High-throughput screening approaches for investigating drug metabolism and pharmacokinetics

1. High-throughput screening approaches have been adopted throughout the pharmaceutical industry to aid in the rapid discovery of new chemical entities. Because it is now well recognized that the selection of a robust candidate requires a balance of potency, safety and pharmacokinetics, the role of drug metabolism departments has widened from their traditional one of supporting drug development to include the screening of compounds during the discovery process. To put drug metabolism and pharmacokinetic (DMPK) studies in context, the evolving role of DMPK screening in the drug discovery strategy of pharmaceutical companies will be discussed and a generalized approach will be presented. 2. With the increasing numbers of compounds requiring screening, DMPK optimization methods have had to be adapted for high throughput. There have been many developments in this field over the past decade and this review will focus on the high-throughput DMPK screening methodologies used today and in the recent past. 3. In vitro and in silico (computer-based) methods have proven most amenable to high-throughput approaches and these will firm the bulk of the review, but some advances with in vivo methods will also be discussed. As there has been a vast increase in published material on the topic of high-throughput DMPK methodologies in the past 10 years, it would be impossible to cover every method in detail, so this review will concentrate on the key areas and refer the reader to other, more detailed reviews wherever possible. 4. Most high-throughput methods would not be possible without the enabling technologies of computing, automation, new sample preparation technologies, and highly sensitive and selective detection systems, and these will also be reviewed. 5. The advantages and disadvantages of the screening methods will be presented, in particular the issue of handling the false-positives and -negatives that arise. 6. In concluding the review, future developments in this field will be discussed along with key issues that will need to be addressed.     

Drug discovery today

In recent years, tools for the development of new drugs have been dramatically improved. These include genomic and proteomic research, numerous biophysical methods, combinatorial chemistry and screening technologies. In addition, early ADMET studies are employed in order to significantly reduce the failure rate in the development of drug candidates. As a consequence, the lead finding, lead optimization and development process has gained marked enhancement in speed and efficiency. In parallel to this development, major pharma companies are increasingly outsourcing many components of drug discovery research to biotech companies. All these measures are designed to address the need for a faster time to market. New screening methodologies have contributed significantly to the efficiency of the drug discovery process. The conventional screening of single compounds or compound libraries has been dramatically accelerated by high throughput screening methods. In addition, in silico screening methods allow the evaluation of virtual compounds. A wide range of new lead finding and lead optimization opportunities result from novel screening methods by NMR, which are the topic of this review article.     

A Discovery Funnel for Nucleic Acid Binding Drug Candidates

Computational approaches are becoming increasingly popular for the discovery of drug candidates against a target of interest. Proteins have historically been the primary targets of many virtual screening efforts. While in silico screens targeting proteins has proven successful, other classes of targets, in particular DNA, remain largely unexplored using virtual screening methods. With the realization of the functional importance of many non-cannonical DNA structures such as G-quadruplexes, increased efforts are underway to discover new small molecules that can bind selectively to DNA structures. Here, we describe efforts to build an integrated in silico and in vitro platform for discovering compounds that may bind to a chosen DNA target. Millions of compounds are initially screened in silico for selective binding to a particular structure and ranked to identify several hundred best hits. An important element of our strategy is the inclusion of an array of possible competing structures in the in silico screen. The best hundred or so hits are validated experimentally for binding to the actual target structure by a high-throughput 96-well thermal denaturation assay to yield the top ten candidates. Finally, these most promising candidates are thoroughly characterized for binding to their DNA target by rigorous biophysical methods, including isothermal titration calorimetry, differential scanning calorimetry, spectroscopy and competition dialysis.This platform was validated using quadruplex DNA as a target and a newly discovered quadruplex binding compound with possible anti-cancer activity was discovered. Some considerations when embarking on virtual screening and in silico experiments are also discussed.     

Integration of virtual and high-throughput screening

High-throughput and virtual screening are important components of modern drug discovery research. Typically, these screening technologies are considered distinct approaches, as one is experimental and the other is theoretical in nature. However, given their similar tasks and goals, these approaches are much more complementary to each other than often thought. Various statistical, informatics and filtering methods have recently been introduced to foster the integration of experimental and in silico screening and maximize their output in drug discovery. Although many of these ideas and efforts have not yet proceeded much beyond the conceptual level, there are several success stories and good indications that early-stage drug discovery will benefit greatly from a more unified and knowledge-based approach to biological screening, despite the many technical advances towards even higher throughput that are made in the screening arena.     

