Tubulosine selectively inhibits JAK3 signalling by binding to the ATP‐binding site of the kinase of JAK3

Abstract Gain‐ or loss‐of‐function mutations in Janus kinase 3 (JAK3) contribute to the pathogenesis of various haematopoietic malignancies and immune disorders, suggesting that aberrant JAK3 signalling is an attractive therapeutic target to treat these disorders. In this study, we performed structure‐based computational database screening using the 3D structure of the JAK3 kinase domain and the National Cancer Institute diversity set and identified tubulosine as a novel JAK3 inhibitor. Tubulosine directly blocked the catalytic activity of JAK3 by selective interacting with the JAK3 kinase domain. Consistently, tubulosine potently inhibited persistently activated and interleukin‐2‐dependent JAK3, and JAK3‐mediated downstream targets. Importantly, it did not affect the activity of other JAK family members, particularly prolactin‐induced JAK2/signal transducer and activator of transcription 5 and interferon alpha‐induced JAK1‐TYK2/STAT1. Tubulosine specifically decreased survival and proliferation of cancer cells, in which persistently active JAK3 is expressed, by inducing apoptotic and necrotic/autophagic cell death without affecting other oncogenic signalling. Collectively, tubulosine is a potential small‐molecule compound that selectively inhibits JAK3 activity, suggesting that it may serve as a promising therapeutic candidate for treating disorders caused by aberrant activation of JAK3 signalling.


| INTRODUC TI ON
Cytokines are critical signalling molecules that regulate various biological responses, including cell proliferation, differentiation, tissue homeostasis, haematopoiesis and immune responses. However, aberrant cytokine signalling can result in a wide variety of human diseases such as cancer and immune disorders. 1  an alpha or beta chain as signalling partners. 2,3 They also thought to be particularly involved in T-cell development and immune homeostasis, as loss-of-function JAK3 mutations in humans have been shown to result in haematopoietic disorders such as severe combined immunodeficiency (SCID). 4,5 Further, gene therapy for autosomal recessive SCID using haematopoietic stem cell transplantation increased the risk of acute T-cell leukaemia due to the direct activation of the γc-mediated JAK3/signal transducer and activator of transcription 5 (STAT5) signalling. 6 Aberrantly activated JAK3/STAT signalling has been implicated in various haematologic cancers. For example, in leukaemic blast cells, JAK3/STAT signalling was persistently activated in 70% of patients with acute myeloid leukaemia (AML). 7 It was also observed in various haematologic cancer cell lines, including anaplastic large cell lymphoma, 8 Burkitt's lymphoma, 9 mantle-cell lymphoma 10 and enteropathy-associated T-cell lymphoma. 11 In addition, constitutively active JAK3/STAT signalling is reported in the mouse model of pre-B-cell leukaemia. This model is established by loss-of-function mutations of the tumour suppressor B-cell linker (BLNK), an inhibitor that binds JAK3 and decreases autocrine JAK3/STAT5 signalling. 12 In this model, BLNK expression was completely lost or drastically reduced in paediatric pre-B-cell acute lymphoblastic leukaemia (ALL) cases. 13 Somatic mutations of JAK3 alleles have also been identified in cancer cell lines, as well as in patients with the following diseases: acute megakaryoblastic leukaemia, 14,15 high-risk childhood ALL, 16,17 Down syndrome AML and ALL, 18 T-cell ALL 19 and cutaneous T-cell lymphomas. 20 In these cases, the patients acquired constitutive-active JAK3/STAT signalling by gain-of-function. This evidence suggests that aberrantly activated JAK3/STAT signalling contributes to the pathogenesis of a subset of haematopoietic malignancies and JAK3 is an attractive therapeutic target for the treatment of patients with these diseases.
In this study, we aimed to discover the small-molecule inhibitors of JAK3 and identified tubulosine as a potent JAK3 inhibitor.
Tubulosine potently inhibited constitutively active and IL-2-induced JAK3/STAT signalling, thereby decreasing proliferation and survival of cancer cells by inducing apoptotic and necrotic/autophagic cell death. These findings indicate that tubulosine may be a promising candidate for therapeutic intervention in diseases caused by abnormal JAK3 activity.

| Structure-based computational database screening
To identify novel small molecules that target JAK3, we employed the AutoDock version 4.2 21 and performed virtual screening using the kinase domain of JAK3 (JAK3-Janus homology 1 [JH1], PDB ID: 1YVJ) 22 with the NCI diversity set of compounds. The docking consists of two stages. In the first stage, the protein coordinates from the complex structure (PDB ID: 1YVJ) between the JAK3-JH1 and its inhibitor staurosporine analog AFN941 22 were chosen as templates. After removing the ligand and solvent molecules, AMBER software 23 added hydrogen atoms, which was based on the PDB2PQR-determined ionizable states in Asp, Glu, His and Lys residues. 24 In the second step, we made detailed calculations with the decreased candidates by the initial stage. It included two JAK3-JH1 structures complex with CP-690,550 (PDB ID: 3LXK) and CMP-6 (PDB ID: 3LXL) in addition. 25 Besides, we generated 30 initial conformations of each small molecule by AMBER package and performed ensemble docking with 90 runs per compound.
The resulting structures were clustered based on the structural similarity that was quantified by the root-mean-square deviation

