maturation, cytokine production, and survival.
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      ). ITK and RLK facilitate a range of downstream signaling from T-cell and NK cell surface receptors, tyrosine kinases, and integrins, including the T-cell receptor (TCR) and Fc receptor (FcR) (
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      The Tec kinases Itk and Rlk regulate NKT cell maturation, cytokine production, and survival.
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      ). Upon surface receptor engagement, ITK mediates signaling by activating phospholipase Cγ1 (PLCγ1), leading to the downstream activation of nuclear factor of activated T-cells (NFAT), nuclear factor κB (NFκB), and mitogen-activated protein kinase (MAPK) pathways (
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      ). ITK knock-out (ITK−/−) mice demonstrate defects in Th2 and Th17 differentiation and function (
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      ). More recently, attention has shifted to autoimmune disease because of the effects of ITK deletion on Th17 function and the concomitant increase in regulatory T-cell (Treg) numbers and function (
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      ). The clinical target validation of IL-17 in psoriasis and psoriatic arthritis adds additional support to the notion that ITK could be a relevant therapeutic target in these diseases (
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      ). In addition, interest remains high in other Th17-driven autoimmune diseases, such as multiple sclerosis and inflammatory bowel disease (
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      ).
      The signaling of RLK is less clear. Its unique N-terminal structure, which lacks the pleckstrin homology domain present in other TEC kinases, renders it free of modulation by phosphoinositide-3 kinase activity (
      • Kannan Y.
      • Wilson M.S.
      TEC and MAPK kinase signalling pathways in T helper (T) cell development, T2 differentiation and allergic asthma.
      ). Deletion of RLK alone has few functional consequences, but deletion of RLK in combination with ITK appears to be important in Th1 cells, where it is preferentially expressed. In addition, the deletion of ITK and RLK has a marked effect on Th17 differentiation and IL-17 production (
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      ).
      To date, no small molecule inhibitors specifically targeting ITK or RLK have progressed past preclinical studies. Conventional approaches to developing reversible competitive inhibitors have been hampered by limited potency and/or selectivity, although efforts continue to be made (
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      ). A recent study utilizing non-competitive binding to an allosteric site of ITK has yielded a compound with high selectivity; however, the absorption, distribution, metabolism, and excretion and physicochemical optimization to produce the extended pharmacokinetics required for reversible compounds has not been completed (
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      ). Because RLK is thought to compensate for ITK in a number of T-cell and NK cell subsets, development of a dual ITK/RLK inhibitor would be advantageous to effectively block TEC kinase-driven activation of these cells (
      • Kannan Y.
      • Wilson M.S.
      TEC and MAPK kinase signalling pathways in T helper (T) cell development, T2 differentiation and allergic asthma.
      ).
      Covalent targeting of cysteine residues within the ATP binding pocket is an effective way to obtain highly potent and selective kinase inhibitors with long target residence time (
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      ). Ibrutinib, for instance, irreversibly binds cysteine 481 in BTK and cysteine 442 in ITK, leading to a blockade of signaling downstream of the B-cell receptor and TCR, respectively. Ibrutinib has demonstrated clinical safety and activity against B-cell malignancies, including mantle cell lymphoma and chronic lymphocytic leukemia (
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      ). All five mammalian TEC kinases have a similar domain organization, including a cysteine-containing kinase domain (
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      Tec family kinases in T lymphocyte development and function.
      ). Herein we describe PRN694, which was developed to irreversibly inhibit ITK and RLK by forming a putative covalent bond with Cys-442 or Cys-350, respectively. We show that this results in in vivo efficacy without the need for an extended plasma half-life. In vitro kinase assays show that PRN694 has potency and selectivity for ITK and RLK. This selectivity is validated in Jurkat T-cells with mutated ITK or overexpressed RLK. We further demonstrate that PRN694 prevents TCR- or FcR-induced cellular and molecular activation, inhibits TCR-induced T-cell proliferation without direct cytotoxicity, and blocks proinflammatory cytokine release. Finally, in vivo experiments demonstrate the pharmacokinetics and pharmacodynamics of PRN694 and show that it attenuates a delayed type hypersensitivity (DTH) reaction in a well established murine model system. These results indicate promising clinical applicability of this ITK/RLK dual inhibitor for the treatments of T-cell or NK cell malignancies as well as inflammatory and autoimmune diseases, such as psoriasis, psoriatic arthritis, rheumatoid arthritis, multiple sclerosis, and irritable bowel disease.

