酰胺酶高通量筛选方法研究进展

酰胺酶是一种合成手性羧酸和酰胺衍生物的重要工具酶,在不对称合成中具有巨大应用潜力.传统的色谱分析方法通过检测底物及相应的产物去测定酰胺酶活性及立体选择性,十分费时费力.根据显色法、荧光法、NMR等原理,较为全面地综述了近年来国内外发展起来的高通量酰胺酶筛选方法.为酰胺酶筛选中筛选效率低、可靠性差等难题提供借鉴,也可为其他水解酶筛选模型的建立提供思路.     

High Throughput Screening with Multiphoton Excitation

Fluorescence detection is extensively used in high throughput screening. In HTS there is a continuous migration toward higher density plates and smaller sample volumes. In the present report we describe the advantages of two-photon or multiphoton excitation for HTS. Multiphoton excitation (MPE) is the simultaneous absorption of two long-wavelength photons to excite the lowest singlet state of the fluorophore. MPE is typically accomplished with short but high-intensity laser pulses, which allows simultaneous absorption of two or more photons. The intensity of the multiphoton-induced fluorescence is proportional to the square, cube, or higher power of the instantneous photon flux. Consequently, two-photon or multiphoton excitation only occurs at the focal point of the incident beam. This property of two-photon excitation allows the excited volume to be very small and to be localized in the center of each well in the HTS plate. We show that two-photon-induced fluorescence of fluorescein can be reliably measured in microwell plates. We also show the use of 6-carboxy fluorescein as a pH probe with two-photon excitation, and measure 4'-6-diamidino-2-phenylindole (DAPI) binding and two-photon-induced fluorescence. In further studies we measure the time-dependent intensity decays of DAPI bound to DNA and of calcium-dependent fluorophores. Finally, we demonstrate the possibility of three-photon excitation of several fluorophores, including indole, in the HTS plate. These results suggest that MPE can be used in high-density multiwell plates.     

高通量筛选技术及其应用

主要介绍了高通量筛选技术(HTS,HighThroughputScreening)的原理,包括非细胞相筛选、细胞相筛选和生物表型筛选及其在生命科学和药学领域中的应用以及高通量筛选技术的发展趋势。     

高通量筛选在抗病毒药物筛选中的应用

高通量筛选(high throughput screening, HTS)是以分子和细胞水平的实验方法为基础,在微孔板上以自动化操作系统执行实验过程,通过灵敏快速的检测仪器采集实验数据、运用计算机对实验数据进行实验结果的分析处理.因此HTS具有大量样品的快速筛选、分子和细胞水平的特异性作用靶点、检测系统的高灵敏度、自动化操作系统和数据采集传输处理系统等优点.     

High Throughput Screening of Gene Expression Signatures

This paper focuses on microarray image analysis and discusses a completely automated approach to image processing, which eliminates human intervention. A system for automated image processing is described, which is capable of processing image files in a batch-mode thus allowing high-throughput of microarray image analysis. Grid-placement and spot finding are achieved without operator's help. The software eliminates noise signals from the data analysis process and minimizes operator's involvement in the procedure.     

High Throughput Screening for Drug Discovery: Continually Transitioning into New Technology

Those working in HTS laboratories, pressured to find increasing numbers of drug leads while containing costs, are seeking larger compound sets, more automated systems to screen them faster, and an integrated set of equipment and consumables. Enabling technologies are continually being developed and suppliers are teaming up to supply integrated equipment and consumable sets. Miniaturization, microfluidic chips, subnanoliter dispensing, fluorescence, homogeneous assays for HTS, and virtual screening are just some of the evolving tools that HTS experts are continually evaluating and incorporating into drug discovery operations.     

