Embryonic stem cell application in drug discovery

Embryonic stem (ES) cells and their differentiated progeny offer tremendous potential for regenerative medicine, even in the field of drug discovery. There is an urgent need for clinically relevant assays that make use of ES cells because of their rich biological utility. Attention has been focused on small molecules that allow the precise manipulation of cells in vitro, which could allow researchers to obtain homogeneous cell types for cell-based therapies and discover drugs for stimulating the regeneration of endogenous cells. Such therapeutics can act on target cells or their niches in vivo to promote cell survival, proliferation, differentiation, and homing. In the present paper, we reviewed the use of ES cell models for high-throughput/content drug screening and toxicity assessment. In addition, we examined the role of stem cells in large pharmaceutical companies' R&D and discussed a novel subject, nicheology, in stem cell-related research fields.     

Human cell systems for drug discovery

In the current drug discovery paradigm, validated recombinant targets form the basis of in vitro high-throughput screening (HTS) assays. Isolated proteins cannot, however, be regarded as representative of complex biological systems; hence, cell-based systems can be employed to complement in vitro data, providing greater confidence in compound activity in an intact biological system. The scarcity of human material and the lack of proliferative capacity of primary cell cultures, combined with problems associated with the use of stem cells, mean that immortalized cell lines are generally used as a source of cells for HTS. While such cell lines have improved proliferative capacity, they often display aberrant genetic and functional characteristics. Consequently, interest has focused on creating new cell lines using defined molecular strategies that overcome senescence signals and telomere shortening. These strategies include expression of viral oncogenes and the catalytic subunit of telomerase (TERT) to generate immortalized cells. However, persistent proliferation induced by oncogenes can have undesirable effects, particularly on differentiation; hence, conditional immortalization approaches have been developed to provide more suitable cell models. The present review discusses the advantages of different technologies available for the generation of cell lines for use in in vitro biology, and describes a representative range of cell lines currently used in drug discovery.     

Affinity-based screening techniques for enhancing lead discovery

Contemporary, rational small-molecule lead discovery methods, comprising target identification, assay development, high-throughput screening (HTS), hit characterization and medicinal chemistry optimization, dominate early-stage drug discovery strategies in many pharmaceutical companies. There is a growing disparity between the increasing cost of funding these methods and the decreasing number of new drugs reaching the market. New strategies must be adopted to reverse this trend. The use of genomics- and proteomics-based target discovery efforts can aid the process by dramatically increasing the number of novel, more highly validated targets entering the discovery process, but HTS must meet this increased demand with faster, cheaper technologies. Although activity-based screening strategies are typically efficient, allowing one scientist to interrogate tens of thousands of compounds per day, affinity-based screening strategies can allow much greater efficiency in the overall process. Affinity-based methods can play a role in both facilitating the screening of a greater number of targets and in efficiently characterizing the primary hits discovered.     

Genotoxicity screening: the slow march to the future

Genetic toxicology testing in drug discovery and development is slowly moving into the age of high-throughput screening (HTS). This has been helped by the development of new tools, as well as validation studies and data analysis to support their use in hit-to-lead or lead optimisation decisions. This review provides an overview of the current genetic toxicology methodologies and a few HTS methodologies. Comparisons are made between the predictivity of carcinogenesis that can be achieved in screening strategies as well as by the battery of regulatory tests. The importance of false-positive and false-negative calls at different stages in development is considered. There is a good prospect that in genetic toxicology, as in other areas of ADME-Tox, HTS will reduce the growing costs of carrying compounds with undesirable characteristics too far along the drug development process.     

How can high-throughput screening deliver drugs to treat atherosclerosis?

