HTRF-powered detection of ubiquitin-like protein (UBL) signaling and interactions

Eduard A. Sergienko

2013

Sanford-Burnham Medical Research Institute, San Diego, CA, USA

5th HTRF Symposium, Avignon, France

Ubiquitin-like proteins (UBL), such as ubiquitin, SUMO and NEDD8, are involved in reversible posttranslational modification of cellular proteins leading to a wide spectrum of biological effects. These UBL modification pathways rely on coordinated efforts of specific multienzyme cascade systems. The complete UBL modification reaction consists of three consecutive steps handled by distinct groups of enzymes: ATP-dependent UBL activation catalyzed by E1 enzymes, UBL conjugation to the active site of E2 enzymes, and eventual ligation of UBL to the epsilon-amino group of specific lysine residues on target proteins, frequently with the help of E3 enzymes. Three biochemical assays, reconstituting ubiquitination, SUMOylation and neddylation pathways in vitro, were developed at Conrad Prebys Center for Chemical Genomics of Sanford-Burnham Medical Research Institute. The assays utilized TR-FRET detection of covalent adducts between UBL and protein targets. The concentrations of E1 and E2 enzymes and of their substrates, the order of addition of reagents and the length of incubation for various assay steps were optimized to ensure robustness of assays and sensitivity to small-molecule inhibitors with desired mechanisms of action. An additional TR-FRET assay was developed to detect binding between SUMO proteins and peptide motifs responsible for their recognition by cellular proteins. All assays were miniaturized into 1536-well plates demonstrating robust performance. All four assays were screened against a small-molecule library of ~300,000 compounds. Detected hits were profiled using the primary HTS data to identify and remove promiscuous compounds from the list. Resulting hits were characterized using panels of additional secondary assays designed to confirm hit specificity, selectivity and mechanism of action. Availability of comprehensive counter screen data and of diverse secondary assays for hit profiling and confirmation was critical for rapid advancement of these projects.

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