Practical High-Throughput Method to Screen Compounds for Anthelmintic Activity against Caenorhabditis elegans

In the present study, we established a practical and cost-effective high throughput screening assay, which relies on the measurement of the motility of Caenorhabditis elegans by infrared light-interference. Using this assay, we screened 14,400 small molecules from the “HitFinder” library (Maybridge), achieving a hit rate of 0.3%. We identified small molecules that reproducibly inhibited the motility of C. elegans (young adults) and assessed dose relationships for a subset of compounds. Future work will critically evaluate the potential of some of these hits as candidates for subsequent optimisation or repurposing as nematocides or nematostats. This high throughput screening assay has the advantage over many previous assays in that it is cost- and time-effective to carry out and achieves a markedly higher throughput (~10,000 compounds per week); therefore, it is suited to the screening of libraries of tens to hundreds of thousands of compounds for subsequent evaluation and development. The present phenotypic whole-worm assay should be readily adaptable to a range of socioeconomically important parasitic nematodes of humans and animals, depending on their dimensions and motility characteristics in vitro, for the discovery of new anthelmintic candidates. This focus is particularly important, given the widespread problems associated with drug resistance in many parasitic worms of livestock animals globally.     

A collaborative hit-to-lead investigation leveraging medicinal chemistry expertise with high throughput library design, synthesis and purification capabilities

High throughput screening (HTS) campaigns, where laboratory automation is used to expose biological targets to large numbers of materials from corporate compound collections, have become commonplace within the lead generation phase of pharmaceutical discovery. Advances in genomics and related fields have afforded a wealth of targets such that screening facilities at larger organizations routinely execute over 100 hit-finding campaigns per year. Often, 10(5) or 10(6) molecules will be tested within a campaign/cycle to locate a large number of actives requiring follow-up investigation. Due to resource constraints at every organization, traditional chemistry methods for validating hits and developing structure activity relationships (SAR) become untenable when challenged with hundreds of hits in multiple chemical families per target. To compound the issue, comparison and prioritization of hits versus multiple screens, or physical chemical property criteria, is made more complex by the informatics issues associated with handling large data sets. This article describes a collaborative research project designed to simultaneously leverage the medicinal chemistry and drug development expertise of the Novartis Institutes for Biomedical Research Inc. (NIBRI) and ArQule Inc.'s high throughput library design, synthesis and purification capabilities. The work processes developed by the team to efficiently design, prepare, purify, assess and prioritize multiple chemical classes that were identified during high throughput screening, cheminformatics and molecular modeling activities will be detailed.     

High‐Throughput Approaches in Carbohydrate‐Active Enzymology: Glycosidase and Glycosyl Transferase Inhibitors,Evolution, and Discovery

Carbohydrates are attached and removed in living systems through the action of carbohydrate‐active enzymes such as glycosyl transferases and glycoside hydrolases. The molecules resulting from these enzymes have many important roles in organisms, such as cellular communication, structural support, and energy metabolism. In general, each carbohydrate transformation requires a separate catalyst, and so these enzyme families are extremely diverse. To make this diversity manageable, high‐throughput approaches look at many enzymes at once. Similarly, high‐throughput approaches can be a powerful way of finding inhibitors that can be used to tune the reactivity of these enzymes, either in an industrial, a laboratory, or a medicinal setting. In this review, we provide an overview of how these enzymes and inhibitors can be sought using techniques such as high‐throughput natural product and combinatorial library screening, phage and mRNA display of (glyco)peptides, fluorescence‐activated cell sorting, and metagenomics.     

High‐Throughput Approaches in Carbohydrate‐Active Enzymology: Glycosidase and Glycosyl Transferase Inhibitors,Evolution, and Discovery

Carbohydrates are attached and removed in living systems through the action of carbohydrate‐active enzymes such as glycosyl transferases and glycoside hydrolases. The molecules resulting from these enzymes have many important roles in organisms, such as cellular communication, structural support, and energy metabolism. In general, each carbohydrate transformation requires a separate catalyst, and so these enzyme families are extremely diverse. To make this diversity manageable, high‐throughput approaches look at many enzymes at once. Similarly, high‐throughput approaches can be a powerful way of finding inhibitors that can be used to tune the reactivity of these enzymes, either in an industrial, a laboratory, or a medicinal setting. In this review, we provide an overview of how these enzymes and inhibitors can be sought using techniques such as high‐throughput natural product and combinatorial library screening, phage and mRNA display of (glyco)peptides, fluorescence‐activated cell sorting, and metagenomics.     