Importance of the field: Atherosclerosis is a progressive disease that is characterized by the accumulation of lipid-rich plaques within the artery walls. Despite the past 3 decades witnessing the most significant advances in the pharmacotherapy of atherosclerosis with statins, atherosclerosis is still one of the leading causes of mortality in industrialized and developing nations. The applications of high-throughput screening (HTS) have retrieved hits and lead compounds which may be further developed to new promising therapeutics to achieve more effective reductions in the risk of cardiovascular morbidity and mortality. Areas covered in this review: The review provides a summary of potential drug targets other than HMG-CoA reductase (primary target of statins) and their application in biochemical or cell-based HTS assays used by pharmaceutical companies and academic laboratories for anti-atherosclerotic drug discovery. What the reader will gain: The reader will gain an overview of the HTS strategies currently used in the development of anti-atherosclerotic agents. The reader is also provided with some abortive examples in anti-atherosclerotic drug discovery as well as the associated limitations and challenges of the process that HTS delivers new drugs to treat atherosclerosis. Take home message: HTS can assist in the efficient discovery of new drugs towards the potential targets involved in the progress of atherosclerosis.     

Stem cells as screening tools in drug discovery

All physiologic processes operate in a cellular setting. Therefore, drug discoverers need the highest quality cells as they pursue the next generation of safe and effective medicines. Recently, investigators have begun to consider stem cells as a new source of predictive, cell-based assays in drug discovery. Stem cell technology still has hurdles to overcome before these cells are fully accepted as decision-making reagents and amenable to high-throughput screening. However, with global research interest in stem cell biology, significant advances in the application of these cells in drug discovery have been reported. These advances are aligned with three important stages of pharmaceutical research: target discovery and validation, identification of efficacious chemical leads, and drug safety pharmacology. This concise review describes the application of stem cells in these areas of drug discovery with emphasis on molecular screening opportunities.     

Bringing primary cells to mainstream drug development and drug testing

Although cell-based screening is already an essential tool in drug discovery, the cell models currently available are fast becoming inadequate. The use of transformed cells as models in almost every step of the discovery pipeline needs to be substituted with more relevant, disease-oriented models, and the use of patient-derived primary cells should logically become the next best strategy. In the past the use of such cells has been restricted by their scarcity and difficulty in manipulation and general handling; however, recent advances in isolation and growth, as well as assay miniaturization, transfection efficiency and assay sensitivity, have enabled their use in the mainstream of drug discovery. This review explores some of these enabling technologies, as well as some of the most critical uses of primary cells that may dramatically alter the landscape of drug discovery and drug testing.     

Embryonic stem cells as a source of models for drug discovery

Embryonic stem cells (ESCs) will become a source of models for a wide range of adult differentiated cells, providing that reliable protocols for directed differentiation can be established. Stem-cell technology has the potential to revolutionize drug discovery, making models available for primary screens, secondary pharmacology, safety pharmacology, metabolic profiling and toxicity evaluation. Models of differentiated cells that are derived from mouse ESCs are already in use in drug discovery, and are beginning to find uses in high-throughput screens. Before analogous human models can be obtained in adequate numbers, reliable methods for the expansion of human ESC cultures will be needed. For applications in drug discovery, involving either species, protocols for directed differentiation will need to be robust and affordable. Here, we explore current challenges and future opportunities in relation to the use of stem-cell technology in drug discovery, and address the use of both mouse and human models.     

Cellular imaging in drug discovery

Traditional screening paradigms often focus on single targets. To facilitate drug discovery in the more complex physiological environment of a cell or organism, powerful cellular imaging systems have been developed. The emergence of these detection technologies allows the quantitative analysis of cellular events and visualization of relevant cellular phenotypes. Cellular imaging facilitates the integration of complex biology into the screening process, and addresses both high-content and high-throughput needs. This review describes how cellular imaging technologies contribute to the drug discovery process.     

Cell microarrays in drug discovery

There is an increasing need for systematic cell-based assays in a high-throughput screening (HTS) format to analyze the phenotypic consequences of perturbing mammalian cells with drugs, genes, interfering RNA. Taking advantage of the recent progress in microtechnology, new cell microarrays are being developed and applied to a large range of issues in metazoan cells. This article compares different approaches and evaluates their potential use in the drug discovery process. Although still an emerging technology, cell microarrays hold great promise to optimize the efficiency:cost ratio in cell-based HTS.     

