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  • 1.

    【2hr】A High Throughput Assay to Identify Small Molecule Modulators of Prostatic Acid Phosphatase

    包量

    机译 一种高通量检测方法，用于鉴定前列腺酸性磷酸酶的小分子调节剂
    摘要：Prostatic acid phosphatase (PAP) is expressed in nociceptive neurons and functions as an ectonucleotidase. Injection of the secretory isoform of PAP has potent antinociceptive effects in mouse models of chronic pain. These data suggested that a small molecule activator of PAP may have utility as a novel therapeutic for chronic pain, while inhibitors could be used to acutely inhibit PAP in vitro and in vivo. To identify small molecule modulators of PAP activity, we validated a high throughput, fluorescence-based biochemical assay and then used this assay to screen a compound library. We decreased the frequency of false positive activators by subtracting compound fluorescence from the final assay fluorescence. This approach significantly reduced the number of false positive activators found in the screen. While no activators were confirmed, seven novel inhibitors of PAP were identified. Our results suggest this high throughput assay could be used to identify small molecule modulators of PAP activity.
  • 2.

    【2hr】An Agarose-Gel Based Method for Transporting Cell Lines

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    机译 基于琼脂糖凝胶的细胞系运输方法
    摘要：Cryopreserved cells stored in dry ice or liquid nitrogen is the classical method for transporting cells between research laboratories in different cities around the world in order to maintain cell viability. An alternative method is to ship the live cells in flasks filled with cell culture medium. Both methods have limitations of either a requirement on special shipping container or short times for the cells to survive on the shipping process. We have recently developed an agarose gel based method for directly transporting the live adherent cells in cell culture plates or dishes in ambient temperature. This convenient method simplifies the transportation of live cells in long distance that can maintain cells in good viability for several days.
  • 3.

    【2hr】Development of Improved Models for Phosphodiesterase-4 Inhibitors with a Multi-Conformational Structure-Based QSAR Method

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    机译 基于多构象结构的QSAR方法开发磷酸二酯酶4抑制剂的改进模型
    摘要：Phosphodiesterase-4 (PDE-4) is an important drug target for several diseases, including COPD (chronic obstructive pulmonary disorder) and neurodegenerative diseases. In this paper, we describe the development of improved QSAR (quantitative structure-activity relationship) models using a novel multi-conformational structure-based pharmacophore key (MC-SBPPK) method. Similar to our previous work, this method calculates molecular descriptors based on the matching of a molecule's pharmacophore features with those of the target binding pocket. Therefore, these descriptors are PDE4-specific, and most relevant to the problem under study. Furthermore, this work expands our previous SBPPK QSAR method by explicitly including multiple conformations of the PDE-4 inhibitors in the regression analysis, and thus addresses the issue of molecular flexibility. The nonlinear regression problem resulted from including multiple conformations has been transformed into a linear equation and solved by an iterative partial least square (iPLS) procedure, according to the Lukacova-Balaz scheme. 35 PDE-4 inhibitors have been analyzed with this new method, and predictive models have been developed. Based on the prediction statistics for both the training set and the test set, these new models are more robust and predictive than those obtained by traditional ligand-based QSAR techniques as well as that obtained with the SBPPK method reported in our previous work. As a result, multiple predictive models have been added to the collection of QSAR models for PDE4 inhibitors. Collectively, these models will be useful for the discovery of new drug candidates targeting the PDE-4 enzyme.
  • 4.

    【2hr】Inhibition of Inducible Nitric Oxide Synthase Expression by a Novel Small Molecule Activator of the Unfolded Protein Response

    包量

    机译 新型的小分子活化剂的未折叠的蛋白质反应的诱导型一氧化氮合酶表达的抑制。
    摘要：The transcription of inducible nitric oxide synthase (iNOS) is activated by a network of proinflammatory signaling pathways. Here we describe the identification of a small molecule that downregulates the expression of iNOS mRNA and protein in cytokine-activated cells and suppresses nitric oxide production in vivo. Mechanistic analysis suggests that this small molecule, erstressin, also activates the unfolded protein response (UPR), a signaling pathway triggered by endoplasmic reticulum stress. Erstressin induces rapid phosphorylation of eIF2α and the alternative splicing of XBP-1, hallmark initiating events of the UPR. Further, erstressin activates the transcription of multiple genes involved in the UPR. These data suggest an inverse relationship between UPR activation and iNOS mRNA and protein expression under proinflammatory conditions.
  • 5.

