共查询到20条相似文献,搜索用时 31 毫秒
1.
Label-free cell-based functional assays 总被引:1,自引:0,他引:1
Minor LK 《Combinatorial chemistry & high throughput screening》2008,11(7):573-580
Label-free technologies based on electrical impedance or refractive index are new tools for measuring a cell-based functional response. Although the technologies are relatively new to high throughput screening cell-based applications, they are rapidly generating interest in that they are able to measure a phenotypic response using cells natively expressing the target protein without using dyes or cellular extracts. In addition, one can measure the cellular response using a kinetic mode resulting in an assay potentially rich in content. This article will describe these technologies and their applications in measuring cell proliferation, cell attachment and spreading, cell apoptosis and their application for several receptor target classes, including receptor tyrosine kinases and G protein-coupled receptors. The potential utility and drawbacks of these tools for high throughput screening, directed screening and compound profiling will also be discussed. 相似文献
2.
Methodologies for rapidly identifying cellular protein interactions resulting in posttranslational modification of one of the partners are lacking. Here, we select for substrates of the v-abl tyrosine kinase from two protein display libraries in which the protein is covalently linked to its encoding mRNA. Successive selection cycles from a randomized peptide library identified a consensus sequence closely matching that previously reported for the v-abl tyrosine kinase. Selections from a proteomic library derived from cellular mRNA identified several novel targets of v-abl, including a new member of a class of SH2 domain-containing adaptor proteins. Upon modification, several of the substrates obtained in these selections were found to be effective inhibitors of v-abl kinase activity in vitro. These experiments establish a novel method for identifying the substrates of tyrosine kinases from synthetic and cellular protein libraries. 相似文献
3.
Videlock EJ Chung VK Hall JM Hines J Agapakis CM Austin DJ 《Journal of the American Chemical Society》2005,127(6):1600-1601
A human cDNA phage display library screen, using a phosphopeptide designed to mimic the activation loop phosphotyrosine of the Src tyrosine kinase, has identified the N-terminal SH2 domain of the p85 regulatory subunit of phosphatidyl inositol-3 kinase (PI3K) as an interacting recognition domain. Activation loop phosphorylation is known to play a conformational role in kinase activation, but is largely not thought to play a role in protein/protein recognition. Affinity chromatography and biochemical evaluation in mouse fibroblast cells has confirmed the dependence of this interaction on both the Src activation loop phosphotyrosine and the N-terminal SH2 domain of PI3K. 相似文献
4.
Although the cell-based assay is becoming more popular for high throughput drug screening and the functional characterization of disease-associated genes, most researchers in these areas do not use it because it is a complex and expensive process. We wanted to create a simple method of performing an on-chip cell-based assay. To do this, we used micro-electro-mechanical systems (MEMS) to fabricate a microwell array chip comprised of a glass substrate covered with a photoresist film patterned to form multiple microwells and tested it in two reverse transfection experiments, an exogenous gene expression study and an endogenous gene knockdown study. It was used effectively in both. Then, using the same MEMS technology, we fabricated a complementary microcolumn array to be used as a drug carrier device to topically apply drugs to cells cultured in the microwell array. We tested the effectiveness of microwell-microcolumn on-chip cell-based assay by using it in experiments to identify epidermal growth factor receptor (EGFR) activity inhibitors, for which it was found to provide effective high throughput and high content functional screening. In conclusion, this new method of cell-based screening proved to be a simple and efficient method of characterizing gene function and discovering drug leads. 相似文献
5.