      DISCUSSION

      Many human diseases and conditions are mediated or exacerbated by aberrant activation of T-cells or NK cells. Because T-cell activation invariably relies on TCR-mediated signaling and NK cells are activated via the FcR, agents that can selectively interrupt these pathways would be of significant clinical benefit. Given that ITK and RLK are key components of both TCR and FcR signaling, a compound with selectivity for these two targets should provide a useful chemical biology tool to impact these important cell populations. Here, we describe the discovery, biochemical characterization, and biological assessment of PRN694, a novel lead that selectively and irreversibly inhibits both of these critical lymphocyte kinases. We demonstrate that PRN694 has the desired potency and selectivity for ITK and RLK, as shown by in vitro kinase assays and experiments in Jurkat T-cells with mutated ITK or overexpressed RLK. We further show that TCR or FcR-induced cellular and molecular activation, TCR-induced T-cell proliferation, and proinflammatory cytokine release are inhibited by PRN694. Moreover, we demonstrate extended in vivo pharmacodynamic effects and efficacy in inhibiting DTH in a well characterized murine model system.
      Consistent with our CD4 and CD8 T-cell data using PRN694, studies from ITK- and RLK-deficient mice demonstrate the critical roles these two TEC kinases play in T-cell and NK cell signaling (
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      • Schwartzberg P.L.
      Requirement for Tec kinases Rlk and Itk in T cell receptor signaling and immunity.
      ). ITK-deficient mice display defects in TCR-induced T-cell responses, including proliferation, cytokine production, and activation of downstream pathways (
      • Liao X.C.
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      Altered T cell receptor signaling and disrupted T cell development in mice lacking Itk.
      ,
      • Liu K.Q.
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      T cell receptor-initiated calcium release is uncoupled from capacitative calcium entry in Itk-deficient T cells.
      ), and these defects are exacerbated by the addition of RLK deficiency, demonstrating the potential for combined inhibition (
      • Schaeffer E.M.
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      • Yap G.
      • McVicar D.
      • Liao X.C.
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      Requirement for Tec kinases Rlk and Itk in T cell receptor signaling and immunity.
      ). In NK cells, ITK functions as a positive regulator of the FcR pathway. FcR stimulation induces the tyrosine phosphorylation of ITK, leading to increases in granule release, calcium mobilization, and cytotoxicity (
      • Khurana D.
      • Arneson L.N.
      • Schoon R.A.
      • Dick C.J.
      • Leibson P.J.
      Differential regulation of human NK cell-mediated cytotoxicity by the tyrosine kinase Itk.
      ). As expected, PRN694 potently blocked FcR activation and the effector functions of NK cells. The relative expression of ITK and RLK appears to be important for regulation of T helper cell differentiation as well (
      • Andreotti A.H.
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      • Berg L.J.
      T-cell signaling regulated by the Tec family kinase, Itk.
      ). Specifically, Th1 cells express both ITK and RLK, whereas Th2 cells express only ITK. This is consistent with early studies with ITK knock-out mice that show that Th2- and IL-4-dependent disease processes, such as ovalbumin-induced asthma, are blocked by ITK deletion, whereas Th1 responses remain intact, presumably due to redundancy of RLK signaling (
      • Ferrara T.J.
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      ,
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      ). RLK expression in Th17 cells is not well studied, but mice that have deletions of both kinases show profound defects in Th17 differentiation and function, a finding that was confirmed in our studies using human Th17 cells (
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      Itk-mediated integration of T cell receptor and cytokine signaling regulates the balance between Th17 and regulatory T cells.
      ). Thus, combined inhibition of ITK and RLK would be expected to target both Th1 and Th17 cell differentiation and function. This could be an optimal therapeutic combination, given the importance of these two T helper cell subsets in the development of autoimmune diseases such as psoriasis, psoriatic arthritis, inflammatory bowel disease, and rheumatoid arthritis (
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      ). Interestingly, recent data utilizing ITK knock-out mice indicate that along with the decrease in Th17 cell differentiation and function, there is an increase in Treg cells that function in suppression of disease in a murine adoptive transfer model of colitis (
      • Gomez-Rodriguez J.
      • Wohlfert E.A.
