A microtitre chemiluminescence sensor for detection of pyrethroids based on dual-dummy-template molecularly imprinted polymer and computational simulation.

The residues of pyrethroids in foods of animal origin are dangerous to the consumers, so this study presented a chemiluminescence sensor for determination of pyrethroids in chicken samples. A dual-dummy-template molecularly imprinted polymer capable of recognizing 10 pyrethroids was synthesized. The results of computation simulation showed that the specific 3D conformations of the templates had important influences on the polymer' recognition ability. The polymer was used to prepare a sensor on conventional 96-well microplates, and the sample solution was added into the wells for direct absorption. The absorbed analytes were initiated with the bis(2,4,6-trichlorophenyl)oxalate-H2 O2 -imidazole system, and the chemiluminescence intensity was used for analyte quantification. Results showed that one assay was finished within 12 min, and this sensor could be reused four times. The limits of detection for the 10 analytes were in the range o0.3-6.0 pg/ml, and the recoveries from the standards of fortified blank chicken samples were in the range 70.5-99.7%.

Dummy molecularly imprinted polymer based microplate chemiluminescence sensor for one-step detection of Sudan dyes in egg.

The objective of this study is to report a molecularly imprinted polymer-based chemiluminescence method for determination of Sudan dyes. A dummy-template molecularly imprinted polymer capable of recognizing seven Sudan dyes was first synthesized and its recognition mechanism was studied by using computation simulation method. The polymer was coated in the wells of conventional microplate to prepare a chemiluminescence sensor and the assay process consisted of only one sample-loading step prior to signal acquisition. The optimized sensor was used to determine the seven dyes in egg yolk and the results were confirmed with a high performance liquid chromatography. Results showed that this sensor achieved ultrahigh sensitivity (1.0-5.0 pg/mL), rapid assay process (10 min) and satisfactory recovery (70.5%-92.2%). Furthermore, the sensor could be reused for 5 times. Therefore, this sensor could be used as a useful tool for screening the residues of Sudan dyes in egg.

Insights into targeting NEMO for pharmacological regulation.

NF-κB essential modulator (NEMO), the non-catalytic regulatory subunit of the IκB kinase (IKK) complex, is essential for the canonical NF-κB activation pathway. It has been identified as a molecular platform for assembling the IKK complex and recruiting upstream IKK activators. However, the exact mechanism for regulating IKK activity has still remained elusive. This review describes structural and functional characteristics of NEMO protein, covers the feasible polyubiquitin-mediated NEMO-dependent IKK complex activation mechanism, and briefly summarizes some proteins that bind to NEMO for enhancing or suppressing IKK complex activity. Furthermore, it also discusses several bioactive compounds that disrupt the protein-protein interactions (PPI) involving NEMO, as these PPI may act as alternative routes to develop novel pharmacological agents for inflammation and cancer therapy.

Recent advances in the structure-based and ligand-based design of IKKß inhibitors as anti-inflammation and anti-cancer agents.

NF-κB is a significant transcription factor that regulates the expression of various pro-survival genes. IKK is a crucial protein kinase that activates NF-κB translocating from cytoplasm to nucleus for DNA binding. It is composed of three subunits, IKKα, IKKß, IKKγ (NEMO), where IKKα and IKKß are catalytic subunits, and IKKγ is the regulatory subunit. Many diseases, such as Hodgkin's disease, Hepatitis-associated hepatocellular carcinoma, colorectal cancer, prostate cancer, rheumatoid arthritis and inflammatory bowel disease, are related to IKK and NF-κB. So far, various IKK inhibitors targeting the ATP binding site have been identified through high throughput screening, rational design or in silico methods, however, only three of them (CHS-828, EB-1627 and IMD-1041) have been under clinical studies, indicating the strategy for the design of IKK inhibitors need to be reinspected. Besides ATP-competitive inhibitors, several other inhibitors have also been disclosed recently, which provide novel concepts to the discovery of IKK inhibitors. In this review, we focus on two parts: 1) the chemotypes and binding patterns of the traditional ATP-competitive IKK inhibitors; 2) novel strategies for the identification of non-ATP-competitive IKK inhibitors as NF-κB modulators. Through these discussions we hope to present inspirations for the development of new IKK inhibitors.

Insight into the intermolecular recognition mechanism between Keap1 and IKKß combining homology modelling, protein-protein docking, molecular dynamics simulations and virtual alanine mutation.

Degradation of certain proteins through the ubiquitin-proteasome pathway is a common strategy taken by the key modulators responsible for stress responses. Kelch-like ECH-associated protein-1(Keap1), a substrate adaptor component of the Cullin3 (Cul3)-based ubiquitin E3 ligase complex, mediates the ubiquitination of two key modulators, NF-E2-related factor 2 (Nrf2) and IκB kinase ß (IKKß), which are involved in the redox control of gene transcription. However, compared to the Keap1-Nrf2 protein-protein interaction (PPI), the intermolecular recognition mechanism of Keap1 and IKKß has been poorly investigated. In order to explore the binding pattern between Keap1 and IKKß, the PPI model of Keap1 and IKKß was investigated. The structure of human IKKß was constructed by means of the homology modeling method and using reported crystal structure of Xenopus laevis IKKß as the template. A protein-protein docking method was applied to develop the Keap1-IKKß complex model. After the refinement and visual analysis of docked proteins, the chosen pose was further optimized through molecular dynamics simulations. The resulting structure was utilized to conduct the virtual alanine mutation for the exploration of hot-spots significant for the intermolecular interaction. Overall, our results provided structural insights into the PPI model of Keap1-IKKß and suggest that the substrate specificity of Keap1 depend on the interaction with the key tyrosines, namely Tyr525, Tyr574 and Tyr334. The study presented in the current project may be useful to design molecules that selectively modulate Keap1. The selective recognition mechanism of Keap1 with IKKß or Nrf2 will be helpful to further know the crosstalk between NF-κB and Nrf2 signaling.

Synthesis and bioevaluation of a series of α-pyrone derivatives as potent activators of Nrf2/ARE pathway (part I).

When exposed to electrophiles, human colorectal cancer cells (HCT116) counteract oxidative stress through activating NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway. To identify new activators, luciferase reporter gene assay was used to screen in-house database of our laboratory, leading to a novel α-pyrone compound 1 as a hit. 2 with 2-fluoro phenyl group exhibited the strongest ARE inductive activity in the first round structure-activity relationship (SAR) study. Biological studies showed the compound induced nuclear translocation of Nrf2 preceded by phosphorylation of ERK1/2. The data encouraged us to use 2 as lead and 20 derivatives were synthesized to discuss a more detailed SAR, leading to a more potent compound 9, which can be the starting compound for further modification.

Novel IKKß inhibitors discovery based on the co-crystal structure by using binding-conformation-based and ligand-based method.

IκB kinase ß (IKKß), an attractive anti-inflammation and anti-cancer target, plays a crucial role in the activation of NF-κB signalling pathway. To identify novel IKKß inhibitors, we combined structure-based and ligand-based methods based on the co-crystal structure of IKKß. According to the chemical similarity, 162 reported IKKß inhibitors were divided into five classes. For each class, a 3D pharmacophore model was established based on the binding conformations of the compounds. The validated models were further used in virtual screening. Twelve drugable compounds were retained for biological test, resulting in two novel inhibitors with IC50 values lower than 10 µM. Compared to other models, our method considers the crystal structure of IKKß for the first time.