Journal of Biomolecular Screening recent issues

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

Society Updates

Thu, 01 Oct 2009 10:19:45 PDT

Mechanism-Based Inhibition: Deriving KI and kinact Directly from Time-Dependent IC50 Values

Krippendorff, B.-F., Neuhaus, R., Lienau, P., Reichel, A., Huisinga, W. — Thu, 01 Oct 2009 10:19:45 PDT

The potential of enzyme inhibition of a drug is frequently quantified in terms of IC₅₀ values. Although this is a suitable quantity for reversible inhibitors, concerns arise when dealing with irreversible or mechanism-based inhibitors (MBIs). IC₅₀ values of MBIs are time dependent, causing serious problems when aiming at ranking different compounds with respect to their inhibitory potential. As a consequence, most studies and ranking schemes related to MBIs rely on the inhibition constant (K_I) and the rate of enzyme inactivation (k_inact) rather than on IC₅₀ values. In this article, the authors derive a novel relation between potentially time-dependent IC₅₀ values and K_I, k_inact parameters for different types of inhibition. This allows for direct estimation of K_I and k_inact values from time-dependent IC₅₀ values, even without the need of additional preincubation experiments. The application of this approach is illustrated using a fluorimetric assay to access the drug-drug interaction potential associated with new chemical entities. The approach can easily be implemented using standard software tools (e.g., XLfit) and may also be suitable for applications where mechanism-based inhibition is a desired mode of action (e.g., at particular pharmacological drug targets). (Journal of Biomolecular Screening 2009:913-923)

Development and Applications of a Broad-Coverage, TR-FRET-Based Kinase Binding Assay Platform

Thu, 01 Oct 2009 10:19:46 PDT

The expansion of kinase assay technologies over the past decade has mirrored the growing interest in kinases as drug targets. As a result, there is no shortage of convenient, fluorescence-based methods available to assay targets that span the kinome. The authors recently reported on the development of a non-activity-based assay to characterize kinase inhibitors that depended on displacement of an Alexa Fluor^® 647 conjugate of staurosporine (a "tracer") from a particular kinase. Kinase inhibitors were characterized by a change in fluorescence lifetime of the tracer when it was bound to a kinase relative to when it was displaced by an inhibitor. Here, the authors report on improvements to this strategy by reconfiguring the assay in a time-resolved fluorescence resonance energy transfer (TR-FRET) format that simplifies instrumentation requirements and allows for the use of a substantially lower concentration of kinase than was required in the fluorescence-lifetime-based format. The authors use this new assay to demonstrate several aspects of the binding assay format that are advantageous relative to traditional activity-based assays. The TR-FRET binding format facilitates the assay of compounds against low-activity kinases, allows for the characterization of type II kinase inhibitors either using nonactivated kinases or by monitoring compound potency over time, and ensures that the signal being detected is specific to the kinase of interest and not a contaminating kinase.

A New Simple Cell-Based Homogeneous Time-Resolved Fluorescence QRET Technique for Receptor-Ligand Interaction Screening

Thu, 01 Oct 2009 10:19:46 PDT

In this article, a single-label separation-free fluorescence technique is presented as a potential screening method for cell-based receptor antagonists and agonists.The time-resolved fluorescence technique, quenching resonance energy transfer (QRET), relies on a single-labeled binding partner in combination with a soluble quencher. The quencher efficiently suppresses the luminescence of the unbound labeled ligand, whereas the luminescence of the bound fraction is not affected. This approach allows the development of cell-based screening assays in a simple and cost-effective manner. The authors have applied the technique to the screening of β₂-adrenoreceptor (β₂AR) antagonists and agonists in intact human embryonic kidney HEK293_i cells overexpressing human β₂-adrenergic receptors. Two antagonists (propranolol, alprenolol) and 2 agonists (metaproterenol, terbutaline) for β₂AR were investigated in a displacement assay using europium(III)-labeled pindolol ligand. The assay Z' values ranged from 0.68 to 0.78, the coefficient of variation was less than 10%, and the K_i values were 19 nM for propranolol and alprenolol and 14 and 5.9 µM for metaproterenol and terbutaline, respectively. The QRET technique with β₂AR was also applied to LOPAC compound library screening, yielding nearly error-free recognition of known binders. This simple and cost-effective technique can be readily adapted to laboratory and industrial-scale screening. (Journal of Biomolecular Screening 2009:936-943)

DetecTiff(C): A Novel Image Analysis Routine for High-Content Screening Microscopy

Gilbert, D. F., Meinhof, T., Pepperkok, R., Runz, H. — Thu, 01 Oct 2009 10:19:46 PDT

In this article, the authors describe the image analysis software DetecTiff©, which allows fully automated object recognition and quantification from digital images. The core module of the LabView©-based routine is an algorithm for structure recognition that employs intensity thresholding and size-dependent particle filtering from microscopic images in an iterative manner. Detected structures are converted into templates, which are used for quantitative image analysis. DetecTiff^© enables processing of multiple detection channels and provides functions for template organization and fast interpretation of acquired data. The authors demonstrate the applicability of DetecTiff^© for automated analysis of cellular uptake of fluorescencelabeled low-density lipoproteins as well as diverse other image data sets from a variety of biomedical applications. Moreover, the performance of DetecTiff^© is compared with preexisting image analysis tools. The results show that DetecTiff^© can be applied with high consistency for automated quantitative analysis of image data (e.g., from large-scale functional RNAi screening projects). (Journal of Biomolecular Screening 2009:944-955)

Live-Cell Imaging of Caspase Activation for High-Content Screening

Antczak, C., Takagi, T., Ramirez, C. N., Radu, C., Djaballah, H. — Thu, 01 Oct 2009 10:19:46 PDT

Caspases are central to the execution of programmed cell death, and their activation constitutes the biochemical hallmark of apoptosis. In this article, the authors report the successful adaptation of a high-content assay method using the DEVDNucView488^TM fluorogenic substrate, and for the first time, they show caspase activation in live cells induced by either drugs or siRNA. The fluorogenic substrate was found to be nontoxic over an exposure period of several days, during which the authors demonstrate automated imaging and quantification of caspase activation of the same cell population as a function of time. Overexpression of the antiapoptotic protein Bcl-XL, alone or in combination with the inhibitor Z-VAD-FMK, attenuated caspase activation in HeLa cells exposed to doxorubicin, etoposide, or cell death siRNA. This method was further validated against 2 well-characterized NSCLC cell lines reported to be sensitive (H3255) or refractory (H2030) to erlotinib, where the authors show a differential time-dependent activation was observed for H3255 and no significant changes in H2030, consistent with their respective chemosensitivity profile. In summary, the results demonstrate the feasibility of using this newly adapted and validated high-content assay to screen chemical or RNAi libraries for the identification of previously uncovered enhancers and suppressors of the apoptotic machinery in live cells. (Journal of Biomolecular Screening 2009:956-969)

