An update of novel screening methods for GPCR in drug discovery

Introduction: G protein-coupled receptors (GPCRs) are the largest and most versatile group of cytomembrane receptors, comprising of approximately 300 non-sensory and druggable members. Traditional GPCR drug screening is based on radiometric competition binding assays, which are expensive and hazardous to human health. Furthermore, the paradox of high investment and low output, in terms of new drugs, highlights the need for more efficient and effective drug screening methods. Areas covered: This review summarizes non-radioactive assays assessing the ligand-receptor binding including: the fluorescence polarization assay, the TR-FRET assay and the surface plasmon resonance assay. It also looks at non-radioactive assays that assess receptor activation and signaling including: second messenger-based assays and β-arrestin recruitment-based assays. This review also looks at assays based on cellular phenotypic change. Expert opinion: GPCR signaling pathways look to be more complicated than previously thought. The existence of receptor allosteric sites and multireceptor downstream effectors restricts the traditional assay methods. The emergence of novel drug screening methods such as those for assessing β-arrestin recruitment and cellular phenotypic change may provide us with improved drug screening efficiency and effect.     

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.     

高内涵药物筛选方法的研究及应用

高通量药物筛选 (high throughputscreening,HTS)是 20世纪 80年代中期产生的为寻找先导物针对大量样品进行药理活性评价分析的一种技术手段,在创新药物的研究和开发中发挥了重要作用。本室于 1998年在国内率先将其用于创新药物的研究,已发现一批具有潜在研究价值的化合物 [1, 2]。近年来在药物发现领域又出现了一个新概念———高内涵药物筛选 (high contentscreening,HCS)。本文就高内涵药物筛选目前的研究和应用情况作一讨论。1　高通量药物筛选与高内涵药物筛选高通量药物筛选是以药物发现的基本规律为基础,应用药理学、生物化学…     

Advances in flow cytometry for drug screening

Importance of the field: Flow cytometry is considered today as a mature technology. Recently, it has become an accurate tool for screening applications. Yet, not many studies have been published emphasizing flow cytometry as a tool of choice for drug screening except multiplex bead assay.

Areas covered in this review: Scanning the literature for technology breakouts in screening by flow is not an easy task. When a private industry has an accurate and fast screening technology on hands, why should they make public a tool precious for their screening applications? On the European academic side, there are regrettably few grants to help develop and publish screening methodologies. So, a less scientific way to find out is a close market survey seeking new instruments and associated kits or new methods. From here, can one expect flow cytometry to be a tool with new potential for drug discovery?

What the reader will gain: As the machines are getting simpler to use, a need for plug-and-analyze software has emerged. New analysis tools remain an important step as they will permit to analyze and compare several parameters in a multi-well format simultaneously and this for several cell types for cytomics: a multiparametric, dynamic approach to cell research as cytomics has a practical role to play in drug discovery within the immediate limitations of cell-based analyses.

Take home message: Developing new software with multi-well comparison capabilities and most importantly real-time interaction on cytograms can easily circumvent the lack of fluorescent channels on small bench top machines.     

Developments with bead-based screening for novel drug discovery

ABSTRACT

Introduction: Combinatorial chemistry provides a cost-effective method for rapid discovery of drug hits/leads. The one-bead-one-compound (OBOC) library method is in principle ideally suited for this application, because it permits a large number of structurally diverse compounds to be rapidly synthesized and simultaneously screened for binding to a target of interest. However, application of OBOC libraries in drug discovery has encountered significant technical challenges.

Areas covered: This Special Report covers the challenges associated with first-generation OBOC libraries (difficulty in structural identification of non-peptidic hits, screening biases and high false positive rates, and poor scalability). It also covers the many strategies developed over the past two decades to overcome these challenges.

Expert opinion: With most of the technical challenges now overcome and the advent of powerful intracellular delivery technologies, OBOC libraries of metabolically stable and conformationally rigidified molecules (macrocyclic peptides and peptidomimetics, rigidified acyclic oligomers, and D-peptides) can be routinely synthesized and screened to discover initial hits against previously undruggable targets such as intracellular protein-protein interactions. On the other hand, further developments are still needed to expand the utility of the OBOC method to non-peptidic chemical scaffolds.     

GPCR heterodimers: asymmetries in ligand binding and signalling output offer new targets for drug discovery

Dimers of GPCRs have held the imagination of researchers for almost 20 years. However, only recently has their value as potentially novel drug targets been increased significantly, and primarily, in the context of GPCR heterodimers. The view of receptor heterodimers as allosteric machines has transformed the way we understand structural and functional asymmetries inherent in their organization. These asymmetries alter both signalling output and how they might be targeted pharmacologically. The paper in this issue of BJP by Siddiquee and colleagues (2013) highlights our growing understanding of such asymmetries and their implications. They show that heterodimers of the angiotensin II AT1 receptor and the apelin receptor recognize and respond to their respective ligands in distinct ways from the parent receptors expressed alone. Further, they demonstrate asymmetric allosteric effects in the context of the heterodimer that may have significant implications for our understanding of such receptor complexes.