| Cell lines and cell culture
The human Hodgkin's lymphoma HDLM-2 and L540 cells and

| In vitro kinase assays
Enzyme samples of the JAK family members were prepared from the HDLM-2 or L540 cells by immunoprecipitation with corresponding JAK antibodies and recombinant His-tagged STAT3α protein was purified as a substrate. The preparation and purification of the enzyme sources and recombinant His-tagged STAT3α protein, as well as in vitro kinase assay, were previously reported. 32 The reaction products were subjected to SDS-PAGE and Western blotting with F I G U R E 1 Schematic modelling of structure-based JAK3 computational database screening. A, The chemical structure of tubulosine (C 29 H 37 N 3 O 3 ). B, Predicted binding model between tubulosine and the JAK3 kinase domain (JAK3-JH1). Tubulosine is coloured in pink. The residues that contact tubulosine with side chain atoms within 3.5 Å are labelled. C, Overlay of different ligands in complex with JAK3-JH1. The following structures are shown: tubulosine (pink), AFN941 (cyan), CP-690,550 (yellow) and CMP-6 (green). These structures were generated from PDB files of 1YVJ, 24 3LXK 26 and 3LXL, 26 respectively. D, Predicted binding model between tubulosine and the kinase domains of JAK family members JAK1 (JAK1-JH1), JAK2 (JAK2-JH1) and JAK3 (JAK3-JH1). JAK1-JH1, JAK2-JH1 and JAK3-JH1 are coloured in pink, white and purple, respectively. All the structural figures were generated using Pymol (http://pymol.sourc eforge.net/). JAK3, Janus kinase 3; JH1, Janus homology 1 appropriate antibodies. Another in vitro kinase assay was performed by KinaseProfiler™ Service (Merck Millipore, Dundee, UK) as previously reported. 33 The IC 50 values of JAK family members were determined by Millipore's standard radiometric assay at various concentrations of tubulosine with 10 μmol/L ATP. The K i and K m values against JAK3 were determined at various concentrations of tubulosine and ATP. The enzyme-specific activity (U/mg) measured in the absence of tubulosine was plotted against the ATP concentration and fitted to the Michaelis-Menten equation, allowing estimation of the K m(app) for ATP.

| Statistical analysis
Results including Western blot analysis, immunoprecipitation and in vitro kinase assay were obtained from at least two independent experiments, and the representative results are shown. Data are represented as mean ± standard error of the mean. Statistical analyses were determined by GraphPad Prism software with two-tailed Student's t test. A P value of less than 0.05 was considered statistically significant.

| Identification of tubulosine as a JAK3 inhibitor
Targeting JAK3/STAT signalling is considered as a valuable therapeutic strategy to treat immune-and inflammation-mediated diseases. 34 JH1 domain is the kinase domain of JAK family proteins, which is essential for the enzymatic activity. 35 To identify novel small molecules that inhibit the kinase activity of JAK3, we performed structurebased computational database screening using the 3D structure of the JAK3 kinase domain (PDB ID: 1YVJ) 22

| Tubulosine inhibits JAK3 kinase activity by binding the ATP-binding site
Because JH1 shares significant structural homology with the seven JH domains of the JAK family, we further investigated the specificity of tubulosine for JAK3-JH1 over the JH1 of other JAKs using in vitro immunoprecipitation kinase assays. Immunoprecipitates were prepared from the lysates of HDLM-2 or L540 cells that have constitutively active forms of JAK1, JAK2, JAK3 or TYK2. 36  To determine the IC 50 values of tubulosine for all JAKs, we performed in vitro kinase assays using the KinaseProfiler™ services.
Tubulosine inhibited JAK3 kinase activity with an IC 50 value of 9.9 nmol/L, and its activity was decreased in the presence of ATP in a concentration-dependent manner, resulting in an EC 50 value of 1.4 μmol/L. Although tubulosine also inhibited the kinase activities of other JAK family members, the extent of inhibition was less than that of JAK3, with IC 50 values of 69.5, 84.9 and 76.3 nmol/L for JAK1, JAK2 and TYK2, respectively ( Figure 2C, Figure S1B). We also determined K i and K m values for JAK3 kinase activity and found a value of 12.9 ± 1.3 nmol/L with tubulosine and 212.4 ± 25.7 μmol/L with ATP ( Figure S1C). Staurosporine was used as a positive control for ATP-competitive inhibition of JAKs. These results strongly indicate that tubulosine is a potent and selective JAK3 inhibitor.

| Tubulosine predominantly inhibits constitutively active JAK3 signalling
We further investigated whether tubulosine could inhibit constitutively active JAK3 signalling in various cancer cell lines. It was previously reported that the cell lines L540, 36

| Tubulosine inhibits cytokine-induced JAK3 signalling
We next determined the inhibitory effect of tubulosine on ligandmediated JAK2 and JAK3 signalling by stimulation with either IL-2 or PRL in Nb2 cells, which have been previously used to study cytokinedependent activation of JAK signalling. 38

| Tubulosine does not affect other oncogenic signalling
We further investigated whether tubulosine could affect the sig-  Figure 5, lanes 1,2). Taken together, these results indicate that tubulosine selectively inhibits the JAK3 activity and subsequently results in the inhibition of its down target signal transduction, but not that of other JAK family members and oncogenic signalling components.