Novel In Vitro Protein Fragment Complementation Assay Applicable to High-Throughput Screening in a 1536-Well Format

Thu, 01 Oct 2009 10:19:46 PDT

Protein-protein interactions (PPIs) play key roles in all cellular processes and hence are useful as potential targets for new drug development. To facilitate the screening of PPI inhibitors as anticancer drugs, the authors have developed a high-throughput screening (HTS) system using an in vitro protein fragment complementation assay (PCA) with monomeric Kusabira-Green fluorescent protein (mKG). The in vitro PCA system was established by the topological formation of a functional complex between 2 split inactive mKG fragments fused to target proteins, which fluoresces when 2 target proteins interact to allow complementation of the mKG fragments. Using this assay system, the authors screened inhibitors for TCF7/β-catenin, PAC1/PAC2, and PAC3 homodimer PPIs from 123,599 samples in their natural product library. Compound TB1 was identified as a specific inhibitor for PPI of PAC3 homodimer. TB1 strongly inhibited the PPI of PAC3 homodimer with an IC₅₀ value of 0.020 µM and did not inhibit PPI between TCF7/β-catenin and PAC1/PAC2 even at a concentration of 250 µM. The authors thus demonstrated that this in vitro PCA system applicable to HTS in a 1536-well format is capable of screening for PPI inhibitors from a huge natural product library. ( Journal of Biomolecular Screening 2009:970-979)

Test System for Trifunctional Antibodies in 3D MCTS Culture

Hirschhaeuser, F., Leidig, T., Rodday, B., Lindemann, C., Mueller-Klieser, W. — Thu, 01 Oct 2009 10:19:46 PDT

The aim of the present study was to assess the feasibility of a 3D tumor cell culture model, that is, multicellular tumor spheroids (MCTSs) as an adequate model for micrometastases and therefore as a pharmacological model for efficacy testing of trifunctional therapeutic antibodies. Unlike conventional monolayer cultures, spheroids allow researchers to study parameters, such as 3D cell shape, 3D cell arrangement and microenvironment, and penetration efficiency of defense cells that may largely influence the efficacy of antibody treatment in vivo. The authors established a long-term coculture of human MCTSs with peripheral blood mononuclear cells (PBMCs) to test the anticancer effect of the trifunctional, bispecific antibody catumaxomab (anti-EpCAM x anti-CD3) or similar therapeutic molecules. The test system is accessible to various analytical methods and thus allows for characterizing multiple parameters, which can help elucidate the mode of action of immunotherapeutic anticancer treatment. For example, the novel approach enables precise, reproducible volume growth analysis of MCTSs under immunotherapeutic treatments. For evaluation of changes within individual spheroids, cryosections can be stained (e.g., for proliferating or apoptotic cells as well as infiltrating PBMCs). Molecular PCR-based assays or flow cytometric analyses allow for discrimination between different cell types, particularly leukocyte subtypes. Furthermore, MCTSs can be disaggregated to form standard monolayers for cell viability or plating efficiency experiments. For these reasons, the MCTS model is a powerful tool to analyze drug efficacy with various endpoints under highly reproducible, standardized conditions. (Journal of Biomolecular Screening 2009:980-990)

Neutralizing Antibodies of Botulinum Neurotoxin Serotype A Screened from a Fully Synthetic Human Antibody Phage Display Library

Yu, R., Wang, S., Yu, Y.-z., Du, W.-s., Yang, F., Yu, W.-y., Sun, Z.-w. — Thu, 01 Oct 2009 10:19:46 PDT

The botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous protein substances known. The neutralizing antibodies against botulinum neurotoxin can effectively prevent and cure the toxicosis. Using purified Hc fragments of botulinum neurotoxin serotype A (BoNT/A-Hc) as antigen, 2 specific neutralizing antibodies mapping different epitopes were selected from a fully synthetic human antibody library. The 2 antibodies can effectively inhibit the binding between BoNT/A-Hc and differentiated PC-12 cells in vitro, and the neutralization was evaluated in vivo. Although no single mAb completely protected mice from toxin, they both could prolong time to death when challenged with 20 LD₅₀s (50% lethal doses) of BoNT/A. When used together, the mAbs completely neutralized 1000 LD₅₀s/mg Ab, suggesting their high neutralizing potency in vivo. The results would lead to further production of neutralizing antibody drugs against BoNT/A. It also proved that it was a quick method to obtain human therapeutic antibodies by selecting from the fully synthetic human antibody phage display library. (Journal of Biomolecular Screening 2009:991-998)

A Primer on Screening Data Management

Palmer, M., Kremer, A., Terstappen, G. C. — Thu, 01 Oct 2009 10:19:46 PDT

A drug discovery startup company or academic lab entering the screening arena faces numerous challenges as it tries to manage the large quantity of data generated by a typical drug discovery screening campaign. Although there are sophisticated off-the-shelf software solutions available, their use requires substantial forethought and attention to detail if the data they capture are to be of sufficient quality to serve the various purposes to which it will be put. For newcomers to the field of screening data management in particular, the problem is compounded by a lack of literature covering the practical aspects of managing screening data. The authors provide some practical advice based on their experience of using a commercially available software suite. They discuss issues ranging from the organizational aspects to examples of how the form and content of metadata can have a big impact on whether results can be easily queried, pivoted, and reported. It is also hoped that their experiences might provide an opportunity for reflection to data management practitioners operating in established environments.

Correction for Interference by Test Samples in High-Throughput Assays

Shapiro, A. B., Walkup, G. K., Keating, T. A. — Thu, 01 Oct 2009 10:19:46 PDT

In high-throughput biochemical assays performed in multiwell plates, the effect of test samples on the activity of the biochemical system is usually measured by optical means such as absorbance, fluorescence, luminescence, or scintillation counting. The test sample often causes detection interference when it remains in the well during the measurement. Interference may be due to light absorption, fluorescence quenching, sample fluorescence, chemical interaction of the sample with a detection reagent, or depression of the meniscus. A simple method is described that corrects for such interference well by well. The interference is measured in a separate artifact assay plate. An appropriate arithmetic correction is then applied to the measurement in the corresponding well of the activity assay plate. The correction procedure can be used for single-point screening or potency measurements on serial dilutions of test samples.

