A quantitative affinity-based technique for the identification of potential lead compounds.

Here, we describe the development and validation of a quantitative analytical method for rapid evaluation of protein-compound interactions. The method uses size-exclusion chromatography in a 96-well format with liquid chromatography/mass spectrometry (qSEC-LC/MS) by which the amount of a compound that was originally in complex with a target protein is determined as an indicator of the binding affinity. Proof of concept of this new analytical approach was performed using a thrombin-inhibitor model. The results showed that the qSEC-LC/MS could be developed into an effective affinity-based analytical technique, despite a few limitations such as difficulty in determining the K(d) value accurately.

A fluorescence polarization-based assay for the identification and evaluation of calmodulin antagonists.

A fluorescence polarization (FP) assay was developed to identify calmodulin (CaM) antagonists. A fluorescent tracer was newly designed by covalently labeling N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which is a well-known CaM antagonist, with the Cy5 dye. In the FP assay, the tracer (Cy5-W-7) was bound to CaM with a dissociation constant (K(d)) of 6.5 microM and demonstrated efficient competitive activity with other CaM antagonists, including W-7, chlorpromazine, trifluoperazine, W-5, and clozapine, indicating that Cy5-W-7 binds to the ligand-binding site of CaM in a specific manner. The inhibitory activities of Cy5-W-7 and CaM antagonists were subsequently measured by the CaM-dependent calcineurin phosphatase assay, and the results were confirmed with those of the FP assays. In addition, assay optimization for high-throughput screening was performed, and a Z' factor of 0.7 was achieved in a 1536-well format. The FP assay was found to be a simple and reliable alternative to conventional assays for evaluating CaM antagonists.

Receptor activator of NF-kappaB ligand (RANKL) activates TAK1 mitogen-activated protein kinase kinase kinase through a signaling complex containing RANK, TAB2, and TRAF6.

The receptor activator of NF-kappaB (RANK) and its ligand RANKL are key molecules for differentiation and activation of osteoclasts. RANKL stimulates transcription factors AP-1 through mitogen-activated protein kinase (MAPK) activation, and NF-kappaB through IkappaB kinase (IKK) activation. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is essential for activation of these kinases. In the interleukin-1 signaling pathway, TAK1 MAPK kinase kinase (MAPKKK) mediates MAPK and IKK activation via interaction with TRAF6, and TAB2 acts as an adapter linking TAK1 and TRAF6. Here, we demonstrate that TAK1 and TAB2 participate in the RANK signaling pathway. Dominant negative forms of TAK1 and TAB2 inhibit NF-kappaB activation induced by overexpression of RANK. In 293 cells stably transfected with full-length RANK, RANKL stimulation facilitates the formation of a complex containing RANK, TRAF6, TAB2, and TAK1, leading to the activation of TAK1. Furthermore, in murine monocyte RAW 264.7 cells, dominant negative forms of TAK1 and TAB2 inhibit NF-kappaB activation induced by RANKL and endogenous TAK1 is activated in response to RANKL stimulation. These results suggest that the formation of the TRAF6-TAB2-TAK1 complex is involved in the RANK signaling pathway and may regulate the development and function of osteoclasts.

The Serratia marcescens bioH gene encodes an esterase.

The 3.9 kb chromosomal DNA was cloned from Serratia marcescens Sr41, which confers on Escherichia coli cells a phenotype of clear halo formation on tributyrin agar plates. Three complete open reading frames (ORFs) were identified in the inserted DNA, and one ORF was demonstrated to encode a 28 kDa protein of 255 amino acids related to esterase activity. Interestingly, the ORF was 70% identical to a product of the E. coli bioH gene, which lies at a locus separated from the bioABFCD operon and acts in the early steps of the biotin synthetic pathway before pimeloyl-CoA synthesis. This gene complemented a bioH-deficient mutation of E. coli. From the sequence analysis, BioH is presumed to be a serine hydrolase, which belongs to the alpha/beta hydrolase-fold family comprising a wide variety of hydrolases including esterases. A catalytic triad composed of a nucleophilic residue (Ser80), an acidic residue (Asp206), and histidine (His234) was conserved in BioH, and the nucleophilic residue Ser, a catalytic center, was situated in the consensus sequence of G-X-S-X-G-G, a nucleophile elbow. Although the enzymatic function of BioH is not yet elucidated, the bioH gene products from S. marcescens and E. coli show esterase activity, which may imply the hydrolysis of a precursor leading to pimeloyl-CoA ester. The esterase activity of BioH and its CoA binding activity recently reported agree with a current hypothesis of pimeloyl-CoA ester synthesis from CoA and acylester derivatives including an acyl-carrier protein.

A quantitative analysis to unveil specific binding proteins for bioactive compounds.

The mechanisms of the action of bioactive compounds discovered via 'black box' assays using phenotypic indicators generally remain unknown, with the major challenges being the identification of target proteins. In this study, we aimed to develop an efficient methodology to unveil target proteins that are rarely characterised. Proof-of-concept experiments were performed using N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a well-known calmodulin (CaM) antagonist, as a bait compound. The results showed that in our methodology CaM was identified as a W-7-specific binding protein in the cytosolic fraction of a rat brain extract, whereas other proteins acquired in the same experiment were recognised as non-specific binding proteins. The binding affinity of CaM to W-7 is not very high (dissociation constant: 1.6 × 10(-5) M), showing that the recognition specificity is applicable to compounds with very low binding affinities.

Novel membrane protein containing glycerophosphodiester phosphodiesterase motif is transiently expressed during osteoblast differentiation.

Osteoblast maturation is a multistep series of events characterized by an integrated cascade of gene expression that are accompanied by specific phenotypic alterations. To find new osteoblast-related genes we cloned differentially expressed cDNAs characteristic of specific differentiation stages in the mouse osteoblast-like MC3T3-E1 cells by a differential display method. We identified a novel cDNA encoding a putative glycerophosphodiester phosphodiesterase, GDE3, which specifically was expressed at the stage of matrix maturation. Interestingly, the deduced amino acid sequence contains 539 amino acids including seven putative transmembrane domains and a glycerophosphodiester phosphodiesterase region in one of the extracellular loops. Northern blot analysis revealed that GDE3 was also expressed in spleen as well as primary calvarial osteoblasts and femur. We next transfected HEK293T cells with GDE3 with green fluorescent protein fused to the C terminus. The green fluorescent protein-fused protein accumulated at the cell periphery, and the transfected cells overexpressing the protein changed from a spread form to rounded form with disappearance of actin filaments. Immunofluorescence staining with GDE3 antibody and phalloidin in MC3T3-E1 cells indicated that endogenous GDE3 might be co-localized with the actin cytoskeleton. To identify a role for GDE3 in osteoblast differentiation, MC3T3-E1 cells stably expressing the full-length protein were constructed. Expression of GDE3 showed morphological changes, resulting in dramatic increases in alkaline phosphatase activity and calcium deposit. These results suggest that GDE3 might be a novel seven-transmembrane protein with a GP-PDE-like extracellular motif expressed during the osteoblast differentiation that dramatically accelerates the program of osteoblast differentiation and is involved in the morphological change of cells.