Journal of Biomolecular Screening current issue

In Memoriam: Tony J. Beugelsdijk (1949--2009)

Thu, 12 Nov 2009 08:41:25 PST

Automated High-Content Screening for Compounds That Disassemble the Perinucleolar Compartment

Norton, J. T., Titus, S. A., Dexter, D., Austin, C. P., Zheng, W., Huang, S. — Thu, 12 Nov 2009 08:41:25 PST

All solid malignancies share characteristic traits, including unlimited cellular proliferation, evasion of immune regulation, and the propensity to metastasize. The authors have previously described that a subnuclear structure, the perinucleolar compartment (PNC), is associated with the metastatic phenotype in solid tumor cancer cells. The percentage of cancer cells that contain PNCs (PNC prevalence) is indicative of the malignancy of a tumor both in vitro and in vivo, and thus PNC prevalence is a marker that reflects metastatic capability in a population of tumor cells. Although the function of the PNC remains to be determined, the PNC is highly enriched with small RNAs and RNA binding proteins. The initial chemical biology studies using a set of anticancer drugs that disassemble PNCs revealed a direct association of the structure with DNA. Therefore, PNC prevalence reduction as a phenotypic marker can be used to identify compounds that target cellular processes required for PNC maintenance and hence used to elucidate the nature of the PNC function. Here the authors report the development of an automated high-content screening assay that is capable of detecting PNC prevalence in prostate cancer cells (PC-3M) stably expressing a green fluorescent protein (GFP)—fusion protein that localizes to the PNC. The assay was optimized using known PNC-reducing drugs and non-PNC-reducing cytotoxic drugs. After optimization, the fidelity of the assay was probed with a collection of 8284 compounds and was shown to be robust and capable of detecting known and novel PNC-reducing compounds, making it the first reported high-content phenotypic screen for small changes in nuclear structure. (Journal of Biomolecular Screening 2009:1045-1053)

Profiling Bioactivity of the ToxCast Chemical Library Using BioMAP Primary Human Cell Systems

Houck, K. A., Dix, D. J., Judson, R. S., Kavlock, R. J., Yang, J., Berg, E. L. — Thu, 12 Nov 2009 08:41:25 PST

The complexity of human biology has made prediction of health effects as a consequence of exposure to environmental chemicals especially challenging. Complex cell systems, such as the Biologically Multiplexed Activity Profiling (BioMAP) primary, human, cell-based disease models, leverage cellular regulatory networks to detect and distinguish chemicals with a broad range of target mechanisms and biological processes relevant to human toxicity. Here the authors use the BioMAP human cell systems to characterize effects relevant to human tissue and inflammatory disease biology following exposure to the 320 environmental chemicals in the Environmental Protection Agency’s (EPA’s) ToxCast phase I library. The ToxCast chemicals were assayed at 4 concentrations in 8 BioMAP cell systems, with a total of 87 assay endpoints resulting in more than 100,000 data points. Within the context of the BioMAP database, ToxCast compounds could be classified based on their ability to cause overt cytotoxicity in primary human cell types or according to toxicity mechanism class derived from comparisons to activity profiles of BioMAP reference compounds. ToxCast chemicals with similarity to inducers of mitochondrial dysfunction, cAMP elevators, inhibitors of tubulin function, inducers of endoplasmic reticulum stress, or NFB pathway inhibitors were identified based on this BioMAP analysis. This data set is being combined with additional ToxCast data sets for development of predictive toxicity models at the EPA. (Journal of Biomolecular Screening 2009:1054-1066)

C5a-Stimulated Recruitment of {beta}-Arrestin2 to the Nonsignaling 7-Transmembrane Decoy Receptor C5L2

Van Lith, L. H.C., Oosterom, J., Van Elsas, A., Zaman, G. J.R. — Thu, 12 Nov 2009 08:41:25 PST

