Enzyme assays for high-throughput screening

Assaying enzyme-catalyzed transformations in high-throughput is crucial to enzyme discovery, enzyme engineering and the drug discovery process. In enzyme assays, catalytic activity is detected using labelled substrates or indirect sensor systems that produce a detectable spectroscopic signal upon reaction. Recent advances in the development of high-throughput enzyme assays have identified new labels and chromophores to detect a wide range of enzymes activities. Enzyme activity profiling and fingerprinting have also been used as tools for identification and classification, while microarray formats have been devised to increase throughput.     

Intracellular detection assays for high-throughput screening

New optical assay methods promise to accelerate the use of living cells in screens for drug discovery. Most of these methods employ either fluorescent or luminescent read-outs and allow cell-based assays for most targets, including receptors, ion channels and intracellular enzymes. Furthermore, genetically encoded probes offer the possibility of custom-engineered biosensors for intracellular biochemistry, specifically localized targets, and protein—protein interactions.     

High-throughput 3-D cell-based proliferation and cytotoxicity assays for drug screening and bioprocess development

We have designed, built and tested a three-dimensional (3-D) cell culture system on modified microplates for high-throughput, real-time, proliferation and cytotoxicity assays. In this 3-D culture system, cells expressing the enhanced green fluorescent protein (EGFP) were cultured in nonwoven polyethylene terephthalate (PET) fibrous scaffolds. Compared to 2-D cultures in conventional microplates, 3-D cultures gave more than 10-fold higher fluorescence signals with significantly increased signal-to-noise ratio (SNR), thus extending the application of conventional fluorescence microplate readers for online monitoring of culture fluorescence. The 3-D system was successfully used to demonstrate the effects of fetal bovine serum, fibronectin coating of PET fibers, and cytotoxicity of dexamethasone on recombinant murine embryonic stem D3 cells. The dosage effects of 5-fluorouracil and gemcitabine on high-density colon cancer HT-29 cells were also tested. These studies demonstrated that the 3-D culture microplate system with EGFP expressing cells can be used as a high-throughput system in drug discovery and bioprocess development.     

Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists

Drug discovery by high-throughput screening is a promising approach to develop new therapies for the most common lethal genetic disease, cystic fibrosis. Because disease-causing mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) protein produce epithelial cells with reduced or absent Cl(-) permeability, the goal of screening is to identify compounds that restore cell Cl(-) transport. We have developed a rapid, quantitative screening procedure for analysis of CFTR-mediated halide transport in cells with the use of a conventional fluorescence plate reader. Doubly transfected cell lines were generated that express wild-type or mutant CFTR together with a yellow fluorescent protein (YFP)-based halide sensor. CFTR function was assayed from the time course of cell fluorescence in response to extracellular addition of 100 mM I(-) followed by forskolin, resulting in decreased YFP fluorescence due to CFTR-mediated I(-) entry. Cell lines were chosen, and conditions were optimized to minimize basal halide transport to maximize assay sensitivity. In cells cultured on 96-well plastic dishes, the assay gave reproducible halide permeabilities from well to well and could reliably detect a 2% activation of CFTR-dependent halide transport produced by low concentrations of forskolin. Applications of the assay are shown, including comparative dose-dependent CFTR activation by genistein, apigenin, 8-cyclopentyl-1,3-dipropylxanthine, IBMX, 8-methoxypsoralen, and milrinone as well as activation of alternative Cl(-) channels. The fluorescence assay and cell lines should facilitate the screening of novel CFTR activators and the characterization of alternative Cl(-) channels and transporters.     

Strategy for analysis and screening of bioactive compounds in traditional Chinese medicines

Traditional Chinese medicines (TCMs), due to their long time clinic test and reliable therapeutic efficacy, are attracting increased global attention served as excellent pools of bioactive compounds for the discovery of new drugs. However, hundreds or even thousands of components are usually contained in traditional Chinese medicines and only a few compounds are responsible for the pharmaceutical and/or toxic effects. The large numbers of other components in traditional Chinese medicines make the screening and analysis of the bioactive components extremely difficult. By the way, the combination effect of bioactive components on the pharmacological activity makes it very difficult to clear the therapeutic mechanism of TCMs. Therefore, some strategies have to design for screening of bioactive compounds in traditional Chinese medicines, which further leads to disclose the therapeutic mechanism of TCMs in molecular level. The review will summarize the present state of the art of screening strategy for active compounds in traditional Chinese medicines, and the chromatography methods for screening and analysis of bioactive compounds in traditional Chinese medicines will be emphasized.     

