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AlphaScreen assay

John R Veloria, Ashwini K Devkota, Eun Jeong Cho, Kevin N Dalby
The use of AlphaScreen® detection has allowed researchers to examine a wide variety of molecular interactions for use in high-throughput screening. However, the cost of Alpha reagents can often be prohibitory for extended screening campaigns or for young investigators with limited funding. To reduce assay costs, many labs have focused on miniaturization, while there have been limited efforts to scale down Alpha reagents. Thus, we describe the optimization of an AlphaScreen detection platform by systematically reducing the Alpha reagents down to 2...
April 2018: BioTechniques
Murugendra Vanarotti, Benjamin J Evison, Marcelo L Actis, Akira Inoue, Ezelle T McDonald, Youming Shao, Richard J Heath, Naoaki Fujii
REV1 protein is a mutagenic DNA damage tolerance (DDT) mediator and encodes two ubiquitin-binding motifs (i.e., UBM1 and UBM2) that are essential for the DDT function. REV1 interacts with K164-monoubiquitinated PCNA (UbPCNA) in cells upon DNA-damaging stress. By using AlphaScreen assays to detect inhibition of REV1 and UbPCNA protein interactions along with an NMR-based strategy, we identified small-molecule compounds that inhibit the REV1/UbPCNA interaction and that directly bind to REV1 UBM2. In cells, one of the compound prevented recruitment of REV1 to PCNA foci on chromatin upon cisplatin treatment, delayed removal of UV-induced cyclopyrimidine dimers from nuclei, prevented UV-induced mutation of HPRT gene, and diminished clonogenic survival of cells that were challenged by cyclophosphamide or cisplatin...
March 19, 2018: Bioorganic & Medicinal Chemistry
Martin Winter, Tom Bretschneider, Carola Kleiner, Robert Ries, Jörg P Hehn, Norbert Redemann, Andreas H Luippold, Daniel Bischoff, Frank H Büttner
Label-free, mass spectrometric (MS) detection is an emerging technology in the field of drug discovery. Unbiased deciphering of enzymatic reactions is a proficient advantage over conventional label-based readouts suffering from compound interference and intricate generation of tailored signal mediators. Significant evolvements of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, as well as associated liquid handling instrumentation, triggered extensive efforts in the drug discovery community to integrate the comprehensive MS readout into the high-throughput screening (HTS) portfolio...
February 1, 2018: SLAS Discovery
Bruna Corominas-Faja, Elisabet Cuyàs, Jesús Lozano-Sánchez, Sílvia Cufí, Sara Verdura, Salvador Fernández-Arroyo, Isabel Borrás-Linares, Begoña Martin-Castillo, Ángel G Martin, Ruth Lupu, Alfons Nonell-Canals, Melchor Sanchez-Martinez, Vicente Micol, Jorge Joven, Antonio Segura-Carretero, Javier A Menendez
Targeting tumor-initiating, drug-resistant populations of cancer stem cells (CSC) with phytochemicals is a novel paradigm for cancer prevention and treatment. We herein employed a phenotypic drug discovery approach coupled to mechanism-of-action profiling and target deconvolution to identify phenolic components of extra virgin olive oil (EVOO) capable of suppressing the functional traits of CSC in breast cancer (BC). In vitro screening revealed that the secoiridoid decarboxymethyl oleuropein aglycone (DOA) could selectively target sub-populations of epithelial-like, aldehyde dehydrogenase (ALDH)-positive and mesenchymal-like, CD44+CD24-/low CSC...
February 14, 2018: Carcinogenesis
Yan Yan, Xin Gu, H Eric Xu, Karsten Melcher
While many methods exist to quantitatively determine protein kinase activities,32 P-based radioactive assays remain the workhorse of many laboratories due to their high sensitivity, high signal to noise ratio, lack of interference by fluorescent and light-absorbing small molecules, and easy quantitation. Here, we demonstrate that the interaction between the yeast Rad53 Forkhead-associated (FHA) domain and a peptide optimized for phosphorylation by AMP-Activated Protein Kinase (AMPK), which has previously been exploited for the generation of intracellular phosphorylation sensors, can serve as a readout for a highly sensitive two-step AMPK AlphaScreen kinase assay with exceptional signal-to-noise ratio...