C5L2 (or GPR77) is a high-affinity receptor for the complement fragment C5a and its desarginated product, C5a-desArg. Unlike the classical C5a receptor CD88, C5L2 does not couple to intracellular G-protein-signaling pathways but is thought to function as a decoy receptor. The authors show that stimulation of C5L2 with C5a and C5a-desArg induces redistribution of green fluorescent protein—labeled β-arrestin2 to cytoplasmic vesicles. C3a and C3a-desArg were inactive in the β-arrestin translocation assay. Direct interaction of ligand-stimulated C5L2 with β-arrestin was confirmed using a novel β-galactosidase fragment complementation assay. In this assay, C5L2 was labeled with a mutationally altered peptide fragment of β-galactosidase, whereas β-arrestin2 was labeled with a corresponding deletion mutant of the enzyme. Stable transfection of the modified C5L2 and subsequent stimulation with C5a or C5a-desArg restored β-galactosidase activity in a dose-dependent manner. The subnanomolar potency of β-arrestin coupling in the β-galactosidase fragment complementation assay is in agreement with the affinity of the receptor-ligand interaction. C5L2 is the first example of a 7-transmembrane decoy receptor that couples to β-arrestin in a ligand-dependent manner. This observation supports the notion that G-protein-signaling and β-arrestin coupling can be 2 separate activities of 7-transmembrane receptors. Furthermore, the β-arrestin assays described in this article provide methods of screening for selective C5L2 modulators. (Journal of Biomolecular Screening 2009:1067-1075)

CXCR2 Inverse Agonism Detected by Arrestin Redistribution

Thu, 12 Nov 2009 08:41:25 PST

To study CXCR2 modulated arrestin redistribution, the authors employed arrestin as a fusion protein containing either the Aequorea victoria—derived enhanced green fluorescent protein (EGFP) or a recently developed mutant of eqFP611, a red fluorescent protein derived from Entacmaea quadricolor. This mutant, referred to as RFP611, had earlier been found to assume a dimeric quarternary structure. It was therefore employed in this work as a "tandem" (td) construct for pseudo monomeric fusion protein labeling. Both arrestin fusion proteins, containing either td RFP611 (Arr td RFP611) or enhanced green fluorescent protein (EGFP; Arr EGFP), were found to colocalize with internalized fluorescently labeled Gro a few minutes after Gro addition. Intriguingly, however, Arr td RFP611 and Arr EGFP displayed distinct cellular distribution patterns in the absence of any CXCR2 activating ligand. Under these conditions, Arr td RFP611 showed a largely homoge neous cytosolic distribution, whereas Arr EGFP segregated, to a large degree, into granular spots. These observations indi cate a higher sensitivity of Arr EGFP to the constitutive activity of CXCR2 and, accordingly, an increased arrestin redistribution to coated pits and endocytic vesicles. In support of this interpretation, the authors found the known CXCR2 antagonist Sch527123 to act as an inverse agonist with respect to Arr EGFP redistribution. The inverse agonistic properties of Sch527123 were confirmed in vitro in a guanine nucleotide binding assay, revealing an IC₅₀ value similar to that observed for Arr EGFP redistribution. Thus, the redistribution assay, when based on Arr EGFP, enables the profiling of antagonistic test compounds with respect to inverse agonism. When based on Arr td RFP611, the assay may be employed to study CXCR2 agonism or neutral antagonism. ( Journal of Biomolecular Screening 2009:1076 1091)

Inhibitors of RecA Activity Discovered by High-Throughput Screening: Cell-Permeable Small Molecules Attenuate the SOS Response in Escherichia coli

Thu, 12 Nov 2009 08:41:25 PST

The phenomenon of antibiotic resistance has created a need for the development of novel antibiotic classes with nonclassical cellular targets. Unfortunately, target-based drug discovery against proteins considered essential for in vitro bacterial viability has yielded few new therapeutic classes of antibiotics. Targeting the large proportion of genes considered nonessential that have yet to be explored by high-throughput screening, for example, RecA, can complement these efforts. Recent evidence suggests that RecA-controlled processes are responsible for tolerance to antibiotic chemotherapy and are involved in pathways that ultimately lead to full-fledged antibiotic resistance. Therefore inhibitors of RecA may serve as therapeutic adjuvants in combination chemotherapy of bacterial infectious diseases. Toward the goal of validating RecA as a novel target in the chemotherapy of bacterial infections, the authors have screened 35,780 small molecules against RecA. In total, 80 small molecules were identified as primary hits and could be clustered in 6 distinct chemotype clades. The most potent class of hits was further examined, and 1 member compound was found to inhibit RecA-mediated strand exchange and prevent ciprofloxacin-induced SOS expression in Escherichia coli. This compound represents the first small molecule demonstrating an ability to inhibit the bacterial SOS response in live bacterial cell cultures. (Journal of Biomolecular Screening 2009:1092-1101)