High Throughput Screening for Anti–Trypanosoma cruzi Drug Discovery

The discovery of new therapeutic options against Trypanosoma cruzi, the causative agent of Chagas disease, stands as a fundamental need. Currently, there are only two drugs available to treat this neglected disease, which represents a major public health problem in Latin America. Both available therapies, benznidazole and nifurtimox, have significant toxic side effects and their efficacy against the life-threatening symptomatic chronic stage of the disease is variable. Thus, there is an urgent need for new, improved anti–T. cruzi drugs. With the objective to reliably accelerate the drug discovery process against Chagas disease, several advances have been made in the last few years. Availability of engineered reporter gene expressing parasites triggered the development of phenotypic in vitro assays suitable for high throughput screening (HTS) as well as the establishment of new in vivo protocols that allow faster experimental outcomes. Recently, automated high content microscopy approaches have also been used to identify new parasitic inhibitors. These in vitro and in vivo early drug discovery approaches, which hopefully will contribute to bring better anti–T. cruzi drug entities in the near future, are reviewed here.     

谷氨酸依赖型氨基转移酶的高通量筛选方法及其应用

目的:建立谷氨酸依赖型氨基转移酶-谷氨酸脱氢酶偶联反应的96孔板高通量筛选方法,并用于大肠杆菌氨基转移酶Wec E突变库的筛选。方法:通过优化偶联指示酶-谷氨酸脱氢酶、信号分子NADH浓度及双酶偶联反应时间,建立了光学法测定氨基转移酶活性的氨基转移酶-谷氨酸脱氢酶偶联反应方法;通过定点饱和突变技术构建了大肠杆菌氨基转移酶WecE的突变库;采用96孔板高通量初筛、摇瓶复筛获得了高活性的转氨酶突变体,并对纯化的突变体进行催化活力分析。结果:建立了谷氨酸依赖型氨基转移酶目标反应与0.5 U/ml L-谷氨酸脱氢酶和0.4 mmol/L NADH信号指示反应相偶联的筛选方法;构建了氨基转移酶WecE Tyr 321饱和突变库,通过96孔板高通量筛选,获得了催化活性比野生型提高3.4倍的突变体Y321F。结论:所建立高通量筛选方法背景干扰小,准确性高,为谷氨酸依赖型氨基转移酶分子进化提供了可行性方案。     

An Assay Suitable for High Throughput Screening of Anti-Influenza Drugs

We developed a novel drug screening system for anti-influenza A virus by targeting the M2 proton channel. In the SPP (Single Protein Production) system, E. coli cell growth occurs only in the presence of effective M2 channel inhibitors, and thus simple measurement of cell growth was used as readouts for drug screening. Two potential inhibitors for M2 (V27A) mutant were verified using this method, which inhibit both the mutant and wild-type M2 channels.     

Complement C5a Receptor Assay for High Throughput Screening

Abstract

The complement C5a receptor on U937 cells, a human histiocytic lymphoma cell line, stimulated with dibutyryl-cAMP have been stabilized for at least 3 months at a diluce, ready to use concentration. [¹²⁵I]-Bolton Hunter labeled C5a, (recombinant, human) has been prepared by reverse phase HPLC to 2200 Ci/mmol. Using a filtration binding assay the Kd from receptor saturation analysis is 10–40 pM and there are 50,000–100,000 receptor sites per cell. These reagents have permitted the development of a reliable, reproducible and convenient drug screening assay, in kit format, for compounds acting at the C5a receptor.     

Design and Implementation of High Throughput Screening Assays

High throughput screening (HTS) is at the core of the drug discovery process, and so it is critical to design and implement HTS assays in a comprehensive fashion involving scientists from the disciplines of biology, chemistry, engineering, and informatics. This requires careful analysis of many variables, starting with the choice of assay target and ending with the discovery of lead compounds. At every step in this process, there are decisions to be made that can greatly impact the outcome of the HTS effort, to the point of making it a success or a failure. Although specific guidelines should be established to insure that the screening assay reaches an acceptable level of quality, many choices require pragmatism and the ability to compromise opposing forces.     

