Escherichia coli acetyl-coenzyme A carboxylase: characterization and development of a high-throughput assay.

Bacterial acetyl-coenzyme A carboxylase (ACCase) is a multicomponent system composed of AccA, AccD, AccC, and AccB (also known as BCCP), which is required for fatty acid biosynthesis. It is essential for cell growth and has been chemically validated as a target for antimicrobial drug discovery. To identify ACCase inhibitors, a simple and robust assay that monitors the overall activity by measuring phosphate production at physiologically relevant concentrations of all protein components was developed. Inorganic phosphate production was demonstrated to directly reflect the coupled activities of AccC and AccA/D with BCCP cycling between the two half-reactions. The K(m) apparent values for ATP, acetyl-coenzyme A, and BCCP were estimated to be 60+/-14 microM, 18+/-4 microM, and 39+/-9 nM, respectively. The stoichiometry between the two half-reactions was measured to be 1:1. Carboxy-biotin produced in the first half-reaction was stable over the time course of the assay. The assay was adapted to a high-throughput screen (HTS) 384-well format using a modified published scintillation proximity method. The optimized HTS assay has acceptable Z' factor values and was validated to report inhibitions of either AccC or AccA/D. The assay is not susceptible to signal quenching due to colored compounds.

Development of a fluorescence polarization bead-based coupled assay to target different activity/conformation states of a protein kinase.

Most of the protein kinase inhibitors being developed are directed toward the adenosine triphosphate (ATP) binding site that is highly conserved in many kinases. A major issue with these inhibitors is the specificity for a given kinase. Structure determination of several kinases has shown that protein kinases adopt distinct conformations in their inactive state, in contrast to their strikingly similar conformations in their active states. Hence, alternative assay formats that can identify compounds targeting the inactive form of a protein kinase are desirable. The authors describe the development and optimization of an Immobilized Metal Assay for Phosphochemicals (IMAP)-based couple d assay using PDK1 and inactive Akt-2 enzymes. PDK1 phosphorylates Akt-2 at Thr 309 in the catalytic domain, leading to enzymatic activation. Activation of Akt by PDK1 is measured by quantitating the phosphorylation of Akt-specific substrate peptide using the IMAP assay format. This IMAP-coupled assay has been formatted in a 384-well microplate format with a Z' of 0.73 suitable for high-throughput screening. This assay was evaluated by screening the biologically active sample set LOPAC trade mark and validated with the protein kinase C inhibitor staurosporine. The IC(50) value generated was comparable to the value obtained by the radioactive (33)P-gamma-ATP flashplate transfer assay. This coupled assay has the potential to identify compounds that target the inactive form of Akt and prevent its activation by PDK1, in addition to finding inhibitors of PDK1 and activated Akt enzymes.

Structure elucidation of Sch 538415, a novel acyl carrier protein synthase inhibitor from a microorganism.

A novel acyl carrier protein synthase inhibitor, Sch 538415 (1), was isolated from an unidentified bacterial microbe. Structure elucidation of 1 was accomplished based on analysis of spectroscopic data including UV, MS and 2D-NMR spectra. Compound 1 exhibited inhibitory activity in the acyl carrier protein synthase (AcpS) assay with an IC(50) value of 4.19 microM and showed antibacterial activity against Staphylococcus aureus in the agar diffusion assay.

2,5-dialkylresorcinol biosynthesis in Pseudomonas aurantiaca: novel head-to-head condensation of two fatty acid-derived precursors.

2-Hexyl-5-propylresorcinol is the predominant analog of several dialkylresorcinols produced by Pseudomonas aurantiaca (Pseudomonas fluorescens BL915). We isolated and characterized three biosynthetic genes that encode an acyl carrier protein, a beta-ketoacyl-acyl carrier protein synthase III, and a protein of unknown function, all of which collectively allow heterologous production of 2-hexyl-5-propylresorcinol in Escherichia coli. Two regulatory genes exhibiting similarity to members of the AraC family of transcriptional regulators are also present in the identified gene cluster. Based on the deduced functions of the proteins encoded by the gene cluster and the observed incorporation of labeled carbons from octanoic acid into 2-hexyl-5-propylresorcinol, we propose that dialkylresorcinols are derived from medium-chain-length fatty acids by an unusual head-to-head condensation of beta-ketoacyl thioester intermediates. Genomic evidence suggests that there is a similar pathway for the biosynthesis of the flexirubin-type pigments in certain bacteria belonging to the order Cytophagales.

Development of a fluorescence polarization AKT serine/threonine kinase assay using an immobilized metal ion affinity-based technology.

A bead-based FP assay methodology, termed IMAP trade mark, has been developed for the serine/threonine kinase, AKT, that allows for direct measurement of product formation. The assay design utilizes a fluoresceinated peptide substrate that, when phosphorylated by the kinase, binds to nanoparticles derivatized with trivalent metal cations through a metal-phospholigand interaction. The result of this bound fluoresceinated phosphorylated product is an increase in polarization signal caused by a decrease in the molecular mobility of the bound product. The AKT IMAP FP assay has been formatted in a 384-well microtiter plate with a Z' of 0.75, suitable for HTS. The assay was validated with six known kinase inhibitors. The IC(50) values generated were comparable to previously reported values using a competitive antibody-based FP assay and a radioactive [(33)P]ATP Flashplate transfer assay. The IMAP assay was further evaluated by screening the biologically active sample set, LOPAC trade mark. It was found that no fluorescent samples interfered with the assay's performance and that one could identify appropriate inhibitors. The advantages of IMAP technology are that it does not require the use of antibodies, the polarization signal generated is large in comparison with most FP assays based on antibodies, and IMAP captures and measures the product formed directly. The higher concentrations of fluorophore used in the assay versus competition assays increase the precision of the data obtained and reduce sample interference from compounds. This work demonstrates that IMAP is a valuable technology that may be used in developing numerous kinase assays.