Crystal Structures of Interleukin-2 Tyrosine Kinase and Their Implications for the Design of Selective Inhibitors

Abstract

Interleukin-2 tyrosine kinase, Itk, is an important member of the Tec family of non-receptor tyrosine kinases that play a central role in signaling through antigen receptors such as the T-cell receptor, B-cell receptor, and Fc⑀. Selective inhibition of Itk may be an important way of modulating many diseases involving heightened or inappropriate activation of the immune system. In addition to an unliganded nonphophorylated Itk catalytic kinase domain, we determined the crystal structures of the phosphorylated and nonphosphorylated kinase domain bound to staurosporine, a potent broad-spectrum kinase inhibitor. These structures are useful for the design of novel, highly potent and selective Itk inhibitors and provide insight into the influence of inhibitor binding and phosphorylation on the conformation of Itk.The Tec kinases are a family of five non-receptor tyrosine kinases that play a central role in signaling through antigenreceptors such as the T-cell receptor (TCR), 1 B-cell receptor, and Fc⑀ (1) and are essential for T-cell activation. Three members of the family, Itk, Rlk, and Tec, are activated downstream of antigen receptor engagement in T-cells and transmit signals to downstream effectors, including PLC-␥. A fourth member, Btk, appears to act independently of T-cell signaling and is essential for B-cell development and activation. Btk-deficient murine mast cells have reduced degranulation and decreased production of proinflammatory cytokines following Fc⑀RI crosslinking (2). Btk deletion in mice has a profound effect on B-cell proliferation induced by anti-IgM and inhibits immune responses to thymus-independent type II antigens (3, 4). A biological role for the final member of this family, Bmx, has not been identified.Itk is a key member of this family, and a number of factors point to the importance of this kinase in immune disease.Deletion of Itk in mice results in reduced TCR-induced proliferation and secretion of the cytokines IL-2, IL-4, IL-5, IL-10, and interferon-␥ (5-7). Also, the immunological symptoms of allergic asthma are attenuated in ItkϪ/Ϫ mice, and lung inflammation, eosinophil infiltration, and mucous production are drastically reduced in response to challenge with the allergen OVA (8). Furthermore, the Itk gene is reported to be more highly expressed in peripheral blood T-cells from patients with moderate or severe atopic dermatitis than in controls or patients with mild atopic dermatitis (9).In certain cell types, the role of Itk may be intricately linked with other members of the family. For example, in mast cells, Btk and Itk are both expressed and activated by Fc⑀RI crosslinking (10). Splenocytes from RlkϪ/Ϫ mice secrete half the IL-2 produced by wild type animals in response to TCR engagement (5), whereas the combined deletion of Itk and Rlk in mice leads to a profound inhibition of TCR-induced responses, including proliferation and production of the cytokines IL-2, IL-4, IL-5, and interferon-␥ (5, 7). Furthermore, intracellular signaling following TCR engagement is affected in Itk...

Loss-of-function mutations within the IL-2 inducible kinase ITK in patients with EBV-associated lymphoproliferative diseases

“…7. Tyr-512 has been shown to be phosphorylated by LCK, 40,41 thought as an important mechanism in ITK regulation. LCK is a specific target of the HVS tyrosine-kinase-interacting protein (Tip).…”

“…Both the proteins could still harbor a basic kinase activity, as the active site of ITK has been mapped to be upstream of the truncations (amino acids 376 --435). 40 The complex regulation of the second messenger Ca 2 þ depends on the relation of the individual TCR signaling components to each other, mode and strength of TCR activation, timing, and the calcium storage pools itself. One possible explanation could be the presence of Ca-flux inhibitory regions, for example, the Tyr-512 residue, located in the truncated region of patient no.…”

Pharmacophore Design for Anti-inflammatory Agent Targeting Interleukin-2 Inducible Tyrosine Kinase (Itk)

“…The X-ray crystal structure of Itk (PDB ID: 1SM2) was used in our molecular docking studies. 31 The binding pocket of Itk has a glycine-rich loop which contains the consensus kinase sequence Gly-X-Gly-X-X-Gly. Previous docking study on Itk states that the binding of the ligand in Itk kinase domain at the interface of the N-and C-terminal lobes mainly shows hydrophobic interactions with I369, V377 and A389 (N-terminal), F435 and Y437 (hinge region) and L489 and C442 (C-terminal lobe) among these residue F435 acts as a gate keeper, makes beneficial edge face interaction with conjugated system of ligand and additional key residues in the active site such as R486, E436, M438.…”

“…These three residues form hydrogen bond (H-bond) interactions with inhibitor. 31 All the compounds including training set of 16 molecules and 40 new hit molecules retrieved from the NCI and Maybridge databases were docked into the active site of Itk. The bound conformations inside the Itk active site were visually examined.…”

Phylogeny of Tec Family Kinases: Identification of a Premetazoan Origin of Btk, Bmx, Itk, Tec, Txk, and the Btk Regulator SH3BP5

“…The kinase domain structure has been solved for BTK (Mao et al, 2001) and ITK (Brown et al, 2004). These domains are highly conserved in all TFK sequences.…”