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PubMed2001

Luminescent proteins from Aequorea victoria: applications in drug discovery and in high throughput analysis.

Recent progress in generating a vast number of drug targets through genomics and large compound libraries through combinatorial chemistry have stimulated advancements in drug discovery through the development of new high throughput screening (HTS) methods. Automation and HTS techniques are also highly desired in fields such as clinical diagnostics. Luminescence-based assays have emerged as an alternative to radiolabel-based assays in HTS as they approach the sensitivity of radioactive detection along with ease of operation, which makes them amenable to miniaturization. Luminescent proteins provide the advantage of reduced reagent and operating costs because they can be produced in unlimited amounts through the use of genetic engineering tools. In that regard, the use of two naturally occurring and recombinantly produced luminescent proteins from the jellyfish Aequorea victoria, namely, aequorin and the green fluorescent protein (GFP), has attracted attention in a number of analytical applications in diverse research areas. Aequorin is naturally bioluminescent and has therefore, virtually no associated background signal, which allows its detection down to attomole levels. GFP has become the reporter of choice in a variety of applications given that it is an autofluorescent protein that does not require addition of any co-factors for fluorescence emission. Furthermore, the generation of various mutants of GFP with differing luminescent and spectral properties has spurred additional interest in this protein. In this review, we focus on the use of aequorin and GFP in the development of highly sensitive assays that find applications in drug discovery and in high throughput analysis.

Aequorin↗
PubMed2005

Description and validation of a four-channel staggered LC-MS/MS system for high-throughput in vitro screens.

A parallel four-channel LC-MS/MS system for quantitative high-throughput in vitro screens is described. The system comprises four pumps, a four-valve autosampler equipped with a nine-port stream selector valve, and a triple quadrupole mass spectrometer. Staggered injections across the sample list are performed by four injector valves onto separate columns in such a manner that the chromatographic window of interest is always eluting from one of the columns into the mass spectrometer. To demonstrate the applicability of the system, three different compounds (verapamil, propanolol, dextromethorphan) were analyzed. The results showed that the sample throughput could be increased more than three times in comparison with a conventional single-column LC-MS/MS system. Validation results for the three compounds showed an accuracy of 85.0-108%, a precision (given as relative standard deviation) of 2.60-10.7%, and a carryover of less than 0.1%.

Caco-2 Cells↗
PubMed2004

Generic three-column parallel LC-MS/MS system for high-throughput in vitro screens.

A parallel three-column LC-MS/MS system for quantitative high-throughput in vitro screens is described. The robust novel system is composed of three LC pumps, an autosampler and a triple quadrupole mass spectrometer used in combination with three valves and three analytical LC columns in parallel configuration. Two of the three valves work in unison to select which column receives the injection, and the third valve selects which column is to be in line with the mass spectrometer. Improved sample throughput is achieved without sacrificing chromatographic separation quality or sensitivity. To demonstrate the applicability of the system, pools of five compounds (phenacetin, S-mephenytoin, bufuralol, midazolam and clomethiazole) were analyzed, together with an internal standard. The results show that the sample throughput can be increased significantly by reducing analysis time to 3 min per sample as compared to 8 min with a general gradient single-column system. Analysis of the five compounds shows an accuracy of 81-108% and a precision (given as relative standard deviation) of 1.5-14%. The system was further applied to samples from a metabolic stability assay in liver microsomes.

Chromatography, High Pressure Liquid↗
PubMed2002

Increasing throughput in lead optimization in vivo toxicity screens.

Lead optimization requires toxicity screening strategies to select a compound with a high likelihood of successful development. As numerous compounds need to be screened and resources to direct toward any single compound are limited, short turnaround times to generate and interpret data are needed. Utilization of in vivo toxicity screens is necessary for an effective screening strategy, however, if not appropriately implemented, they may consume excessive resources and prolong selection of a developable compound. Optimization of in vivo studies requires identifying effective placement into the screening strategy, selecting the appropriate study designs, implementing processes that allow rapid data generation and interpretation, and understanding the utilities of in vivo data. When implemented, an effective, high-throughput screening strategy will utilize adequate but minimal amounts of resources, and will prioritize processing near technical time limits. These require generating only the data from which decisions will be made and can be best achieved using as few animals as possible per study.

Animals↗
PubMed2003

Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells.

We used synthetic lethal high-throughput screening to interrogate 23,550 compounds for their ability to kill engineered tumorigenic cells but not their isogenic normal cell counterparts. We identified known and novel compounds with genotype-selective activity, including doxorubicin, daunorubicin, mitoxantrone, camptothecin, sangivamycin, echinomycin, bouvardin, NSC146109, and a novel compound that we named erastin. These compounds have increased activity in the presence of hTERT, the SV40 large and small T oncoproteins, the human papillomavirus type 16 (HPV) E6 and E7 oncoproteins, and oncogenic HRAS. We found that overexpressing hTERT and either E7 or LT increased expression of topoisomerase 2alpha and that overexpressing RAS(V12) and ST both increased expression of topoisomerase 1 and sensitized cells to a nonapoptotic cell death process initiated by erastin.