Applications of Activity‐Based Protein Profiling (ABPP) and Bioimaging in Drug Discovery

Activity‐based protein profiling (ABPP) and bioimaging have been developed in recent years as powerful technologies in drug discovery. Specifically, both approaches can be applied in critical steps of drug development, such as therapy target discovery, high‐throughput drug screening and target identification of bioactive molecules. We have been focused on the development of various strategies that enable simultaneous activity‐based protein profiling and bioimaging studies, thus facilitating an understanding of drug actions and potential toxicities. In this Minireview, we summarize these novel strategies and applications, with the aim of promoting these technologies in drug discovery.     

临近闪烁分析法在高通量筛选中的应用

起源于放射性免疫分析的临近闪烁分析法(scintillation proximity assay,SPA)是一种均相、灵敏、快速和简便的基于闪烁载体的分析平台。该平台可用于筛选药物靶点的先导化合物和研究其生理过程。由于无需分离,易于固定药物靶点和检测其活性,SPA成为一种重要的高通量筛选方法。由于放射性标记分子和亲和标签分子的多样化和商业化、以及液闪计数器和液相操作等技术的发展,SPA已经广泛用于受体结合、高通量药物筛选、酶分析、放射性免疫分析、蛋白质-蛋白质相互作用和细胞水平分析等方面。本文阐述了SPA原理,讨论了其关键技术(包括闪烁载体、液闪计数器和放射性标记分子),分析了其评价体系;同时简述了SPA分析的发展, 并介绍了其在高通量筛选中的应用实例, 归纳了存在的问题,给出了未来的发展趋势。目前,基于SPA和荧光分析方法已成为高通量药物筛选的热点研究领域, 这些筛选技术的革新必然提升我们对细胞体系生物学的全面理解和促进先导化合物筛选过程的显著进步。     

Cheminformatics aspects of high throughput screening: from robots to models: symposium summary

The “Cheminformatics aspects of high throughput screening (HTS): from robots to models” symposium was part of the computers in chemistry technical program at the American Chemical Society National Meeting in Denver, Colorado during the fall of 2011. This symposium brought together researchers from high throughput screening centers and molecular modelers from academia and industry to discuss the integration of currently available high throughput screening data and assays with computational analysis. The topics discussed at this symposium covered the data-infrastructure at various academic, hospital, and National Institutes of Health-funded high throughput screening centers, the cheminformatics and molecular modeling methods used in real world examples to guide screening and hit-finding, and how academic and non-profit organizations can benefit from current high throughput screening cheminformatics resources. Specifically, this article also covers the remarks and discussions in the open panel discussion of the symposium and summarizes the following talks on “Accurate Kinase virtual screening: biochemical, cellular and selectivity”, “Selective, privileged and promiscuous chemical patterns in high-throughput screening” and “Visualizing and exploring relationships among HTS hits using network graphs”.     

ADLOC: An Aptamer‐Displacement Assay Based on Luminescent Oxygen Channeling

Functional nucleic acids, such as aptamers and allosteric ribozymes, can sense their ligands specifically, thereby undergoing structural alterations that can be converted into a detectable signal. The direct coupling of molecular recognition to signal generation enables the production of versatile reporters that can be applied as molecular probes for various purposes, including high‐throughput screening. Here we describe an unprecedented type of a nucleic acid‐based sensor system and show that it is amenable to high‐throughput screening (HTS) applications. The approach detects the displacement of an aptamer from its bound protein partner by means of luminescent oxygen channeling. In a proof‐of‐principle study we demonstrate that the format is feasible for efficient identification of small drug‐like molecules that bind to a protein target, in this case to the Sec7 domain of cytohesin. We extended the approach to a new cytohesin‐specific single chain DNA aptamer, C10.41, which exhibits a similar binding behavior to cytohesins but has the advantage of being more stable and easier to synthesize and to modify than the RNA‐aptamer M69. The results obtained with both aptamers indicate the general suitability of the aptamer‐displacement assay based on luminescent oxygen channelling (ADLOC) for HTS. We also analyzed the potential for false positive hits and identified from a library of 18 000 drug‐like small molecules two compounds as strong singlet‐oxygen quenchers. With full automation and the use of commercially available plate readers, we estimate that the ADLOC‐based assay described here could be used to screen at least 100 000 compounds per day.     