Chemoproteomics-driven drug discovery: addressing high attrition rates

The advent of multiple high-throughput technologies has brought drug discovery round almost full circle, from pharmacological testing of compounds in vivo to engineered molecular target assays and back to integrated phenotypic screens in cells and organisms. In the past, primary screens to identify new pharmacological agents involved administering compounds to an animal and monitoring a pharmacologic endpoint. For example, antihypertensive agents were identified by dosing spontaneously hypertensive rats with compounds and observing whether their blood pressure dropped. In taking this phenomenological approach, scientists were focused on the final goal, in this example lowering of blood pressure, rather than developing an understanding of the target, or targets, the compounds were impacting. With the evolution of rational target-based approaches, scientists were able to study the direct interaction of compounds with their intended targets, expecting that this would lead to more-selective and safer therapeutics. With the industrialization of screening, referred to as HTS, hundreds of thousands of compounds were screened in robot-driven assays against targets of interest (with this goal in mind). However, an unintentional outcome of the migration from in vivo primary screens to highly target-specific HTS assays was a reduction in biological context caused by the separation of the target from other cellular proteins and processes that might impact its function. Recognition of the potential consequences of this over-simplification drove the modification of HTS processes and equipment to be compatible with cellular assays.     

How can high-throughput screening deliver drugs to treat atherosclerosis?

Importance of the field: Atherosclerosis is a progressive disease that is characterized by the accumulation of lipid-rich plaques within the artery walls. Despite the past 3 decades witnessing the most significant advances in the pharmacotherapy of atherosclerosis with statins, atherosclerosis is still one of the leading causes of mortality in industrialized and developing nations. The applications of high-throughput screening (HTS) have retrieved hits and lead compounds which may be further developed to new promising therapeutics to achieve more effective reductions in the risk of cardiovascular morbidity and mortality.

Areas covered in this review: The review provides a summary of potential drug targets other than HMG-CoA reductase (primary target of statins) and their application in biochemical or cell-based HTS assays used by pharmaceutical companies and academic laboratories for anti-atherosclerotic drug discovery.

What the reader will gain: The reader will gain an overview of the HTS strategies currently used in the development of anti-atherosclerotic agents. The reader is also provided with some abortive examples in anti-atherosclerotic drug discovery as well as the associated limitations and challenges of the process that HTS delivers new drugs to treat atherosclerosis.

Take home message: HTS can assist in the efficient discovery of new drugs towards the potential targets involved in the progress of atherosclerosis.     

Disease modeling and drug screening for neurological diseases using human induced pluripotent stem cells

With the general decline of pharmaceutical research productivity, there are concerns that many components of the drug discovery process need to be redesigned and optimized. For example, the human immortalized cell lines or animal primary cells commonly used in traditional drug screening may not faithfully recapitulate the pathological mechanisms of human diseases, leading to biases in assays, targets, or compounds that do not effectively address disease mechanisms. Recent advances in stem cell research, especially in the development of induced pluripotent stem cell (iPSC) technology, provide a new paradigm for drug screening by permitting the use of human cells with the same genetic makeup as the patients without the typical quantity constraints associated with patient primary cells. In this article, we will review the progress made to date on cellular disease models using human stem cells, with a focus on patient-specific iPSCs for neurological diseases. We will discuss the key challenges and the factors that associated with the success of using stem cell models for drug discovery through examples from monogenic diseases, diseases with various known genetic components, and complex diseases caused by a combination of genetic, environmental and other factors.     

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.     

A dual-readout F2 assay that combines fluorescence resonance energy transfer and fluorescence polarization for monitoring bimolecular interactions

F?rster (fluorescence) resonance energy transfer (FRET) and fluorescence polarization (FP) are widely used technologies for monitoring bimolecular interactions and have been extensively used in high-throughput screening (HTS) for probe and drug discovery. Despite their popularity in HTS, it has been recognized that different assay technologies may generate different hit lists for the same biochemical interaction. Due to the high cost of large-scale HTS campaigns, one has to make a critical choice to employee one assay platform for a particular HTS. Here we report the design and development of a dual-readout HTS assay that combines two assay technologies into one system using the Mcl-1 and Noxa BH3 peptide interaction as a model system. In this system, both FP and FRET signals were simultaneously monitored from one reaction, which is termed "Dual-Readout F(2) assay" with F(2) for FP and FRET. This dual-readout technology has been optimized in a 1,536-well ultra-HTS format for the discovery of Mcl-1 protein inhibitors and achieved a robust performance. This F(2) assay was further validated by screening a library of 102,255 compounds. As two assay platforms are utilized for the same target simultaneously, hit information is enriched without increasing the screening cost. This strategy can be generally extended to other FP-based assays and is expected to enrich primary HTS information and enhance the hit quality of HTS campaigns.     