Establishing Quality Assurance Criteria for Serial Dilution Operations on Liquid-Handling Equipment

Thu, 01 Oct 2009 10:19:46 PDT

Since the advent of high-throughput screening (HTS) in the early 1990s, parallel multichannel liquid handlers have become a mainstay in every drug discovery setting. Although several peer-reviewed publications have discussed methods and criteria for stamping multiwell copies, there is very little information about establishing a standard operating procedure (SOP) for standard (microliter-level) serial dilutions of compounds used in dose-response experiments. The authors discuss the 4 main criteria any serial dilution process must pass (accuracy, precision, fold dilution, and outliers) and the process for establishing thresholds for all of these values in a compound management or biological screening laboratory. The thresholds need to be both low enough to be acceptable from a biological potency variability perspective and high enough to allow the instruments to pass the quality assurance (QA) analysis on a regular basis. In this article, the authors suggest suitable thresholds arrived at by a variety of methods, including trend analysis of QA data, survey questionnaire from the main stakeholders (screening scientists, chemists), and published criteria for single-shot stamping. A mathematical analysis of the effect of threshold values on estimated XC₅₀s was performed to ensure that the variability introduced by the serial dilution step is within acceptable overall variability limits.

Product Focus: Software, Databases, and Information Services

Thu, 01 Oct 2009 10:19:46 PDT

2009-2010 Meetings Calendar

Thu, 01 Oct 2009 10:19:46 PDT

Society Updates

Tue, 01 Sep 2009 15:40:25 PDT

Stem Cells in Drug Discovery, Tissue Engineering, and Regenerative Medicine: Emerging Opportunities and Challenges

Nirmalanandhan, V. S., Sittampalam, G. S. — Tue, 01 Sep 2009 15:40:25 PDT

Stem cells, irrespective of their origin, have emerged as valuable reagents or tools in human health in the past 2 decades. Initially, a research tool to study fundamental aspects of developmental biology is now the central focus of generating transgenic animals, drug discovery, and regenerative medicine to address degenerative diseases of multiple organ systems. This is because stem cells are pluripotent or multipotent cells that can recapitulate developmental paths to repair damaged tissues. However, it is becoming clear that stem cell therapy alone may not be adequate to reverse tissue and organ damage in degenerative diseases. Existing small-molecule drugs and biologicals may be needed as "molecular adjuvants" or enhancers of stem cells administered in therapy or adult stem cells in the diseased tissues. Hence, a combination of stem cell-based, high-throughput screening and 3D tissue engineering approaches is necessary to advance the next wave of tools in preclinical drug discovery. In this review, the authors have attempted to provide a basic account of various stem cells types, as well as their biology and signaling, in the context of research in regenerative medicine. An attempt is made to link stem cells as reagents, pharmacology, and tissue engineering as converging fields of research for the next decade. (Journal of Biomolecular Screening 2009:755-768)

Population Patch-Clamp Electrophysiology Analysis of Recombinant GABAA {alpha}1{beta}3{gamma}2 Channels Expressed in HEK-293 Cells

Tue, 01 Sep 2009 15:40:25 PDT

-Amino butyric acid (GABA)—activated Cl^— channels are critical mediators of inhibitory postsynaptic potentials in the CNS. To date, rational design efforts to identify potent and selective GABA_A subtype ligands have been hampered by the absence of suitable high-throughput screening approaches. The authors describe 384-well population patch-clamp (PPC) planar array electrophysiology methods for the study of GABA_A receptor pharmacology. In HEK293 cells stably expressing human 1β32 GABA_A channels, GABA evoked outward currents at 0 mV of 1.05 ± 0.08 nA, measured 8 s post GABA addition. The I_GABA was linear and reversed close to the theoretical E_Cl (—56 mV). Concentration-response curve analysis yielded a mean pEC₅₀ value of 5.4 and Hill slope of 1.5, and for a series of agonists, the rank order of potency was muscimol > GABA > isoguvacine. A range of known positive modulators, including diazepam and pentobarbital, produced concentration-dependent augmentation of the GABA EC₂₀ response (1 µM). The competitive antagonists bicuculline and gabazine produced concentration-dependent, parallel, rightward displacement of GABA curves with pA₂ and slope values of 5.7 and 1.0 and 6.7 and 1.0, respectively. In contrast, picrotoxin (0.2-150 µM) depressed the maximal GABA response, implying a non-competitive antagonism. Overall, the pharmacology of human 1β32 GABA_A determined by PPC was highly similar to that obtained by conventional patch-clamp methods. In small-scale single-shot screens, Z' values of >0.5 were obtained in agonist, modulator, and antagonist formats with hit rates of 0% to 3%. The authors conclude that despite the inability of the method to resolve the peak agonist responses, PPC can rapidly and usefully quantify pharmacology for the 1β32 GABA_A isoform. These data suggest that PPC may be a valuable approach for a focused set and secondary screening of GABA_A receptors and other slow ligand-gated ion channels. ( Journal of Biomolecular Screening 2009:769-780)

Development of Generic Calcium Imaging Assay for Monitoring Gi-Coupled Receptors and G-Protein Interaction

Ueda, T., Ugawa, S., Ishida, Y., Hondoh, A., Shimada, S. — Tue, 01 Sep 2009 15:40:25 PDT

G-protein-coupled receptors (GPCRs) are important therapeutic targets for many areas of drug research and development. Although chimeric G₁₆ proteins are valuable tools for detecting the activation of G_i/o-coupled receptors, the details of the activation process remain unclear. The authors introduce a series of chimeras that combine both G₁₆ and G_i/o (G_16/o, G_16/i2, and G_16/i3) into a well-established transient expression system to examine the ability of these chimeras to interact with D₂ long-form (D_2L) dopamine and 5-HT_1A serotonin receptors. The pEC₅₀ data obtained for known agonists were similar to results from previous studies that used other cell-based assays, thus indicating sufficient sensitivity for the assay. Moreover, quinpirole exhibited similar intrinsic activity to dopamine at the D_2L receptor, whereas S-(—)-3-PPP displayed partial activity of dopamine and quinpirole in the presence of the G_16/o chimera. The potency of dopamine for D_2L receptors was similar among G_16/o, G_16/i2, and G_16/i3. In contrast, the 5-HT_1A receptor exhibited a significantly preferential coupling for G_16/i3 compared with G_16/i2 when serotonin was used as a ligand. This finding was in close agreement with the results of previous reports. The present system could therefore be used as a rapid functional assay for high-throughput screening and deorphanization. (Journal of Biomolecular Screening 2009:781-788)