Label-free cell-based functional assays

Label-free technologies based on electrical impedance or refractive index are new tools for measuring a cell-based functional response. Although the technologies are relatively new to high throughput screening cell-based applications, they are rapidly generating interest in that they are able to measure a phenotypic response using cells natively expressing the target protein without using dyes or cellular extracts. In addition, one can measure the cellular response using a kinetic mode resulting in an assay potentially rich in content. This article will describe these technologies and their applications in measuring cell proliferation, cell attachment and spreading, cell apoptosis and their application for several receptor target classes, including receptor tyrosine kinases and G protein-coupled receptors. The potential utility and drawbacks of these tools for high throughput screening, directed screening and compound profiling will also be discussed.     

Application and utilization of chemoinformatics tools in lead generation and optimization

The process of Drug Discovery is a complex and high risk endeavor that requires focused attention on experimental hypotheses, the application of diverse sets of technologies and data to facilitate high quality decision-making. All is aimed at enhancing the quality of the chemical development candidate(s) through clinical evaluation and into the market. In support of the lead generation and optimization phases of this endeavor, high throughput technologies such as combinatorial/high throughput synthesis and high throughput and ultra-high throughput screening, have allowed the rapid analysis and generation of large number of compounds and data. Today, for every analog synthesized 100 or more data points can be collected and captured in various centralized databases. The analysis of thousands of compounds can very quickly become a daunting task. In this article we present the process we have developed for both analyzing and prioritizing large sets of data starting from diversity and focused uHTS in support of lead generation and secondary screens supporting lead optimization. We will describe how we use informatics and computational chemistry to focus our efforts on asking relevant questions about the desired attributes of a specific library, and subsequently in guiding the generation of more information-rich sets of analogs in support of both processes.     

High throughput screening for neurodegeneration and complex disease phenotypes

High throughput screening (HTS) for complex diseases is challenging. This stems from the fact that complex phenotypes are difficult to adapt to rapid, high throughput assays. We describe the recent development of high throughput and high-content screens (HCS) for neurodegenerative diseases, with a focus on inherited neurodegenerative disorders, such as Huntington's disease. We describe, among others, HTS assays based on protein aggregation, neuronal death, caspase activation and mutant protein clearance. Furthermore, we describe high-content screens that are being used to prioritize hits identified in such HTS assays. These assays and screening approaches should accelerate drug discovery for neurodegenerative disorders and guide the development of screening approaches for other complex disease phenotypes.     

Surrogate docking: structure-based virtual screening at high throughput speed

Summary Structure-based screening using fully flexible docking is still too slow for large molecular libraries. High quality docking of a million molecule library can take days even on a cluster with hundreds of CPUs. This performance issue prohibits the use of fully flexible docking in the design of large combinatorial libraries. We have developed a fast structure-based screening method, which utilizes docking of a limited number of compounds to build a 2D QSAR model used to rapidly score the rest of the database. We compare here a model based on radial basis functions and a Bayesian categorization model. The number of compounds that need to be actually docked depends on the number of docking hits found. In our case studies reasonable quality models are built after docking of the number of molecules containing 50 docking hits. The rest of the library is screened by the QSAR model. Optionally a fraction of the QSAR-prioritized library can be docked in order to find the true docking hits. The quality of the model only depends on the training set size – not on the size of the library to be screened. Therefore, for larger libraries the method yields higher gain in speed no change in performance. Prioritizing a large library with these models provides a significant enrichment with docking hits: it attains the values of 13 and 35 at the beginning of the score-sorted libraries in our two case studies: screening of the NCI collection and a combinatorial libraries on CDK2 kinase structure. With such enrichments, only a fraction of the database must actually be docked to find many of the true hits. The throughput of the method allows its use in screening of large compound collections and in the design of large combinatorial libraries. The strategy proposed has an important effect on efficiency but does not affect retrieval of actives, the latter being determined by the quality of the docking method itself. Electronic supplementary material is available at http://dx.doi.org/10.1007/s10822-005-9002-6.     