High-throughput techniques for compound characterization and purification

A new paradigm in drug discovery is the synthesis of structurally diverse collections of compounds, so-called libraries, followed by high-throughput biological screening. High-throughput characterization and purification techniques are required to provide high-quality compounds and reliable biological data, which has led to the development of faster methods, system automation and parallel approaches. This review summarizes recent advances in support of analytical characterization and preparative purification technologies. Notably, mass spectrometry (MS) and supercritical fluid chromatography (SFC) are among the areas where new developments have had a major impact on defining these high-throughput applications.     

In silico approaches for predicting ADME properties of drugs

Combinatorial chemistry and high-throughput screening have increased the possibility of finding new lead compounds at much shorter time periods than conventional medicinal chemistry. However, too much promising drug candidates often fail because of unsatisfactory ADME properties. In silico ADME studies are expected to reduce the risk of late-stage attrition of drug development and to optimize screening and testing by looking at only the promising compounds. To this end, many in silico approaches for predicting ADME properties of compounds from their chemical structure have been developed, ranging from data-based approaches such as quantitative structure-activity relationship (QSAR), similarity searches, and 3-dimensional QSAR, to structure-based methods such as ligand-protein docking and pharmacophore modelling. In addition, several methods of integrating ADME properties to predict pharmacokinetics at the organ or body level have been studied. In this article, we briefly summarize in silico ADME approaches.     

High-throughput and in silico screenings in drug discovery

Background: In the current situation of weak drug pipelines, impending patent expiration of several blockbuster drugs, industry consolidation and changing business models that target special diseases like cancer, diabetes, Alzheimer's and obesity, the pharmaceutical industry is under intense pressure to generate a strong drug pipeline distinguished by better productivity, diversity and cost effectiveness. The goal is discovering high-quality leads in the initial stages of the development cycle, to minimize the costs associated with failures at later ones. Objective: Thus, there is a great amount of interest in further developing and optimizing high-throughput screening and in silico screening, the two methods responsible for generating most of the lead compounds. Although high-throughput screening is the predominant starting point for discovery programs, in silico methods have gradually made inroads by their more rational approach, to expedite the drug discovery and development process. Conclusion: Modern drug discovery strategies include both methods in tandem or in an iterative way. This review primarily provides a succinct overview and comparison of experimental and in silico screening techniques, selected case studies where both methods were used in concert to investigate their performance and complementary nature and a statement on the developments in experimental and in silico approaches in the near future.     

High-throughput screening approaches for investigating drug metabolism and pharmacokinetics

1. High-throughput screening approaches have been adopted throughout the pharmaceutical industry to aid in the rapid discovery of new chemical entities. Because it is now well recognized that the selection of a robust candidate requires a balance of potency, safety and pharmacokinetics, the role of drug metabolism departments has widened from their traditional one of supporting drug development to include the screening of compounds during the discovery process. To put drug metabolism and pharmacokinetic (DMPK) studies in context, the evolving role of DMPK screening in the drug discovery strategy of pharmaceutical companies will be discussed and a generalized approach will be presented. 2. With the increasing numbers of compounds requiring screening, DMPK optimization methods have had to be adapted for high throughput. There have been many developments in this field over the past decade and this review will focus on the high-throughput DMPK screening methodologies used today and in the recent past. 3. In vitro and in silico (computer-based) methods have proven most amenable to high-throughput approaches and these will form the bulk of the review, but some advances with in vivo methods will also be discussed. As there has been a vast increase in published material on the topic of high-throughput DMPK methodologies in the past 10 years, it would be impossible to cover every method in detail, so this review will concentrate on the key areas and refer the reader to other, more detailed reviews wherever possible. 4. Most high-throughput methods would not be possible without the enabling technologies of computing, automation, new sample preparation technologies, and highly sensitive and selective detection systems, and these will also be reviewed. 5. The advantages and disadvantages of the screening methods will be presented, in particular the issue of handling the false-positives and-negatives that arise. 6. In concluding the review, future developments in this field will be discussed along with key issues that will need to be addressed.     

