G-protein-coupled receptor oligomerization and its potential for drug discovery

G-protein-coupled receptors (GPCRs) represent by far the largest class of targets for modern drugs. Virtually all therapeutics that are directed towards GPCRs have been designed using assays that presume that these receptors are monomeric. The recent realization that these receptors form homo-oligomeric and hetero-oligomeric complexes has added a new dimension to rational drug design. However, this important aspect of GPCR biology remains largely unincorporated into schemes to search for new therapeutics. This review provides a synopsis of the current thinking surrounding GPCR homo-oligomerization and hetero-oligomerization and shows how new models point towards unexplored avenues in the development of new therapies.     

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.     

抑制蛋白介导的G蛋白偶联受体偏向性信号途径及其在新药发现中的应用（英文）

G protein coupled receptors(GPCR)are important transmembrane proteins which account for more than 30%of direct clinical drug targets.Two main signaling pathways,either mediated by different G protein subtype or arrestins,underlies most of 800 GPCR functions in human genome.Selective ligands targeting to one of the G protein or arrestin signaling through specific receptor,which is also called biased ligands,may have beneficial effects and delete the unwanted side effects compared with traditional full agonists or antagonists.However,the mechanism governing the arrestin mediated GPCR biased signaling is still unclear.In recent years,our research group have combined animal models,cell biology and biophysical approaches to address the arrestin mediated GPCR function and its underlying mechanism,which is a key issue for GPCR targeted drug discovery.We have identified that downstream of β2 adrenergic receptor,arrestin mediated signaling plays critical roles in maintaining the pancreatic islet homeostasis and promotes the learning and memory through regulation of astrocyteneuron lactate transportation cycle.Targetingβ-arrestin-1 signaling rather than Gq signaling downstream of CCK1R receptor may provide a better therapy for diabetes.Although arrestin mediated signaling was traditionally recognized as the second wave of GPCR signaling,our recent results indicated thatβ-arrestin-1 was able to induce the first wave signaling in to regulate the catecholamine secretion from adrenal gland,by directly mediating AT1R/TRPC3 coupling.This result provided new mode for the connection of GPCR to activation of ion channels.Moreover,all above arrestin mediated signaling are regulated by receptor phosphorylation barcode,a hypothesis brought up by Prof.Lefkowitz and Prof.Andrew Tobin.Using biophysical and cellular approaches,we have identified that the 10 distinct phosphorylation interacting sites along the N-terminal of arrestin is the "phospho-code" reader of the arrestin,which recognized the information passed by GPCR,then translated to more than 1000 distinct arrestin conformations,and recruit distinct downstream signaling molecule.We therefore proposed "a flute model" working mechanism for arrestin mediated GPCR signaling.Using this flute model combined with GPCR ligand identification,we were able to regulate specific signaling and therefore arrestin mediated physiological functions by activation of the receptor and operation of the receptor phosphorylation barcode simultaneously(unpublished results).These knowledge advances in arrestin mediated GPCR signaling may facilitate further drug development targeting to GPCR family members.     

Molecular interaction fingerprint approaches for GPCR drug discovery

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The compromise of virtual screening and its impact on drug discovery

Introduction: Docking and structure-based virtual screening (VS) have been standard approaches in structure-based design for over two decades. However, our understanding of the limitations, potential, and strength of these techniques has enhanced, raising expectations.

Areas covered: Based on a survey of reports in the past five years, we assess whether VS: (1) predicts binding poses in agreement with crystallographic data (when available); (2) is a superior screening tool, as often claimed; (3) is successful in identifying chemical scaffolds that can be starting points for subsequent lead optimization cycles. Data shows that knowledge of the target and its chemotypes in postprocessing lead to viable hits in early drug discovery endeavors.

