Imaged-based high-throughput screening for anti-angiogenic drug discovery

Recent developments in high-content screening (HCS) technologies make it an attractive alternative for anti-angiogenic drug discovery. HCS integrates high-throughput methodologies with automated multicolor fluorescence microscopy to collect quantitative morphological and molecular data from complex biological systems. Organotypic systems based on primary vascular cells model many facets of angiogenesis. The adaptation of these complex in vitro assay systems to high-throughput HCS formats with automated image acquisition enables large-scale chemical library screening campaigns. These HCS principles can be extended further to allow small molecule compounds in in vivo model organisms such as zebrafish. In this review we discuss the latest developments within automated image-based high-throughput screening of chemical libraries for anti-angiogenic compounds.     

Flow cytometry for drug discovery, receptor pharmacology and high-throughput screening

Although flow cytometry is viewed as a mature technology, there have been dramatic advances in analysis capabilities, sorting, sample handling and sensitivity in the past decade. These advances contribute to its application in biological and chemical diversity, sample throughput, high content, and complex systems biology. This article will evaluate the new opportunities for flow cytometry relating to receptor assembly and pharmacology, as well as a range of screening applications.     

Exploiting high-throughput ion channel screening technologies in integrated drug discovery

Ion channels are increasingly being implicated in disease. Although existing drugs that modulate channel function currently represent a key class of pharmaceutical agents, future ion channel drugs could help to treat an even wider variety of diseases. Despite their disease relevance, ion channels remain largely under exploited as drug targets, chiefly resulting from the absence of screening technologies that provide the throughput and quality of data required to support medicinal chemistry. Although some technical challenges still lie ahead, this historic bottleneck in drug discovery is now being bypassed by newer technologies that can be fully integrated into the early stages of drug discovery and will allow the discovery of novel therapeutic agents. Sequencing the human genome has greatly added to the number of potential drug targets but selecting suitable ion channels for drug discovery research should be based on the potential therapeutic relevance of the channel and not just the availability of suitable screens. Currently, ion channel drug discovery is focused on the need to identify compounds that can provide tractable starting points for medicinal chemistry. Advances in laboratory automation have brought significant opportunities to increase screening throughput for ion channel assays but careful assay configuration to model drug-target interactions in a physiological manner remains an essential consideration. Ion channel screening platforms are described in this review to provide some insight into the variety of technologies available for screening, together with some of their inherent advantages and limitations.     

Validating a firefly luciferase-based high-throughput screening assay for antimalarial drug discovery

The emergence and spread of multidrug-resistant Plasmodium falciparum and recent detection of potential artemisinin-resistant strains in Southeast Asia highlight the importance of developing novel antimalarial therapies. Using a previously generated stable transgenic P. falciparum line with high-level firefly luciferase expression, we report the adaptation, miniaturization, optimization, and validation of a high-throughput screening assay in 384-well plates. Assay conditions, including the percentage of parasitemia and hematocrit, were optimized. Parameters of assay robustness, including Z'-value, coefficient variation (CV), and signal-to-background (S/B) ratio, were determined. The LOPAC(1280) small-compound library was used to validate this assay. Our results demonstrated that this assay is robust and reliable, with an average Z'-value of >0.7 and CV of <10%. Moreover, this assay showed a very low background, with the S/B ratio up to 71. Further, identified hits were selected and confirmed using a SYBR Green I-based confirmatory assay. It is evident that this assay is suitable for large-scale screening of chemical libraries for antimalarial drug discovery.     

Application of LC/MS and related techniques to high-throughput drug discovery

The broad coordinated implementation of common platform technologies, such as LC/MS, can be a key factor in attaining increased throughput, sensitivity and data quality for pharmaceutical discovery. These platform technologies are the key tools that Medicinal Analytical Chemists rely upon to address the ever-changing needs of a drug discovery team. Despite the recent advances in key areas of sensitivity and speed for LC/MS, additional progress to address these never-ending analytical problems can be anticipated. This review will highlight current status and future advances for the applications of LC/MS and related techniques to high-throughput drug discovery.     

高通量筛选药动学模型的研究进展

高通量筛选体系在创新药物药动学筛选中的应用是当前新药开发研究的一个重要领域。建立合理的药动学筛选模型不仅可以降低新药开发的成本,并且可以在新药研究早期对其作出正确的评价。本文对当前常用的高通量筛选药动学模型作了简单的回顾,指出了合理的药动学筛选模型在寻找新药过程中的重要性,以及建立药动学筛选模型的紧迫性。     

Application of NMR screening in drug discovery

The application of NMR screening in drug discovery has recently attained heightened importance throughout the pharmaceutical industry. NMR screening can be applied at various points in a drug discovery program, ranging from very early in the program, when new targets can be screened long before an HTS enzymatic assay is developed, to later in the program, as in the case where no useful hits have been detected by HTS using biological assays. The binders determined in primary NMR screens are used to guide secondary screens, which can be either completely NMR driven or use NMR in combination with other biophysical techniques. In this review we briefly discuss the methods and techniques used in NMR screening. Then, we describe in detail the NMR screening strategies and their applications to specific targets, including successful examples from actual drug design programs at our own and other pharmaceutical companies.     