Complex phenotypic assays in high-throughput screening

High-throughput screening (HTS), systematically testing thousands of small molecules to find candidates for lead optimization, primarily involves exposure of purified proteins to arrayed collections of small molecules. More complex phenotypic assays, such as cell-based or whole-organism assays, traditionally have flanked HTS, preceding it to validate new therapeutic targets, and following it to characterize new lead compounds in cellular contexts. Recently, however, cell- and organism-based phenotypic assays have increasingly been adopted as a primary screening platform for annotating small molecules.     

Cell lysis methods for high-throughput screening or miniaturized assays

The widely and routinely used cell lysis techniques are still being evolved to address the technical needs of micro-scale sample processing, for example in high-throughput screening or miniaturized assays. This review focuses on several recently emerged mechanical, physical and biological methods designed for lysing cells on small scales for screening or analysis.     

Determining appropriate substrate conversion for enzymatic assays in high-throughput screening

It is generally accepted that the conversion of substrate should be kept at less than 10% of the total substrate used when studying enzyme kinetics. However, 10% or less substrate conversion often will not produce sufficient signal changes required for robust high-throughput screening (HTS). To increase the signal-to-background ratio, HTS is often performed at higher than 10% substrate conversion. Because the consequences of high substrate conversion are poorly understood, the screening results are sometimes questioned by enzymologists. The quality of an assay is judged by the ability to detect an inhibitor under HTS conditions, which depends on the robustness of the primary detection signal (Z factor) and the sensitivity to an inhibitor. The assay sensitivity to an inhibitor is reflected in the observed IC(50) value or percent inhibition at a fixed compound concentration when single-point data are collected. The major concern for an enzymatic assay under high substrate conversion is that the sensitivity of the screen may be compromised. Here we derive the relationship between the IC(50) value for a given inhibitor and the percentage of substrate conversion using a first-order kinetic model under conditions that obey Henri-Michaelis-Menten kinetics. The derived theory was further verified experimentally with a cAMP-dependent protein kinase. This model provides guidance for assay developers to choose an appropriate substrate conversion in designing an enzymatic assay, balancing the needs for robust signal and sensitivity to inhibitors.     

10种中草药提取物体外抗铜绿假单胞菌作用研究

通过对粗糠柴等10种中草药采用80%乙醇室温下浸渍制备的提取物进行体外抗铜绿假单胞菌及其耐药菌活性研究,并采取药敏纸片法测定临床分离菌株的耐药性。结果表明:这10种中草药80%乙醇提取物中,粗糠柴的乙酸乙酯层对铜绿假单胞菌标准菌及其耐药菌的抑菌效果最好,其抑菌圈直径范围在10~17 mm之间,MIC范围在0.125~0.5 mg·mL~(-1)之间,MBC范围在0.5~1 mg·mL~(-1)之间;正丁醇层、水层的抑菌活性较乙酸乙酯层弱,石油醚层对铜绿假单胞菌没有效果。而小叶藤黄、滇南红厚壳、续随子的乙酸乙酯层,巴豆、罗汉松、肉桂醇提物对铜绿假单胞菌及其耐药菌株有较弱抗菌活性;滇南红厚壳的正丁醇层、续随子乙酸乙酯层以及大八角和郁金的醇提物对铜绿假单胞菌及其耐药菌株均无活性。从这些数据中可以得出,粗糠柴的乙酸乙酯层、正丁醇层和水层对铜绿假单胞菌及其耐药菌有较好的抑菌活性,尤以乙酸乙酯层活性最好,而粗糠柴的石油醚层没有活性。     

Fluorescence polarization assays for high-throughput screening of neuropeptide FF receptors

We have developed the first fluorescence polarization assays of human neuropeptide FF2 receptors in 384-well microtiter plates. Assays are completed in a single well with no transfer, separation, or wash steps. The performance is suitable for high-throughput drug screening applications with regard to speed of analysis, magnitude of displaceable signal, precision, and sensitivity of various reagents. The rank order of potency of agonists and antagonists agrees well relative to the published radiometric filtration assays: DMe NPFF > NPFF > frog PP (Rana temporaria pancreatic polypeptide) > PQRFamide > BIBP 3226. The effect of highly colored compounds is very small on the polarization signal up to micromolar concentrations. The method serves as a simple and fast alternative to radioligand binding assays of antiobesity drug candidates related to NPFF receptors.     