Identification of Surrogate Agonists and Antagonists for Orphan G-Protein-Coupled Receptor GPR139

Hu, L. A., Tang, P. M., Eslahi, N. K., Zhou, T., Barbosa, J., Liu, Q. — Tue, 01 Sep 2009 15:40:25 PDT

GPR139 is an orphan G-protein-coupled receptor (GPCR) that is expressed nearly exclusively in the central nervous system and may play a role in the control of locomotor activity. The signal transduction pathway and pharmacological function of GPR139, however, are still controversial due to the lack of natural or synthetic ligands. The authors report the characterization of human GPR139 signaling pathway and identification of surrogate agonists and antagonists. In both transient and stable transfections of HEK293F cells, overexpression of GPR139 increased basal intracellular cAMP concentrations compared to control cells. Furthermore, forskolin and isoproterenol-stimulated cAMP responses were enhanced in GPR139-expressing cells, suggesting that GPR139 is predominantly coupled to G_s. The authors screened a large library of small molecules for compounds that increase cAMP levels in GPR139-expressing cells and identified a compound with GPR139 agonist activity. This compound increased cAMP production specifically in cells expressing GPR139 but not in cells expressing its highly homologous receptor GPR142. Furthermore, this compound did not induce calcium mobilization in GPR139 cells, indicating no G_q-mediated response. In addition, antagonist screening with the identified agonist yielded 2 classes of compounds as antagonists. The identification of surrogate agonists and antagonists of human GPR139 provides important tools for further study of this orphan GPCR. (Journal of Biomolecular Screening 2009:789-797)

A Homogeneous Fluorescent Live-Cell Assay for Measuring 7-Transmembrane Receptor Activity and Agonist Functional Selectivity Through Beta-Arrestin Recruitment

Tue, 01 Sep 2009 15:40:25 PDT

Seven-transmembrane (7TM) receptors play an essential role in the regulation of a wide variety of physiological processes, making them one of the top target classes for pharmaceuticals. 7TM receptor function is mediated and modulated through 2 primary processes: G-protein and beta-arrestin signaling. Classically, it has been recognized that these 2 processes can interact with one another during 7TM receptor desensitization, but it has more recently been recognized that these 2 processes can also act independently of one another and can activate parallel signaling pathways. As such, the methods used to interrogate 7TM receptor signaling, both from a biological and a pharmaceutical perspective, may need to be reevaluated and the question of whether functionally selective compounds (compounds that selectively activate one pathway over another) can be rationally developed must be raised. Although numerous high-throughput screening (HTS) compatible assays exist for studying second messengers arising from G-protein signaling, far fewer HTS compatible assays exist for studying beta-arrestin recruitment. The authors report on the Tango^TM 7TM receptor assay technology, a high-throughput homogeneous assay method for monitoring beta-arrestin recruitment that uses a live-cell fluorescent readout. This assay format is broadly applicable to 7TM receptors, independent of G-protein coupling and, as such, has been used to produce assays for over 70 7TM receptor targets. The authors also show how flow cytometry can be used to select clones with desired pharmacological profiles and how an inducible expression system can increase the assay window for targets with high levels of constitutive activity. Finally, they demonstrate how the Tango^TM system can be used in parallel with assays aimed at second-messenger signaling to enable functional selectivity studies. (Journal of Biomolecular Screening 2009:798-810)

Pharmacological Characterization of Receptor Redistribution and {beta}-Arrestin Recruitment Assays for the Cannabinoid Receptor 1

Tue, 01 Sep 2009 15:40:26 PDT

Receptor redistribution and β-arrestin recruitment assays provide a G-protein-subtype-independent method to measure ligand-stimulated activation of G-protein-coupled receptors. In particular β-arrestin assays are becoming an increasingly popular tool for drug discovery. The authors have compared a high-content-imaging-based Redistribution^® assay and 2 nonimaging-based β-arrestin recruitment assays, Tango^{^TM} and PathHunter^{^TM}, for the cannabinoid receptor 1. Inasmuch as all 3 assays use receptors that are modified at the C-terminus, the authors verified their pharmacology via detection of G_i coupling of the receptor in cAMP assays using reference ligands. The potencies and efficacies of the cannabinoid receptor agonists CP55,940 and WIN55,212-2 correlated well between the 3 assays, and are comparable with the measured ligand binding affinities. The inverse agonist SR141716 decreased basal signal in all 3 assays, but only in the Tango bla assay a reliable EC₅₀ could be determined for this compound, suggesting that Tango is the most suitable assay for the identification of new inverse agonists. Both the Redistribution and the PathHunter assay could discriminate partial agonists from full agonists, whereas in the Tango assay partial agonists behaved as full agonists. Only the PathHunter cells allowed detection of cannabinoid receptor activation via β-arrestin recruitment and G_i-protein-mediated inhibition of cAMP, thus enabling the identification of biased ligands that differ in these cellular effects. The characteristics and limitations of the different assays are discussed. (Journal of Biomolecular Screening 2009:811-823)

Identification and Characterization of Novel Tissue-Nonspecific Alkaline Phosphatase Inhibitors with Diverse Modes of Action

Tue, 01 Sep 2009 15:40:26 PDT

Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitous enzyme expressed at high levels in bone, liver, and kidney. It appears involved in dephosphorylation of numerous phosphate monoesters, but only 2 of them, pyrophosphate and pyridoxal phosphate, have yet been unequivocally documented. Discovery and characterization of other substrates could be considerably facilitated if specific and potent modulators of TNAP activity with various modes of action were available. Here, the authors describe in detail a high-throughput screening campaign to identify inhibitors of TNAP, performed within the Molecular Library Screening Center Network (MLSCN). A novel homogeneous luminescent TNAP assay was developed and optimized with respect to the enzyme and substrate concentrations, enabling identification of a large number of compounds overlooked by a conventional colorimetric assay. Several new chemical series were identified from screening the Molecular Libraries Small Molecule Repository (MLSMR) collection and demonstrated to have diverse selectivity and mode of inhibition profiles. The nanomolar potency of some of these scaffolds surpasses currently known inhibitors. This article provides an example of a success where the Roadmap Initiative collaborative model, sponsored by the National Institutes of Health, brought together a deep knowledge of target biology from a principal investigator’s laboratory, a well-designed compound collection from the MLSMR, and an industrial-level screening facility and staff at the MLSCN center to identify pharmacologically active compounds, with outstanding selectivity data from a panel of more than 200 publicly accessible assays, through a high-throughput screen. (Journal of Biomolecular Screening 2009:824-837)