Optimization of Assay Conditions fo r Dengue Virus Protease: Effect of Various Polyols and Nonionic Detergents

Steuer, C., Heinonen, K. H., Kattner, L., Klein, C. D. — Thu, 12 Nov 2009 08:41:25 PST

The aim of this work was to perform a systematic study of the effect of nonionic detergents on the activity of the dengue virus NS2B-NS3 protease. To ensure a high activity of the protease, the assay procedures for the dengue virus and other flaviviral proteases published to date are performed in the presence of up to 35% glycerol, which does not represent the cellular physicochemical environment. In addition, the high viscosity of glycerol-containing solutions leads to various experimental problems in miniaturized assays. Using an internally quenched peptide substrate, the authors show that glycerol is not essential for enzymatic activity if certain nonionic detergents are added to the assay buffer. In addition, nonionic detergents may help to avoid false-positive screening results caused by "promiscuous" inhibitors. Other polyalcohols can substitute glycerol and have less effect on the viscosity of the assay buffer. The assay was used to screen a compound library and allowed the identification of small-molecular nonpeptidic inhibitors of dengue NS3 protease. Finally, the authors discuss the mode of action of nonionic detergents and the influence that they may have on the conformational properties of the NS2B-NS3 protease. (Journal of Biomolecular Screening 2009:1102-1108)

Virtual Screening Against {alpha}-Cobratoxin

Utsintong, M., Talley, T. T., Taylor, P. W., Olson, A. J., Vajragupta, O. — Thu, 12 Nov 2009 08:41:25 PST

-Cobratoxin (Cbtx), the neurotoxin isolated from the venom of the Thai cobra Naja kaouthia , causes paralysis by preventing acetylcholine (ACh) binding to nicotinic acetylcholine receptors (nAChRs). In the current study, the region of the Cbtx molecule that is directly involved in binding to nAChRs is used as the target for anticobratoxin drug design. The crystal structure (1YI5) of Cbtx in complex with the acetylcholine binding protein (AChBP), a soluble homolog of the extracellular binding domain of nAChRs, was selected to prepare an -cobratoxin active binding site for docking. The amino acid residues (Ser182-Tyr192) of the AChBP structure, the binding site of Cbtx, were used as the positive control to validate the prepared Cbtx active binding site (root mean square deviation < 1.2 Å). Virtual screening of the National Cancer Institute diversity set, a library of 1990 compounds with nonredundant pharmacophore profiles, using AutoDock against the Cbtx active site, revealed 39 potential inhibitor candidates. The adapted in vitro radioligand competition assays using [³H]epibatidine and [¹²⁵I]bungarotoxin against the AChBPs from the marine species, Aplysia californica ( Ac), and from the freshwater snails, Lymnaea stagnalis (Ls ) and Bolinus truncates (Bt), revealed 4 compounds from the list of inhibitor candidates that had micromolar to nanomolar interferences for the toxin binding to AChBPs. Three hits (NSC42258, NSC121865, and NSC134754) can prolong the survival time of the mice if administered 30 min before injection with Cbtx, but only NSC121865 and NSC134754 can prolong the survival time if injected immediately after injection with Cbtx. These inhibitors serve as novel templates/scaffolds for the development of more potent and specific anticobratoxin. (Journal of Biomolecular Screening 2009:1109-1118)

A Novel High-Throughput Screening Assay for HCN Channel Blocker Using Membrane Potential-Sensitive Dye and FLIPR