Y Liu A Bishop L Witucki B Kraybill E Shimizu J Tsien J Ubersax J Blethrow D O Morgan K M Shokat 《Chemistry & biology》1999,6(9):671-678
BACKGROUND: Small-molecule inhibitors that can target individual kinases are powerful tools for use in signal transduction research. It is difficult to find such compounds because of the enormous number of protein kinases and the highly conserved nature of their catalytic domains. Recently, a novel, potent, Src family selective tyrosine kinase inhibitor was reported (PP1). Here, we study the structural basis for this inhibitor's specificity for Src family kinases. RESULTS: A single residue corresponding to Ile338 (v-Src numbering; Thr338 in c-Src) in Src family tyrosine kinases largely controls PP1's ability to inhibit protein kinases. Mutation of Ile338 to a larger residue such as methionine or phenylalanine in v-Src makes this inhibitor less potent. Conversely, mutation of Ile338 to alanine or glycine increases PP1's potency. PP1 can inhibit Ser/Thr kinases if the residue corresponding to Ile338 in v-Src is mutated to glycine. We have accurately predicted several non-Src family kinases that are moderately (IC(50) approximately 1 microM) inhibited by PP1, including c-Abl and the MAP kinase p38. CONCLUSIONS: Our mutagenesis studies of the ATP-binding site in both tyrosine kinases and Ser/Thr kinases explain why PP1 is a specific inhibitor of Src family tyrosine kinases. Determination of the structural basis of inhibitor specificity will aid in the design of more potent and more selective protein kinase inhibitors. The ability to desensitize a particular kinase to PP1 inhibition of residue 338 or conversely to sensitize a kinase to PP1 inhibition by mutation should provide a useful basis for chemical genetic studies of kinase signal transduction. 相似文献
6.
Protein tyrosine phosphatases, SH2 and PTB domains are crucial elements for cellular signal transduction and regulation. Much effort has been directed towards elucidating their specificity in the past decade using a variety of approaches. Combinatorial library methods have contributed significantly to the understanding of substrate and ligand specificity of phosphoprotein recognizing domains. This review gives a brief overview of the structural characteristics of protein tyrosine phosphatases, SH2 and PTB domains and their binding to phosphopeptides. The chemical synthesis of peptides containing phosphotyrosine or phosphotyrosine mimics and the various formats of synthesis and deconvolution of combinatorial libraries are explained in detail. Examples are given as how different combinatorial libraries have been used to study the interaction of phosphopeptides with SH2 domains and phosphatases. The intrinsic advantages and difficulties of library synthesis, screening and deconvolution are pointed out. Finally, some experimental results on the substrate specificity of protein tyrosine phosphatase 1B and the SH2 domain of the adaptor protein Grb-2 are summarized and discussed. 相似文献
7.
8.
Shin KS Shin EY Lee CS Quan SH Woo KN Soung NK Kwak SJ Kim SR Kim EG 《Experimental & molecular medicine》2002,34(2):172-176
p21-activated kinase (PAK) targeting to the plasma membrane is essential for PC12 cell neurite outgrowth. Phospholipase C-gamma1 (PLC-gamma1) can mediate the PAK translocation in response to growth factors, since PLC-gamma1 binds to both tyrosine-phosphorylated receptor tyrosine kinases and PAK through its SH2 and SH3 domain, respectively. In the present study, we examined a potential role for PLC-gamma1 in the basic fibroblast growth factor (bFGF)-induced PAK translocation using stable PC12 cell lines that overexpress in a tetracycline-inducible manner either the wild-type FGFR-1 or the Y766F FGFR-1 mutant. Phosphatidylinositol hydrolysis was increased 6.5-fold in response to bFGF in the wild type cells but negligible in the mutant cells. The recombinant GST-PLC-gamma1 SH3 was able to bind to PAK1 but not GST alone. However, examination of PLC-gamma1 as an adaptor for translocation of PAK1 in cells showed that both cells transfected with pEGFP-PAK1 was able to differentiate for 24 h, as visualized by laser confocal microscopy. Translocation of PAK1 to growth cones occurs at similar levels in both wild and mutant cells. These results suggest that a protein(s) other than PLC-gamma1 is functionally relevant for PAK targeting. 相似文献
9.