High-Throughput, Cell-Free, Liposome-Based Approach for Assessing In Vitro Activity of Lipid Kinases

Tue, 01 Sep 2009 15:40:26 PDT

Lipid kinases are central players in lipid signaling pathways involved in inflammation, tumorigenesis, and metabolic syndrome. A number of these kinase targets have proven difficult to investigate in higher throughput cell-free assay systems. This challenge is partially due to specific substrate interaction requirements for several of the lipid kinase family members and the resulting incompatibility of these substrates with most established, homogeneous assay formats. Traditional, cell-free in vitro investigational methods for members of the lipid kinase family typically involve substrate incorporation of [-³²P] and resolution of signal by thin-layer chromatography (TLC) and autoradiograph densitometry. This approach, although highly sensitive, does not lend itself to high-throughput testing of large numbers of small molecules (100 s to 1 MM+). The authors present the development and implementation of a fully synthetic, liposome-based assay for assessing in vitro activity of phosphatidylinositol-5-phosphate-4-kinase isoforms (PIP4KIIβ and ) in 2 commonly used homogeneous technologies. They have validated these assays through compound testing in both traditional TLC and radioactive filterplate approaches as well as binding validation using isothermic calorimetry. A directed library representing known kinase pharmacophores was screened against type IIβ phosphatidylinositol-phosphate kinase (PIPK) to identify small-molecule inhibitors. This assay system can be applied to other types and isoforms of PIPKs as well as a variety of other lipid kinase targets. (Journal of Biomolecular Screening 2009:838-844)

Multiplexing of Pathway-Specific {beta}-Lactamase Reporter Gene Assays by Optical Coding With Qtracker(R) Nanocrystals

Machleidt, T., Whitney, P., Bi, K. — Tue, 01 Sep 2009 15:40:26 PDT

Reporter assays are widely used in research and drug discovery for analysis of signaling pathways in a cell-based format. Traditionally, reporter gene assays are run in a single-parameter mode, interrogating only 1 pathway per sample. To enable more complex assay formats for pathway analysis, the authors developed a multiplexed reporter cell-based assay that combines optical encoding with a β-lactamase reporter gene readout. The optical encoding is achieved by peptide-mediated delivery of quantum dots into reporter cell lines. Using different quantum dots, the authors were able to simultaneously analyze multiple signaling pathways in the same sample using fluorescence microscopy or flow cytometry. They selected 3 β-lactamase reporter cell lines for the analysis of tumor necrosis factor alpha (TNF-), interleukin 6 (IL-6), and interferon gamma (IFN-) induced signaling to perform proof-of-principle experiments. The analysis demonstrates that this multiplexed assay allows the reliable detection of ligand-specific activation patterns as well as pathway-specific inhibitors. This method provides a template for the development of novel assay designs that enable the analysis of complex signaling networks involving multiple signaling pathways as well as cell-specific pathways in heterotypic cell models. (Journal of Biomolecular Screening 2009:845-852)

A High-Throughput Method to Identify Novel Senescence-Inducing Compounds

Ewald, J. A., Peters, N., Desotelle, J. A., Hoffmann, F. M., Jarrard, D. F. — Tue, 01 Sep 2009 15:40:26 PDT

Cellular senescence is a persistently growth-arrested phenotype in normal and transformed cells induced by noncytotoxic stress. Cytostasis as a method of cancer treatment has recently generated significant interest. Research into the induction of cellular senescence as cancer therapy has been hindered by a lack of compounds that efficiently induce this response. The authors describe a semiautomated high-throughput method to identify library compounds that induce senescence using prostate cancer cells cultured in 96-well plates. Primary hits are identified by low cell numbers after 3 days in culture, measured by Hoechst 33342 fluorescence. A secondary visual assessment of senescence-associated β-galactosidase staining and cellular morphology in the same wells distinguishes senescence from quiescence, apoptosis, and other false positives. This method was used to screen a 4160-compound library of known bioactive compounds and natural products at a 10-µM dose. Candidate compounds were further selected based on persistent growth arrest after drug removal and increased expression of previously described senescence marker genes. Four lead compounds not previously associated with senescence were identified for further investigation. This is the first successful assay to identify novel agents from compound libraries based on senescence induction in cancer cells. (Journal of Biomolecular Screening 2009:853-858)

Product Focus: Microplates, Assay Reagents, Screening Consumables, and Kits

Tue, 01 Sep 2009 15:40:26 PDT

2009 Meetings Calendar

Tue, 01 Sep 2009 15:40:26 PDT

Oral Abstracts from the Society of Biomolecular Sciences 15th Annual Conference and Exhibition

Tue, 01 Sep 2009 15:40:26 PDT

Detection of Intracellular Granularity Induction in Prostate Cancer Cell Lines by Small Molecules Using the HyperCyt(R) High-Throughput Flow Cytometry System

Fri, 17 Jul 2009 12:52:35 PDT

Prostate cancer is a leading cause of death among men due to the limited number of treatment strategies available for advanced disease. Discovery of effective chemotherapeutics involves the identification of agents that inhibit cancer cell growth. Increases in intracellular granularity have been observed during physiological processes that include senescence, apoptosis, and autophagy, making this phenotypic change a useful marker for identifying small molecules that induce cellular growth arrest or death. In this regard, epithelial-derived cancer cell lines appear uniquely susceptible to increased intracellular granularity following exposure to chemotherapeutics. We have established a novel flow cytometry approach that detects increases in side light scatter in response to morphological changes associated with intracellular granularity in the androgen-sensitive LNCaP and androgen-independent PC3 human prostate cancer cell lines. A cell-based assay was developed to screen for small molecule inducers of intracellular granularity using the HyperCyt^® high-throughput flow cytometry platform. Validation was performed using the Prestwick Chemical Library, where known modulators of LNCaP intracellular granularity, such as testosterone, were identified. Nonandrogenic inducers of granularity were also detected. A further screen of ~25,000 small molecules led to the identification of a class of aryl-oxazoles that increased intracellular granularity in both cell lines, often leading to cell death. The most potent agents exhibited submicromolar efficacy in LNCaP and PC3 cells. (Journal of Biomolecular Screening. 2009:596-609)

