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.     

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.     

Targeting opioid receptor heterodimers: Strategies for screening and drug development

G-protein-coupled receptors are a major target for the development of new marketable drugs. A growing number of studies have shown that these receptors could bind to their ligands, signal, and be internalized as dimers. Most of the evidence comes from in vitro studies, but recent studies using animal models support an important role for dimerization in vivo and in human pathologies. It is therefore becoming highly relevant to include dimerization in screening campaigns: the increased complexity reached by the ability to target 2 receptors should lead to the identification of more specific hits that could be developed into drugs with fewer side effects. In this review, we have summarized results from a series of studies characterizing the properties of G-protein-coupled receptor dimers using both in vitro and in vivo systems. Since opioid receptors exist as dimers and heterodimerization modulates their pharmacology, we have used them as a model system to develop strategies for the identification of compounds that will specifically bind and activate opioid receptor heterodimers: such compounds could represent the next generation of pain relievers with decreased side effects, including reduced drug abuse liability.     

G-protein-coupled receptors as therapeutic targets for glioblastoma

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumour in adults. Treatments include surgical resection, radiotherapy, and chemotherapy. Despite this, the prognosis remains poor, with an impacted quality of life during treatment coupled with brain tumour recurrence; thus, new treatments are desperately needed. In this review, we focus on recent advances in G-protein-coupled receptor (GPCR) targets. To date, the most promising targets are the chemokine, cannabinoid, and dopamine receptors, but future work should further examine the melanocortin receptor-4 (MC4R), adhesion, lysophosphatidic acid (LPA) and smoothened (Smo) receptors to initiate new drug-screening strategies and targeted delivery of safe and effective GBM therapies.     

High-throughput drug screens for amyotrophic lateral sclerosis drug discovery

ABSTRACT

Introduction: Amyotrophic lateral sclerosis (ALS) is a rapid adult-onset neurodegenerative disorder characterised by the progressive loss of upper and lower motor neurons. Current treatment options are limited for ALS, with very modest effects on survival. Therefore, there is a unmet need for novel therapeutics to treat ALS.

Areas covered: This review highlights the many diverse high-throughput screening platforms that have been implemented in ALS drug discovery. The authors discuss cell free assays including in silico and protein interaction models. The review also covers classical in vitro cell studies and new cell technologies, such as patient derived cell lines. Finally, the review looks at novel in vivo models and their use in high-throughput ALS drug discovery

Expert opinion: Greater use of patient-derived in vitro cell models and development of better animal models of ALS will improve translation of lead compounds into clinic. Furthermore, AI technology is being developed to digest and interpret obtained data and to make ‘hidden knowledge’ usable to researchers. As a result, AI will improve target selection for high-throughput drug screening (HTDS) and aid lead compound optimisation. Furthermore, with greater genetic characterisation of ALS patients recruited to clinical trials, AI may help identify responsive genetic subtypes of patients from clinical trials.     

G蛋白偶联受体固有活性研究进展与新药开发

G蛋白偶联受体(G-prote in-coup led receptor,GPCR)是与G蛋白有信号连接的一大类受体家族,是人体内最大的膜受体蛋白家族,是一类具有7个跨膜螺旋的跨膜蛋白受体。GPCR的结构特征和在信号传导中的重要作用决定了其可以作为很好的药物靶标。目前世界药物市场上有三分之一的小分子药物是GPCR的激活剂(agon ist)或拮抗剂(antagon ist)。以其为靶点的药物在医药产业中占据显著地位。在当今前50种最畅销的上市药物中,20%属于G蛋白受体相关药物。近来的研究发现,大多数G蛋白偶联受体具有一个很重要的特性,就是具有固有活性(Constitutive ac-tivity),即无激动剂条件受体自发的维持激活并维持下游信号传导通路的活性。固有活性涉及受体、G蛋白及下游信号通路之间的关系。该文就G蛋白偶联受体固有活性概念、研究进展、反相激动剂与固有活性研究、固有活性与新药开发4个方面,进行以下论述。     

Application of high-throughput affinity-selection mass spectrometry for screening of chemical compound libraries in lead discovery

High-throughput screening of chemical libraries for compounds that interfere with a particular molecular target is among the most powerful methodologies applied in lead discovery at present. In this review, the authors describe a label-free, homogeneous, affinity-selection-based technology developed at Novartis, termed SpeedScreen, which is compared with similar technologies used for high-throughput screening in the pharmaceutical and biotechnology industries. The focus at present of SpeedScreen is twofold: first, this technology is applied to orphan genomic targets and to those targets that are non-tractable by a functional assay; second, this technology is applied complementary to the well-established traditional methodologies for the screening of molecular targets. In summary, the authors discuss the value of affinity-selection-based high-throughput screening as a complementary technology to the common functional screening platforms and the benefits as well as the limitations of this new technology are outlined.     