Linked Article

This article is a commentary on the research paper by Siddiquee et al., pp. 1104–1117 of this issue. To view this paper visit http://dx.doi.org/10.1111/j.1476-5381.2012.02192.x     

Neural stem cells as tools for drug discovery: novel platforms and approaches

Introduction: Neural stem cells catalyze strong interests for the development of systems to screen for effective drugs to treat neurodegenerative conditions and/or improve neurogenesis, fields where the classical approaches have so far failed in discovering successful drugs.

Areas covered: The authors review the known biology of NSCs, their normal function in development, the adult brain, and in vitro culture systems. The authors also discuss the scientific and technological progress which will aid wider applications of NSCs for drug screening/development purposes. The authors base this article on literature searches performed through PubMed and Google Scholar.

Expert opinion: NSC systems present unique opportunities that are starting to be successfully explored for genetic and chemical screening. These systems provide the possibility of identifying and optimizing molecules/drugs that could lead to the tighter control in self-renewal and lineage specification of NSCs as well as their functional maturation. This could be crucial in moving forward NSC-based therapies. It is expected that recent advances in the method of producing NSCs from patient-specific human induced pluripotent stem (iPS) cells and in the technologies to grow them in vitro, while preserving their full developmental potential, will allow a full exploitation of NSCs both in drug discovery programs and in predictive toxicology studies.     

Emerging concepts and approaches for chemokine-receptor drug discovery

Importance of the field: Chemokine receptors are most noted for their role in cell migration. However, inappropriate utilization or regulation of these receptors is implicated in many inflammatory diseases, cancer and HIV, making them important drug targets.

Areas covered in this review: Allostery, oligomerization and ligand bias are presented as they pertain to chemokine receptors and their associated pathologies. Specific examples of each are described from the recent literature and their implications are discussed in terms of drug discovery efforts targeting chemokine receptors.

What the reader will gain: Insight into the expanding view of the multitude of pharmacological variables that need to be considered or that may be exploited in chemokine receptor drug discovery.

Take home message: Since 2007, two drugs targeting chemokine receptors have been approved by the FDA, Maraviroc for preventing HIV infection and Mozobil^? for hematopoietic stem cell mobilization. While these successes permit optimism for chemokine receptors as drug targets, only recently has the complexity of this system begun to be appreciated. The concepts of allosteric inhibitors, biased ligands and functional selectivity raise the possibility that drugs with precisely-defined properties can be developed. Other complexities such as receptor oligomerization and tissue-specific functional states of receptors also offer opportunities for increased target and response specificity, although it will be more challenging to translate these ideas into approved therapeutics compared to traditional approaches.     

Signaling bias in drug discovery

Introduction: The availability of different functional pharmacological assays has revealed that agonists for receptors that are pleiotropically coupled to multiple signaling pathways in the cell can emphasize signals to some pathways over others, i.e. can be biased toward certain signals. This, in turn, opens the possibility that molecules can be made to emphasize favorable signals, de-emphasize harmful signals or selectively block the ability of the natural agonist to produce unfavorable signals.

Areas covered: This paper discusses the mechanism of biased signaling, the possible therapeutic implications of this effect, methods to quantify and measure bias and the current literature describing the translation of biased measure in vitro to in vivo systems. In addition, the challenges of exploiting this mechanism for therapy are outlined.

Expert opinion: While this mechanism is well established and ubiquitous in pharmacology and easily measured in vitro, there are theoretical and practical hurdles to overcome to the fruitful utilization of signaling bias in therapeutic systems. There will be failures in the translation of biased molecules in vivo because of these challenges but hopefully also success and these latter translations hopefully will provide guidance in exploiting this effect further for therapy.     

An overview of Ca2+ mobilization assays in GPCR drug discovery

Introduction: Calcium ions (Ca²⁺) serve as a second messenger or universal signal transducer implicated in the regulation of a wide range of physiological processes. A change in the concentration of intracellular Ca²⁺ is an important step in intracellular signal transduction. G protein-coupled receptors (GPCRs), the largest and most versatile group of cell surface receptors, transduce extracellular signals into intracellular responses via their coupling to heterotrimeric G proteins. Since Ca²⁺ plays a crucial role in GPCR-induced signaling, measurement of intracellular Ca²⁺ has attracted more and more attention in GPCR-targeted drug discovery.

Areas covered: This review focuses on the most popular functional assays measuring GPCRs-induced intracellular Ca²⁺ signaling. These include photoprotein-based, synthetic fluorescent indicator-based and genetically encoded calcium indicator (GECI)-based Ca²⁺ mobilization assays. A brief discussion of the design strategy of fluorescent probes in GPCR studies is also presented.

