title: Development of a fluorescence polarization based high-throughput assay to identify Casitas B-lineage lymphoma RING domain regulators.
abstract: The E3 ubiquitin protein ligase Casitas B-lineage Lymphoma (Cbl) proteins and their binding partners play an important role in regulating signal transduction pathways. It is important to utilize regulators to study the protein-protein interactions (PPIs) between these proteins. However, finding specific small-molecule regulators of PPIs remains a significant challenge due to the fact that the interfaces involved in PPIs are not well suited for effective small molecule binding. We report the development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule regulators of Cbl (RING) domain. The FP assay was used to measure binding affinities and inhibition constants of UbCH7 peptides and small molecule regulators of Cbl (RING) domains, respectively. In order to rule out promiscuous, aggregation-based inhibition, two assay conditions were developed and compared side by side. Under optimized conditions, we screened a 10,000 natural compound library in detergent-free and detergent-present (0.01% Triton X-100) systems. The results indicate that the detergent-present system is more suitable for high-throughput screens. Three potential compounds, methylprotodioscin, leonuride and catalpol, have been identified that bind to Cbl (RING) domain and interfere with the Cbl (RING)-UbCH7 protein-protein interaction.
authors.affiliation: Department of Pharmacy, Chengdu Medical College, Chengdu, Sichuan Province, China.
authors.forename: Xingliang
authors.initials: X
authors.lastname: Xie
authors.forename: Lin
authors.initials: L
authors.lastname: Sun
authors.forename: Ziyan Yuan
authors.initials: ZY
authors.lastname: Pessetto
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhao
authors.forename: Zhihe
authors.initials: Z
authors.lastname: Zang
authors.forename: Ling
authors.initials: L
authors.lastname: Zhong
authors.forename: Min
authors.initials: M
authors.lastname: Wu
authors.forename: Qing
authors.initials: Q
authors.lastname: Su
authors.forename: Xiurong
authors.initials: X
authors.lastname: Gao
authors.forename: Wang
authors.initials: W
authors.lastname: Zan
authors.forename: Yiyi
authors.initials: Y
authors.lastname: Sun
mesh_terms: D005454:Fluorescence Polarization / Q000379:methods*; D008223:Lymphoma / Q000201:enzymology*; D011485:Protein Binding; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism
chemical_list: D044763:Ubiquitin-Conjugating Enzymes; D050721:Proto-Oncogene Proteins c-cbl