Antineoplastic Agents↗
PubMed2006

A screening method for chiral selectors that does not require covalent attachment.

A high-throughput screening protocol is proposed for chiral selector discovery. It is modeled after the protocol for biological screening of candidate drugs from chemical libraries. The procedure works based on target distribution between an aqueous phase and an organic phase. The target may be a racemate or separate enantiomers. Screening for noncovalent intermolecular association between target and candidate selectors is carried out by partitioning experiments in the presence and absence of the candidate chiral selectors in the organic phase (plasticized poly(vinyl chloride)). The partition ratio measurement uses 96-well plates for high throughput. The feasibility of this approach is validated by working with a known target/chiral selector pair, N-(3,5-dinitrobenzoyl)-alpha-phenylglycine and 2,2,2-trifluoro-1-(9-anthryl)ethanol. The validated protocol is applied to a small library of 12 cyclopropyl dipeptide isosteres. Eight bind the racemic target, econazole. Among them, one has measurable chiral selectivity. The advantage of the method is that it does not require the covalent attachment of either the analyte or the selector, and the required amount of the potential chiral selector is about 100 mug.

Anthracenes↗
PubMed2006

Application of fragment screening and fragment linking to the discovery of novel thrombin inhibitors.

The screening of fragments is an alternative approach to high-throughput screening for the identification of leads for therapeutic targets. Fragment hits have been discovered using X-ray crystallographic screening of protein crystals of the serine protease enzyme thrombin. The fragment library was designed to avoid any well-precedented, strongly basic functionality. Screening hits included a novel ligand (3), which binds exclusively to the S2-S4 pocket, in addition to smaller fragments which bind to the S1 pocket. The structure of these protein-ligand complexes are presented. A chemistry strategy to link two such fragments together and to synthesize larger drug-sized compounds resulted in the efficient identification of hybrid inhibitors with nanomolar potency (e.g., 7, IC50 = 3.7 nM). These potent ligands occupy the same area of the active site as previously described peptidic inhibitors, while having very different chemical architecture.

Carbamates↗
PubMed2004

Dynamic ion channel activation scheduling in patch clamp on a chip.

In 2002, Fertig et al. made a remarkable invention: the first successful demonstration of a patch clamp on a chip--a planar quartz-based biological chip that contains up to several hundred ion channels. This patch-clamp chip can be used in massively parallel screens for ion channel activity, thereby providing a high-throughput screening tool for drug discovery efforts. In this paper, we propose computationally efficient dynamic stochastic scheduling algorithms for activating individual ion channels in the patch-clamp chip. By formulating the ion channel activation scheduling problem as a partially observed Markov decision process with a multiarmed bandit structure, near-optimal dynamic scheduling for activation of the individual channels is achieved to optimize the information gained from the patch-clamp chip. Numerical examples using state-of-the-art algorithms developed recently in artificial intelligence and operations research are presented to illustrate these dynamic ion channel (macromolecule) activation scheduling algorithms.

Algorithms↗
PubMed2005

G-protein-coupled receptors in drug discovery: nanosizing using cell-free technologies and molecular biology approaches.

Signal transduction by G-protein-coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. Signal transduction has been studied extensively with both cell-based systems and assays comprising isolated signaling components. Interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high throughput screening, biosensors, and so on will focus greater attention on assay development to allow for miniaturization, ultra-high throughput and, eventually, microarray/biochip assay formats. Although cell-based assays are adequate for many GPCRs, it is likely that these formats will limit the development of higher density GPCR assay platforms mandatory for other applications. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR assay platforms adaptable for such applications as microarrays. The authors review current cell-free GPCR assay technologies and molecular biological approaches for construction of novel, functional GPCR assays.

Biological Assay↗
PubMed2006

Purification and assay of kinase-active EGF receptor from mammalian cells by immunoaffinity chromatography.

The epidermal growth factor (EGF) receptor possesses intrinsic protein-tyrosine kinase activity, and both overexpressed wild-type and mutated forms have been associated with many types of cancers. Therefore, understanding the mechanisms that modulate receptor activity and function is essential to the development of treatments for many of these cancers. However, to address this issue by either conventional or high-throughput screening methods requires the availability of large amounts of highly purified and active EGF receptor. The technique described in this chapter utilizes immunoaffinity chromatography, which allows for the isolation of highly purified and active preparations of EGF receptor. By immobilizing an antibody that recognizes the ligand-binding domain of the receptor to Sepharose beads, the receptor can be eluted specifically from the antibody by the addition of EGF. This association establishes a unique interaction that ensures the isolation of a highly enriched preparation of EGF receptor. This protocol allows for the purification of large or small batches of receptor that retain their kinase activity. Additionally, this chapter reports on the subsequent steps necessary to characterize the receptor: kinase activity, mass, purity, and the ability of the receptor to undergo autophosphorylation.

