Biomimetic screening of class-B G protein-coupled receptors

The 41-amino acid peptide corticotropin releasing factor (CRF) is a major modulator of the mammalian stress response. Upon stressful stimuli, it binds to the corticotropin releasing factor receptor 1 (CRF(1)R), a typical member of the class-B G-protein-coupled receptors (GPCRs) and a prime target in the treatment of mood disorders. To chemically probe the molecular interaction of CRF with the transmembrane domain of its cognate receptor, we developed a high-throughput conjugation approach that mimics the natural activation mechanism of class-B GPCRs. An acetylene-tagged peptide library was synthesized and conjugated to an azide-modified high-affinity carrier peptide derived from the CRF C-terminus using copper-catalyzed dipolar cycloaddition. The resulting conjugates reconstituted potent agonists and were tested in situ for activation of the CRF(1) receptor in a cell-based assay. By use of this approach we (i) defined the minimal sequence motif that is required for full receptor activation, (ii) identified the critical functional groups and structure-activity relationships, (iii) developed an optimized, highly modified peptide probe with high potency (EC(50) = 4 nM) that is specific for the activation domain of the receptor, and (iv) probed the behavioral role of CRF receptors in living mice. The membrane recruitment by a high-affinity carrier enhanced the potency of the tethered peptides by >4 orders of magnitude and thus allowed the testing of very weak initial fragments that otherwise would have been inactive on their own. As no chromatography purification of the test peptides was necessary, a substantial increase in screening throughput was achieved. Importantly, the peptide conjugates can be used to probe the endogenous receptor in its native environment in vivo.     

High throughput screening (HTS) in identification new ligands and drugable targets of G protein-coupled receptors (GPCRs)

G protein-coupled receptors (GPCRs) which constitute one of the largest and most versatile families of cell surface receptors are involved in a wide spectrum of physiological functions, such as, neuronal transmission, chemotaxis, pacemaker activity and embryonic development. Therefore, in the past a few years GPCR families have become very important targets in pharmaceutical design. However, according to the human genome project, there are approximately 1000 genes encoding GPCRs, only about 200 of GPCRs have known ligands and functions. Searching for ligands of the unknown GPCRs and better modulators of known GPCRs are currently attracting lots of interest. High throughput screening (HTS), which is commonly defined as an automatic process of testing potential drug candidates efficiently, is widely used in drug discovery. In this review, the use of high throughput screening (HTS) in studying GPCRs and the choice of screening technology in different G-protein signaling pathways were summarized.     

A Seoul-Fluor-based bioprobe for lipid droplets and its application in image-based high throughput screening

We developed a novel fluorescent bioprobe (SF44) that can specifically visualize the cellular lipid droplets in in vitro and in vivo systems and illustrated the mechanistic rationale of its fluorogenic property. Its application to image-based high throughput screening led us to the identification of a new small-molecule modulator of lipid droplet formation.     

The application of high throughput siRNA screening technology to study host-pathogen interactions

Recent advances in high throughput screening technologies have accelerated the identification and characterization of potential factors involved in host-virus interactions, facilitating early detection and diagnosis of diseases, as well as providing promising drug targets. The last decade has seen a plethora of successful examples of high throughput screening approaches, especially siRNA screening. With support from protein interaction studies, mRNA expression profiling, and bioinformatics, siRNA screening has also been successfully utilized to identify host factors required for a number of viruses including HIV, West Nile virus and H1N1 virus. Such studies have raised the awareness of virologists, and have opened a new chapter of global analysis of host-pathogen interactions. However, to play a more defining role in prognostics, diagnostics and therapeutics for virus diseases, acknowledged drawbacks, including false positives and negatives, inherent in this technology, must be successfully addressed.     

Application of affinity selection-mass spectrometry assays to purification and affinity-based screening of the chemokine receptor CXCR4

Affinity selection-mass spectrometry (AS-MS) is a sensitive technology for identifying small molecules that bind to target proteins, and assays enabled by AS-MS can be used to delineate relative binding affinities of ligands for proteins. 'Indirect' AS-MS assays employ size-exclusion techniques to separate target-ligand complexes from unbound ligands, and target-associated ligands are then specifically detected by liquid chromatography mass spectrometry. We report how indirect AS-MS binding assays with known reference control compounds were used as guideposts for development of an optimized purification method for CXCR4, a G-protein coupled chemokine receptor, for which we sought novel antagonists. The CXCR4 purification method that was developed was amenable to scale-up and enabled the screening of purified recombinant human CXCR4 against a large combinatorial library of small molecules by high throughput indirect AS-MS. The screen resulted in the discovery of new ligands that competed off binding of reference compounds to CXCR4 in AS-MS binding assays and that antagonized SDF1α-triggered responses and CXCR4-mediated HIV1 viral uptake in cell-based assays. This report provides a methodological paradigm whereby indirect AS-MS-based ligand binding assays may be used to guide optimal integral membrane protein purification methods that enable downstream affinity selection-based applications such as high throughput AS-MS screens.     