Rokosz LL Huang CY Reader JC Stauffer TM Southwick EC Li G Chelsky D Baldwin JJ 《Combinatorial chemistry & high throughput screening》2006,9(7):545-558
The development of structure-activity relationships (SARs) relating to the function of a biological protein is often a long and protracted undertaking when using an iterative medicinal chemistry approach. High throughput screening of ECLiPS (Encoded Combinatorial Libraries on Polymeric Support) libraries can be used to simplify this process. In this paper, we illustrate how a large ECLiPS library of 26,908 compounds, based on a tricyclic core structure, was used to define a multitude of SARs for the oncogenic target, farnesyltransferase (FTase). This library, FT-2, was prepared using a split-and-pool approach in which small molecules are constructed on resin that contains tag/linker constructs to track the synthetic process [1-5] Highly defined SARs were produced from this screen that enhanced our understanding of FTase binding site interactions. The pivotal compounds culled from this library were potent in both cell-free and cell-based FTase assays, selective over the closely related enzyme, geranylgeranyltransferase I (GGTase I), and inhibited the adherent-independent growth of a transformed cell line. 相似文献
10.
Using an ATP analog that is a specific substrate for an analog-specific allele of v-Src, we identified several novel cytoskeletal substrates that control actin assembly processes. A screen for less abundant v-Src substrates revealed the scaffolding protein Dok-1 as a direct substrate of v-Src. Further studies suggest that v-Src phosphorylation sites on Dok-1 are critical for its binding to RasGAP and Csk, negative regulators of Src signaling. This results in the downregulation of growth-promoting signals of the Src family kinases and the Ras pathway. Identification of the direct substrates of v-Src leads to a model for the precise order of assembly of a retrograde signaling pathway in v-Src-transformed cells and has provided new insight into the balance between those signals that promote cell transformation mediated by v-Src catalyzed tyrosine phosphorylation and those that inhibit it. 相似文献
11.
Suda M Ishii T Sootome H King AJ Shibahara M Noro N Yamashita K Noumi T 《Analytical and bioanalytical chemistry》2008,390(1):343-348
No universal approach has been reported for specific monitoring of the catalytic activity of a wide range of kinases in cells.
The present study describes an original platform for detecting the autonomous activity of serine/threonine kinases in cells
through the introduction of expression vectors encoding modified substrate kinase fusion proteins. The surrogate substrate
used consists of the p53 tumor suppressor protein fused with individual kinase domains (Chk1, DYRK3, and Cdk5) at its carboxy-terminal
through four tandem Gly-Gly-Gly-Gly-Ser repeats. After transfection into cells, phosphorylation of the p53 moiety could be
specifically induced by the catalytic activity of kinases contained in the fusion protein. Moreover, p53 phosphorylation was
significantly blocked when a kinase-inactive mutant was used as the fusion partner instead of the active kinase. Using this
system, the cell-based evaluation of several Cdk5 inhibitors was demonstrated. Thus, this approach provides a novel platform
for the specific, cell-based screening of inhibitors of a wide prospective range of protein kinases and is of tremendous potential
for drug discovery efforts. 相似文献
12.
Fang YQ Wong WQ Yap YW Orner BP 《Combinatorial chemistry & high throughput screening》2007,10(8):635-651
Stem cell-based technologies have the potential to help cure a number of cell degenerative diseases. Combinatorial and high throughput screening techniques could provide tools to control and manipulate the self-renewal and differentiation of stem cells. This review chronicles historic and recent progress in the stem cell field involving both pluripotent and multipotent cells, and it highlights relevant cellular signal transduction pathways. This review further describes screens using libraries of soluble, small-molecule ligands, and arrays of molecules immobilized onto surfaces while proposing future trends in similar studies. It is hoped that by reviewing both the stem cell and the relevant high throughput screening literature, this paper can act as a resource to the combinatorial science community. 相似文献
13.