      • Handon R.
      • Meylan F.
      • Wu J.Z.
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      • Kirby M.R.
      • Belkaid Y.
      • Schwartzberg P.L.
      Itk-mediated integration of T cell receptor and cytokine signaling regulates the balance between Th17 and regulatory T cells.
      ). Thus, an added value of regulating the pathways targeted by PRN694 is potentially an increase in regulatory T-cells that also are important in control of autoimmune disease.
      Although our studies reveal an NK cell dependence on RLK and ITK for FcR stimulation, this was not observed in monocyte-derived macrophages or dendritic cells. Moreover, our studies using the RBL-2H3 mast cell line demonstrated no effects of PRN694 on FcRϵ-induced activation, indicating that myeloid-derived immune cells are not uniquely dependent upon RLK and ITK. This concept is supported by recent literature indicating that BTK is a dominant regulator of mast cell FcRϵ degranulation and that ITK plays an accessory role, and only combined genetic ablation can eliminate degranulation (
      • Iyer A.S.
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      Absence of Tec family kinases interleukin-2 inducible T cell kinase (Itk) and Bruton's tyrosine kinase (Btk) severely impairs FcϵRI-dependent mast cell responses.
      ). Notably, however, we did not observe significant expression of ITK in our RBL-2H3 cell line, indicating some potential molecular differences that warrant further study (
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      ).
      Medicinal chemistry discovery and optimization of ITK inhibitors have resulted in the identification of many chemical series with potent biochemical activities. However, due to the conserved topology and sequence composition of the ATP binding site of the TEC family and kinases in general, achieving high specificity has proven difficult (
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      ). A new chemical design strategy that can address the potency and selectivity challenges of kinase inhibition is based on covalent binding to cysteine residues within the ATP binding site. With the recent Food and Drug Administration approval of ibrutinib and the late stage development status of several other covalent kinase inhibitors, this approach is gaining widespread acceptance in the oncology arena (
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      ,
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      Discovery of selective irreversible inhibitors for Bruton's tyrosine kinase.
      ). Of primary importance for our novel approach was the identification a selective hinge-binding molecular scaffold and compatible linker chemistry to covalently bond to Cys-442. Although covalent ITK inhibitors targeting Cys-442 have been reported previously, there was limited success in achieving high selectivity (
      • Harling J.D.
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      Discovery of novel irreversible inhibitors of interleukin (IL)-2-inducible tyrosine kinase (Itk) by targeting cysteine 442 in the ATP pocket.
      ). Cys-442 of ITK is located at the end of the C-lobe α-helix, a conserved location shared with only 10 other kinases, including all members of the TEC family (ITK, RLK, BTK, BMX, and TEC). As a consequence, a strategy to achieve high selectivity required more than simply targeting a covalent interaction with Cys-442. This approach optimizes the interactions with the targets of interest while limiting the interactions with off-target cysteine-containing proteins. Thus, small molecules with high potency, selectivity, and prolonged target residence time are produced, which may lead to superior safety and tolerability of clinical leads.
      In addition to the potential utility for autoimmune diseases, our results support potential applications of PRN694 for the treatment of T-cell and NK cell-related cancers. Our ex vivo studies on T-PLL signaling demonstrate the potential for therapeutic utility for PRN694 in these hematopoietic malignancies. Although the molecular pathogenesis of aggressive T-cell and NK cell malignancies remains unclear, ITK and RLK have been shown to be critical mediators of intracellular signaling that support the survival and growth of these malignancies (
      • Kaukonen J.
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      ,
      • Shin J.
      • Monti S.
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      • Golub T.
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      Lesional gene expression profiling in cutaneous T-cell lymphoma reveals natural clusters associated with disease outcome.
      ,
      • Zhao W.L.
      Targeted therapy in T-cell malignancies: dysregulation of the cellular signaling pathways.
      ). Our studies reveal that ITK- and RLK-based signaling in T-PLL can be blocked by PRN694, thereby starving these cells of an essential activation pathway. This effect is similar to how ibrutinib starves malignant leukemic B-cells by blocking BTK signaling. Further preclinical studies focusing on novel ITK- and/or RLK-relevant clinical applications for PRN694 are currently being pursued.

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      1. K. A. Brameld, T. A. Owens (March 6, 2014) International Patent WO 2014/036016 A1