Development and comparison of nonradioactive in vitro kinase assays for NIMA-related kinase 2

NIMA (never in mitosis arrest)-related kinase 2 (Nek2) is a serine/threonine kinase required for centrosome splitting and bipolar spindle formation during mitosis. Currently, two in vitro kinase assays are commercially available: (i) a radioactive assay from Upstate Biotechnology and (ii) a nonradioactive fluorescence resonance energy transfer (FRET) assay from Invitrogen. However, due to several limitations such as radioactive waste management and lower sensitivity, a need for more robust nonradioactive assays would be ideal. Accordingly, we have developed four quantitative and sensitive nonradioactive Nek2 in vitro kinase assays: (i) a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) using peptides identified from a physiologically relevant protein substrate, (ii) DELFIA using Nek2 itself, (iii) a homogeneous time-resolved FRET assay termed LANCE, and (iv) A method of detecting phosphorylated products by HPLC. The DELFIA and LANCE assays are robust in that they generated more than 10-fold and 20-fold increases in signal-to-noise ratios, respectively, and are amenable to robotic high-throughput screening platforms. Validation of all four assays was confirmed by identifying a panel of small molecule ATP competitive inhibitors from an internal corporate library. The most potent compounds consistently demonstrated less than 100 nM activity regardless of the assay format and therefore were complementary. In summary, the Nek2 in vitro time-resolved FRET kinase assays reported are sensitive, quantitative, reproducible and amenable to high-throughput screening with improved waste management over radioactive assays.     

Imidazo[4,5-d]thiazolo[5,4-b]pyridine based inhibitors of IKK2: synthesis, SAR, PK/PD and activity in a preclinical model of rheumatoid arthritis

The synthesis, structure-activity relationships (SAR) and biological evaluation of thiazole based tricyclic inhibitors of IKK2 are described. Compound 9 was determined to be orally efficacious in a murine model of rheumatoid arthritis.     

Novel tricyclic inhibitors of IKK2: discovery and SAR leading to the identification of 2-methoxy-N-((6-(1-methyl-4-(methylamino)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-7-yl)pyridin-2-yl)methyl)acetamide (BMS-066)

The synthesis, structure-activity relationships (SAR), and biological results of pyridyl-substituted azaindole based tricyclic inhibitors of IKK2 are described. Compound 4m demonstrated potent in vitro potency, acceptable pharmacokinetic and physicochemical properties, and efficacy when dosed orally in a mouse model of inflammatory bowel disease.     

Development of nonradioactive microtiter plate assays for nuclease activity

We have developed two microtiter plate assays for the detection of DNA cleavage by nucleases, using 3'-biotinylated oligonucleotide substrates. In the covalently linked oligonucleotide nuclease assay (CLONA), the biotinylated substrates are phosphorylated at the 5' end to facilitate their covalent immobilization on CovaLink NH plates. The cleavage of the covalently immobilized substrate by nucleases results in biotin release. The uncleaved substrate molecules are detected with an enzyme-avidin conjugate. The affinity-linked oligonucleotide nuclease assay (ALONA) makes use of substrates with a digoxigenin on the 5' end of the 3'-biotinylated DNA strand. The substrate binds specifically to the wells of streptavidin-coated microtiter plates, in which the nuclease reaction takes place. Uncleaved substrate retains the digoxigenin label, which is detected with an enzyme-labeled anti-digoxigenin antibody. We assessed the efficiency of these two assays by measuring S1 nuclease and DNase I activities, and the inhibitory effect of EDTA and aurintricarboxylic acid on the reaction. Both methods are more convenient than the standard radioactive nuclease assay and are suitable for high-throughput screening of potential nuclease inhibitors, nucleases, and catalytic antibodies. The ALONA assay was found to be more sensitive than the CLONA assay, with a performance similar to that of the standard nuclease assay.     