Tools for GPCR drug discovery

G-protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic targets for a broad spectrum of diseases. The design and implementation of high-throughput GPCR assays that allow the cost-effective screening of large compound libraries to identify novel drug candidates are critical in early drug discovery. Early functional GPCR assays depend primarily on the measurement of G-protein-mediated 2nd messenger generation. Taking advantage of the continuously deepening understanding of GPCR signal transduction, many G-protein-independent pathways are utilized to detect the activity of GPCRs, and may provide additional information on functional selectivity of candidate compounds. With the combination of automated imaging systems and label-free detection systems, such assays are now suitable for high-throughput screening (HTS). In this review, we summarize the most widely used GPCR assays and recent advances in HTS technologies for GPCR drug discovery.     

Advances in methods for therapeutic peptide discovery, design and development

Drug discovery and development are intense, lengthy and interdisciplinary processes. Traditionally, drugs were discovered by synthesizing compounds in time-consuming multi-step experimental investigations followed by in vitro and in vivo biological screening. Promising candidates were then further studied for their pharmacokinetic properties, metabolism and potential toxicity. Today, the process of drug discovery has been revolutionized due to the advances in genomics, proteomics, and bioinformatics. Efficient technologies such as combinatorial chemistry, high throughput screening (HTS), virtual screening, de novo design and structure-based drug design contribute greatly to drug discovery. Peptides are emerging as a novel class of drugs for cancer therapy, and many efforts have been made to develop peptide-based pharmacologically active compounds. This paper presents a review of current advances and novel approaches in experimental and computational drug discovery and design. We also present a novel bioactive peptide analogue, designed using the Resonant Recognition Model (RRM), and discuss its potential use for cancer therapeutics.     

Induced pluripotent stem cell research: A revolutionary approach to face the challenges in drug screening

Discovery of induced pluripotent stem (iPS) cells in 2006 provided a new path for cell transplantation and drug screening. The iPS cells are stem cells derived from somatic cells that have been genetically reprogrammed into a pluripotent state. Similar to embryonic stem (ES) cells, iPS cells are capable of differentiating into three germ layers, eliminating some of the hurdles in ES cell technology. Further progress and advances in iPS cell technology, from viral to non-viral systems and from integrating to non-integrating approaches of foreign genes into the host genome, have enhanced the existing technology, making it more feasible for clinical applications. In particular, advances in iPS cell technology should enable autologous transplantation and more efficient drug discovery. Cell transplantation may lead to improved treatments for various diseases, including neurological, endocrine, and hepatic diseases. In studies on drug discovery, iPS cells generated from patient-derived somatic cells could be differentiated into specific cells expressing specific phenotypes, which could then be used as disease models. Thus, iPS cells can be helpful in understanding the mechanisms of disease progression and in cell-based efficient drug screening. Here, we summarize the history and progress of iPS cell technology, provide support for the growing interest in iPS cell applications with emphasis on practical uses in cell-based drug screening, and discuss some challenges faced in the use of this technology.     

Integrative functional assays, chemical genomics and high throughput screening: harnessing signal transduction pathways to a common HTS readout

Chemical genomics is a drug discovery strategy that relies heavily on high-throughput screening (HTS) and therefore benefits from functional assay platforms that allow HTS against all relevant genomic targets. Receptor Selection and Amplification Technology (R-SAT) is a cell-based, high-throughput functional assay where the receptor stimulus is translated into a measurable cellular response through an extensive signaling cascade occurring over several days. The large biological and chronological separation of stimulus from response provides numerous opportunities for enabling assays and increasing assay sensitivity. Here we review strategies for building homogeneous assay platforms across large gene families by redirecting and/or amplifying signal transduction pathways.