Polyplexed Flow Cytometry Protein Interaction Assay: A Novel High-Throughput Screening Paradigm for RGS Protein Inhibitors

Roman, D. L., Ota, S., Neubig, R. R. — Fri, 17 Jul 2009 12:52:35 PDT

Intracellular signaling cascades are a series of regulated protein-protein interactions that may provide a number of targets for potential drug discovery. Here, the authors examine the interaction of regulators of G-protein signaling (RGS) proteins with the G-protein Go, using a flow cytometry protein interaction assay (FCPIA). FCPIA accurately measures nanomolar binding constants of this protein-protein interaction and has been used in high-throughput screening. This report focuses on 5 RGS proteins (4, 6, 7, 8, and 16). To increase the content of screens, the authors assessed high-throughput screening of these RGS proteins in multiplex, by establishing binding constants of each RGS with Go in isolation, and then in a multiplex format with 5 RGS proteins present. To use this methodology as a higher-content multiplex protein-protein interaction screen, they established Z-factor values for RGS proteins in multiplex of 0.73 to 0.92, indicating this method is suitable for screening using FCPIA. To increase throughput, they also compressed a set of 8000 compounds by combining 4 compounds in a single assay well. Subsequent deconvolution of the compounds mixtures verified the identification of active compounds at specific RGS targets in their mixtures using the polyplexed FCPIA method. (Journal of Biomolecular Screening 2009: 610-619)

Development and Evaluation of a FACS-Based Medium-Throughput Assay for HCV Entry Inhibitors

Fri, 17 Jul 2009 12:52:35 PDT

The interaction between the hepatitis C virus (HCV) envelope glycoprotein E2 and the human tetraspanin protein CD81 is one of the key events involved in HCV cell entry. Therefore, compounds that interfere with this interaction may be useful tools for basic research and potential drugs for the treatment of HCV infection. The authors describe a medium-throughput assay for ligands of the E2 binding site on the CD81 receptor. In the assay, human hepatoma cells are incubated with the test compounds and stained with a fluorescently labeled anti-CD81 antibody (JS81). Flow cytometry is used to detect the level of bound antibody, reflecting the inhibitory potencies of the compounds. Eighty percent of compounds active in the assay show efficacy in an infection assay using luciferase reporter genome in cell culture. Thus, the assay can be used as a fast screening system for inhibitors of interaction of viral E2 to host cell CD81-LELs. (Journal of Biomolecular Screening 2009;620-626)

Characterization of a Robust Enzymatic Assay for Inhibitors of 2-Oxoglutarate-Dependent Hydroxylases

Fri, 17 Jul 2009 12:52:35 PDT

The prolyl-4-hydroxylase proteins regulate the hypoxia-inducible transcription factors (HIFs) by hydroxylation of proline residues targeting HIF-1 for proteasomal degradation. Using the purified catalytic domain of prolyl hydroxylase 2 (PHD2_181-417), an enzymatic assay has been developed to test inhibitors of the enzyme in vitro. Because PHD2 hydroxylates HIF-1, with succinic acid produced as an end product, radiolabeled [5-¹⁴C]-2-oxoglutaric acid was used and formation of [¹⁴C]-succinic acid was measured to quantify PHD2_181-417 enzymatic activity. Comparison of the separation of 2-oxoglutaric acid and succinic acid by either ion exchange chromatography or precipitation with phenylhydrazine showed similar results, but the quantification and throughput were vastly increased using the latter method. The PHD2 reaction was substrate and concentration dependent. The addition of iron to the enzyme reaction mix resulted in an increase in enzymatic activity. The K_m value for 2-oxoglutaric acid was determined to be 0.9 µM, and known PHD2 inhibitors were used to validate the assay. In addition, the authors demonstrate that this assay can be applied to other 2-oxoglutaric acid-dependent enzymes, including the asparaginyl hydroxylase, factor-inhibiting HIF-1 (FIH). A concentration-dependent increase in succinic acid production using recombinant FIH enzyme with a synthetic peptide substrate was observed. The authors conclude that a by-product enzyme assay measuring the conversion of 2-oxoglutaric acid to succinic acid using the catalytic domain of the human PHD2 provides a convenient method for the biochemical evaluation of inhibitors of the 2-oxoglutaric acid-dependent hydroxylases. (Journal of Biomolecular Screening 2009:627-635)

A Simplified Scintillation Proximity Assay for Fatty Acid Synthase Activity: Development and Comparison with Other FAS Activity Assays

Bays, N. W., Hill, A. D., Kariv, I. — Fri, 17 Jul 2009 12:52:35 PDT

Fatty acid synthase (FAS), an essential enzyme for de novo lipogenesis, has been implicated in a number of disease states, including obesity, dyslipidemia, and cancer. To identify small-molecule inhibitors of FAS, the authors developed a bead-based scintillation proximity assay (SPA) to detect the fatty acid products of FAS enzymatic activity. This homogeneous SPA assay discriminates between a radiolabeled hydrophilic substrate of FAS (acetyl-coenzyme A) and the labeled lipophilic products of FAS (fatty acids), generating signal only when labeled fatty acids are present. The assay requires a single addition of unmodified polystyrene imaging SPA beads and can be miniaturized to 384- or 1536-well density with appropriate assay statistics for high-throughput screening. High-potency FAS inhibitors were used to compare the sensitivity of the SPA bead assay with previously described assays that measure FAS reaction intermediates (CoA-SH and NADP⁺). The advantages and disadvantages of these different FAS assays in small-molecule inhibitor discovery are discussed. (Journal of Biomolecular Screening 2009:636-642)

Dequalinium, a New Inhibitor of Mycobacterium tuberculosis Mycothiol Ligase Identified by High-Throughput Screening

Gutierrez-Lugo, M.-T., Baker, H., Shiloach, J., Boshoff, H., Bewley, C. A. — Fri, 17 Jul 2009 12:52:35 PDT