Viral proteins of gamma-2 herpesviruses, such as LMP2A of Epstein Barr virus (EBV) and Tip of herpesvirus saimiri (HVS) dysregulate lymphocyte signaling by interacting with Src family kinases. K15 open reading frame of Kaposi's sarcoma associated herpesvirus (KSHV), located at the right end of the viral genome, encodes several splicing variants differing in numbers of transmembrane domains. Previously, we demonstrated that the cytoplasmic tail of the K15 protein interfered with B cell receptor signal transduction to cellular tyrosine phosphorylation and calcium mobilization. However, the detailed mechanism underlying this phenomenon was not understood. In the C-terminal cytoplasmic region of K15, putative binding domains for Src-SH2 and -SH3 were identified. In this study, we attempted to characterize these modular elements and cellular binding protein(s) by GST pull down and co-immunoprecipitation assays. These studies revealed that K15 interacted with the major B cell tyrosine kinase Lyn. In vitro kinase and transient co-expression assays showed that the expression of K15 protein resulted in activation of Lyn kinase activity. In addition, GST pull down assay suggested that the SH2 domain of Lyn alone was necessary for interaction with the C-terminal SH2B (YEEV) of K15, but the addition of Lyn SH3 to the SH2 domain increases the binding affinity to K15 protein. The data from luciferase assays indicate that K15 expression in BJAB cells induced NFAT and AP1 activities. The tyrosine residue in the C-terminal end of K15 required for the Lyn interaction appeared to be essential for NFAT/AP1 activation, highlighting the significance of the C-terminal SH2B of K15 as a modular element in interfering with B lymphocyte signaling through interaction with Lyn kinase. 相似文献
14.
The use of carbohydrate microarrays to study carbohydrate-cell interactions and to detect pathogens 总被引:3,自引:0,他引:3
The use of carbohydrate microarrays to investigate the carbohydrate binding specificities of bacteria, to detect pathogens, and to screen antiadhesion therapeutics is reported. This system is ideal for whole-cell applications because microarrays present carbohydrate ligands in a manner that mimics interactions at cell-cell interfaces. Other advantages include assay miniaturization, since minimal amounts (approximately picomoles) of a ligand are required to observe binding, and high throughput, since thousands of compounds can be placed on an array and analyzed in parallel. Pathogen detection experiments can be completed in complex mixtures of cells or protein using the known carbohydrate binding epitopes of the pathogens in question. The nondestructive nature of the arrays allows the pathogen to be harvested and tested for antibacterial susceptibility. These investigations allow microarray-based screening of biological samples for contaminants and combinatorial libraries for antiadhesion therapeutics. 相似文献
15.
Transfection of siRNA and plasmid nucleic molecules to animal, microbial and plant cell cultures is a critical process in various research areas, including drug discovery, functional genomics and basic life science research. Till recent times, transfection of these exogenous molecules have been global in nature i.e. targeting all the cells in a culture and lacking capability to spatially confine the transfection to small populations of cells within a single culture. However, in emerging areas like high-throughput screening of large molecule libraries, there is a critical need to transfect multiple different molecules to locally specified regions of a single cell culture and monitor phenotypical changes in these different cell populations. In this study, we present a cell-based biochip that utilizes a microelectrode array to generate localized current density fields that induce electroporation to a targeted group of cells for site-specific transfection of exogenous molecules. More specifically, we optimize the transfection efficiency and viabilities for spatially controlled transfection of Alexa-Fluor-488 conjugated siRNA molecules into NIH3T3 fibroblast cell cultures. Optimal electroporation parameters are established at current density values ranging between 0.05-0.07 microA microm(-2) for high transfection efficiencies (>60%) while maintaining viability (>80%) on individual microelectrodes. Additionally, exogenous plasmid molecules are electroporated for site-specific GFP expression and monitored over 48 h in-situ. The microelectrode array technology reported here can therefore be potentially used for targeting specific cells in a culture with spatial precision and transfecting siRNA and plasmids. The microfabrication approach lends itself to significant high-throughput applications in drug-discovery research. 相似文献
16.