4SCan/vADME: intelligent library screening as a shortcut from hits to lead compounds

Managing to solve the first step in drug discovery - the hit finding - can be a quite elaborate task, but it is only the initial step to the final goal; hit-to-lead optimisation still lies ahead and consumes even more time and resources. The solution is rather simple, that is, to take only the most promising compounds into account; but who is going to decide which ones are the most promising among a list of tens of millions of compounds in a virtual combinatorial library? 4SCan/vADME helps by bridging the gap between virtual (combinatorial) libraries designed by chemists and the in silico methods, docking and alignment, for screening databases. After choosing a random starting set, the implemented learning and prediction algorithm iteratively considers only combinations of fragments that have shown to result in more suitable interactions by the chosen method. ADME properties of the final list are then calculated via several in silico methods, resulting in a combined evaluation of the individual compound's target-specific, as well as ADME, properties. Based on the latter list of evaluated compounds, medicinal chemists can then decide which compounds might be the best ones to synthesise first and to serve as possible lead candidates. Following a brief introduction to virtual high-throughput screening techniques, the 4SCan/vADME method is outlined and discussed in this paper, using an example coming out of the 4SC pipeline.     

A decade of fragment-based drug design: strategic advances and lessons learned

Since the early 1990s, several technological and scientific advances - such as combinatorial chemistry, high-throughput screening and the sequencing of the human genome - have been heralded as remedies to the problems facing the pharmaceutical industry. The use of these technologies in some form is now well established at most pharmaceutical companies; however, the return on investment in terms of marketed products has not met expectations. Fragment-based drug design is another tool for drug discovery that has emerged in the past decade. Here, we describe the development and evolution of fragment-based drug design, analyse the role that this approach can have in combination with other discovery technologies and highlight the impact that fragment-based methods have made in progressing new medicines into the clinic.     

SPR-based fragment screening: advantages and applications

Fragment-based screening has recently evolved into a promising strategy in drug discovery, and a range of biophysical methods can be employed for fragment library screening. Relevant approaches, such as X-ray, NMR and tethering are briefly introduced focussing on their suitability for fragment-based drug discovery. In particular the application of surface plasmon resonance (SPR) techniques to the primary screening of large libraries comprising small molecules is discussed in detail. SPR is known to be a powerful tool for studying biomolecular interactions in a sensitive and label-free detection format. Advantages of SPR methods over more traditional assay formats are discussed and the application of available channel and array based SPR systems to biosensing are reviewed. Today, SPR protocols have been applied to secondary screening of compound libraries and hit conformation, but primary screening of large fragment libraries for drug discovery is often hampered by the throughput of available systems. Chemical microarrays, in combination with SPR imaging, can simultaneously generate affinity data for protein targets with up to 9,216 immobilized fragments per array. This approach has proven to be suitable for screening fragment libraries of up to 110,000 compounds in a high throughput fashion. The design of fragment libraries and appropriate immobilization chemistries are discussed, as well as suitable follow-up strategies for fragment hit optimization. Finally, described case studies demonstrate the successful identification of selective low molecular weight inhibitors for pharmacologically relevant drug targets through the SPR screening of fragment libraries.     

Structure-based screening of low-affinity compounds

Conventional bioassay-based screening remains a mainstream approach for lead discovery. However, its limitations have meant that other, more biophysical methods, such as X-ray crystallography and NMR, are now being developed as lead discovery tools. These methods are particularly effective at detecting the binding of low affinity, low molecular weight compounds and transforming them into novel potent leads using structure-guided chemistry. Here, we describe some of the technologies and approaches that are being developed in structure-based screening using X-ray crystallography, which promise to have a major impact on lead discovery.     

ADMET in silico modelling: towards prediction paradise?

Following studies in the late 1990s that indicated that poor pharmacokinetics and toxicity were important causes of costly late-stage failures in drug development, it has become widely appreciated that these areas should be considered as early as possible in the drug discovery process. However, in recent years, combinatorial chemistry and high-throughput screening have significantly increased the number of compounds for which early data on absorption, distribution, metabolism, excretion (ADME) and toxicity (T) are needed, which has in turn driven the development of a variety of medium and high-throughput in vitro ADMET screens. Here, we describe how in silico approaches will further increase our ability to predict and model the most relevant pharmacokinetic, metabolic and toxicity endpoints, thereby accelerating the drug discovery process.     

Proteomics. Making sense of genomic information for drug discovery

As an increasing number of available genomes triggers a gold rush in modern biology, the scientific challenge shifts towards understanding the total of the encoded information, most notably the proteins, their structures, functions and interactions. Currently this work is in its early stages but the near future will bring a merger of biology, engineering and informatics with a far broader impact on society than pure genomics has had so far. The challenge of characterizing the structures and functions of all proteins in a given cell demands technological advances beyond the classical methodologies of protein biochemistry. Mass spectrometry techniques for high-throughput protein identification, including peptide mass fingerprinting, sequence tagging and mass spectrometry on full-length proteins are providing the driving force behind proteomics endeavors. New technologies are needed to move high-resolution protein structure determination to an industrial scale. Nonetheless, improvements in techniques for the separation of intrinsic membrane proteins are enabling proteomics efforts towards identifying drug targets within this important class of biomolecules. Beyond the acquisition of data on sequences, structures and interactions, however, the major work in drug discovery remains: the screening of large candidate compound libraries combined with clever medicinal chemistry that guarantees selective action and defined delivery of the drug.     