中草药生物质炼制工程

目前我国中药产业面临资源紧张、药材利用率低、加工过程浪费严重等问题,究其原因主要是单一药效成分利用、加工转化技术落后所致。针对上述问题,从生物质炼制角度,综述了实现中草药资源高效利用的原料预处理、提取、转化及残渣后处理等4个关键单元操作中主要技术的研究进展,并指出中草药生物质炼制工程发展趋势与前景。     

Multiplexed G-protein-coupled receptor Ca2+ flux assays for high-throughput screening

An early drug discovery approach focusing on gene families can benefit from strategies that exploit common signaling mechanisms to more effectively identify and characterize novel chemical lead structures. Multiplexing, defined as the screening of multiple targets within the same experiment, is an example of this strategy. Here, the authors describe a technique that allows multiplexing of a common assay type used to study G-protein-coupled receptors: changes in intracellular Ca2+ levels as measured by Molecular Device's fluorometric imaging plate reader (FLIPR). The multiplexed FLIPR assays showed the expected pharmacological properties of single assays, with good reproducibility and Z* factors. The authors used them to screen large compound libraries in 2 multiplexed assay designs. The 1st used a single-cell line expressing 2 different receptors and the 2nd a mixture of 2 cell lines of the same type each expressing distinct receptors. Screening using these multiplexed assays produced significant savings in reagents, time, and human resources and allowed the authors to quickly identify specific and selective hits.     

Microwell bioreactor system for cell-based high throughput proliferation and cytotoxicity assays

Cell-based high throughput proliferation and cytotoxicity assays are increasingly used in drug screening and bioprocess development. However, online monitoring of cell proliferation, pH, and dissolved oxygen (DO) has been a challenge in 3D cell-based assays. In this work, a 40-microwell bioreactor (40-MBR) system was developed from a 384-well plate for real-time, noninvasive monitoring of pH, DO, and cell proliferation in 3D microenvironments. Chinese hamster ovary (CHO) and MCF-07 breast cancer cells cultured in 40-MBR confirmed that the 40-MBR was capable of simultaneously monitoring DO and cell proliferation based on culture fluorescence and pH by measuring the absorbance of phenol red. Cytotoxicity studies of sodium butyrate on CHO cells demonstrated that 40-MBR with dynamic background fluorescence correction gave more reliable and highly reproducible growth kinetic data compared to conventional multiwells with static background correction. Furthermore, the dosage effects of two new anticancer drug candidates, 5,7-dihydroxy-2-(4-hydroxyphenyl)-8-[(E)-2-phenylethenyl]-3,4-dihydro-2H-1-benzopyran-4-one (DH-8P-DB) and 5,7-dihydroxy-2-(4-hydroxyphenyl)-6-[(E)-2-phenylethenyl]-3,4-dihydro-2H-1-benzopyran-4-one (DH-6P-DB), on HT-29 colon cancer cells were assessed using the 40-MBR, and the results indicated that DH-6P-DB would be a more potent drug in treating colon cancer than DH-8P-DB. These studies demonstrated that 40-MBR could serve as a high throughput platform for screening potential cancer drugs in early-stage drug discovery.     

Novel fluorescent technology platform for high throughput cytotoxicity and proliferation assays

We have developed a novel fluorescent Oxygen BioSensor technology platform adaptable to many applications in the area of drug discovery and development, particularly cell-based assays. This biosensor technology requires no additional reagents or incubations, and affords continuous real-time readout of dissolved oxygen concentrations. Since the level of oxygen dissolved in an assay's medium correlates to the number and viability of the cells in the medium, this technology is ideally suited for monitoring cell viability, proliferation, or death. The technology is particularly well suited to investigating cells' kinetic responses to proliferative or toxic stimuli, such as drugs. When incorporated into a 96- or 384-well microplate format, it is compatible with standard laboratory automation systems. Here we present data illustrating the application of the Oxygen BioSensor technology for rapid, homogeneous detection and evaluation of metabolic activity of a variety of eukaryotic and prokaryotic cells, including mammalian cells, insect cells, yeast, and bacteria. In the absence of toxic substances, we find a good correlation between cell number and signal over a wide range of cell concentrations and growth times. To evaluate the usefulness of the Oxygen BioSensor for cytotoxicity assays, we have performed a series of experiments using a range of toxic agents and cell types, including both bacteria and mammalian cell lines. In a side-by-side comparison to standard MTT assays using HL60 cells, comparable IC(50) values were found with the Oxygen BioSensor for five different toxins or drugs. This assay method does not have the need for additional reagents, handling steps, or incubation periods required by the MTT assays.     

