Emdogain-Regulated Gene Expression in Palatal Fibroblasts Requires TGF-βRI Kinase Signaling

Genome-wide microarrays have suggested that Emdogain regulates TGF-β target genes in gingival and palatal fibroblasts. However, definitive support for this contention and the extent to which TGF-β signaling contributes to the effects of Emdogain has remained elusive. We therefore studied the role of the TGF-β receptor I (TGF-βRI) kinase to mediate the effect of Emdogain on palatal fibroblasts. Palatal fibroblasts were exposed to Emdogain with and without the inhibitor for TGF-βRI kinase, SB431542. Emdogain caused 39 coding genes to be differentially expressed in palatal fibroblasts by microarray analysis (p<0.05; >10-fold). Importantly, in the presence of the TGF-βRI kinase inhibitor SB431542, Emdogain failed to cause any significant changes in gene expression. Consistent with this mechanism, three independent TGF-βRI kinase inhibitors and a TGF-β neutralizing antibody abrogated the increased expression of IL-11, a selected Emdogain target gene. The MAPK inhibitors SB203580 and U0126 lowered the impact of Emdogain on IL-11 expression. The data support that TGF-βRI kinase activity is necessary to mediate the effects of Emdogain on gene expression in vitro.     

Transforming Growth Factor-�� (TGF-��1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-�� Receptor Kinase Activity in Mesangial Cells

Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that signals through the interaction of type I (TβRI) and type II (TβRII) receptors to activate distinct intracellular pathways. TAK1 is a serine/threonine kinase that is rapidly activated by TGF-β1. However, the molecular mechanism of TAK1 activation is incompletely understood. Here, we propose a mechanism whereby TAK1 is activated by TGF-β1 in primary mouse mesangial cells. Under unstimulated conditions, endogenous TAK1 is stably associated with TβRI. TGF-β1 stimulation causes rapid dissociation from the receptor and induces TAK1 phosphorylation. Deletion mutant analysis indicates that the juxtamembrane region including the GS domain of TβRI is crucial for its interaction with TAK1. Both TβRI-mediated TAK1 phosphorylation and TGF-β1-induced TAK1 phosphorylation do not require kinase activity of TβRI. Moreover, TβRI-mediated TAK1 phosphorylation correlates with the degree of its association with TβRI and requires kinase activity of TAK1. TAB1 does not interact with TGF-β receptors, but TAB1 is indispensable for TGF-β1-induced TAK1 activation. We also show that TRAF6 and TAB2 are required for the interaction of TAK1 with TβRI and TGF-β1-induced TAK1 activation in mouse mesangial cells. Taken together, our data indicate that TGF-β1-induced interaction of TβRI and TβRII triggers dissociation of TAK1 from TβRI, and subsequently TAK1 is phosphorylated through TAB1-mediated autophosphorylation and not by the receptor kinase activity of TβRI.Members of the transforming growth factor-β (TGF-β)³ superfamily are key regulators of various biological processes such as cellular differentiation, proliferation, apoptosis, and wound healing (1, 2). TGF-β1, the prototype of TGF-β family, is a potent inducer of extracellular matrix synthesis and is well established as a central mediator in the final common pathway of fibrosis associated with progressive kidney diseases (3, 4). Upon ligand stimulation, TGF-β type I (TβRI) and type II (TβRII) receptors form heterotetrameric complexes, by which TβRI is phosphorylated in the GS domain and activated. Smad signaling pathway is well established as a canonical pathway induced by TGF-β1 (5, 6). Receptor-regulated Smads (Smad2 and Smad3) are recruited and activated by the activated TβRI. The phosphorylation in the GS domain (7) and L45 loop (8) of TβRI are crucial for its interaction with receptor-regulated Smads. After phosphorylation, receptor-regulated Smads are rapidly dissociated from TβRI and interact with common Smad (Smad4) followed by nuclear translocation. In addition to the Smad pathway, a recently emerging body of evidence has demonstrated that TGF-β1 also induces various Smad-independent signaling pathways (9–17) by which mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinase (JNK) (18, 19), p38 MAPK (20–22), and extracellular signal-regulated kinase 1/2 (23, 24) can be activated by TGF-β1.TAK1, initially identified as a MAPK kinase kinase 7 (MKKK7 or MAP3K7) in the TGF-β signaling pathway (11, 12), also can be activated by environmental stress (25), proinflammatory cytokines such as IL-1 and TNF-α (26, 27) and lipopolysaccharide (28). For TAK1 activation, phosphorylation at Thr-187 and Ser-192 in the activation loop of TAK1 is essentially required (29–31). TAK1 can transduce signals to several downstream signaling cascades, including the MAPK kinase (MKK) 4/7-JNK cascade, MKK3/6-p38 MAPK cascade, and nuclear factor κB (NF-κB)-inducing kinase-IκB kinase cascade (26–28). A recent report has shown that TAK1 is also activated by agonists of AMP-activated kinase (AMPK) and ischemia, which in turn activates the LKB1/AMPK pathway, a pivotal energy-sensor pathway (32). TAK1 is also involved in Wnt signaling (33). We and others have previously demonstrated that TAK1 is a major mediator of TGF-β1-induced type I collagen and fibronectin expression through activation of the MKK3-p38 MAPK and MKK4-JNK signaling cascades, respectively (34–37). Furthermore, increased expression and activation of TAK1 enhance p38 phosphorylation and promote interstitial fibrosis in the myocardium from 9-day-old TAK1 transgenic mice (37). These data implicate a crucial role of TAK1 in extracellular matrix production and tissue fibrosis. TAK1 is also implicated in regulation of cell cycle (38), cell apoptosis (39–41), and the Smad signaling pathway (42–44). Thus, TAK1 may function as an important regulator and mediator of TGF-β1-induced Smad-dependent and Smad-independent signaling pathways.It has been demonstrated that TAK1 can be activated by the interaction with TAK1-binding protein 1 (TAB1) by in vitro binding assays and in overexpression studies (29–31); however, it is not clear whether TAB1 plays a crucial role in ligand-induced TAK1 activation. In embryonic fibroblasts from TAB1 null mice, IL-1 and TNF-α could induce TAK1-mediated NF-κB and JNK activation (45). TAK1 activation induced by TNF-α, IL-1, and T-cell receptor requires TAB2 or its homologous protein TAB3 (46–50). Although many questions still remain, much progress has been made in understanding the activation mechanism of TAK1 by inflammatory cytokines (46, 47, 51–53). Ligand binding of IL-1 receptor (IL-1R) results in recruitment of MyD88, which serves as an adaptor for IL-1 receptor-associated kinase (IRAK) 1 and 4. Subsequently IRAK1 is hyperphosphorylated and induces interaction with TNF-α receptor-associated factor 6 (TRAF6), resulting in TRAF6 oligomerization. After oligomerization of TRAF6, IRAK1-TRAF6 complex is dissociated from the receptor and associated with TAK1, which is mediated by TAB2 (or TAB3). In this process polyubiquitination of TRAF6 by Ubc13/Uev1A is thought to be critical for the association with TAB2 (or TAB3), which links TAK1 activation (46, 54, 55). In the case of TNF-α stimulation, TNF-α receptors form trimers and recruit adaptor proteins, TRAF2/5, and receptor-interacting protein 1 on the membrane. Ubc13/Uev1A- and TRAF2-dependent polyubiquitination of receptor-interacting protein 1 induce association of TAB2 (or TAB3), which then activates TAK1. Thus, TAB2 is required for ubiquitin-dependent activation of TAK1 by TRAFs. On the other hand, it has been demonstrated that hematopoietic progenitor kinase 1 plays a role as an upstream mediator of TGF-β-induced TAK1 activation, which in turn activates the MKK4-JNK signaling cascade in 293T cells (56, 57). Besides hematopoietic progenitor kinase 1, it has been also suggested that X-linked inhibitor of apoptosis (XIAP) might link TAK1 to TGF-β/BMP receptors through the capability of XIAP to interact with TGF-β/BMP receptors and TAB1 (58). Thus, although various molecules participate in the activation of TAK1, the precise mechanism by which TGF-β1 induces TAK1 activation is incompletely understood. Here, we provide evidence that the association of TAK1 with TGF-β receptors is important for TGF-β1-induced activation of TAK1 in mouse mesangial cells. TGF-β1 stimulation induces interaction of TβRI and TβRII, triggering dissociation of TAK1 from TβRI, and subsequently TAK1 is phosphorylated through TAB1-mediated autophosphorylation, independent of receptor kinase activity of TβRI.     

