TY - JOUR AB - The ubiquitin-proteasome system plays an essential role in a broad range of cellular signaling pathways. Ubiquitination is a posttranslational protein modification that involves the action of an enzymatic cascade (E1, E2, and E3 enzymes) for the covalent attachment of ubiquitin to target proteins. The emerging knowledge of the molecular mechanisms and correlation of deregulation of the ubiquitin system in human diseases is uncovering new opportunities for therapeutics development. The E3 ligase RNF8 acts in cooperation with the heterodimeric E2 enzyme Ubc13/Uev1a to generate ubiquitin conjugates at the sides of DNA double-strand breaks, and recent findings suggest RNF8 as a potential therapeutic target for the treatment of breast cancer. Here, we present a novel high-throughput screening (HTS)–compatible assay based on the AlphaScreen technology to identify inhibitors of the RNF8-Ubc13 protein–protein interaction, along with a follow-up strategy for subsequent validation. We have adapted the AlphaScreen assay to a 384-well format and demonstrate its reliability, reproducibility, and suitability for automated HTS campaigns. In addition, we have established a biochemical orthogonal homogeneous time-resolved fluorescence (HTRF) assay in HTS format and a cellular microscopy-based assay allowing verification of the primary hits. This strategy will be useful for drug screening programs aimed at RNF8-Ubc13 modulation. AU - Weber, E. AU - Rothenaigner, I. AU - Brandner, S. AU - Hadian, K. AU - Schorpp, K.K. C1 - 50440 C2 - 42235 CY - Thousand Oaks SP - 1-8 TI - A high-throughput screening strategy for development of RNF8-Ubc13 protein–protein interaction inhibitors.   JO - J. Biomol. Screen. VL - 22 IS - 3 PB - Sage Publications Inc PY - 2017 SN - 1087-0571 ER - TY - JOUR AB - In high-throughput screening (HTS) campaigns, the binding of glutathione S-transferase (GST) to glutathione (GSH) is used for detection of GST-tagged proteins in protein-protein interactions or enzyme assays. However, many false-positives, so-called frequent hitters (FH), arise that either prevent GST/GSH interaction or interfere with assay signal generation or detection. To identify GST-FH compounds, we analyzed the data of five independent AlphaScreen-based screening campaigns to classify compounds that inhibit the GST/GSH interaction. We identified 53 compounds affecting GST/GSH binding but not influencing His-tag/Ni(2+)-NTA interaction and general AlphaScreen signals. The structures of these 53 experimentally identified GST-FHs were analyzed in chemoinformatic studies to categorize substructural features that promote interference with GST/GSH binding. Here, we confirmed several existing chemoinformatic filters and more importantly extended them as well as added novel filters that specify compounds with anti-GST/GSH activity. Selected compounds were also tested using different antibody-based GST detection technologies and exhibited no interference clearly demonstrating specificity toward their GST/GSH interaction. Thus, these newly described GST-FH will further contribute to the identification of FH compounds containing promiscuous substructures. The developed filters were uploaded to the OCHEM website (http://ochem.eu) and are publicly accessible for analysis of future HTS results. AU - Brenke, J.K. AU - Salmina, E.S.* AU - Ringelstetter, L. AU - Dornauer, S. AU - Kuzikov, M.* AU - Rothenaigner, I. AU - Schorpp, K.K. AU - Giehler, F. AU - Gopalakrishnan, J.* AU - Kieser, A. AU - Gul, S.* AU - Tetko, I.V. AU - Hadian, K. C1 - 48250 C2 - 41007 CY - Thousand Oaks SP - 596-607 TI - Identification of small-molecule frequent hitters of glutathione S-transferase-glutathione interaction. JO - J. Biomol. Screen. VL - 21 IS - 6 PB - Sage Publications Inc PY - 2016 SN - 1087-0571 ER - TY - JOUR AB - Many screening hits show relatively poor quality regarding later efficacy and safety. Therefore, small-molecule screening efforts shift toward high-content analysis providing more detailed information. Here, we describe a novel screening approach to identify cell cycle modulators with low toxicity by combining the Cell Cycle Chromobody (CCC) technology with the CytoTox-Glo (CTG) cytotoxicity assay. The CCC technology employs intracellularly functional single-domain antibodies coupled to a fluorescent protein (chromobodies) to visualize the cell cycle-dependent redistribution of the proliferating cell nuclear antigen (PCNA) in living cells. This image-based cell cycle analysis was combined with determination of dead-cell protease activity in cell culture supernatants by the CTG assay. We adopted this multiplex approach to high-throughput format and screened 960 Food and Drug Administration (FDA)-approved drugs. By this, we identified nontoxic compounds, which modulate different cell cycle stages, and validated selected hits in diverse cell lines stably expressing CCC. Additionally, we independently validated these hits by flow cytometry as the current state-of-the-art format for cell cycle analysis. This study demonstrates that CCC imaging is a versatile high-content screening approach to identify cell cycle modulators, which can be multiplexed with cytotoxicity assays for early elimination of toxic compounds during screening. AU - Schorpp, K.K. AU - Rothenaigner, I. AU - Maier, J.