Animals↗
PubMed2006

Fluorescence activated cell sorting for enzymatic activity.

Directed evolution is a reliable method for protein engineering and as a tool for investigating structure/function relationships. A key for a successful directed evolution experiment is oftentimes the screen. Fluorescence activated cell sorting (FACS) is powerful high-throughput screening approach to isolate and identify mutants from large protein libraries. FACS has been successful in isolating proteins with improved or altered binding affinity. However, FACS screening for mutants with enhanced catalytic activity has been met with limited success. This review focuses on the FACS screening of protein libraries for enzymatic activity.

Catalysis↗
PubMedDate not supplied

Development of an XTT tetrazolium salt-based assay for detection of specific hyperthermia sensitizers in a high-flux screening programme.

It is now possible to search for new drugs using high-throughput screening of chemical libraries accumulated over the past few years. To detect potential new hyperthermia sensitizers, we are screening for chemical inhibitors of thermotolerance. For the screening of a large chemical library, a rapid and simple assay based on the XTT-tetrazolium salt with the addition of intermediate electron acceptor, phenazine methosulphate (PMS) as a promoter, was developed. It was found that the sensitivity of the XTT/PMS assay is sufficient for assessing thermal cell killing and thermotolerance, although it was highly dependent on cell number and type. When the formazan assay system was challenged with the bioflavonoid drug quercetin (up to 25mm) and validated against the clonogenic cell survival assay, significant decreases in thermotolerant cell viability were observed, directly reflecting inhibition of thermotolerance. Although short-term assays can, in some instances, underestimate overall cell killing, the dose dependency of inhibition of thermotolerance by quercetin recorded in this study by clonogenic and XTT/PMS assays was similar. Application of the XTT/PMS assay in chemical library screening was highly effective in differentiating potential thermotolerance inhibitors from both chemicals with lack of efficacy and from toxic compounds. Taken together, these results show that the XTT/PMS assay, when carried out under careful conditions, is well suited for primary high-flux screen of many thousands of compounds, thus opening up new areas for discovery of hyperthermia sensitizers.

3T3 Cells↗
PubMed2005

Here be dragons: docking and screening in an uncharted region of chemical space.

To compare virtual and high-throughput screening in an unbiased way, 50,000 compounds were docked into the 3-dimensional structure of dihydrofolate reductase prospectively, and the results were compared to a subsequent experimental screening of the same library. Undertaking these calculations demanded careful database curation and control calculations with annotated inhibitors. These ultimately led to a ranked list of more likely and less likely inhibitors and to the prediction that relatively few inhibitors would be found in the empirical screen. The latter prediction turned out to be correct, with arguably no validated inhibitors found experimentally. Subsequent retesting of high-scoring docked molecules may have found 2 true inhibitors, although this remains uncertain due to experimental ambiguities. The implications of this study for screening campaigns are considered.

Computational Biology↗
PubMed2001

Cost-effective whole-cell assay for laboratory evolution of hydroxylases in Escherichia coli.

Cytochrome P450 BM-3 from Bacillus megaterium catalyzes the subterminal hydroxylation of medium- and longchain fatty acids at the omega-1, omega-2, and omega-3 positions. A continuous spectrophotometric assay for P450 BM-3 based on the conversion of p-nitrophenoxycarboxylic acids (pNCA) to omega-oxycarboxylic acids and the chromophore p-nitrophenolate was reported recently. However, this pNCA assay procedure contained steps that limited its application in high throughput screening, including expression of P450 BM-3 variant F87A in 4-ml cultures, centrifugation, resuspension of the cell pellet, and cell lysis. We have shown that permeabilization of the outer membrane of Escherichia coli DH5alpha with polymyxin B sulfate, EDTA, polyethylenimine, or sodium hexametaphosphate results in rapid conversion of 12-pNCA. A NADPH-generating system consisting of NADP(+), D/L-isocitric acid, and the D/L-isocitrate dehydrogenase of E. coli DH5alpha reduced the cofactor expense more than 10-fold. By avoiding cell lysis, resuspension, and centrifugation, the high throughput protocol allows screening of thousands of samples per day.

Automation↗
PubMed2002

Homogeneous time-resolved fluorescence quenching assay (LANCE) for caspase-3.

