Development of a Fluorescence Polarization Based High-Throughput Assay to Identify Casitas B-Lineage Lymphoma RING Domain Regulators
Open accessAbstract
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.
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- Authors
- Xie, X., Sun, L., Pessetto, Z. Y., Zhao, Y., Zang, Z., Zhong, L., Wu, M., Su, Q., Gao, X., Zan, W., & Sun, Y.
- Affiliations
- Chengdu Medical College (CN)
- Sichuan University (CN)
- University of Kansas Medical Center (US)
- Corresponding author
- Sun, Yiyi
- Venue
- PLoS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2013-10-31
- Volume
- 8
- Issue
- 10
- Pages
- e78042
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC3814989
- OpenAlex
- W2171225865
- Cited by
- 8
- References
- 26
- Citations by year
- 2026: 2 · 2021: 2 · 2015: 3 · 2014: 1
- FWCI
- 0.75
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- PLoS ONE (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, other-oa)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Figshare (submittedVersion, cc-by)
- Funders
- Chengdu Medical College
- National Natural Science Foundation of China
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chemical Synthesis and Analysis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
- Keywords
- RING domain, E3 ubiquitin ligase, protein-protein interaction inhibitors, small molecule modulators, fluorescence polarization assay, high-throughput screening, catalpol, natural product library, binding affinity
- MeSH terms
- Fluorescence Polarization, Lymphoma, Protein Binding, Ubiquitin-Conjugating Enzymes, Proto-Oncogene Proteins c-cbl
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