Current chemical genomics最新文献

{"title":"Homogenous fluorescent assays for characterizing small-molecule activators of AMP-activated protein kinase (AMPK).","authors":"Laurie J Reichling,&nbsp;Steven M Riddle,&nbsp;Baigen Mei,&nbsp;Rica Bruinsma,&nbsp;Tony A Goossens,&nbsp;Kristin G Huwiler,&nbsp;Mark Maffitt,&nbsp;Alyssa M G Newport,&nbsp;Xiao-Dong Qian,&nbsp;Carmen Ruttimann-Johnson,&nbsp;Kurt W Vogel","doi":"10.2174/1875397300801010034","DOIUrl":"https://doi.org/10.2174/1875397300801010034","url":null,"abstract":"<p><p>AMP activated protein kinase (AMPK) is a key regulator of cellular metabolism. AMPK activity is modulated in part by binding of AMP to the gamma-subunit of the kinase, which increases the activity of the catalytic alpha-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 alpha(1)beta(1)gamma(1) and alpha(2)beta(1)gamma(1) isoforms in response to small molecule activators and inhibitors.</p>","PeriodicalId":88232,"journal":{"name":"Current chemical genomics","volume":"1 ","pages":"34-42"},"PeriodicalIF":0.0,"publicationDate":"2008-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2774621/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28717999","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The use of AlphaScreen technology in HTS: current status.","authors":"Richard M Eglen,&nbsp;Terry Reisine,&nbsp;Philippe Roby,&nbsp;Nathalie Rouleau,&nbsp;Chantal Illy,&nbsp;Roger Bossé,&nbsp;Martina Bielefeld","doi":"10.2174/1875397300801010002","DOIUrl":"https://doi.org/10.2174/1875397300801010002","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":88232,"journal":{"name":"Current chemical genomics","volume":"1 ","pages":"2-10"},"PeriodicalIF":0.0,"publicationDate":"2008-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.2174/1875397300801010002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28717994","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Multiplexing bioluminescent and fluorescent reporters to monitor live cells.","authors":"Michael Haugwitz,&nbsp;Omar Nourzaie,&nbsp;Tatiana Garachtchenko,&nbsp;Lanrong Hu,&nbsp;Suvarna Gandlur,&nbsp;Cathy Olsen,&nbsp;Andrew Farmer,&nbsp;Grigoriy Chaga,&nbsp;Hiroaki Sagawa","doi":"10.2174/1875397300801010011","DOIUrl":"https://doi.org/10.2174/1875397300801010011","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":88232,"journal":{"name":"Current chemical genomics","volume":"1 ","pages":"11-9"},"PeriodicalIF":0.0,"publicationDate":"2008-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2774656/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28717996","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cellular assays for high-throughput screening for modulators of Trk receptor tyrosine kinases.","authors":"Jun Wang,&nbsp;Michael K Hancock,&nbsp;Jeanne M Dudek,&nbsp;Kun Bi","doi":"10.2174/1875397300801010027","DOIUrl":"https://doi.org/10.2174/1875397300801010027","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":88232,"journal":{"name":"Current chemical genomics","volume":"1 ","pages":"27-33"},"PeriodicalIF":0.0,"publicationDate":"2008-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2774616/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28717998","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fluorescent cascade and direct assays for characterization of RAF signaling pathway inhibitors.","authors":"Kevin R Kupcho,&nbsp;Rica Bruinsma,&nbsp;Tina M Hallis,&nbsp;David A Lasky,&nbsp;Richard L Somberg,&nbsp;Tammy Turek-Etienne,&nbsp;Kurt W Vogel,&nbsp;Kristin G Huwiler","doi":"10.2174/1875397300801010043","DOIUrl":"https://doi.org/10.2174/1875397300801010043","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":88232,"journal":{"name":"Current chemical genomics","volume":"1 ","pages":"43-53"},"PeriodicalIF":0.0,"publicationDate":"2008-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2774691/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28717912","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Editorial.","authors":"Wei Zheng","doi":"10.2174/1875397300801010001","DOIUrl":"https://doi.org/10.2174/1875397300801010001","url":null,"abstract":"EDITORIAL We are pleased to launch this inaugural issue of Current Chemical Genomics. As an open access online journal, all the papers from this journal can be read free of charge by our readers without any restrictions to access of the full content. This peerreviewed journal publishes research letters/articles, reviews, and technology notes on all aspects of the research and development focusing on integrative approaches at the interface of chemistry and genomics. The journal reports the newly identified small molecule probes and their applications in chemical genomic research. It also reports on the methods and technologies used to identify and optimize chemical probes that interact with proteins of unknown function and with proteins of cellular signaling pathways. Coverage also includes reports on the new chemoinformatics methods for chemical genomics. We hope that these approaches will facilitate the identification of new drug targets and the development of novel therapeutic strategies leading to a new generation of medicines.","PeriodicalId":88232,"journal":{"name":"Current chemical genomics","volume":"1 ","pages":"1"},"PeriodicalIF":0.0,"publicationDate":"2008-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2774618/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28717993","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Glutamatergic Synaptic Dysfunction and Obsessive-Compulsive Disorder.","authors":"Jonathan T Ting, Guoping Feng","doi":"10.2174/1875397300802010062","DOIUrl":"10.2174/1875397300802010062","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":88232,"journal":{"name":"Current chemical genomics","volume":"2 ","pages":"62-75"},"PeriodicalIF":0.0,"publicationDate":"2008-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/d2/78/TOCHGENJ-2-62.PMC2746669.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28414520","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}