Thu, 12 Nov 2009 08:41:25 PST

Hyperpolarization-activated cation nonselective (HCN) channels represent an interesting group of targets for drug development. In this study, the authors report the development of a novel membrane potential-sensitive dye (MPSD) assay for HCN channel modulators that has been miniaturized into 384-well fluorescent imaging plate reader (FLIPR) high-throughput screening (HTS) format. When optimized (by cell plating density, plate type, cell recovery from cryopreservation), the wellto-well signal variability was low, with a Z' = 0.73 and coefficient of variation = 6.4%, whereas the MPSD fluorescence signal amplitude was -23,700 ± 1500 FLIPR³ relative fluorescence units (a linear relationship was found between HCN1 MPSD fluorescence signal and the cell plating density) and was completely blocked by 30 µM ZD7288. The assay tolerated up to 1% DMSO, inclusion of which did not significantly change the signal kinetics or amplitude. A single-concentration screening of an ion channel-focused library composed of 4855 compounds resulted in 89 HCN1 blocker hits, 51 of which were subsequently analyzed with an 8-point concentration-response analysis on the IonWorks HT electrophysiology platform. The correlation between MPSD and the electrophysiology assay was moderate, as shown by the linear regression analysis (r² = 0.56) between the respective IC₅₀s obtained using these 2 assays. The reported HTS-compatible HCN channel blocker assay can serve as a tool in drug discovery in the pursuit of HCN channel isoform-selective small molecules that could be used in the development of clinically relevant compounds. (Journal of Biomolecular Screening 2009:1119-1128)

A Continuous Protein Methyltransferase (G9a) Assay for Enzyme Activity Measurement and Inhibitor Screening

Dhayalan, A., Dimitrova, E., Rathert, P., Jeltsch, A. — Thu, 12 Nov 2009 08:41:25 PST

The authors describe a continuous protein methylation assay using the G9a protein lysine methyltransferase and its substrate protein WIZ (widely interspaced zinc finger motifs). The assay is based on the coupling of the biotinylated substrate protein to streptavidin-coated FlashPlates and the transfer of radioactive methyl groups from the S-adenosyl-L-methionine to the substrate. The reaction progress is monitored continuously by proximity scintillation counting. The assay is very accurate, convenient, well suited for automation, and highly reproducible with standard errors in the range of 5%. Because of few pipetting steps and continuous data readout, it is ideal for high-throughput applications such as screening of inhibitors, testing many enzyme variants, or analyzing differences in methylation rates of different substrates under various conditions. By using this new assay, the IC₅₀ of AdoHcy and the G9a inhibitor BIX-01294 were determined for methylation of the G9a nonhistone substrate WIZ. (Journal of Biomolecular Screening 2009:1129-1133)

Utilization of the TangoTM {beta}-Arrestin Recruitment Technology for Cell-Based EDG Receptor Assay Development and Interrogation

Wetter, J. A., Revankar, C., Hanson, B. J. — Thu, 12 Nov 2009 08:41:25 PST

Cellular assay development for the endothelial differentiation gene (EDG) family of G-protein-coupled receptors (GPCRs) and related lysophospholipid (LP) receptors is complicated by endogenous receptor expression and divergent receptor signaling. Endogenously expressed LP receptors exist in most tissue culture cell lines. these LP receptors, along with other endogenously expressed GPCRs, contribute to off-target signaling that can complicate interpretation of second-messenger-based cellular assay results. these receptors also activate a diverse and divergent set of cellular signaling pathways, necessitating the use of a variety of assay formats with mismatched procedures and functional readouts. this complicates examination and comparison of these receptors across the entire family. the tango^TM technology uses the conserved β-arrestin-dependent receptor deactivation process to allow interrogation of the EDG and related receptors with a single functional assay. this method also isolates the target receptor signal, allowing the use of tissue culture cell lines regardless of their endogenous receptor expression. the authors describe the use of this technique to build cell-based receptor-specific assays for all 8 members of the EDG receptor family as well as the related LPA receptors GPR23, GPR92, and GPR87. In addition, they demonstrate the value of this technology for identification and investigation of functionally selective receptor compounds as demonstrated by the immunosuppressive compound FtY720-P and its action at the EDG₁ and EDG₃ receptors. (Journal of Biomolecular Screening 2009:1134-1141)

Product Focus: Analytical and Preparative Instrumentation

Thu, 12 Nov 2009 08:41:25 PST

2009 Meetings Calendar

Thu, 12 Nov 2009 08:41:25 PST