| Tubulosine induces apoptotic and necrotic/ autophagic cell death
Previous reports have demonstrated that treatment with JAK3specific inhibitors or siRNA induce apoptotic cell death by blocking JAK3 signalling in persistently JAK3-activated cancer cells. 32 Therefore, we performed TUNEL assay to determine apoptotic cell population, followed by treatment with vehicle alone, tubulosine or AG-490 for 72 hours in L540 cells. We observed that tubulo-

| D ISCUSS I ON
The identification of active pharmacological compounds that target specific signalling pathways is a part of early-stage drug discovery. In this study, we identified tubulosine as a potent and selective JAK3 inhibitor that predominantly targeted the enzyme activity of JAK3 kinase, as compared to other JAK family members. Tubulosine was F I G U R E 6 Tubulosine selectively decreases the survival and proliferation of cancer cells that constitutively express active JAK3. A and B, L540, BKO-84 and BaF3/JAK3V674A cells (A and C) and HDLM-2, DU145, A431 and BaF3/TEL-JAK2 cells (B and D) were incubated with either vehicle (0.1% DMSO) alone, tubulosine (25,50 and 100 nmol/L) or the pan-JAK inhibitor AG-490 (150 μmol/L) for the indicated time intervals. Cell viability and proliferation were determined using a WST-1 reagent or by counting viable cells using the trypan blue exclusion assay. Data are represented as a % control compared to the vehicle alone-treated group (n = 3). *P < 0.05 and **P < 0.005 compared to the vehicle-treated group. JAK3, Janus kinase 3 Structure-based computational database screening is currently a popular approach in early-stage drug discovery and the progressive optimization of lead compounds because it is faster and more cost-efficient than traditional screening methods. It is also possible to perform large-scale screening using chemical compound libraries. This tool is a computer-aided method that conducts molecular docking of chemical compounds into the 3D structure of the target protein. 42 3D structure of target protein is generally retrieved from the PDB determined by X-ray crystallography or NMR spectroscopy. 43 In this study, we chose the kinase domain of JAK3 (PDB ID: 1YVJ) as the biological target and used the NCI diversity set of compounds as a library. JAK3 is a member of the Janus family of tyrosine kinases that is mainly associated with immune, inflammatory and haematopoietic disorders because they are commonly expressed in haematopoietic cells such as T cells and NK cells. 2,34,44 To perform structure-based virtual screening against JAK3, we modified the conventional docking methods by generating 30 initial conformations of each compound, performed ensemble docking and then calculated the binding energies. We compared the ligand-binding pockets in all JAK proteins and superimposed the ligand structures onto the pockets to identify JAK3-specifc inhibitors. Our data showed that the selectivity of tubulosine towards JAK3-JH1 over JAK1-JH1 and JAK2-JH1 in the experimental assays is quantitatively consistent with the results from in silico calculations that show lower binding free energies for JAK3-JH1 (−11.79 kcal/mol), compared to JAK1-JH1 (−11.10 kcal/ mol) and JAK2-JH1 (−10.83 kcal/mol), respectively. This is thought to be due to the contact around Asp-847 because it plays an essential role in increasing the contact area between tubulosine and the protein, which resulted in a tight fit into the region of JAK3-JH1.

F I G U R E 7
Various types of leukaemia and lymphoma have been recently identified from the extensive studies of human patients and mouse models that had persistent activation of JAK3 signalling with somatic mutations. In these cases, the mutations are generally associated with gain-of-function. On the contrary, loss-of-function mutations of JAK3 in human and mouse models caused immunodeficiency such as SCID. 45,46 This evidence indicates the importance of JAK3 in the immune system and its contribution to the pathogenesis of various haematopoietic malignancies. In addition, targeting JAK3 signalling is a valuable thera- Tubulosine is an Alangium alkaloid, which was first isolated from the bark of Pogonopus tubulosus. 48 Although its isolation and structure have been known for a long time, the biological function and mechanism of action have not been clearly elucidated. The exceptions, in this regard, are its effects on amebicidal activity and inhibition of protein biosynthesis, 49 inhibition of eukaryotic elongation F I G U R E 8 A schematic diagram illustrating the proposed action mechanism of tubulosine. Tubulosine specifically targets the signalling cascades of JAK3-mediated STATs and their downregulated targets. JAK3, Janus kinase 3 factor 2-dependent peptide chain elongation 50 and hypoxia-inducible factor-1α transcriptional activity, 51 and anti-tumour activity. 52,53 The anti-tumour activity was evaluated by the cytotoxicity in cancer cell lines. Recently, we reported that tubulosine inhibited JAK2/STAT3 signalling in IL-6-induced breast cancer. 54

CO N FLI C T O F I NTE R E S T
The authors declare that they have no financial interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support findings of the study are available from the corresponding author upon request.