基于小分子化合物库筛选协同多腺苷二磷酸核糖聚合酶抑制剂杀伤卵巢癌细胞的药物

多腺苷二磷酸核糖聚合酶(poly(ADP-ribose) polymerase, PARP)抑制剂是一类靶向 DNA 修复缺陷癌细胞的新型药物。早期研究表明 PARP 抑制剂取得了令人满意的结果,然而药物治疗后出现的耐药机制尚未完全揭露。因此,有必要寻找更多的靶向药物与PARP 抑制剂联用,以达到杀伤肿瘤细胞的目的。本文基于379种小分子化合物和PARP抑制剂尼拉帕尼(Niraparib)的联合用药筛选,通过细胞增殖实验、克隆存活实验和免疫荧光染色等方法筛选潜在的具有协同PARP抑制剂杀伤卵巢癌细胞的药物。结果表明,其中有8种小分子化合物具有较好的联合用药效果,包括2种已经报道的与PARP抑制剂具有联用效果的小分子化合物STF-118804和Disulfiram。我们从中选取原肌球蛋白受体激酶 A (tropomyosin receptor kinase A,TrKA)的抑制剂GW441756,进行了多种肿瘤细胞的验证以及初步机制的探究。Niraparib和TrKA抑制剂的联合用药显著增加肿瘤细胞对PARP抑制剂的敏感性(P<0.05)。从机制上分析,联合用药组细胞内γH2AX foci的数目显著增加(P<0.05),说明TrKA抑制剂阻碍损伤后细胞的DNA损伤修复能力;同时,联合用药显著降低细胞内同源重组修复(homologous recombination repair,HRR)标志物RAD51 foci(P<0.05)的形成,说明TrKA抑制剂可能通过抑制细胞的HRR效率阻碍细胞的DNA损伤修复。本研究的结果提示,TrKA抑制剂可以作为一种与PARP抑制剂联用杀伤卵巢癌细胞的潜在药物。     

Identification of novel autophagy regulators by a luciferase-based assay for the kinetics of autophagic flux

Macroautophagy (hereafter referred to as autophagy) has recently emerged as an attractive target for the treatment of various degenerative diseases and cancer. The discovery of effective pharmaceutical regulators of autophagy has, however, been hindered by a lack of feasible assay systems for autophagic flux. Here, we present a luciferase-based reporter assay that measures autophagic flux in real time in living cells and demonstrate that this assay system is apt for the detection of dose- and stimulus-dependent differences in autophagy kinetics. Furthermore, by screening a small molecule kinase inhibitor library containing 80 compounds we identified 12 compounds as inducers of autophagic flux. Importantly, six inhibitors of the class I phosphoinositide 3-kinase – protein kinase B – mammalian target of rapamycin complex 1 axis, the central signaling pathway repressing autophagy, scored as autophagy inducers adequately validating the screen. We conclude that the assay system presented here allows easy and rapid monitoring of autophagy kinetics and is suitable for screening of small molecule libraries.     

A novel and efficient ligand-based virtual screening approach using the HWZ scoring function and an enhanced shape-density model

In this work, we extend our previous ligand shape-based virtual screening approach by using the scoring function Hamza–Wei–Zhan (HWZ) score and an enhanced molecular shape-density model for the ligands. The performance of the method has been tested against the 40 targets in the Database of Useful Decoys and compared with the performance of our previous HWZ score method. The virtual screening results using the novel ligand shape-based approach demonstrated a favorable improvement (area under the receiver operator characteristics curve AUC?=?.89?±?.02) and effectiveness (hit rate HR^1%?=?53.0%?±?6.3 and HR^10%?=?71.1%?±?4.9). The comparison of the overall performance of our ligand shape-based method with the highest ligand shape-based virtual screening approach using the data fusion of multi queries showed that our strategy takes into account deeper the chemical information of the set of active ligands. Furthermore, the results indicated that our method are suitable for virtual screening and yields superior prediction accuracy than the other study derived from the data fusion using five queries. Therefore, our novel ligand shape-based screening method constitutes a robust and efficient approach to the 3D similarity screening of small compounds and open the door to a whole new approach to drug design by implementing the method in the structure-based virtual screening.     