Expert opinion: VS is capable of accurate placements in the pocket for the most part, but does not consistently score screening collections accurately. What matters is capitalization on available resources to get closer to a viable lead or optimizable series. Integration of approaches, subjective hit selection guided by knowledge of the receptor or endogenous ligand, libraries driven by experimental guides, validation studies to identify the best docking/scoring that reproduces experimental findings, constraints regarding receptor–ligand interactions, thoroughly designed methodologies, and predefined cutoff scoring criteria strengthen VS’s position in pharmaceutical research.     

Human metabolic network reconstruction and its impact on drug discovery and development

This paper describes the process for the reconstruction of a high quality human metabolic network from the genome information, the existing problems in the reconstruction and why a time-consuming literature based consolidation process is needed. The reconstructed metabolic network provides a unified platform to integrate all the biological and medical information on genes, proteins, metabolites, disease, drugs and drug targets for a system level study of the relationship between metabolism and disease. System analysis of metabolic networks will help us, not only in identifying new drug targets but also in developing a system-oriented drug design strategy.     

Subtleties in GPCR drug discovery: a medicinal chemistry perspective

Therapeutic effects through G protein-coupled receptors (GPCRs) are promoted by a full agonist, partial agonist, neutral antagonist or inverse agonist. Dramatic change of function such as from a neutral antagonist to a full agonist with minimal variation of ligand structure is a phenomenon that medicinal chemists often encounter. This is also influenced by a change of assay format. The subtle nature of structure-function relationships is difficult to grasp unless carefully considered from both chemistry and assay perspectives. In this article we discuss the subtle aspects of GPCR drug discovery from the medicinal chemistry perspective.     

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.     

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.     

Stabilized helical peptides: overview of the technologies and its impact on drug discovery

Introduction: Protein-protein interactions are predominant in the workings of all cells. Until now, there have been a few successes in targeting protein-protein interactions with small molecules. Peptides may overcome some of the challenges of small molecules in disrupting protein-protein interactions. However, peptides present a new set of challenges in drug discovery. Thus, the study of the stabilization of helical peptides has been extensive.

Areas covered: Several technological approaches to helical peptide stabilization have been studied. In this review, stapled peptides, foldamers, and hydrogen bond surrogates are discussed. Issues regarding design principles are also discussed. Furthermore, this review introduces select computational techniques used to aid peptide design and discusses clinical trials of peptides in a more advanced stage of development.

Expert opinion: Stabilized helical peptides hold great promise in a wide array of diseases. However, the field is still relatively new and new design principles are emerging. The possibilities of peptide modification are quite extensive and expanding, so the design of stabilized peptides requires great attention to detail in order to avoid a large number of failed lead peptides. The start of clinical trials with stapled peptides is a promising sign for the future.     

The impact of pharmacogenetics and pharmacogenomics on drug discovery

It is widely perceived at present that pharmacogenetics and pharmacogenomics are about to revolutionize the face of medicine. In a more realistic assessment, the implementation of molecular genetics and biology will provide us with better ways to treat illnesses, and has already begun to do so in an incremental and evolutionary fashion. However, it is unlikely to change fundamentally the direction of medical progress. Advances are most likely to be made in the area of pharmacodynamics, as we learn to differentiate broader conventional clinical diagnoses into separate molecular subtypes.     

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.     

The impact of microwave synthesis on drug discovery

In the past few years, using microwave energy to heat and drive chemical reactions has become increasingly popular in the medicinal chemistry community. First described 20 years ago, this non-classical heating method has matured from a laboratory curiosity to an established technique that is heavily used in academia and industry. One of the many advantages of using rapid 'microwave flash heating' for chemical synthesis is the dramatic reduction in reaction times--from days and hours to minutes and seconds. As will be discussed here, there are good reasons why many pharmaceutical companies are incorporating microwave chemistry into their drug discovery efforts.     

The impact of combinatorial chemistry on drug discovery

This review will firstly present recent examples in the fields of immobilized arrays and dynamic combinatorial chemistry. The article will then emphasize the impact of combinatorial chemistry on target-focused libraries, demonstrating the usefulness of matrix-formatting for structure-activity relationship analysis and/or the advancement in library design and evaluation.