Rapid-access, high-throughput synchrotron crystallography for drug discovery

Synchrotron X-ray sources provide the highest quality crystallographic data for structure-guided drug design. In general, industrial utilization of such sources has been intermittent and occasionally limited. The Lilly Research Laboratories Collaborative Access Team (LRL-CAT) beamline provides a unique alternative to traditional synchrotron use by pharmaceutical and biotechnology companies. Crystallographic experiments at LRL-CAT and the results therefrom are integrated directly into the drug discovery process, permitting structural data, including screening of fragment libraries, to be routinely and rapidly used on a daily basis as part of pharmaceutical lead discovery and optimization. Here we describe how LRL-CAT acquires and disseminates the results from protein crystallography to maximize their impact on the development of new potential medicines.     

Advancing automation in high-throughput screening: Modular unguarded systems enable adaptable drug discovery

Challenged by ageing infrastructure and increasingly demanding screening cascades, AstraZeneca High Throughput Screening department has developed advanced automation systems that can support both current needs and future strategies in drug discovery. Through collaboration with HighRes Biosolutions and other third-party vendors, highly versatile automated modular platforms have been designed. Safety features such as collaborative robots allow enhanced system accessibility, and adaptive scheduling software has improved protocol design and system recovery. These innovations have led to significant improvements in system flexibility while maintaining screening productivity.     

Current targets for anticancer drug discovery

The call for the discovery of less toxic, more selective, and more effective agents to treat cancer has become more urgent. Inhibition of angiogenesis continues to be one of the main streams in the current cancer drug discovery activity. Insights into tumor angiogenesis biology have led to the identification of a number of molecules, which are important for the progression of these processes. Of particular interest is a group of growth factors including fibroblast growth factor, platelet-derived growth factor, and vascular endothelial growth factor. These growth factors and their corresponding receptor tyrosine kinases have become important targets for inhibition of the proliferation of endothelial cells, the main component of blood vessels. The validated targets for inhibition of angiogenesis also include a family of matrix metalloproteinases and cell adhesion molecules. In the closely related area, protein kinases have emerged as one of the most important targets for drug discovery. Besides growth factor receptor tyrosine kinases, numerous other protein kinases implicated in malignancies have been identified including non-receptor kinases such as Bcl-Abl and Src kinases. In addition, the cell cycle regulators (cyclin-dependent kinases, p21 gene) and apoptosis modulators (Bcl-2 oncoprotein, p53 tumor suppressor gene, survivin protein, etc) have also attracted renewed interest as potential targets for anticancer drug discovery. Other molecular targets include protein farnesyltransferase (FTase), histone deacetylase (HDAC), and telomerase, which have essential roles in cellular signal transduction pathways (FTase, HDAC) and cell life-span (telomerase). This review presents a comprehensive summary and discussion on the most important targets currently attracting a great deal of interest in contemporary anticancer drug design and discovery. Recent advances complementing these targets are also highlighted.     

Primary cells and stem cells in drug discovery: emerging tools for high-throughput screening

Many drug discovery screening programs employ immortalized cells, recombinantly engineered to express a defined molecular target. Several technologies are now emerging that render it feasible to employ more physiologically, and clinically relevant, cell phenotypes. Consequently, numerous approaches use primary cells, which retain many functions seen in vivo, as well as endogenously expressing the target of interest. Furthermore, stem cells, of either embryonic or adult origin, as well as those derived from differentiated cells, are now finding a place in drug discovery. Collectively, these cells are expanding the utility of authentic human cells, either as screening tools or as therapeutics, as well as providing cells derived directly from patients. Nonetheless, the growing use of phenotypically relevant cells (including primary cells or stem cells) is not without technical difficulties, particularly when their envisioned use lies in high-throughput screening (HTS) protocols. In particular, the limited availability of homogeneous primary or stem cell populations for HTS mandates that novel technologies be developed to accelerate their adoption. These technologies include detection of responses with very few cells as well as protocols to generate cell lines in abundant, homogeneous populations. In parallel, the growing use of changes in cell phenotype as the assay readout is driving greater use of high-throughput imaging techniques in screening. Taken together, the greater availability of novel primary and stem cell phenotypes as well as new detection technologies is heralding a new era of cellular screening. This convergence offers unique opportunities to identify drug candidates for disorders at which few therapeutics are presently available.     