An aminopyridazine-based inhibitor of a pro-apoptotic protein kinase attenuates hypoxia-ischemia induced acute brain injury

Death associated protein kinase (DAPK) is a calcium and calmodulin regulated enzyme that functions early in eukaryotic programmed cell death, or apoptosis. To validate DAPK as a potential drug discovery target for acute brain injury, the first small molecule DAPK inhibitor was synthesized and tested in vivo. A single injection of the aminopyridazine-based inhibitor administered 6 h after injury attenuated brain tissue or neuronal biomarker loss measured, respectively, 1 week and 3 days later. Because aminopyridazine is a privileged structure in neuropharmacology, we determined the high-resolution crystal structure of a binary complex between the kinase domain and a molecular fragment of the DAPK inhibitor. The co-crystal structure describes a structural basis for interaction and provides a firm foundation for structure-assisted design of lead compounds with appropriate molecular properties for future drug development.     

Parallel chemical genetic and genome-wide RNAi screens identify cytokinesis inhibitors and targets

Cytokinesis involves temporally and spatially coordinated action of the cell cycle and cytoskeletal and membrane systems to achieve separation of daughter cells. To dissect cytokinesis mechanisms it would be useful to have a complete catalog of the proteins involved, and small molecule tools for specifically inhibiting them with tight temporal control. Finding active small molecules by cell-based screening entails the difficult step of identifying their targets. We performed parallel chemical genetic and genome-wide RNA interference screens in Drosophila cells, identifying 50 small molecule inhibitors of cytokinesis and 214 genes important for cytokinesis, including a new protein in the Aurora B pathway (Borr). By comparing small molecule and RNAi phenotypes, we identified a small molecule that inhibits the Aurora B kinase pathway. Our protein list provides a starting point for systematic dissection of cytokinesis, a direction that will be greatly facilitated by also having diverse small molecule inhibitors, which we have identified. Dissection of the Aurora B pathway, where we found a new gene and a specific small molecule inhibitor, should benefit particularly. Our study shows that parallel RNA interference and small molecule screening is a generally useful approach to identifying active small molecules and their target pathways.     

Design of two new chemotypes for inhibiting the Janus kinase 2 by scaffold morphing

JAK2 is a target of high interest in chronic myeloproliferative disorders drug research. Starting from a screening hit, two new JAK2 inhibitor chemotypes were designed by scaffold morphing. The prototype compounds of these new series showed nanomolar inhibition of the kinase.     

High-throughput screening assay for identification of small molecule inhibitors of Aurora2/STK15 kinase

STK15/Aurora2 is a centrosome-associated serine/threonine kinase, the protein levels and kinase activity of which rise during G2 and mitosis. STK15 overexpression induces tumorigenesis and is amplified in various human cancers and tumor cell lines. Thus, STK15 represents an important therapeutic target for small molecule inhibitors that would disrupt its activity and block cell proliferation. The availability of a robust and selective small molecule inhibitor would also provide a useful tool for identification of the potential role of STK15 in cell cycle regulation and tumor development. The authors report the development of a novel, fast, simple microplate assay for STK15 activity suitable for high-throughput screening. In the assay, gamma-(33)P-ATP and STK15 were incubated in a myelin basic protein (MBP)-coated FlashPlate(R) to generate a scintillation signal. The assay was reproducible, the signal-to-noise ratio was high (11) and the Z' factor was 0.69. The assay was easily adapted to a robotic system for drug discovery programs targeting STK15. The authors also demonstrate that STK15 is regulated by phosphorylation and the N-amino terminal domain of the protein. Treatment with phosphatase inhibitors (okadaic acid) or deletion of the N-amino terminal domain results in a significant increase in the enzymatic activity.     

LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5

The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9 kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co-expressed and activated LmxMPK4 in a dose-dependent manner. To our knowledge this is the first time that an in vitro activator of an essential Leishmania MAP kinase was identified and our findings form the basis for the development of drug screening assays to identify small molecule inhibitors of LmxMPK4 in the search for new therapeutic drugs against leishmaniasis.     