    【2hr】Improvements in Live Cell Analysis of G Protein Coupled Receptors using Second Generation BD Calcium Assay Kits

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    机译 使用第二代BD钙测定试剂盒对G蛋白偶联受体进行活细胞分析的改进
    摘要：BD™ Calcium Assay Kits are designed for cell-based calcium mobilization high-throughput screening assays. The kits use a proprietary formulation including a non-fluorescent calcium indicator that becomes activated inside the cell and shows increased fluorescence upon calcium binding. The formulation includes a signal-enhancing reagent to maximize the signal over background in a homogeneous, no-wash assay format, based on a technology developed at BD. We have compared the next generation BD calcium assay kit product family to previous versions of the formulation, and to other commercially available homogeneous calcium assay kits. The improvements have enabled better performance on the cell lines and receptors that we have tested in all plate formats including 1536.
  • 6.

    【2hr】A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase

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    机译 使用工程萤火虫荧光素酶的新型生物发光蛋白酶测定
    摘要：Proteases play important roles in a variety of disease processes. Understanding their biological functions underpins the efforts of drug discovery. We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the native enzyme termini were joined by a peptide containing a protease site of interest. Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold. The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased. The assay has broad applicability using a variety of proteases and their cognate sites and can sensitively detect protease activity. In this report we further demonstrate its utility for the evaluation of protease recognition sequence specificity and subsequent establishment of an optimized assay for the identification and characterization of protease inhibitors using high throughput screening.
  • 7.

    【2hr】A New Structure-Based QSAR Method Affords both Descriptive and Predictive Models for Phosphodiesterase-4 Inhibitors

    包量

    机译 一种新的基于结构的QSAR方法同时满足磷酸二酯酶4抑制剂的描述性模型和预测模型。
    摘要：We describe the application of a new QSAR (quantitative structure-activity relationship) formalism to the analysis and modeling of PDE-4 inhibitors. This new method takes advantage of the X-ray structural information of the PDE-4 enzyme to characterize the small molecule inhibitors. It calculates molecular descriptors based on the matching of their pharmacophore feature pairs with those (the reference) of the target binding pocket. Since the reference is derived from the X-ray crystal structures of the target under study, these descriptors are target-specific and easy to interpret. We have analyzed 35 indole derivative-based PDE-4 inhibitors where Partial Least Square (PLS) analysis has been employed to obtain the predictive models. Compared to traditional QSAR methods such as CoMFA and CoMSIA, our models are more robust and predictive measured by statistics for both the training and test sets of molecules. Our method can also identify critical pharmacophore features that are responsible for the inhibitory potency of the small molecules. Thus, this structure-based QSAR method affords both descriptive and predictive models for phosphodiesterase-4 inhibitors. The success of this study has also laid a solid foundation for systematic QSAR modeling of the PDE family of enzymes, which will ultimately contribute to chemical genomics research and drug discovery targeting the PDE enzymes.
  • 8.

    【2hr】A Homogenous Luminescent Proximity Assay for 14-3-3 Interactions with Both Phosphorylated and Nonphosphorylated Client Peptides

    包量

    机译 14-3-3与磷酸化和非磷酸化客户肽相互作用的同质发光法
    摘要：The 14-3-3 proteins are a family of dimeric eukaryotic proteins that mediate both phosphorylation-dependent and -independent protein-protein interactions. Through these interactions, 14-3-3 proteins participate in the regulation of a wide range of cellular processes, including cell proliferation, cell cycle progression, and apoptosis. Because of their fundamental importance, 14-3-3 proteins have also been implicated in a variety of diseases, including cancer and neurodegenerative disorders. In order to monitor 14-3-3/client protein interactions for the discovery of small molecule 14-3-3 modulators, we have designed and optimized 14-3-3 protein binding assays based on the amplified luminescent proximity homogeneous assay (AlphaScreen) technology. Using the interaction of 14-3-3 with a phosphorylated Raf-1 peptide and a nonphosphorylated R18 peptide as model systems, we have established homogenous “add-and-measure” high-throughput screening assays. Both assays achieved robust performance with S/B ratios above 7 and Z’ factors above 0.7. Application of the known antagonistic peptides in our studies further validated the assay for screening of chemical compound libraries to identify small molecules that can modulate 14-3-3 protein-protein interactions.
  • 9.