March 2018: Methods and protocols
Qiuping Xiang, Chao Wang, Yan Zhang, Xiaoqian Xue, Ming Song, Cheng Zhang, Chenchang Li, Chun Wu, Kuai Li, Xiaoyan Hui, Yulai Zhou, Jeff B Smaill, Adam V Patterson, Donghai Wu, Ke Ding, Yong Xu
The CREB (cAMP responsive element binding protein) binding protein (CBP) and its homolog EP300 have emerged as new therapeutic targets for the treatment of cancer and inflammatory diseases. Here we report the identification, optimization and evaluation of 1-(1H-indol-1-yl)ethanone derivatives as CBP/EP300 inhibitors starting from fragment-based virtual screening (FBVS). A cocrystal structure of the inhibitor (22e) in complex with CBP provides a solid structural basis for further optimization. The most potent compound 32h binds to the CBP bromodomain and has an IC 50 value of 0...
February 6, 2018: European Journal of Medicinal Chemistry
Jian Chen, Diana Bellin, Elodie Vandelle
Cyclic guanosine-3',5'-monophosphate (cGMP) is recognized as an important second messenger in plants, mediating intracellular signal in important physiological processes, including the hypersensitive disease resistance response induced by avirulent pathogens. In this context, the analysis of cGMP levels in infected plants requires an accurate and specific detection method allowing its quantification. Here, we describe an assay based on the Alphascreen technology, developed for animal cells and further adapted and optimized for the detection of cGMP in plants...
2018: Methods in Molecular Biology
Torun Ekblad, Patricia Verheugd, Anders E Lindgren, Tomas Nyman, Mikael Elofsson, Herwig Schüler
Macrodomains recognize intracellular adenosine diphosphate (ADP)-ribosylation resulting in either removal of the modification or a protein interaction event. Research into compounds that modulate macrodomain functions could make important contributions. We investigated the interactions of all seven individual macrodomains of the human poly(ADP-ribose) polymerase (PARP) family members PARP9, PARP14, and PARP15 with five mono-ADP-ribosylated (automodified) ADP-ribosyltransferase domains using an AlphaScreen assay...
April 2018: SLAS Discovery
Xiayun Huang, Tannin A Schmidt, Claire Shortt, Shivani Arora, Akira Asari, Thorsten Kirsch, Mary K Cowman
A method for specific quantification of hyaluronan (HA) concentration using AlphaScreen® (Amplified Luminescent Proximity Homogeneous Assay) technology is described. Two types of hydrogel-coated and chromophore-loaded latex nanobeads are employed. The proximity of the beads in solution is detected by excitation of the donor bead leading to the production of singlet oxygen, and chemiluminescence from the acceptor bead upon exposure to singlet oxygen. In the HA assay, the donor bead is modified with streptavidin, and binds biotin-labeled HA...
March 1, 2018: Glycobiology
Stefania Gessi, Serena Bencivenni, Enrica Battistello, Fabrizio Vincenzi, Vittoria Colotta, Daniela Catarzi, Flavia Varano, Stefania Merighi, Pier Andrea Borea, Katia Varani
Several evidences indicate that the ubiquitous nucleoside adenosine, acting through A1 , A2A , A2B , and A3 receptor (AR) subtypes, plays crucial roles in tumor development. Adenosine has contrasting effects on cell proliferation depending on the engagement of different receptor subtypes in various tumors. The involvement of A2A ARs in human A375 melanoma, as well as in human A549 lung and rat MRMT1 breast carcinoma proliferation has been evaluated in view of the availability of a novel A2A AR antagonist, with high affinity and selectivity, named as 2-(2-furanyl)-N5 -(2-methoxybenzyl)[1,3]thiazolo[5,4-d]pyrimidine-5,7-diammine (TP455)...