Enamel Matrix Derivative Inhibits Adipocyte Differentiation of 3T3-L1 Cells via Activation of TGF-βRI Kinase Activity

Enamel matrix derivative (EMD), an extract of fetal porcine enamel, and TGF-β can both suppress adipogenic differentiation. However, there have been no studies that functionally link the role of EMD and TGF-β in vitro. Herein, we examined whether TGF-β signaling contributes to EMD-induced suppression of adipogenic differentiation. Adipogenesis was studied with 3T3-L1 preadipocytes in the presence of SB431542, an inhibitor of TGF-βRI kinase activity. SB431542 reversed the inhibitory effect of EMD on adipogenic differentiation, based on Oil Red O staining and mRNA expression of lipid regulated genes. SB431542 also reduced EMD-stimulated expression of connective tissue growth factor (CTGF), an autocrine inhibitor of adipogenic differentiation. Moreover, short interfering (si)RNAs for CTGF partially reversed the EMD-induced suppression of lipid regulated genes. We conclude that the TGF-βRI - CTGF axis is involved in the anti-adipogenic effects of EMD in vitro.     

Establishment of the active catalytic domain of human PDGFRβ tyrosine kinase-based ELISA assay for inhibitor screening

Tyrosine kinases are emerging as frequent targets of primary oncogenic events and therefore represent an optimal focus of therapeutic intervention. In an effort towards therapeutic PDGFR inactivation, we expressed the catalytic domain of PDGFRβ as a soluble active kinase using Bac-to-Bac expression system, and studied the correlations between PDGFRβ activity and enzyme concentration, ATP concentration, substrate concentration and divalent cation type. And a convenient, effective and non-radioactive ELISA screening model is then established for identification of the potential inhibitors targeting PDGFRβ kinase. Of 500 RTK target-based compounds, TKI-30 was identified as a small molecule potential inhibitor of PDGFRβ  (IC₅₀ = 0.34 μM). Further studies indicated that TKI-30 blocked PDGF-BB-induced autophosphorylation of PDGFRβ in a dose-dependent manner in Swiss 3T3 cells and human umbilical vein smooth muscle cells (HUVSMCs). Moreover, it dose-dependently suppressed the PDGF-BB-induced proliferation in HUVSMCs and tube formation of HUVEC. Our data collectively indicated that PDGFRβ-based ELISA assay is a new method available for screening inhibitors targeting PDGFRβ kinase and TKI-30 is a potential novel anti-cancer agent worthy of being further investigated.     

From cosubstrate similarity to inhibitor diversity--inhibitors of ADP-ribosyltransferases from kinase inhibitor screening

ADP-ribosyltransferases (ADP-RTs) use NAD(+) to transfer an ADP-ribosyl group to target proteins. Although some ADP-RTs are bacterial toxins only few inhibitors are known. Here we present the development of fluorescence-based assays and a focussed library screening using kinase inhibitors as a new approach towards inhibitors of ADP-RTs. Different screening setups were established using surrogate small molecule substrates or the quantitation of the cofactor NAD(+). Proof-of-principle screening experiments were performed using a kinase inhibitor library in order to target the NAD(+) binding pockets. This led to the discovery of structurally different lead inhibitors for the mono-ADP-ribosyltransferases Mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1, C3bot toxin from Clostridium botulinum and CDTa from Clostridium difficile. The interaction of the inhibitors with the toxin proteins was analyzed by means of docking and binding free energy calculations. Binding at the nicotinamide subpocket, which shows a significant difference in the three enzymes, is used to explain the selectivity of the identified inhibitors and offers an opportunity for further development of potent and selective inhibitors.     

Tyrosine kinases. New target of anticancer therapy

Recently, clinical studies of new drugs development to target specific forms of cancer were reported. Herceptin, a monoclonal antibody against the Her2/neu receptor tyrosine kinase, prolonged the survival of women with Her2/neu positive metastatic breast cancer. STI571, a small molecule inhibitor of the BCR/ABL, c-Kit and platelet derived growth factor receptor tyrosine kinase, produced pronounced clinical responses in patients with BCR/ABL positive chronic myeloid leukemia and c-Kit positive gastrointestial stromal tumors. In order to consider the use of the inhibitor of tyrosine kinases activity as anticancer drug, their mechanisms of the oncogenic activation and their impact on tumor transformation should be studied. The treatment with tyrosine kinase inhibitors such as STI571 or herceptin was a spectacular clinical success which stimulated research on the structure and function of both kinases and their inhibitors.