Stem cells and combinatorial science

Stem cell-based technologies have the potential to help cure a number of cell degenerative diseases. Combinatorial and high throughput screening techniques could provide tools to control and manipulate the self-renewal and differentiation of stem cells. This review chronicles historic and recent progress in the stem cell field involving both pluripotent and multipotent cells, and it highlights relevant cellular signal transduction pathways. This review further describes screens using libraries of soluble, small-molecule ligands, and arrays of molecules immobilized onto surfaces while proposing future trends in similar studies. It is hoped that by reviewing both the stem cell and the relevant high throughput screening literature, this paper can act as a resource to the combinatorial science community.     

The use of immortalized cell lines in GPCR screening: the good, bad and ugly

For most membrane-bound molecular targets, including G protein linked receptors (GPCRs), the optimal approach in drug discovery involves the use of cell based high throughput screening (HTS) technologies to identify compounds that modulate target activity. Most GPCRs have been cloned and can therefore be routinely expressed in immortalized cell lines. These cells can be easily and rapidly grown in unlimited quantities making them ideal for use in current HTS technologies. A significant advantage of this approach is that immortalized recombinant cells provide a homogenous background for expression of the target which greatly facilitates consistency in screening, thus allowing for a better understanding of the mechanism of action of the interacting compound or drug. Nonetheless, it is now evident that numerous disparities exist between the physiological environment of screening systems using recombinant cells and natural tissues. This has lead to a problem in the validity of the pharmacological data obtained using immortalized cells in as much as such cells do not always reflect the desired clinical efficacy and safety of the compounds under examination. This brief review discusses these issues and describes how they influence the discovery of drugs using modern HTS.     

High-throughput screening for bioactive components from traditional Chinese medicine

Throughout the centuries, traditional Chinese medicine has been a rich resource in the development of new drugs. Modern drug discovery, which relies increasingly on automated high throughput screening and quick hit-to-lead development, however, is confronted with the challenges of the chemical complexity associated with natural products. New technologies for biological screening as well as library building are in great demand in order to meet the requirements. Here we review the developments in these techniques under the perspective of their applicability in natural product drug discovery. Methods in library building, component characterizing, biological evaluation, and other screening methods including NMR and X-ray diffraction are discussed.     

Developing the molecular modelling of diffusion in zeolites as a high throughput catalyst screening technique

Molecular modelling techniques have been used to screen zeolite catalysts for their suitability for organic synthesis. For example, we have used these techniques to study the alkylation of aromatic molecules which are important in the fine-chemical and drug industries. A survey of all such efforts is reviewed in this article. The application of molecular modelling techniques in a systematic manner is an efficient first step in the design of zeolite catalysts. As a qualitative screening tool, molecular graphics is used to visualize how well the reactant and product molecules fit inside the pores of the zeolites. Using a hybrid of several molecular modelling methods, which combines molecular dynamics (MD) and Monte Carlo methods with energy minimization, it is possible to determine the minimum energy locations of the molecules inside the zeolites cages. The minimum energy configurations determined by this hybrid method are taken as a starting point for diffusion of the molecules through the zeolite channels. When a molecule is allowed to diffuse through zeolite channel, the molecule attains some maxima and minima in its diffusion energy profile. From the differences between a maximum and a minimum energy configuration, the diffusion energy barrier for the molecule can be calculated in the zeolites. By comparing the diffusion energy barriers for various isomers of a molecule in different zeolites, it is possible to find out the most suitable zeolite for achieving the required shape-selectivity. In addition, factors influencing the diffusivity of the molecules and consequently the shape selectivity are derived. The list of factors and their relative importance are analysed to derive valuable guidelines to design shape-selective zeolite catalysts for a given reaction. Thus, the ultimate aim of these studies is to develop a high throughput computational screening process for the selection of shape-selective zeolite catalysts for various reactions. The dynamic behaviour of molecules inside the pores of zeolites can be studied using MD methods. Since MD is computationally time consuming, it is more efficient to screen the possible zeolite catalysts by energy minimization methods and then perform MD in specific zeolites. More accurate values of diffusivity of the molecules can be calculated using MD methods, and these values can be correlated with the shape-selectivity observed experimentally and /or derived from diffusion energy barrier calculations.     