High-throughput flow cytometry for drug discovery

High-throughput flow cytometry exploits a novel many-samples/one-file approach to dramatically speed data acquisition, limit aspirated sample volume to as little as 2 μl/well and produce multisample data sets that facilitate automated analysis of samples in groups as well as individually. It has been successfully applied to both cell- and microsphere-based bioassays in 96- and 384-well formats, to screen tens-of-thousands of compounds and identify novel bioactive structures. High-content multiparametric analysis capabilities have been exploited for assay multiplexing, allowing the assessment of biologic selectivity and specificity to be an integral component of primary screens. These and other advances in the last decade have contributed to the application of flow cytometry as a uniquely powerful tool for probing biologic and chemical diversity and complex systems biology.     

Steroid-binding G-protein-coupled receptors: new drug discovery targets for old ligands

Steroid-binding receptors have long been a successful target class for the pharmaceutical industry. Clinical applications for steroids range from contraception and hormone replacement therapy to immune regulation and cancer therapy. With the recent demonstration that the orphan GPCR, GPR30 binds and is activated by estrogen, as well as the identification of a GPR30-selective agonist, it is likely that GPR30 represents a novel drug target with many potential clinical applications. This review discusses the role of GPR30 in mediating the effects of estrogen, as well as recent efforts to isolate GPR30-specific ligands using a combination of virtual and biomolecular screening. Finally, comments are made on the future directions regarding GPCRs, steroids and drug discovery.     

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.     

A perspective on more effective GPCR-targeted drug discovery efforts

Background: G-protein-coupled receptors (GPCRs) form the largest receptor family in mammalian genomes and over the years they have repeatedly proven themselves to be druggable targets. The activation of a GPCR (usually by ligand binding) results in conformational changes that lead to G-protein coupling with subsequent activation of downstream effectors and ultimately a cellular response. GPCRs can be modulated at several strategic signaling positions including ligand binding, G-protein coupling, phosphorylation, internalization, and recycling. Objective: As the complexities of GPCR activation and signaling become more recognized, new technologies have been sought for the identification of ligands that can alter not only receptor activation but also receptor action. Methods: Taking into consideration the key contact sites, this review will cover several points to bear in mind both when developing a screening campaign for a GPCR and when validating the hits obtained from those primary screens. Results/conclusions: The final selection of which screen to use for a given GPCR should take into account the end goal of the drug, such as inflammation, and work towards utilizing screens that allow physiological end points in this case chemotaxis, relevant cell types, and endogenous receptors whenever possible.     

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     

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.     

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.     

Preclinical discovery of duloxetine for the treatment of depression

Introduction: Affective disorders, including major depressive disorder (MDD), are among the most severely disabling mental disorders, and in many cases are associated with poor treatment outcomes. From the emergence of the monoamine hypothesis of depression, the first-line treatment for MDD had mainly acted by inhibiting monoamine reuptake, and thereby increasing these levels in the synaptic cleft. However, in recent years, several new antidepressant drugs have appeared, including duloxetine, a dual serotonin (5-HT) and noradrenaline (NA) reuptake inhibitor recommended for the treatment of MDD.

Areas covered: The article reviews and discusses the biochemical and functional profile of duloxetine splitting the review into acute and long-term treatment with this dual monoamine reuptake inhibitor. In addition, the authors summarize available preclinical behavioral research data, which have demonstrated among other effects, the antidepressant-like activity of duloxetine in several animal models. The authors focus on the most recent literature on synaptic neuroplasticity modulation of this antidepressant drug. Finally, the authors briefly mention other approved indications of duloxetine.

Expert opinion: Duloxetine inhibits 5-HT and NA reuptake, effectively desensitizes various autoreceptors and promotes neuroplasticity. Clinically, duloxetine is an effective antidepressant that is well tolerated and has significant efficacy in the treatment of MDD.     

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.     

The development of label-free cellular assays for drug discovery

Introduction: The need to improve drug research and development productivity continues to drive innovation in pharmacological assays. Technologies that can leverage the advantages of both molecular and phenotypic assays would hold great promise for discovery of new medicines.

Areas covered: This article briefly reviews current label-free platforms for cell-based assays and is primarily focused on fundamental aspects of these assays using dynamic mass redistribution technology as an example. The article also presents strategies for relating label-free profiles to molecular modes of actions of drugs.

Expert opinion: Emerging evidence suggests that label-free cellular assays are phenotypic in nature, yet permit molecular mechanistic deconvolution. Together with unique competency in throughput, sensitivity and pathway coverages, label-free cellular assays allow users to screen drugs against endogenous receptors in native cells (including disease relevant primary cells) and determine the molecular modes of action of drug molecules. However, there are challenges for label-free in both basic research and drug discovery: the deconvolution of the cellular and molecular mechanisms for the biosensor signatures of receptor–drug interactions, new methodologies for data analysis and the development of new biosensor technologies. These challenges will need to be met for the wide adoption of these assays in drug discovery.