* AU - Traenkle, B.* AU - Rothbauer, U.* AU - Hadian, K. C1 - 48249 C2 - 41011 CY - Thousand Oaks SP - 965-977 TI - A multiplexed high-content screening approach using the chromobody technology to identify cell cycle modulators in living cells. JO - J. Biomol. Screen. VL - 21 IS - 9 PB - Sage Publications Inc PY - 2016 SN - 1087-0571 ER - TY - JOUR AB - Although small-molecule drug discovery efforts have focused largely on enzyme, receptor, and ion-channel targets, there has been an increase in such activities to search for protein-protein interaction (PPI) disruptors by applying high-throughout screening (HTS)-compatible protein-binding assays. However, a disadvantage of these assays is that many primary hits are frequent hitters regardless of the PPI being investigated. We have used the AlphaScreen technology to screen four different robust PPI assays each against 25,000 compounds. These activities led to the identification of 137 compounds that demonstrated repeated activity in all PPI assays. These compounds were subsequently evaluated in two AlphaScreen counter assays, leading to classification of compounds that either interfered with the AlphaScreen chemistry (60 compounds) or prevented the binding of the protein His-tag moiety to nickel chelate (Ni(2+)-NTA) beads of the AlphaScreen detection system (77 compounds). To further triage the 137 frequent hitters, we subsequently confirmed by a time-resolved fluorescence resonance energy transfer assay that most of these compounds were only frequent hitters in AlphaScreen assays. A chemoinformatics analysis of the apparent hits provided details of the compounds that can be flagged as frequent hitters of the AlphaScreen technology, and these data have broad applicability for users of these detection technologies. AU - Schorpp, K.K. AU - Rothenaigner, I. AU - Salmina, E. AU - Reinshagen, J.* AU - Low, T.* AU - Brenke, J.K. AU - Gopalakrishnan, J.* AU - Tetko, I.V. AU - Gul, S.* AU - Hadian, K. C1 - 29089 C2 - 33632 CY - Thousand Oaks SP - 715-726 TI - Identification of small-molecule frequent hitters from AlphaScreen high-throughput screens. JO - J. Biomol. Screen. VL - 19 IS - 5 PB - Sage Publications Inc PY - 2013 SN - 1087-0571 ER - TY - JOUR AB - The fatty acid binding protein 4 (FABP4) belongs to the family of lipid chaperones that control intracellular fluxes and compartmentalization of their respective ligands (e.g., fatty acids). FABP4, which is almost exclusively expressed in adipocytes and macrophages, contributes to the development of insulin resistance and atherosclerosis in mice. Lack of FABP4 protects against the development of insulin resistance associated with genetic or diet-induced obesity in mice. Furthermore, total or macrophage-specific FABP4 deficiency is protective against atherosclerosis in apolipoprotein E-deficient mice. The FABP4 small-molecule inhibitor BMS309403 has demonstrated efficacy in mouse models for type 2 diabetes mellitus and atherosclerosis, resembling phenotypes of mice with FABP4 deficiency. However, despite the therapeutically attractive long-term effects of FABP4 inhibition, an acute biomarker for drug action is lacking. The authors applied mass spectrometry lipidomics analysis to in vitro and in vivo (plasma and adipose tissue) samples upon inhibitor treatment. They report the identification of a potential biomarker for acute in vivo FABP4 inhibition that is applicable for further investigations and can be implemented in simple and fast-flow injection mass spectrometry assays. In addition, this approach can be considered a proof-of-principle study that can be applied to other lipid-pathway targeting mechanisms. AU - Suhre, K. AU - Römisch-Margl, W. AU - Hrabě de Angelis, M. AU - Adamski, J. AU - Luippold, G.* AU - Augustin, R.* C1 - 5746 C2 - 28543 SP - 467-475 TI - Identification of a potential biomarker for FABP4 inhibition: The power of lipidomics in preclinical drug testing. JO - J. Biomol. Screen. VL - 16 IS - 5 PB - Sage Publications Inc. PY - 2011 SN - 1087-0571 ER -