High content screening for G protein-coupled receptors using cell-based protein translocation assays

G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR discovery is described, and proof-of-concept data from a pilot screen with a CXCR4 assay are presented. This chemokine receptor is a highly relevant drug target which plays an important role in the pathogenesis of inflammatory disease and also has been shown to be a co-receptor for entry of HIV into cells as well as to play a role in metastasis of certain cancer cells.     

Ligand and decoy sets for docking to G protein-coupled receptors

We compiled a G protein-coupled receptor (GPCR) ligand library (GLL) for 147 targets, selecting for each ligand 39 decoy molecules, collected in the GPCR Decoy Database (GDD). Decoys were chosen ensuring a ligand-decoy similarity of six physical properties, while enforcing ligand-decoy chemical dissimilarity. The performance in docking of the GDD was evaluated on 19 GPCRs, showing a marked decrease in enrichment compared to bias-uncorrected decoy sets. Both the GLL and GDD are freely available for the scientific community.     

Retroviral display and high throughput screening

Retroviruses distinguish themselves from all other mammalian viruses by their abilities to infect and propagate in mammalian cells without causing a cytopathic effect and to stably integrate their genetic information into the genome of the host cell. These unique properties make them an ideal platform for the display and directed evolution of proteins in a mammalian cell environment. This review will describe the essentials about retrovirus biology and then discuss in detail display and screening strategies that have been developed during the past 15 years of retroviral display technology.     

Photo-affinity labeling strategy to study the binding site of G protein-coupled receptors

G protein-coupled receptors (GPCRs) are involved in the control of every aspect of our behavior and physiology. GPCR can be involved in pathological processes as well and are linked to numerous diseases, including cardiovascular and mental disorders, retinal degeneration, cancer, and AIDS. This article reviews the methods of approaching photo-affinity labeling strategy to obtain the possible G protein-coupled receptors’s binding site.     

Fluorescence-based adenylyl cyclase assay adaptable to high throughput screening

The second messenger cAMP has been implicated in numerous cellular processes such as glycogen metabolism, muscle contraction, learning and memory, and differentiation and development. Genetic evidence suggests that the enzyme that produces cAMP, adenylyl cyclase (AC), may be involved in pathogenesis in many of these cellular processes. In addition, these data suggest that membrane-bound ACs may be valuable targets for therapeutics to treat pathogenesis of these processes. The development of a robust real-time adenylyl cyclase assay that can be scalable to high-throughput screening could help in the development of novel therapeutics. Here we report a novel fluorescence-based cyclase assay using Bodipy FL GTPgammaS (BGTPgammaS). The fluorescence of the Bodipy moiety of BGTPgammaS was dramatically enhanced by incubation with the minimal catalytic core of wild-type-AC (wt-AC) and a mutant with decreased purine selectivity (mut-AC), in an AC activation-dependent manner. No increase in fluorescence was observed using Bodipy FL ATPgammaS (BATPgammaS) as substrate for either wt-AC or mut-AC. Using BGTPgammaS, forskolin, Gsalpha.GTPgammaS and the divalent cation Mn(2+) potently enhanced the rate of fluorescence increase in a concentration-dependent manner. The fluorescence enhancement of the Bodipy moiety was inhibited by known inhibitors of AC such as 2'deoxy,3'AMP and 2',5'-dideoxy-3'ATP. Furthermore, the fluorescence assay is adaptable to 96-well and 384-well multiplate format and is thus applicable to high throughput screening methodologies.     

Biophysical approaches to G protein-coupled receptors: Structure, function and dynamics

G protein-coupled receptors (GPCR) represent a large family of drug targets for which there is no high-resolution structural information. In order to understand the mechanisms of ligand recognition and receptor activation, there is a strong need for novel biophysical methods. In this Perspective we provide an overview of recent experimental approaches used to explore the molecular architecture and dynamics of GPCR and their interactions with ligands and G proteins using biophysical, non-crystallographic, methods.     

Computational prediction of the coupling specificity of G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent one of the most important categories of membrane proteins that play important roles in signaling pathways. GPCRs transduce the extracellular stimuli into intracellular second messengers via their coupling to specific class of heterotrimeric GTP-binding proteins (G proteins) and the subsequent regulation of a diverse variety of effectors. Understanding the coupling specificity of GPCRs is critical for further comprehending their function, and is of tremendous clinical significance because GPCRs are the most successful drug targets. This minireview addresses the computational approaches that have been created for the prediction of coupling specificity of GPCRs and highlights the perspective of bioinformatics strategies that may be used to tackle this important task. In addition, some of the important resources of this field are also provided.     