    【2hr】A Novel Fluorogenic Coumarin Substrate for Monitoring Acid Phosphatase Activity at Low pH Environment

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    机译 用于监测低pH环境下酸性磷酸酶活性的新型荧光香豆素底物
    摘要：This article described the synthesis and application of 6-chloro-8-fluoro-4-methylumbelliferone phosphate (CF-MUP) in analyzing acid phosphatase activity. Compared to the existing MUP, the new coumarin phosphate, CF-MUP, demonstrateed much higher sensitivity and was more robust for detecting the activity of acid phosphatase than the classic substrate 4-methylumbelliferone phosphate (MUP). The product of enzyme reaction, 6-chloro-8-fluoro-4-methylumbelliferone (CF-MU) possesses strong fluorescence at ~450 nm with low pKa (4.7), high fluorescence quantum yield and pH independence in the physiological pH range. This new fluorescence dye, CF-MU, is a convenient tool for assays with buffer pH between 4.5 and 8.
  • 10.

    【2hr】A High Throughput Serum Paraoxonase Assay for Discovery of Small Molecule Modulators of PON1 Activity

    包量

    机译 高通量血清对氧磷酶测定发现PON1活性的小分子调节剂。
    摘要：PON1 has been demonstrated to be the serum enzyme responsible for detoxifying organophosphate chemical weapons and plays a protective role against atherosclerosis. In order to identify small molecules that modulate PON1 activity in serum, we developed a high throughput kinetic absorbance assay using mouse serum and the organophosphate paraoxon. The IC50 value obtained for the known PON1 inhibitor, 2-hydroxyquinoline, matched the value reported for purified PON1. A compound library was screened resulting in no confirmed activators, but 12 confirmed inhibitors. Seven of these hits also inhibited purified human PON1. One compound was only two-fold less potent than 2-hydroxyquinoline in the serum assay, but 10-fold more potent against purified PON1. This compound (IC50 = 420 nM) may be useful towards a chemical probe for PON1. Therefore, this assay has utility as a high throughput assay for discovery of small molecule modulators of PON1 activity that maintain activity in serum.
  • 11.

    【2hr】Glutamatergic Synaptic Dysfunction and Obsessive-Compulsive Disorder

    包量

    机译 谷氨酸能突触功能障碍和强迫症
    摘要：Obsessive-compulsive disorder (OCD) is a debilitating neuropsychiatric condition estimated to afflict 1-3% of the world population. The estimated financial impact in the treatment and management of OCD is in the billions of dollars annually in the US alone. At present there is a marked lack of evidence on the specific causes of OCD. Current hypotheses largely focus on the serotonin (5-HT) system on the basis of the effectiveness of selective serotonin reuptake inhibitors (SSRIs) in alleviating symptoms of patients with OCD, yet a considerable fraction of patients are non-responsive or minimally responsive to these agents. Despite this fact, SSRIs have remained the primary pharmacological treatment avenue for OCD. In recent years, multiple lines of evidence have implicated glutamatergic synaptic dysfunction within the cortico-striatal-thalamo-cortical (CSTC) brain circuit in the etiology of OCD and related disorders, thereby prompting intensified effort in the development and evaluation of agents that modulate glutamatergic neurotransmission for the treatment of OCD. With this in mind, here we review the following topics with respect to synaptic dysfunction and the neural circuitry underlying OCD: (1) evidence supporting the critical involvement of the CSTC circuit, (2) genetic studies supporting the involvement of glutamatergic dysfunction, (3) insights from genetic animal models of OCD, and (4) preliminary findings with glutamatergic neurotransmission-modulating agents in the treatment of OCD. Given the putative mechanistic overlap between OCD and the broader OC-spectrum of disorders, unraveling the synaptic basis of OCD has potential to translate into more effective treatments for an array of poorly understood human disorders.
  • 12.

    【2hr】Comparative Docking Assessment of Glucokinase Interactions with its Allosteric Activators

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    机译 葡萄糖激酶与其变构活化剂相互作用的对接比较评估
    摘要：Glucokinase (GK) is expressed in multiple organs and plays a key role in hepatic glucose metabolism and pancreatic insulin secretion. GK could indeed serve as pacemaker of glycolysis and could be an attractive target for type 2 diabetes (T2D). The recent preclinical data of first GK activator RO-28-1675 has opened up a new field of GK activation as a powerful tool in T2D therapies. The GK allosteric site is located ~20Å away from glucose binding site. Chemical structure of Glucokinase activators (GKA) includes three chemical arms; all consisting of cyclic moiety and joined in a shape resembling the letter Y. In this study, comparative docking assessment using Autodock4 revealed that the three arms bind to three aromatic/hydrophobic subpockets at the allosteric site. Our dockings have overall consistency with experimental data in both docking modes and simulated binding free energies, and offer insights on understanding GK/GKA interactions and further GKA design. Specifically, for the first pocket, involvement of Arg63 as key residue in two specific hydrogen-bond formations with all allosteric activators defines the binding feature; for the second pocket, it has the most diverse binding interactions, mostly aromatic, hydrophobic and multiple hydrogen bonds. The site has the best potential for further GKA optimization by utilizing aromatic heterocycles and hydrogen bond forming linkers to build the GKA 2nd arm.
  • 13.