New fluorescence techniques for high-throughput drug discovery

The rapid increase of compound libraries as well as new targets emerging from the Human Genome Project require constant progress in pharmaceutical research. An important tool is High-Throughput Screening (HTS), which has evolved as an indispensable instrument in the pre-clinical target-to-IND (Investigational New Drug) discovery process. HTS requires machinery, which is able to test more than 100,000 potential drug candidates per day with respect to a specific biological activity. This calls for certain experimental demands especially with respect to sensitivity, speed, and statistical accuracy, which are fulfilled by using fluorescence technology instrumentation. In particular the recently developed family of fluorescence techniques, FIDA (Fluorescence Intensity Distribution Analysis), which is based on confocal single-molecule detection, has opened up a new field of HTS applications. This report describes the application of these new techniques as well as of common fluorescence techniques--such as confocal fluorescence lifetime and anisotropy--to HTS. It gives experimental examples and presents advantages and disadvantages of each method. In addition the most common artifacts (auto-fluorescence or quenching by the drug candidates) emerging from the fluorescence detection techniques are highlighted and correction methods for confocal fluorescence read-outs are presented, which are able to circumvent this deficiency.     

High-throughput screening, metabolomics and drug discovery

The science of metabolomics has the potential to deliver wide-reaching benefits to the currently embattled pharmaceutical industry. Current applications for this field center around toxicological profiling and biomarker studies; however, the ability of metabolomics to quantitatively assess pharmacologically induced changes in biological systems at the phenotype level suggests that there would be value in its adoption at much earlier phases of the drug-discovery process. As is argued herein, this approach could be coupled with a re-organization of early drug-discovery paradigms to reduce both the rates of attrition and the costs of bringing a drug to market.     

The impact of diversity-based, high-throughput screening on drug discovery: "chance favours the prepared mind"

Since its modest beginnings in support of natural product discovery in the early 1980s, diversity-based high-throughput screening (dHTS) has developed within the pharmaceutical, biotechnology and academic sectors to become one of the most widely used hit identification screening paradigms in early drug discovery. Advances in key component technologies, specifically in diversity collection design, high-throughput assay development and screening informatics, continue to improve the economics and successes of dHTS hit discovery from large screening collections. Through the application of these components in concert, dHTS has evolved from an expensive technology-centric process that was used to screen collections of randomly acquired compounds, into a process that balances chemical, biological and technological inputs to purposefully build diverse compound collections through planned synthesis and purchasing strategies, and that screens these collections efficiently and economically. As a backlash to the 1990s hype that placed the HTS paradigm at the center of attempts to improve overall R&D productivity, sceptics predicted an undignified demise for this approach. Nevertheless, the use of key component technologies in tandem with sophisticated process and quality control systems is now beginning to deliver the success rates promised by the early proponents of the approach. These results indicate that, given continued 'preparedness', dHTS will remain as the principal hit identification tool for early drug discovery well into the next decade.     

Third anticancer drug discovery and development symposium

Third anticancer drug discovery and development symposium     

The potential of high-content high-throughput microscopy in drug discovery

Fluorescence microscopy is a powerful method to study protein function in its natural habitat, the living cell. With the availability of the green fluorescent protein and its spectral variants, almost any gene of interest can be fluorescently labelled in living cells opening the possibility to study protein localization, dynamics and interactions. The emergence of automated cellular systems allows rapid visualization of large groups of cells and phenotypic analysis in a quantitative manner. Here, we discuss recent advances in high-content high-throughput microscopy and its potential application to several steps of the drug discovery process.     

Toward high-throughput drug screening using mass spectrometry

A critical overview on the potential of mass spectrometry-based methods regarding high-throughput screening analysis is presented. Within this scope, screening procedures will be discussed for simultaneous detection of several drug classes relevant to clinical and forensic toxicology or doping control in urine or blood using gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry.     

The role of atelocollagen-based cell transfection array in high-throughput screening of gene functions and in drug discovery

The human genome project has been completed, but the function of many genes is unknown. It is, therefore, necessary to elucidate the function of a large number of genes within a short time. To achieve this goal, materials are needed that condense or package DNA into nano-particles that can easily be taken up by cells and would allow DNA to be retained without degradation. Atelocollagen is a reliable carrier for gene delivery because it is considered safe and appropriate for practical use. We developed a basic technique for high-throughput gene transfer and expression screening by pre-coating a multi-well plate with an Atelocollagen/DNA complex in which cells are then seeded. Complexes with a nano-particle form were efficiently transduced into cells without the use of additional transfection reagents, and they allowed for long-term gene expression. The complex spotted onto the well of a plate was stable for a long period and allowed the cells to transduce and express reporter genes. We also showed that the present method with Atelocollagen-based gene transfer is applicable to gene medicines, such as antisense ODNs, siRNA, and adenovirus vectors. These results suggest that an Atelocollagen-based cell transfection array may be appropriate for general use in the high-throughput screening of large sets of gene medicines with functions in mammalian cells.