A recessive mutant cell line with a constitutive IkappaB kinase activity

To search for negative regulatory components of the NF-kappaB activation pathways, we mutagenized Rat-1 fibroblasts and established a stable mutant cell line with a constitutive NF-kappaB activity. This mutant cell line, designated as TK26, showed permanently elevated I kappa B kinase (IKK) activity and a genetically recessive phenotype revealed by somatic cell hybridization between TK26 and Rat-1. Our results suggested that lack of a negative regulation of IKK could lead to permanent NF-kappaB activation. The TK26 cell line will be useful to genetically identify a component necessary for keeping the IKK complex under an inactive form in resting cells.     

Discovery of imidazo[1,2-b]pyridazines as IKKβ inhibitors. Part 2: improvement of potency in vitro and in vivo

We have increased the potency of imidazo[1,2-b]pyridazine derivatives as IKKβ inhibitors with two strategies. One is to enhance the activity in cell-based assay by adjusting the polarity of molecules to improve permeability. Another is to increase the affinity for IKKβ by the introduction of additional substituents based on the hypothesis derived from an interaction model study. These improved compounds showed inhibitory activity of TNFα production in mice.     

The human IKKbeta subunit kinase domain displays CK2-like phosphorylation specificity

NF-κB activation in response to pro-inflammatory stimuli relies upon phosphorylation of IκBα at serines 32 and 36 by the β subunit of the IκB kinase complex (IKK). In this study, we build upon the observation that highly purified human IKKβ subunit preparations retain this specificity in vitro. We show that IKKβ constructs that lack their carboxy-terminus beginning at the leucine zipper motif fail to phosphorylate IκBα at Ser-32 and Ser-36. Rather, these constructs, which contain the entire IKKβ subunit kinase domain, phosphorylate serine and threonine residues contained within the IκBα carboxy-terminal PEST region. Furthermore, removal of the leucine zipper and helix-loop-helix regions converts IKKβ to monomer. We propose that the helix-loop-helix of the human IKKβ subunit is necessary for restricting substrate specificity toward Ser-32 and Ser-36 in IκBα and that in the absence of its carboxy-terminal protein structural motifs the human IKKβ subunit kinase domain exhibits a CK2-like phosphorylation specificity.     

NF-κB、IκB、IKK在非小细胞肺癌中的表达及意义

目的检测非小细胞肺癌(non-small cell lung cancer,NSCLC)中NF-κB P65、p-IκBα(IκBα磷酸化)、p-IKKβ(IKKβ磷酸化)的表达情况及其与NSCLC临床特征的关系。方法采用免疫组化Elivision法检测NF-κB P65、p-IκBα、p-KKβ在56例NSCLC中表达情况,以20例癌旁组织作为对照。结果在NSCLC中NF-κB P65、p-IκBα、p-IKKβ的表达阳性率分别为83.9%(47/56)、55.7%(31/56)、69.6%(39/56),癌旁组织三者分别为20%(4/20)、25%(5/20)、30%(6/20),NF-κB P65、p-IκBα、p-IKKβ的表达与吸烟史、TNM分期、淋巴结转移相关,差异有统计学意义(P<0.05)。结论 NF-κB P65、p-IκBα、p-IKKβ高表达与NSCLC的发生、发展起着重要作用。     

针对核因子-κBP65基因的短发夹干扰RNA逆转录病毒载体构建与鉴定

目的：构建针对人核因子κB亚基P65基因mRNA的短发夹干扰RNA（shRNA）逆转录病毒表达载体,并探讨小干扰RNA（siRNA）靶向抑制NF—κB P65基因表达的作用。方法：根据shRNA设计原则,在人NF-κB P65全长序列中选取含19个核苷酸靶序列,设计形成siRNA的DNA模板并克隆到shRNA表达载体pSUPER．retro．neo中,构建针对NF—κB P65基因的shRNA表达载体。经293A细胞包装,并感染NIH3T3细胞进行病毒滴度测定后,感染THP-1细胞。分别采用RT—PCR和Western blot从mRNA和蛋白水平检测干扰效果。结果：限制性酶切和基因测序证实针对人NF-κB P65亚基的shRNA表达逆转录病毒载体成功构建;其感染THP-1细胞后,NF—κB P65的mRNA和蛋白表达明显抑制。结论：成功构建了NF-κB P65 shRNA逆转录病毒表达载体,该载体能高效感染THP-1并明显抑制NF—κB P65的表达。     