中草药活性成分的高通量筛选技术研究进展

随着高通量筛选技术的不断发展,该技术已经成为发现新药物的重要途径之一。高通量筛选技术已大量应用于筛选药物活性成分的领域中,但是其中大部分为从化合物库中筛选活性成分,仅有十几篇文献应用于中药活性成分的筛选,而中国传统中草药却是探索和发展新药物的丰富来源。本文通过综述国内外2008年到2017年的相关文献,阐述了分子和细胞水平上的高通量筛选技术在中草药活性成分的筛选及其应用进展,为今后中草药新药研发提供参考。     

Pharmacophore and docking-based virtual screening approach for the design of new dual inhibitors of Janus kinase 1 and Janus kinase 2

Janus kinase 1 and 2, non-receptor protein tyrosine kinases, are implicated in various cancerous diseases. Involvement of these two enzymes in the pathways that stimulate cell proliferation in cancerous conditions makes them potential therapeutic targets for designing new dual-targeted agents for the treatment of cancer. In the present study, two separate pharmacophore models were developed and the best models for JAK1 (AAADH.25) and JAK2 (ADRR.92) were selected on the basis of their external predictive ability. Both models were subjected to a systematic virtual screening (VS) protocol using a PHASE database of 1.5 million molecules. The hits retrieved in VS were investigated for ADME properties to avoid selection of molecules with a poor pharmacokinetic profile. The molecules considered to be within the range of acceptable limits of ADME properties were further employed for docking simulations with JAK1 and JAK2 proteins to explore the final hits that possess structural features of both pharmacophore models as well as display essential interactions with both of them. Thus, the new molecules obtained in this way should show inhibitory activity against JAK1 and JAK2 and may serve as novel therapeutic agents for the treatment of cancerous disease conditions.     

How large does a compound screening collection need to be?

Increasingly, chemical libraries are being produced which are focused on a biological target or group of related targets, rather than simply being constructed in a combinatorial fashion. A screening collection compiled from such libraries will contain multiple analogues of a number of discrete series of compounds. The question arises as to how many analogues are necessary to represent each series in order to ensure that an active series will be identified. Based on a simple probabilistic argument and supported by in-house screening data, guidelines are given for the number of compounds necessary to achieve a "hit", or series of hits, at various levels of certainty. Obtaining more than one hit from the same series is useful since this gives early acquisition of SAR (structure-activity relationship) and confirms a hit is not a singleton. We show that screening collections composed of only small numbers of analogues of each series are sub-optimal for SAR acquisition. Based on these studies, we recommend a minimum series size of about 200 compounds. This gives a high probability of confirmatory SAR (i.e. at least two hits from the same series). More substantial early SAR (at least 5 hits from the same series) can be gained by using series of about 650 compounds each. With this level of information being generated, more accurate assessment of the likely success of the series in hit-to-lead and later stage development becomes possible.     

Measuring intracellular calcium fluxes in high throughput mode

The measurement of intracellular calcium fluxes in real time is widely applied within the pharmaceutical industry to measure the activation of G-protein coupled receptors (GPCRhyp;s), either for pharmacological characterisation or to screen for new surrogate ligands. Initially restricted to G(q) coupled GPCRs, the introduction of promiscuous and chimeric G-proteins has further widened the application of these assays. The development of new calcium sensitive dyes and assays has provided sensitive, homogeneous assays which can be readily applied to high throughput screening (HTS). In this paper we describe the full automation of this assay type using a fluorometric imaging plate reader (FLIPR ) integrated into a Beckman/Sagian system to establish a simple robotic system that is well suited for the current medium throughput screening in this area of lead discovery. Using a recently completed HTS we discuss important determinants for FLIPR based screening, highlight some limitations of the current approach, and look at the requirements for future automated systems capable of keeping up with expanding compound files.