Time-Resolved Fluorescence Energy Transfer DNA Helicase Assays for High Throughput Screening

DNA helicases are responsible for the unwinding of double-stranded DNA, facilitated by the binding and hydrolysis of 5'-nucleoside triphosphates. These enzymes represent an important class of targets for the development of novel anti-infective agents particularly because opportunity exists for synergy with existing therapies targeted at other enzymes involved in DNA replication. Unwinding reactions are conventionally monitored by low throughput, gel-based radiochemical assays; to overcome the limitations of low throughput to achieve comprehensive characterization of adenosine triphosphate (ATP)-dependent unwinding by viral and bacterial helicases and the screening for unwinding inhibitors, we have developed and validated homogeneous time-resolved fluorescence energy transfer (TRET) assays. Rapid characterization and screening of DNA helicase has been performed in 96- and 384-well plate densities, and the ability to assay in 1536-well format also demonstrated. We have successfully validated and are running full high throughput runs using 384-well TRET helicase assays, culminating in the identification of a range of chemically diverse inhibitors of viral and bacterial helicases. For screening in mixtures, we used a combination of quench correction routines and confirmatory scintillation proximity (SP) assays to eliminate false-positives due to the relatively high levels of compound quenching (unlike other Ln(3+)-based assays). This strategy was successful yet emphasised the need for further improvements in helicase assays.     

Ultra-High Efficient Colony PCR for High Throughput Screening of Bacterial Genes

Current colony PCR methods are not suitable for screening genes encoded in genomic DNA and are limited to E. coli host strains. Here, we describe an ultra-high efficient colony PCR method for high throughput screening of bacterial genes embedded in the genomic DNA of any bacterial species. This new technique expands colony PCR method to several hosts as well as offers a rapid, less expensive and reliable bacterial genomic DNA extraction.     

Natural Products in High Throughput Screening: Automated High-Quality Sample Preparation

At present, compound libraries from combinatorial chemistry are the major source for high throughput screening (HTS) programs in drug discovery. On the other hand, nature has been proven to be an outstanding source for new and innovative drugs. Secondary metabolites from plants, animals, and microorganisms show a striking structural diversity that supplements chemically synthesized compounds or libraries in drug discovery programs. Unfortunately, extracts from natural sources are usually complex mixtures of compounds, often generated in time-consuming and, for the most part, manual processes. Because quality and quantity of the provided samples play a pivotal role in the success of HTS programs, this poses serious problems. In order to make samples of natural origin competitive with synthetic compound libraries, we devised a novel, automated sample preparation procedure based on solid-phase extraction (SPE). By making use of modified Zymark (Hopkinton, MA) RapidTrace? SPE workstations, we developed an easy-to-handle and effective fractionation method that generates high-quality samples from natural origin, fulfilling the requirements for an integration in high throughput drug discovery programs.     

Minireview: Challenges of High Throughput Screening Against Cell Surface Receptors

Abstract

Excessive or inappropriate activation of cell surface receptors can mediate the development of disease. Receptors, therefore, are a focus for drug discovery activities. Empirical screening is important in the search for novel compounds acting at receptors. Technical developments and the application of molecular biology have facilitated access to receptors of interest and have provided efficient screening methods capable of very high throughput. Reliability in high throughput screening requires the use of appropriate methodology, good screen design and effective validation and quality control processes. Validation should aim to establish that the basic experimental design is sound. In developing software to handle high throughput screening data, a fundamental requirement is to provide performance monitoring and error trapping facilities. Additional requirements are automatic data capture from instruments, on-line data reduction and analysis and transfer of results to central databases. As data volumes increase through effective high throughput screening, conventional interrogation methods become less appropriate and are being augmented by newer computing techniques referred to as knowledge mapping or database mining. Targeting cell surface receptors has been very successful as an approach to drug discovery. If the challenges of high throughput empirical screening are addressed effectively, cell surface receptors will provide new opportunities for improved therapy in the coming years.