17.
The discovery/development of novel drug candidates has witnessed dramatic changes over the last two decades. Old methods to identify lead compounds are not suitable to screen wide libraries generated by combinatorial chemistry techniques. High throughput screening (HTS) has become irreplaceable and hundreds of different approaches have been described. Assays based on purified components are flanked by whole cell-based assays, in which reporter genes are used to monitor, directly or indirectly, the influence of a chemical over the metabolism of living cells. The most convenient and widely used reporters for real-time measurements are luciferases, light emitting enzymes from evolutionarily distant organisms. Autofluorescent proteins have been also extensively employed, but proved to be more suitable for end-point measurements, in situ applications - such as the localization of fusion proteins in specific subcellular compartments - or environmental studies on microbial populations. The trend toward miniaturization and the technical advances in detection and liquid handling systems will allow to reach an ultra high throughput screening (uHTS), with 100,000 of compounds routinely screened each day. Here we show how similar approaches may be applied also to the search for new and potent antimicrobial agents. 相似文献
18.
Joseph A. Loo Peifeng Hu Patrick McConnell W. Tom Mueller Tomi K. Sawyer Venkataraman Thanabal 《Journal of the American Society for Mass Spectrometry》1997,8(3):234-243
The noncovalent binding of various peptide ligands to pp60src (Src) SH2 (Src homology 2) domain protein (12.9 ku) has been used as a model system for development of electrospray ionization mass spectrometry (ESI-MS) as a tool to study noncovalently bound complexes. SH2 motifs in proteins are critical in the signal transduction pathways of the tyrosine kinase growth factor receptors and recognize phosphotyrosine-containing proteins and peptides. ESI-MS with a magnetic sector instrument and array detection has been used to detect the protein-peptide complex with low-picomole sensitivity. The relative abundances of the multiply charged ions for the complex formed between Src SH2 protein and several nonphosphorylated and phosphorylated peptides have been compared. The mass spectrometry data correlate well to the measured binding constants derived from solution-based methods, indicating that the mass spectrometry-based method can be used to assess the affinity of such interactions. Solution-phase equilibrium constants may be determined by measuring the amount of bound and unbound species as a function of concentration for construction of a Scatchard graph. ESI-MS of a solution containing Src SH2 with a mixture of phosphopeptides showed the expected protein-phosphopeptide complex as the dominant species in the mass spectrum, demonstrating the method’s potential for screening mixtures from peptide libraries. 相似文献
19.
20.
Spleen tyrosine kinase (Syk) is essential for high affinity IgE receptor (FcεRI) mediated mast cell degranulation. Once FcεRI is stimulated, intracellular ITAM motifs of the receptor are diphosphorylated (dpITAM) and Syk is recruited to the receptor by binding of the Syk tandem SH2 domain to dpITAM, resulting in activation of Syk and, eventually, degranulation. To investigate intracellular effects of ITAM mimics, constructs were synthesized with ITAM mimics conjugated to different cell penetrating peptides, i.e. Tat, TP10, octa-Arg and K(Myr)KKK, or a lipophilic C(12)-chain. In most constructs the cargo and carrier were linked to each other through a disulfide bridge, which is convenient for combining different cargos with different carriers and has the advantage that the cargo and the carrier may be separated by reduction of the disulfide once it is intracellular. The ability of these ITAM constructs to label RBL-2H3 cells was assessed using flow cytometry. Fluorescence microscopy showed that the octa-Arg-SS-Flu-ITAM construct was present in various parts of the cells, although it was not homogeneously distributed. In addition, cell penetrating constructs without fluorescent labels were synthesized to examine degranulation in RBL-2H3 cells. Octa-Arg-SS-ITAM stimulated the mediator release up to 140%, indicating that ITAM mimics may have the ability to activate non-receptor bound Syk. 相似文献