Cell‐based screening of traditional chinese medicines for proliferation enhancers of mouse embryonic stem cells

A high‐throughput cell‐based method was developed for screening traditional Chinese herbal medicines (TCHMs) for potential stem cell growth promoters. Mouse embryonic stem (mES) cells expressing enhanced green fluorescent protein (EGFP) were cultured in growth media supplemented with various TCHM extracts. The dosage‐dependent effects of TCHM extracts on cell growth, including proliferation and cytotoxicity, were assessed via EGFP fluorescence measurement. Seven TCHMs were investigated, and among them Panax notoginseng (PN), Rhizoma Atractylodis macrocephalae, Rhizoma chuanxiong, and Ganoderma lucidum spores (GLS) showed potential to improve mES cell proliferation. Eleven mixtures of these four TCHMs were then studied, and the results showed that the mixture of PN and GLS had the strongest growth promoting effect, increasing the specific growth rate of mES cells by 29.5% at a low dosage of 0.01% (wt/vol) PN/GLS (P < 0.01) and 34.2% at 0.1% (wt/vol) PN/GLS (P < 0.05) compared to the control. The growth promoting effect of PN/GLS was further confirmed with ES cells cultured in spinner flasks. A 29.3‐fold increase in the total cell number was achieved in the medium supplemented with 0.01% PN/GLS after 5 days, while the control culture only gave a 16.8‐fold increase. This cell‐based screening method thus can provide an efficient and high‐throughput way to explore potential stem cell growth promoters from TCHMs. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:738–744, 2013     

High-yield synthesis and purification of recombinant human GABA transaminase for high-throughput screening assays

Many studies have focussed on modulating the activity of γ-aminobutyric acid transaminase (GABA-T), a GABA-catabolizing enzyme, for treating neurological diseases, such as epilepsy and drug addiction. Nevertheless, human GABA-T synthesis and purification have not been established. Thus, biochemical and drug design studies on GABA-T have been performed by using porcine GABA-T mostly and even bacterial GABA-T. Here we report an optimised protocol for overexpression of 6xHis-tagged human GABA-T in human cells followed by a two-step protein purification. Then, we established an optimised human GABA-T (0.5 U/mg) activity assay. Finally, we compared the difference between human and bacterial GABA-T in sensitivity to two irreversible GABA-T inhibitors, gabaculine and vigabatrin. Human GABA-T in homodimeric form showed 70-fold higher sensitivity to vigabatrin than bacterial GABA-T in multimeric form, indicating the importance of using human GABA-T. In summary, our newly developed protocol can be an important first step in developing more effective human GABA-T modulators.     

Cell-based high-throughput screening assay system for monitoring G protein-coupled receptor activation using beta-galactosidase enzyme complementation technology

A novel cell-based functional assay to directly monitor G protein-coupled receptor (GPCR) activation in a high-throughput format, based on a common GPCR regulation mechanism, the interaction between beta-arrestin and ligand-activated GPCR, is described. A protein-protein interaction technology, the InteraX trade mark system, uses a pair of inactive beta-galactosidase (beta-gal) deletion mutants as fusion partners to the protein targets of interest. To monitor GPCR activation, stable cell lines expressing both GPCR- and beta-arrestin-beta-gal fusion proteins are generated. Following ligand stimulation, beta-arrestin binds to the activated GPCR, and this interaction drives functional complementation of the beta-gal mutant fragments. GPCR activation is measured directly by quantitating restored beta-gal activity. The authors have validated this assay system with two functionally divergent GPCRs: the beta2-adrenergic amine receptor and the CXCR2 chemokine-binding receptor. Both receptors are activated or blocked with known agonists and antagonists in a dose-dependent manner. The beta2-adrenergic receptor cell line was screened with the LOPAC trade mark compound library to identify both agonists and antagonists, validating this system for high-throughput screening performance in a 96-well microplate format. Hit specificity was confirmed by quantitating the level of cAMP. This assay system has also been performed in a high-density (384-well) microplate format. This system provides a specific, sensitive, and robust methodology for studying and screening GPCR-mediated signaling pathways.