Targeting cancer using fragment based drug discovery

Over the past decade, fragment-based drug discovery has developed significantly and has gained increasing popularity in the pharmaceutical industry as a powerful alternative and complement to traditional high-throughput screening approaches for hit identification. Fragment-based methods are capable of rapidly identifying starting points for structure-based drug design from relatively small libraries of low molecular weight compounds. The main constraints are the need for sensitive methods that can reliably detect the typically weak interactions between fragments and the target protein, and strategies for transforming fragments into higher molecular weight drug candidates. This approach has recently been validated as series of compounds from various programs have entered clinical trials.     

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.     

Screening methods for influenza antiviral drug discovery

INTRODUCTION: Influenza antiviral high-throughput screens have been extensive, and yet no approved influenza antivirals have been identified through high-throughput screening. This underscores the idea that development of successful screens should focus on the exploitation of the underrepresented viral targets and novel, therapeutic host targets. AREAS COVERED: The authors review conventional screening applications and emerging technologies with the potential to enhance influenza antiviral discovery. Real-world examples from the authors' work in biocontained environments are also provided. Future innovations are discussed, including the use of targeted libraries, multiplexed assays, proximity-based endpoint methods, non-laboratory-adapted virus strains, and primary cells, for immediate physiological relevance and translational applications. EXPERT OPINION: The lack of successful anti-influenza drug discovery using high-throughput screening should not deter future efforts. Increased understanding of the functions of viral targets and host-pathogen interactions has broadened the target reservoir. Future screening efforts should focus on identifying new drugs against unexploited viral and host targets using currently developed assays, and on the development of novel, innovative assays to discover new drugs with novel mechanisms. Innovative screens must be designed to identify compounds that specifically inhibit protein-protein or protein-RNA interactions or other virus/host factor interactions that are crucial for viral replication. Finally, the use of recent viral isolates, increased biocontainment (for highly-pathogenic strains), primary cell lines, and targeted compound libraries must converge in efficient high-throughput primary screens to generate high-content, physiologically-relevant data on compounds with robust antiviral activity.     

A novel high-throughput automated chip-based nanoelectrospray tandem mass spectrometric method for PAMPA sample analysis

Parallel artificial membrane permeability assay (PAMPA) has recently gained popularity as a novel, high-throughput assay capable of rapidly screening compounds for their permeability characteristics in early drug discovery. The analytical techniques typically used for PAMPA sample analysis are HPLC-UV, LC/MS or more recently UV-plate reader. The LC techniques, though sturdy and accurate, are often labor and time intensive and are not ideal for high-throughput. On the other hand, UV-plate reader technique is amenable to high-throughput but is not sensitive enough to detect the lower concentrations that are often encountered in early drug discovery work. This article investigates a novel analytical method, a chip-based automated nanoelectrospray mass spectrometric method for its ability to rapidly analyze PAMPA permeability samples. The utility and advantages of this novel analytical method is demonstrated by comparing PAMPA permeability values obtained from nanoelectrospray to those from conventional analytical methods. Ten marketed drugs having a broad range of structural space, physico-chemical properties and extent of intestinal absorption were selected as test compounds for this investigation. PAMPA permeability and recovery experiments were conducted with model compounds followed by analysis by UV-plate reader, UV-HPLC as well as the automated nanoelectrospray technique (nanoESI-MS/MS). There was a very good correlation (r(2) > 0.9) between the results obtained using nanoelectrospray and the other analytical techniques tested. Moreover, the nanoelectrospray approach presented several advantages over the standard techniques such as higher sensitivity and ability to detect individual compounds in cassette studies, making it an attractive high-throughput analytical technique. Thus, it has been demonstrated that nanoelectrospray analysis provides a highly efficient and accurate analytical methodology to analyze PAMPA samples generated in early drug discovery.     

Discovery of β2 Adrenergic Receptor Ligands Using Biosensor Fragment Screening of Tagged Wild-Type Receptor

相似文献