Celastrol: Molecular targets of Thunder God Vine

Celastrol, a quinone methide triterpene, is a pharmacologically active compound present in Thunder God Vine root extracts used as a remedy of inflammatory and autoimmune diseases, e.g. rheumatoid arthritis. Celastrol is one of the most promising medicinal molecules isolated from the plant extracts of traditional medicines. Molecular studies have identified several molecular targets which are mostly centered on the inhibition of IKK-NF-κB signaling. Celastrol (i) inhibits directly the IKKα and β kinases, (ii) inactivates the Cdc37 and p23 proteins which are co-chaperones of HSP90, (iii) inhibits the function of proteasomes, and (iv) activates the HSF1 and subsequently triggers the heat shock response. It seems that the quinone methide structure present in celastrol can react with the thiol groups of cysteine residues, forming covalent protein adducts. In laboratory experiments, celastrol has proved to be a potent inhibitor of inflammatory responses and cancer formation as well as alleviating diseases of proteostasis deficiency. Celastrol needs still to pass several hurdles, e.g. ADMET assays, before it can enter the armoury of western drugs.     

Small molecule inhibitors of IκB kinase β: A chip-based screening and molecular docking simulation

A chip-based screening system for IκB kinase β (IKKβ) has been developed by physically immobilizing the substrate IκBα on a glass matrix using a calixarene linker. Phosphorylation of IκBα by IKKβ and ATP was quantitated using a fluorescently labeled antibody. Using this efficient assay system a chemical library of 2000 bioactive compounds was screened against IKKβ and four were identified as good inhibitors, namely, aurintricarboxylic acid, diosmin, ellagic acid, and hematein. None of them have been reported to be an inhibitor of IKKβ although they were implicated in various NFκB-mediated biological processes. Our enzyme-based assay showed that IC50 of the four inhibitors is comparable with that of IKK-16, a previously known strong inhibitor. Molecular docking simulation shows that the hydrophobic moiety of an inhibitor interacts with the four hydrophobic residues (Leu21, Val29, Val152, and Ile165) of the active site. The MM-PBSA calculation suggests that these hydrophobic interactions appear to be the predominant contributor to the binding free energy. As IKKβ is ubiquitously expressed in various cell types and executes many biological functions, the enzyme and cell specificity of the four inhibitors need to be rigorously tested before accepted as a drug candidate.     

Overexpression of PPM1B inhibited chemoresistance to temozolomide and proliferation in glioma cells

Protein phosphatase magnesium-dependent 1B (PPM1B) functions as IKKβ phosphatases to terminate nuclear factor kappa B (NF-κB) signaling. NF-κB signaling was constitutively activated in glioma cells. At present, little is known about the role of PPM1B in glioma. In the current study, we found that the expression of PPM1B was reduced in glioma tissues and cells, and decreased expression of PPM1B was related to poor overall survival of patients. Overexpression of PPM1B inhibited the proliferation and promoted apoptosis of glioma cells. Moreover, PPM1B overexpression reduced the phosphorylation of IKKβ and inhibited the nuclear localization of NF-κBp65. PDTC, an inhibitor of NF-κB signaling, reversed PPM1B-knockdown-induced cell proliferation. Furthermore, overexpression of PPM1B enhanced the sensitivity of glioma cells to temozolomide. In vivo experiments showed that overexpression of PPM1B could inhibit tumor growth, improve the survival rate of nude mice, and enhance the sensitivity to temozolomide. In conclusion, PPM1B suppressed glioma cell proliferation and the IKKβ-NF-κB signaling pathway, and enhanced temozolomide sensitivity of glioma cells.