The use of proton transfer reaction mass spectrometry for high throughput screening of terpene synthases

In this work, we introduce the application of proton transfer reaction mass spectrometry (PTR-MS) for the selection of improved terpene synthase mutants. In comparison with gas chromatography mass spectrometry (GC-MS)-based methods, PTR-MS could offer advantages by reduction of sample preparation steps and analysis time. The method we propose here allows for minimal sample preparation and analysis time and provides a promising platform for the high throughput screening (HTS) of large enzyme mutant libraries. To investigate the feasibility of a PTR-MS-based screening method, we employed a small library of Callitropsis nootkatensis valencene synthase (CnVS) mutants. Bacterial cultures expressing enzyme mutants were subjected to different growth formats, and headspace terpenes concentrations measured by PTR-Qi-ToF-MS were compared with GC-MS, to rank the activity of the enzyme mutants. For all cultivation formats, including 96 deep well plates, PTR-Qi-ToF-MS resulted in the same ranking of the enzyme variants, compared with the canonical format using 100 mL flasks and GC-MS analysis. This study provides a first basis for the application of rapid PTR-Qi-ToF-MS detection, in combination with multi-well formats, in HTS screening methods for the selection of highly productive terpene synthases.     

Virtual screen for ligands of orphan G protein-coupled receptors

This paper describes a virtual screening methodology that generates a ranked list of high-binding small molecule ligands for orphan G protein-coupled receptors (oGPCRs), circumventing the requirement for receptor three-dimensional structure determination. Features representing the receptor are based only on physicochemical properties of primary amino acid sequence, and ligand features use the two-dimensional atomic connection topology and atomic properties. An experimental screen comprised nearly 2 million hypothetical oGPCR-ligand complexes, from which it was observed that the top 1.96% predicted affinity scores corresponded to "highly active" ligands against orphan receptors. Results representing predicted high-scoring novel ligands for many oGPCRs are presented here. Validation of the method was carried out in several ways: (1) A random permutation of the structure-activity relationship of the training data was carried out; by comparing test statistic values of the randomized and nonshuffled data, we conclude that the value obtained with nonshuffled data is unlikely to have been encountered by chance. (2) Biological activities linked to the compounds with high cross-target binding affinity were analyzed using computed log-odds from a structure-based program. This information was correlated with literature citations where GPCR-related pathways or processes were linked to the bioactivity in question. (3) Anecdotal, out-of-sample predictions for nicotinic targets and known ligands were performed, with good accuracy in the low-to-high "active" binding range. (4) An out-of-sample consistency check using the commercial antipsychotic drug olanzapine produced "active" to "highly-active" predicted affinities for all oGPCRs in our study, an observation that is consistent with documented findings of cross-target affinity of this compound for many different GPCRs. It is suggested that this virtual screening approach may be used in support of the functional characterization of oGPCRs by identifying potential cognate ligands. Ultimately, this approach may have implications for pharmaceutical therapies to modulate the activity of faulty or disease-related cellular signaling pathways. In addition to application to cell surface receptors, this approach is a generalized strategy for discovery of small molecules that may bind intracellular enzymes and involve protein-protein interactions.     

Functional G protein-coupled receptor assays for primary and secondary screening

Screening technologies for G protein-coupled receptors comprise two major approaches; homogeneous assays, conducted in microtiter plate formats, and protein redistribution assays that require imaged-based analysis using automated confocal systems. Generally, the former are used in primary screening campaigns for lead identification, while the latter are used in secondary screens for lead optimization. Homogeneous assays measure changes in G protein-coupled receptor second messengers such as cAMP, Ins P3 or calcium. Protein redistribution assays assess internalization of the receptor ligand complex or translocation of G-protein coupled receptor-associated proteins, such as beta-arrestin. In the present review functional cell based approaches are discussed, emphasizing the variety of non radiometric technologies now in use for HTS.     

基于报告基因技术的MDR1转录抑制剂高通量筛选模型的建立和应用

MDR1基因是引起肿瘤多药耐药的主要基因,其编码的P-gp蛋白可持续将药物由胞内排出胞外以降低胞内药物浓度导致多药耐药,MDR1基因的转录抑制剂可抑制MDR1基因在癌细胞中的表达,从而逆转肿瘤多药耐药.通过克隆MDR1基因的启动子,将其插入pGL3-basic质粒构建MDR1-luc＋报告基因载体,再将重组载体转染入HepG2肝癌细胞并筛选单克隆细胞株,构建了MDR1启动子的高通量筛选模型,Z′因子为0.75;通过对中药样品库的筛选,得到两种中药提取物高良姜水提物、红豆蔻醇提物有明显耐药逆转效果,EC50值分别为高良姜水提物16.37mgL-1和红豆蔻醇提物14.96mgL-1,RT-PCR验证上述两种阳性样品具有明显的抑制MDR1基因表达的作用.以上结果为MDR1基因的转录抑制剂高通量筛选奠定了基础.