An impedimetric immunosensor for determination of porcine epidemic diarrhea virus based on the nanocomposite consisting of molybdenum disulfide/reduced graphene oxide decorated with gold nanoparticles.

An electrochemical immunosensor for the determination of porcine epidemic diarrhea virus (PEDV) is described. It was manufactured by using gold nanoparticles/molybdenum disulfide/reduced graphene oxide nanocomposites modified on the surface of a glassy carbon electrode (GCE). The independently developed monoclonal antibody of PEDV-2C11 was immobilized on the modified electrode at site of gold nanoparticles provided in the nanocomposites. The concentration of PEDV was quantified by measuring the changes in the charge transfer resistance of the electrode before and after the immunoreaction between antigen-antibody by using hexacyanoferrate(II)/(III) as the redox probe. The frequency range was 10-1 to 105 Hz at the amplitude of 10 mV and an applied potential of + 0.180 V. Based on the immunoreaction between PEDV antigen and PEDV-2C11 antibody in 0.1 M phosphate buffer containing 0.1 M KCl at 37.5 °C for 140 min, the relative change in impedance was proportional to the logarithmic value of PEDV concentrations in the range of 82.5 to 1.65 × 104 TCID50 mL-1. Good reproducibility, stability, and specificity of the proposed immunosensor were obtained. It was successfully applied to the determination of PEDV in the spiked sample. Graphical abstractSchematic representation. a The preparation of AuNP/MoS2/rGO composites. b Representation of modification and functioning of the label-free electrochemical immunosensor and the electrochemical impedimetric response obtained before (a) and after (b) incubation of PEDV.

Dissecting the phenotypes of Plk1 inhibition in cancer cells using novel kinase inhibitory chemical CBB2001.

Polo-like kinase 1 (Plk1) is a mitotic serine/threonine kinase and its kinase activity is closely interrelated to cell cycle progression, various types of cancer development and often correlates with poor prognosis. Thus, it is of prime importance to characterize the phenotypes of Plk1 inhibition in cells for drug development and clinical application. Here, we report a novel kinase inhibitory chemical, CBB2001, which specifically inhibited Plk1 kinase activity in vitro with an IC(50) of 0.39 µM. In cervical carcinoma HeLa cells, we found that treatment of CBB2001 caused mitotic cell cycle arrest (EC(50)=0.72 µM) and induction of 'polo' cells (EC(50)=0.32 µM). Interestingly, the cell cycle arrest induced by CBB2001 was associated with accumulation of Plk1 (EC(50)=0.61 µM) and Geminin (EC(50)=0.43 µM) proteins, but distinct from the phenotypes induced by Aurora kinase inhibitors. The inhibitory effects of CBB2001 were phenocopied by RNA interferences of Plk1. We also confirmed the cell cycle inhibitory effects of CBB2001 in other cancer cells. Moreover, CBB2001 inhibited the growth of HeLa cells with an IC(50) of 0.85 µM in MTT assays, which is better than that of reported Plk1 inhibitory chemicals ON01910 (IC(50)=6.46 µM) and LFM-A13 (IC(50)=37.36 µM). CBB2001 also inhibited mouse xenograft tumor growth. Furthermore, CBB2001 inhibited mitotic exit and delayed degradation of APC/C substrates, Geminin, Cyclin B1 and Aurora A. These specific phenotypes may serve as specific features for Plk1 inhibition and for Plk1-based clinic trials.

Identification of allosteric PIF-pocket ligands for PDK1 using NMR-based fragment screening and 1H-15N TROSY experiments.

Aberrant activation of the phosphoinositide 3-kinase pathway because of genetic mutations of essential signalling proteins has been associated with human diseases including cancer and diabetes. The pivotal role of 3-phosphoinositide-dependent kinase-1 in the PI3K signalling cascade has made it an attractive target for therapeutic intervention. The N-terminal lobe of the 3-phosphoinositide-dependent kinase-1 catalytic domain contains a docking site which recognizes the non-catalytic C-terminal hydrophobic motifs of certain substrate kinases. The binding of substrate in this so-called PDK1 Interacting Fragment pocket allows interaction with 3-phosphoinositide-dependent kinase-1 and enhanced phosphorylation of downstream kinases. NMR spectroscopy was used to a screen 3-phosphoinositide-dependent kinase-1 domain construct against a library of chemically diverse fragments in order to identify small, ligand-efficient fragments that might interact at either the ATP site or the allosteric PDK1 Interacting Fragment pocket. While majority of the fragment hits were determined to be ATP-site binders, several fragments appeared to interact with the PDK1 Interacting Fragment pocket. Ligand-induced changes in 1H-15N TROSY spectra acquired using uniformly 15N-enriched PDK1 provided evidence to distinguish ATP-site from PDK1 Interacting Fragment-site binding. Caliper assay data and 19F NMR assay data on the PDK1 Interacting Fragment pocket fragments and structurally related compounds identified them as potential allosteric activators of PDK1 function.

Selectivity-determining residues in Plk1.

Polo-like kinase 1 is an important regulator of cell cycle progression whose over-expression is often associated with oncogenesis. Polo-like kinase 1 hence represents an attractive target for cancer intervention. BI 2536 (Boehringer Ingelheim, Ingelheim, Germany), a Polo-like kinase 1 inhibitor currently in clinical trials, exhibits nanomolar potency against Polo-like kinase isoforms and high selectivity against other kinases. We have previously published the crystal structures of the Polo-like kinase 1 domain in complex with AMPPNP and an Aurora A inhibitor. In this work, we present the co-crystal structure of Polo-like kinase 1 with BI 2536. The structure, in combination with selectivity data for BI 2536 and related compounds, illustrates important features for potency and selectivity. In particular, we show that the methoxy group of BI 2536 is an important specificity determinant against non-Polo-like kinases by taking advantage of a small pocket generated by Leu 132 in the hinge region of Polo-like kinase 1. The work presented here provides a framework for structure-based drug design of Polo-like kinase 1-specific inhibitors.

Structure of the catalytic domain of human polo-like kinase 1.

Polo-like kinase 1 (Plk1) is an attractive target for the development of anticancer agents due to its importance in regulating cell-cycle progression. Overexpression of Plk1 has been detected in a variety of cancers, and expression levels often correlate with poor prognosis. Despite high interest in Plk1-targeted therapeutics, there is currently no structure publicly available to guide structure-based drug design of specific inhibitors. We determined the crystal structures of the T210V mutant of the kinase domain of human Plk1 complexed with the nonhydrolyzable ATP analogue adenylylimidodiphosphate (AMPPNP) or the pyrrolo-pyrazole inhibitor PHA-680626 at 2.4 and 2.1 A resolution, respectively. Plk1 adopts the typical kinase domain fold and crystallized in a conformation resembling the active state of other kinases. Comparison of the kinetic parameters determined for the (unphosphorylated) wild-type enzyme, as well as the T210V and T210D mutants, shows that the mutations primarily affect the kcat of the reaction, with little change in the apparent Km for the protein or nucleotide substrates (kcat = 0.0094, 0.0376, and 0.0049 s-1 and Km(ATP) = 3.2, 4.0, and 3.0 microM for WT, T210D, and T210V, respectively). The structure highlights features of the active site that can be exploited to obtain Plk1-specific inhibitors with selectivity over other kinases and Plk isoforms. These include the presence of a phenylalanine at the bottom of the ATP pocket, combined with a cysteine (as opposed to the more commonly found leucine) in the roof of the binding site, a pocket created by Leu132 in the hinge region, and a cluster of positively charged residues in the solvent-exposed area outside of the adenine pocket adjacent to the hinge region.