Mycothiol ligase (MshC) is a key enzyme in the biosynthesis of mycothiol, a small molecular weight thiol that is unique to actinomycetes and whose primary role is to maintain intracellular redox balance and remove toxins. MshC catalyzes the adenosine triphosphate (ATP)—dependent condensation of cysteine and glucosamine-inositol (GI) to produce cysteine-glucosamine-inositol (CGI). MshC is essential to Mycobacterium tuberculosis and therefore represents an attractive target for chemotherapeutic intervention. A screening protocol was developed to identify MshC inhibitors based on quantification of residual ATP using a coupled luminescent assay. The protocol was used to screen a library of 3100 compounds in a 384-well plate format (Z' ≥ 0.65). Fifteen hits (0.48%) were identified from the screen, and 2 hits were confirmed in a secondary assay that measures production of CGI. The structures of both hits contain N-substituted quinolinium moieties, and the more potent of the 2—namely, dequalinium chloride—inhibits MshC with an IC₅₀ value of 24 ± 1 µM. Further studies showed dequalinium to be an ATP-competitive inhibitor of MshC, to bind MshC with a K_D of 0.22 µM, and to inhibit the growth of M. tuberculosis under aerobic and anaerobic conditions with minimum inhibitory and anaerobic bactericidal concentrations of 1.2 and 0.3 µg/mL, respectively. The screening protocol described is robust and has enabled the identification of new MshC inhibitors. (Journal of Biomolecular Screening 2009:643-652)

Can Phage Display Be Used as a Tool to Functionally Identify Endogenous Eat-Me Signals in Phagocytosis?

Caberoy, N. B., Zhou, Y., Li, W. — Fri, 17 Jul 2009 12:52:35 PDT

Removal of apoptotic cells and cellular debris by phagocytosis is essential for development, tissue homeostasis, and resolution of inflammation. Eat-me signals control the initiation of phagocytosis, holding a key to the understanding of phagocyte biology. Because of a lack of functional cloning strategy, eat-me signals are conventionally identified and characterized on a case-by-case basis. The feasibility of functional cloning of eat-me signals by phage display is investigated by characterizing the biological behavior of T7 phages displaying 2 well-known eat-me signals: growth arrest—specific gene 6 (Gas6) and milk fat globule—EGF8 (MFG-E8). Gas6-phage binds to all 3 known Gas6 receptors: Mer, Axl, and Tyro3 receptor tyrosine kinases. Gas6-phage and MFG-E8-phage are capable of binding to phagocytes and nonphagocytes. However, both phages stimulate phage uptake only in phagocytes, including macrophages, microglia, and retinal pigment epithelium cells, but not in nonphagocytes. Furthermore, functional phage selection by phagocytosis in phagocytes enriches both Gas6-phage and MFG-E8-phage, suggesting that phage display can be used as a tool to functionally identify unknown eat-me signals from phage display cDNA library. (Journal of Biomolecular Screening 2009:653-661)

Novel Temperature Activation Cell-Based Assay on Thermo-TRP Ion Channels

Aneiros, E., Dabrowski, M. — Fri, 17 Jul 2009 12:52:35 PDT

The precise temperature control of the ABI Prism^® 7900HT Sequence Detection System designed for detection of fluorescence of a biological sample in real-time PCR assays (TaqMan assays) was used to activate Thermo-TRP ion channels, enabling a novel 384-/96-well plate-based assay. Functional pharmacology was verified against the temperature activation using intracellular calcium fluorescence as a measure of ion channel activity. The assay is applicable to both heterologous expression systems and dorsal root ganglia primary cells. This will benefit several analgesic drug discovery programs searching for new Thermo-TRP modulators. (Journal of Biomolecular Screening 2009:662-667)

Development of a Fluorescence-Based, Ultra High-Throughput Screening Platform for Nanoliter-Scale Cytochrome P450 Microarrays

Sukumaran, S. M., Potsaid, B., Lee, M.-Y., Clark, D. S., Dordick, J. S. — Fri, 17 Jul 2009 12:52:35 PDT

Cytochrome P450 enzyme (CYP450s) assays are critical enzymes in early-stage lead discovery and optimization in drug development. Currently available fluorescence-based reaction assays provide a rapid and reliable method for monitoring CYP450 enzyme activity but are confined to medium-throughput well-plate systems. The authors present a high-throughput, integrated screening platform for CYP450 assays combining enzyme encapsulation techniques, microarraying methods, and wide-field imaging. Alginate-containing microarrays consisting of up to 1134 CYP450 reaction elements were fabricated on functionalized glass slides (reaction volumes 20 to 80 nL, total enzyme content in pg) and imaged to yield endpoint activity, stability, and kinetic data. A charge-coupled device imager acquired quantitative, high-resolution images of a 20 x 20 mm area/snapshot using custom-built wide-field optics with telecentric lenses and easily interchangeable filter sets. The imaging system offered a broad dynamic intensity range (linear over 3 orders of magnitude) and sensitivity down to fluorochrome quantities of <5 fmols, with read accuracy similar to a laser scanner or a fluorescence plate reader but with higher throughput. Rapid image acquisition enabled analysis of CYP450 kinetics. Fluorogenic assays with CYP3A4, CYP2C9, and CYP2D6 on the alginate microarrays exhibited Z' factors ranging from 0.75 to 0.85, sensitive detection of inhibitory compounds, and reactivity comparable to that in solution, thereby demonstrating the reliability and accuracy of the microarray platform. This system enables for the first time a significant miniaturization of CYP enzyme assays with significant conservation of assay reagents, greatly increased throughput, and no apparent loss of enzyme activity or assay sensitivity. (Journal of Biomolecular Screening 2009:668-678)

Efficient Elimination of Nonstoichiometric Enzyme Inhibitors from HTS Hit Lists

Fri, 17 Jul 2009 12:52:35 PDT

High-throughput screening often identifies not only specific, stoichiometrically binding inhibitors but also undesired compounds that unspecifically interfere with the targeted activity by nonstoichiometrically binding, unfolding, and/or inactivating proteins. In this study, the effect of such unwanted inhibitors on several different enzyme targets was assessed based on screening results for over a million compounds. In particular, the shift in potency on variation of enzyme concentration was used as a means to identify nonstoichiometric inhibitors among the screening hits. These potency shifts depended on both compound structure and target enzyme. The approach was confirmed by statistical analysis of thousands of dose-response curves, which showed that the potency of competitive and therefore clearly stoichiometric inhibitors was not affected by increasing enzyme concentration. Light-scattering measurements of thermal protein unfolding further verified that compounds that stabilize protein structure by stoichiometric binding show the same potency irrespective of enzyme concentration. In summary, measuring inhibitor IC₅₀ values at different enzyme concentrations is a simple, cost-effective, and reliable method to identify and eliminate compounds that inhibit a specific target enzyme via nonstoichiometric mechanisms. (Journal of Biomolecular Screening 2009:679-689)