    【2hr】Development of Fluorescence Polarization Assays for the Molecular Chaperone Hsp70 Family Members: Hsp72 and DnaK

    包量

    机译 分子伴侣Hsp70家族成员：Hsp72和DnaK的荧光偏振测定的发展。
    摘要：The heat shock protein 70 (Hsp70) family of chaperones play crucial roles in protein folding and have been linked to numerous diseases. We were interested in developing a generally applicable assay format for the Hsp70 family and have developed fluorescence polarization based assays for both the mammalian Hsp72 and its bacterial counterpart, DnaK. These assays are comparable in assay set-up, incubation conditions and buffer components. Both unfolded polypeptides and synthetic peptides can be utilized as tracers to detect binding although peptides meeting the minimum seven residue length for Hsp70 binders have weaken binding when modified with fluorescein presumably due to steric effects. Although we did not identify a suitable general substrate for all Hsp70 proteins, fluorescein tagged peptide substrates that gave high affinity binding were identified for both DnaK and hsp72. We would predict that these assays will be suitable for identifying both selective chemical probes of Hsp70 family members and “pan” Hsp70 inhibitors.
  • 14.

    【2hr】EDITORIAL

    包量

    机译 社论
    • 作者：Wei Zheng
    • 刊名：Current Chemical Genomics and Translational Medicine
    • 2008年第期
    摘要：
  • 15.

    【2hr】The Use of AlphaScreen Technology in HTS: Current Status

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    机译 HTS中AlphaScreen技术的使用：当前状态
    摘要：AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) is versatile assay technology developed to measuring analytes using a homogenous protocol. This technology is an example of a bead-based proximity assay and was developed from a diagnostic assay technology known as LOCI (Luminescent Oxygen Channeling Assay). Here, singlet oxygen molecules, generated by high energy irradiation of Donor beads, travel over a constrained distance (approx. 200 nm) to Acceptor beads. This results in excitation of a cascading series of chemical reactions, ultimately causing generation of a chemiluminescent signal.In the past decade, a wide variety of applications has been reported, ranging from detection of analytes involved in cell signaling, including protein:protein, protein:peptide, protein:small molecule or peptide:peptide interactions. Numerous homogeneous HTS-optimized assays have been reported using the approach, including generation of second messengers (such as accumulation of cyclic AMP, cyclic GMP, inositol [1, 4, 5] trisphosphate or phosphorylated ERK) from liganded GPCRs or tyrosine kinase receptors, post-translational modification of proteins (such as proteolytic cleavage, phosphorylation, ubiquination and sumoylation) as well as protein-protein and protein-nucleic acid interactions.Recently, the basic AlphaScreen technology was extended in that the chemistry of the Acceptor bead was modified such that emitted light is more intense and spectrally defined, thereby markedly reducing interference from biological fluid matrices (such as trace hemolysis in serum and plasma). In this format, referred to as AlphaLISA, it provides an alternative technology to classical ELISA assays and is suitable for high throughput automated fluid dispensing and detection systems.Collectively, AlphaScreen and AlphaLISA technologies provide a facile assay platform with which one can quantitate complex cellular processes using simple no-wash microtiter plate based assays. They provide the means by which large compound libraries can be screened in a high throughput fashion at a diverse range of therapeutically important targets, often not readily undertaken using other homogeneous assay technologies. This review assesses the current status of the technology in drug discovery, in general, and high throughput screening (HTS), in particular.
  • 16.

    【2hr】Multiplexing Bioluminescent and Fluorescent Reporters to Monitor Live Cells

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    机译 复用生物发光和荧光记者监测活细胞
    摘要：Reporter proteins are valuable tools to monitor promoter activities and characterize signal transduction pathways. Many of the currently available promoter reporters have drawbacks that compromise their performance. Enzyme-based reporter systems using cytosolic luciferases are highly sensitive, but require a cell lysis step that prevents their use in long-term monitoring. By contrast, secreted bioluminescent reporters like Metridia luciferase and Secreted Alkaline Phosphatase can be assayed repeatedly, using supernatant from the same live cell population to produce many sets of data over time. This is crucial for studies with limited amounts of cells, as in the case of stem cells. The use of secreted bioluminescent reporters also enables broader applications to provide more detailed information using live cells; for example, multiplexing with fluorescent proteins. Here, data is presented describing the characteristics of secreted Metridia luciferase and its use in multiplexing applications with either Secreted Alkaline Phosphatase or a fluorescent protein.
  • 17.