2017: Frontiers in Pharmacology
Yi Lu, Weili Zheng, Shengchen Lin, Fusheng Guo, Yanlin Zhu, Yijuan Wei, Xi Liu, Shikai Jin, Lihua Jin, Yong Li
Farnesoid X receptor (FXR) and G-protein-coupled bile acid receptor 1 (GPBAR1) are two important bile acid (BA) receptors. As non-BAs drug template for GPBAR1, none of the natural oleanane-type triterpenes have been reported as FXR ligands, despite FXR and GPBAR1 having similar binding pockets for BAs. Here, we report the natural triterpene hedragonic acid that has been isolated from the stem and root of Celastrus orbiculatus Thunb. (COT) as an effective agonist for FXR. Both biochemical amplified luminescent proximity homogeneous assay and cell-based reporter assays showed that hedragonic acid regulated the transcriptional activity of FXR...
February 2018: Molecular Pharmacology
De-Si Pan, Wei Wang, Nan-Song Liu, Qian-Jiao Yang, Kun Zhang, Jing-Zhong Zhu, Song Shan, Zhi-Bin Li, Zhi-Qiang Ning, Laiqiang Huang, Xian-Ping Lu
Type 2 diabetes mellitus is often treated with insulin-sensitizing drugs called thiazolidinediones (TZD), which improve insulin resistance and glycemic control. Despite their effectiveness in treating diabetes, these drugs provide little protection from eminent cardiovascular disease associated with diabetes. Here we demonstrate how chiglitazar, a configuration-restricted non-TZD peroxisome proliferator-activated receptor (PPAR) pan agonist with moderate transcription activity, preferentially regulates ANGPTL4 and PDK4 , which are involved in glucose and lipid metabolism...
2017: PPAR Research
Teemu Haikarainen, Mirko M Maksimainen, Ezeogo Obaji, Lari Lehtiö
Protein mono-ADP-ribosylation is a posttranslational modification involved in the regulation of several cellular signaling pathways. Cellular ADP-ribosylation is regulated by ADP-ribose hydrolases via a hydrolysis of the protein-linked ADP-ribose. Most of the ADP-ribose hydrolases share a macrodomain fold. Macrodomains have been linked to several diseases, such as cancer, but their cellular roles are mostly unknown. Currently, there are no inhibitors available targeting the mono-ADP-ribose hydrolyzing macrodomains...
October 1, 2017: SLAS Discovery
Yukako Senga, Hiroshi Imamura, Takamitsu Miyafusa, Hideki Watanabe, Shinya Honda
Therapeutic IgG becomes unstable under various stresses in the manufacturing process. The resulting non-native IgG molecules tend to associate with each other and form aggregates. Because such aggregates not only decrease the pharmacological effect but also become a potential risk factor for immunogenicity, rapid analysis of aggregation is required for quality control of therapeutic IgG. In this study, we developed a homogeneous assay using AlphaScreen and AF.2A1. AF.2A1 is a 25-residue artificial protein that binds specifically to non-native IgG generated under chemical and physical stresses...
September 29, 2017: Scientific Reports
Stanleyson V Hato, Carl G Figdor, Susumu Takahashi, Anja E Pen, Altuna Halilovic, Kalijn F Bol, Angela Vasaturo, Yukie Inoue, Nienke de Haas, Dagmar Verweij, Carla M L Van Herpen, Johannes H Kaanders, Johan H J M van Krieken, Hanneke W M Van Laarhoven, Gerrit K J Hooijer, Cornelis J A Punt, Akira Asai, I Jolanda M de Vries, W Joost Lesterhuis
Platinum-based chemotherapeutics are amongst the most powerful anti-cancer drugs. Although their exact mechanism of action is not well understood, it is thought to be mediated through covalent DNA binding. We investigated the effect of platinum-based chemotherapeutics on signaling through signal transducer and activator of transcription (STAT) proteins, which are involved in many oncogenic signaling pathways. We performed in vitro experiments in various cancer cell lines, investigating the effects of platinum chemotherapeutics on STAT phosphorylation and nuclear translocation, the expression of STAT-modulating proteins and downstream signaling pathways...