Plate-Based Diversity Selection Based on Empirical HTS Data to Enhance the Number of Hits and Their Chemical Diversity

Fri, 17 Jul 2009 12:52:35 PDT

Typically, screening collections of pharmaceutical companies contain more than a million compounds today. However, for certain high-throughput screening (HTS) campaigns, constraints posed by the assay throughput and/or the reagent costs make it impractical to screen the entire deck. Therefore, it is desirable to effectively screen subsets of the collection based on a hypothesis or a diversity selection. How to select compound subsets is a subject of ongoing debate. The authors present an approach based on extended connectivity fingerprints to carry out diversity selection on a per plate basis (instead of a per compound basis). HTS data from 35 Novartis screens spanning 5 target classes were investigated to assess the performance of this approach. The analysis shows that selecting a fingerprint-diverse subset of 250K compounds, representing 20% of the screening deck, would have achieved significantly higher hit rates for 86% of the screens. This measure also outperforms the Murcko scaffold-based plate selection described previously, where only 49% of the screens showed similar improvements. Strikingly, the 2-fold improvement in average hit rates observed for 3 of 5 target classes in the data set indicates a target bias of the plate (and thus compound) selection method. Even though the diverse subset selection lacks any target hypothesis, its application shows significantly better results for some targets—namely, G-protein-coupled receptors, proteases, and protein-protein interactions—but not for kinase and pathway screens. The synthetic origin of the compounds in the diverse subset appears to influence the screening hit rates. Natural products were the most diverse compound class, with significantly higher hit rates compared to the compounds from the traditional synthetic and combinatorial libraries. These results offer empirical guidelines for plate-based diversity selection to enhance hit rates, based on target class and the library type being screened. (Journal of Biomolecular Screening 2009:690-699)

Buffer Optimization of Thermal Melt Assays of Plasmodium Proteins for Detection of Small-Molecule Ligands

Fri, 17 Jul 2009 12:52:35 PDT

In the past decade, thermal melt/thermal shift assays have become a common tool for identifying ligands and other factors that stabilize specific proteins. Increased stability is indicated by an increase in the protein's melting temperature (T_m). In optimizing the assays for subsequent screening of compound libraries, it is important to minimize the variability of T_m measurements so as to maximize the assay's ability to detect potential ligands. The authors present an investigation of T_m variability in recombinant proteins from Plasmodium parasites. Ligands of Plasmodium proteins are particularly interesting as potential starting points for drugs for malaria, and new drugs are urgently needed. A single standard buffer (100 mM HEPES [pH 7.5], 150 mM NaCl) permitted estimation of T_m for 58 of 61 Plasmodium proteins tested. However, with several proteins, T_m could not be measured with a consistency suitable for high-throughput screening unless alternative protein-specific buffers were employed. The authors conclude that buffer optimization to minimize variability in T_m measurements increases the success of thermal melt screens involving proteins for which a standard buffer is suboptimal. (Journal of Biomolecular Screening 2009:700-707)

Overcoming Problems of Compound Storage in DMSO: Solvent and Process Alternatives

Waybright, T. J., Britt, J. R., McCloud, T. G. — Fri, 17 Jul 2009 12:52:35 PDT

The common practice of preparing storage libraries of compounds in 100% DMSO solution well in advance of bioassay brings with it difficulties that affect the accuracy of the data obtained. This publication presents a series of studies done on a subset of compounds that are difficult to bioassay because they precipitate from DMSO solution. These compounds are members of a frequently used, diverse compound library of the sort commonly used in the high-throughput screening (HTS) environment. Experiments were performed to determine the concentration of drug in solution above the precipitate, observe the time course and effect of various mixtures of solvents upon precipitation, measure the viscosity of cosolvents to determine compatibility with HTS, determine water absorption rates for various solvent combinations, and investigate resolubilization techniques to ensure proper drug solution for HTS. Recommendations are made on how to best maximize the probability that problem compounds will remain in solution, be accurately transferred during assay plate production, and, as a result, be accurately bioassayed at the specified molar concentration. (Journal of Biomolecular Screening 2009:708-715)

Use of Reduced Temperature Cell Pausing to Enhance Flexibility of Cell-Based Assays

Wise, H., Abel, P. W., Cawkill, D. — Fri, 17 Jul 2009 12:52:35 PDT

Construction and supply of cell-based reagents for in vitro plate-based screens are often highlighted as a bottleneck within drug discovery. Recent years have seen the successful application of both cryopreservation and automation to increase the capacity and flexibility of cell provision. However, routine cell culture remains a fixed experimental process that requires cells to be prepared and used at specific times. We have investigated the potential of reduced temperature incubation to be used as a simple methodology for stopping and starting cell growth and introduce further flexibility into cell provision. Our results show that incubation of CHOK1, HEK293, and 1321N1 cells at 23 °C arrested growth while maintaining cell viability. Recovery of these paused cells at 37 °C resulted in resumption of normal cell growth and target protein function. Experiments demonstrated that paused cells, expressing either a recombinant G-protein-coupled receptor or an ion channel, performed comparably with the equivalent continuously cultured cells in a 384-well cell-based assay. This simple technique offers the potential to introduce flexibility into cell culture experiments and processes that were previously considered to be fixed. (Journal of Biomolecular Screening 2009:716-722)

Primary Leukocyte Screens for Innate Immune Agonists

Fri, 17 Jul 2009 12:52:35 PDT

The innate immune system of mammals is a key defense mechanism against invading foreign pathogens. Innate immune stimulants may have applications as vaccine adjuvants as well as in the treatment of cancer and some viral diseases, and clinical studies have been performed using agonists of Toll-like receptors (TLRs) 7, 8, and 9. The high-throughput screens for such agonists have typically relied on the overexpression of a single TLR gene in an immortalized cell line and are inherently artificial systems that are restricted to the identification of agonists for a single receptor. The authors describe 2 assays for the identification of immunostimulants that employ primary human leukocytes cocultured with hepatitis C virus (HCV) replicon-expressing cells. In these assays, stimulation of innate immune pathways in leukocytes induces interferon (IFN) expression, which acts to inhibit HCV replication, providing a high-throughput and low-cost readout for leukocyte activation. These assays are highly sensitive and provide a physiologically relevant system for the identification of a broad range of immunostimulant agents. (Journal of Biomolecular Screening 2009:723-730)

Product Focus: Screening Robotics and Automation

Fri, 17 Jul 2009 12:52:35 PDT

2009 Meetings Calendar

Fri, 17 Jul 2009 12:52:35 PDT