    【2hr】Development of a HTRF® Kinase Assay for Determination of Syk Activity

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    机译 开发用于确定Syk活性的HTRF®激酶测定法
    摘要：Regulation of protein phosphorylation is a primary cellular signaling mechanism. Many cellular responses to internal and external events are mitigated by protein kinase signaling cascades. Dysfunction of protein kinase activity has been linked to a variety of human pathologies, in the areas of cancer, inflammation, metabolism, cell cycle, apoptosis, as well as cardiovascular, neurodegenerative and autoimmune diseases [1-3]. As such, there is an important need for protein kinase activity detection methodologies for researchers engaged in Drug Discovery. A number of different technologies have been employed for the measurement of protein kinase activity, including radioactive methods, luminescent methods, and fluorescent methods. More recently, Homogeneous Time Resolved Fluorescence technology (HTRF®), based on the principle of time-resolved fluorescent resonance energy transfer (TR-FRET), has been developed and applied for the measurement of protein kinase activity in vitro. This technology note describes the development of an HTRF® assay for detection of Syk enzyme activity in a format consistent with the requirements of High-Throughput Screening (HTS) campaigns currently used in drug discovery.
  • 18.

    【2hr】Cellular Assays for High-Throughput Screening for Modulators of Trk Receptor Tyrosine Kinases

    包量

    机译 Trk受体酪氨酸激酶调节剂的高通量筛选的细胞分析
    摘要：Trk receptor tyrosine kinases are required for signal transduction initiated by neurotrophins leading to cell proliferation, differentiation, survival and death. Alterations in Trk kinase activity have been linked to various diseases. To address the need for cell-based assays for screening and studying the selectivity of Trk kinase modulators, we developed high-throughput cell-based assays for Trk receptor kinases using nuclear factor of activated T-cells (NFAT) beta-lactamase reporter lines stably expressing full length human Trk kinases. These assays were functionally validated with cognate neurotrophin(s), inhibitors and TRK RNAi oligos and demonstrated for their utility in identifying potent and selective modulators of Trk receptor kinases.
  • 19.

    【2hr】Homogenous Fluorescent Assays for Characterizing Small-Molecule Activators of AMP-Activated Protein Kinase (AMPK)

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    机译 表征AMP活化蛋白激酶（AMPK）小分子活化剂的均相荧光测定
    摘要：AMP activated protein kinase (AMPK) is a key regulator of cellular metabolism. AMPK activity is modulated in part by binding of AMP to the γ-subunit of the kinase, which increases the activity of the catalytic α-subunit. Because increased AMPK activity in the liver and in skeletal muscle leads to increased fatty acid oxidation and decreased cholesterol and fatty acid biosynthesis, activators of AMPK are being sought for treatment of type-2 diabetes and other metabolic disorders. The unique mechanism of AMPK activation offers an opportunity to develop small molecules that directly upregulate AMPK activity, and there exists a need for simplified methods to identify and characterize small-molecules that show isoform-specific effects on AMPK. We have developed a suite of fluorescence-based assays to identify and characterize such compounds, and have used these to characterize and compare activity of recombinant AMPK α1β1γ1 and α2β1γ1 isoforms in response to small molecule activators and inhibitors.
  • 20.

    【2hr】Fluorescent Cascade and Direct Assays for Characterization of RAF Signaling Pathway Inhibitors

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    机译 荧光级联反应和RAF信号通路抑制剂的表征的直接测定。
    摘要：RAF kinases are part of a conserved signaling pathway that impacts cell growth, differentiation, and survival, and RAF pathway dysregulation is an attractive target for therapeutic intervention. We describe two homogeneous fluorescent formats that distinguish RAF pathway inhibitors from direct RAF kinase inhibitors, using B-RAF, B-RAF V599E, and C-RAF. A Förster-resonance energy transfer (FRET) based method was used to develop RAF and MEK cascade assays as well as a direct ERK kinase assay. This method uses a peptide substrate, that is terminally labeled with a FRET-pair of fluorophores, and that is more sensitive to proteolysis relative to the phosphorylated peptide. A second time-resolved FRET-based assay using fluorescently labeled MEK substrate was used to detect direct inhibitors of RAF kinase activity. The cascade assays detect compounds that interact with activated and unactivated kinases within the recapitulated RAF pathway, and the direct assays isolate the point of action for an inhibitor.
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