August 15, 2017: Oncotarget
Wei Yang, Søren Berg Padkjær, Jishu Wang, Zhe Sun, Bing Shan, Li Yang, Haibin Chen, Lishan Kang, Dennis Madsen, Xun Li, Chenxi Shen, Bingke Yu, Haisun Zhu, Tzu-Yuan Chao, Zhuoxiao Cao, Dapeng Li, Wei Liu, Yanping Du, Jinjing Xu, Dongxia Hao, Fengting Xu, Lujia Peng, Tengkun Li, Lin Wang, Lin Li, Haimei Xing, Di Liu, Zibing Liu, Zhishuang Guan, Wan Wang, Hong Cheng, Henrik Østergaard, Chihchuan Chang, Zhiru Yang, Esper Boel, Jing Su
Interactions between protein ligands and receptors play crucial roles in cell-cell signalling. Most of the human cell surface receptors have been identified in the post-Human Genome Project era but many of their corresponding ligands remain unknown. To facilitate the pairing of orphan receptors, 2762 sequences encoding all human single-pass transmembrane proteins were selected for inclusion into a mammalian-cell expression library. This expression library, consisting of all the individual extracellular domains (ECDs), was constructed as a Fab fusion for each protein...
September 1, 2017: Journal of Biotechnology
Chad J Miller, Yagmur Muftuoglu, Benjamin E Turk
Extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylate a variety of substrates important for survival and proliferation, and their activity is frequently deregulated in tumors. ERK pathway inhibitors have shown clinical efficacy as anti-cancer drugs, but most patients eventually relapse due to reactivation of the pathway. One factor limiting the efficacy of current therapeutics is the difficulty in reaching clinically effective inhibition of the ERK pathway in the absence of on-target toxicities...
October 15, 2017: Biochemical Pharmacology
Peng Huang, Hideki Yoshida, Kenji Yano, Shunsuke Kinoshita, Kyosuke Kawai, Eriko Koketsu, Masako Hattori, Sayaka Takehara, Ji Huang, Ko Hirano, Reynante Lacsamana Ordonio, Makoto Matsuoka, Miyako Ueguchi-Tanaka
Previously, we found 123 transcription factors (TFs) as candidate regulators of secondary cell wall (SCW) formation in rice by using phylogenetic and co-expression network analyses. Among them, we examined in this work the role of OsIDD2, a zinc finger and indeterminate domain (IDD) family TF. Its overexpressors showed dwarfism, fragile leaves, and decreased lignin content, which are typical phenotypes of plants defective in SCW formation, whereas its knockout plants showed slightly increased lignin content...
June 2, 2017: Journal of Integrative Plant Biology
Andrew F Voter, Michael P Killoran, Gene E Ananiev, Scott A Wildman, F Michael Hoffmann, James L Keck
Antibiotic-resistant bacterial infections are increasingly prevalent worldwide, and there is an urgent need for novel classes of antibiotics capable of overcoming existing resistance mechanisms. One potential antibiotic target is the bacterial single-stranded DNA binding protein (SSB), which serves as a hub for DNA repair, recombination, and replication. Eight highly conserved residues at the C-terminus of SSB use direct protein-protein interactions (PPIs) to recruit more than a dozen important genome maintenance proteins to single-stranded DNA...
January 2018: SLAS Discovery
Lies Schoonaert, Laura Rué, Bart Roucourt, Mieke Timmers, Susan Little, Lucía Chávez-Gutiérrez, Maarten Dewilde, Peter Joyce, Adam Curnock, Peter Weber, Jurgen Haustraete, Gholamreza Hassanzadeh-Ghassabeh, Bart De Strooper, Ludo Van Den Bosch, Philip Van Damme, Robin Lemmens, Wim Robberecht
The ephrin receptor A4 (EphA4) is one of the receptors in the ephrin system that plays a pivotal role in a variety of cell-cell interactions, mostly studied during development. In addition, EphA4 has been found to play a role in cancer biology as well as in the pathogenesis of several neurological disorders such as stroke, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease. Pharmacological blocking of EphA4 has been suggested to be a therapeutic strategy for these disorders...
July 7, 2017: Journal of Biological Chemistry
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