Expert opinion: GPCR-mediated intracellular signaling is multidimensional. There is an urgent need for the development of multiple-readout screening assays capable of simultaneous detection of biased signaling and screening of both agonists and antagonists in the same assay. It is also necessary to develop GECIs offering low cost and consistent assays suitable for investigating GPCR activation in vivo.     

药物筛选技术的最新进展--高内涵筛选

化合物活性筛选是创新药物研究过程的起点和具有决定意义的关键步骤。基于细胞的高内涵药物筛选技术实现了对化合物多靶点多参数的同时检测，代表着创新药物研究技术发展的必然趋势，将在未来的新药研发过程中发挥重要作用。笔者介绍了高内涵筛选技术的概念、系统组成，分析了其优势特点，并简要讨论了其在新药研究尤其是抗肿瘤药物研究中的实际应用。     

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.     

Navigating tuberculosis drug discovery with target-based screening

Introduction: Mycobacterium tuberculosis kills more people than any other bacterial pathogen. New drugs are required to shorten the treatment time and provide a viable therapy for drug-resistant and latent forms of tuberculosis. The tuberculosis field has advanced considerably since the publication of the M. tuberculosis genome sequence. Today, researchers can build a high definition map of the pathogen's traits and behavior and select individual targets for chemical disruption.

Areas covered: This review examines the discovery of current clinical and candidate tuberculosis drugs. It outlines recent developments in the selection of molecular targets for the discovery of new anti-mycobacterial agents. It appraises techniques that incorporate target knowledge into the screening protocol. These techniques include in silico, in vitro enzyme-based, differential antisense sensitivity and gene expression screening systems. The review also looks ahead to further techniques that may be applied in tuberculosis drug discovery.

Expert opinion: The adoption of an ‘either/or’ approach to targeted or random tuberculosis drug screening is not expected. The historical success of random screening in providing the tuberculosis drugs currently in clinical use is likely to ensure that non-targeted protocols retain an important role in drug screening. However, a number of M. tuberculosis inhibitors in lead optimization and preclinical development have been discovered using targeted methods. Realization of the first clinically-approved tuberculosis drugs derived from targeted screening and continued refinements in targeted screening technologies are likely to increase the adoption of targeted approaches in the future.     

Molecular dynamics simulations and novel drug discovery

Introduction: Molecular dynamics (MD) simulations can provide not only plentiful dynamical structural information on biomacromolecules but also a wealth of energetic information about protein and ligand interactions. Such information is very important to understanding the structure-function relationship of the target and the essence of protein–ligand interactions and to guiding the drug discovery and design process. Thus, MD simulations have been applied widely and successfully in each step of modern drug discovery.

Areas covered: In this review, the authors review the applications of MD simulations in novel drug discovery, including the pathogenic mechanisms of amyloidosis diseases, virtual screening and the interaction mechanisms between drugs and targets.

Expert opinion: MD simulations have been used widely in investigating the pathogenic mechanisms of diseases caused by protein misfolding, in virtual screening, and in investigating drug resistance mechanisms caused by mutations of the target. These issues are very difficult to solve by experimental methods alone. Thus, in the future, MD simulations will have wider application with the further improvement of computational capacity and the development of better sampling methods and more accurate force fields together with more efficient analysis methods.     

An update on the advancing high-throughput screening techniques for patch clamp-based ion channel screens: implications for drug discovery

Introduction: Automated patch clamp (APC) devices have become commonplace in many industrial and academic labs. Their ease-of-use and flexibility have ensured that users can perform routine screening experiments and complex kinetic experiments on the same device without the need for months of training and experience. APC devices are being developed to increase throughput and flexibility.

Areas covered: Experimental options such as temperature control, internal solution exchange and current clamp have been available on some APC devices for some time, and are being introduced on other devices. A comprehensive review of the literature pertaining to these features for the Patchliner, QPatch and Qube and data for these features for the SyncroPatch 384/768PE, is given. In addition, novel features such as dynamic clamp on the Patchliner and light stimulation of action potentials using channelrhodosin-2 is discussed.

Expert opinion: APC devices will continue to play an important role in drug discovery. The instruments will be continually developed to meet the needs of HTS laboratories and for basic research. The use of stem cells and recordings in current clamp mode will increase, as will the development of complex add-ons such as dynamic clamp and optical stimulation on high throughput devices.     

Future trends in screening technology for drug discovery

The last decade showed a further upsurge in screening technology advance and innovation. Notably, the establishment of ultra high-throughput screening facilities led to an explosion of screening capacities. However, in the last 2 years, a turning point in screening philosophy can be observed worldwide. Increasingly more companies are reducing the size of screening campaigns, while increasing the emphasis on data quality and relevance. This article tries to investigate how screening technologies will develop in the ever-changing landscape of 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。