In addition to kinases and G protein-coupled receptors, proteases are one of the main targets in modern drug discovery. Caspases and viral proteases, for instance, are potential targets for new drugs. To satisfy the current need for fast and sensitive high-throughput screening for inhibitors, new homogeneous protease assays are needed. We used a caspase-3 assay as a model to develop a homogeneous time-resolved fluorescence quenching assay technology. The assay utilizes a peptide labeled with both a luminescent europium chelate and a quencher. Cleavage of the peptide by caspase-3 separates the quencher from the chelate and thus recovers europium fluorescence. The sensitivity of the assay was 1 pg/microl for active caspase-3 and 200 pM for the substrate. We evaluated the assay for high-throughput usage by screening 9600 small-molecule compounds. We also evaluated this format for absorption/distribution/metabolism/excretion assays with cell lysates. Additionally, the assay was compared to a commercial fluorescence caspase-3 assay.

Biological Assay↗
PubMed2001

Development of SPC-ELISA: a new assay principle for the study of sulfated polysaccharide-protein interactions.

A sulfated polysaccharide-coating enzyme-linked immunosorbent assay (SPC-ELISA), a new screening assay for the study of interactions between sulfated polysaccharides and proteins, has been developed. Fibrinogen was used as representative for the protein. A microplate is coated with the sulfated polysaccharide to be tested and then incubated with various concentrations of fibrinogen. The bound fibrinogen is quantified by ELISA technique. The assay has been optimized with respect to coating procedure, incubation times, antibody concentrations, and detection conditions. Its capacity was demonstrated using three different sulfated polysaccharides: heparin, a sulfated glucuronogalactan extracted from a red algae, and a semisynthetic xanthan sulfate. Furthermore, the fibrinogen binding of semisynthetic laminarin sulfates with different degrees of sulfation showed good correlation with their anticoagulant effect as measured by fibrinogen clotting time. The intraassay as well as the interassay variations were lower than 8%. The binding properties observed in the SPC-ELISA correlated well with those found utilizing conventional gel permeation chromatography and fibrinogen affinity gel electrophoresis. Compared to these methods, the SPC-ELISA has several advantages: It is more rapid and far easier to perform, allows high throughput screening, and is suitable for automation. Furthermore, it is inexpensive, highly sensitive, and reproducible and has no special equipment requirements. Finally, the method represents the basis for multiple variations with regard to the target proteins as well as the detection methods.

Chromatography, Gel↗
PubMed2003

Design of small molecule libraries for NMR screening and other applications in drug discovery.

There are conceptual differences between high-throughput screening (HTS) and fragment-based screening by NMR. The number of compounds in libraries for NMR screening may be significantly smaller than those used for HTS. Because one relies on a small library its design is significantly important and is the object of this article. A short introduction on fragment-based NMR screening approaches will be provided. Although there are currently very few reports describing the design of libraries of small molecules for NMR screening, aspects of the question of how to compile diverse collections of small molecular fragments useful for drug design were previously addressed for the purposes of combinatorial library design and de novo drug design. As these disciplines are highly interrelated and are applied in an interconnected manner with NMR screening within the drug discovery process, a review of combinatorial library design and especially the building block or fragment selection strategies applied for combinatorial library design and de novo design is well suited to reveal fundamental strategies and potential techniques for the design of NMR screening libraries. This section will be rounded off by a report on hands-on-experience with the design of the Novartis second-site NMR screening library and practical considerations for the design of compound mixtures. Rather than providing an exact protocol general guidelines will be indicated.

Chemistry, Pharmaceutical↗
PubMed2007

Automated multiple ligand screening by frontal affinity chromatography-mass spectrometry (FAC-MS).

High-throughput screening (HTS) efforts to discover "hits" typically rely on the large-scale parallel screening of individual compounds with attempts to screen mixtures of compounds typically and, unfortunately, giving rise to false positives and false negatives due to the nature of the HTS readout (% inhibition/activation above a defined threshold) that makes deconvolution virtually intractable. Bioaffinity screening methods have emerged as an alternative or orthogonal method to classic HTS. One of these methods, frontal affinity chromatography coupled to mass spectrometry detection (FAC-MS), although still a relatively new technique, is turning out to be a viable screening tool. However, to push FAC-MS more to the forefront as a moderate primary HTS system (or a secondary screening assay), automation needs to be addressed. An automated FAC-MS system is described using 2 columns containing immobilized hERbeta, whereby while 1 column is being regenerated, the other is being used. The authors are extrapolating that in a continuous 24-h operation, the number of ligands screened could potentially approach 10,000. In addition, preliminary structure-activity relationship binding information (typically not seen in early primary HTS) can be obtained by observing the rank order of the library members in the various mixtures.

Amino Acid Sequence↗