Related Experiment Video
Updated: May 13, 2026
Oligopeptide Competition Assay for Phosphorylation Site Determination
Published on: May 18, 2017
Discovery of Phosphorylated Peptidomimetics Targeting Cbl-b SH2 Domain as Orthosteric Cbl-b Inhibitors by an
Erdong Li1, Xiuqi Hu1, Lvbin Hu1
1Jiang Su Key Laboratory of Drug Design and Optimization and Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China.
Abstract:
Casitas B-lineage lymphoma-b (Cbl-b), a pivotal negative regulator of TCR signaling, is highly expressed in immune cells. Its inhibition potentiates immune-mediated antitumor effects. Herein, we constructed a potent fluorescent tracer, Tracer 2, based on the Cbl-b peptide inhibitor Pep 1. Utilizing this tracer, we established a fluorescence polarization (FP) assay with enhanced sensitivity, discriminative capacity, and precision for the activity evaluation and high-throughput screening of orthosteric Cbl-b Src homology 2 (SH2) domain inhibitors. Leveraging this FP assay, we derived a potent phosphorylated peptidomimetic Cbl-b inhibitor, Pep 19, which significantly enhanced TCR signaling and promoted IL-2 secretion in Jurkat cells. Furthermore, in vivo studies demonstrated that Pep 19 exhibited robust immune-mediated antitumor effects in the CT26 syngeneic mouse model. This study not only established a validated FP-based screening platform but also identified Pep 19 as a lead compound for developing orthosteric Cbl-b SH2 domain small-molecule inhibitors for cancer immunotherapy.
Insights
Researchers developed a new assay to screen for cancer immunotherapy drugs. They identified a compound, Pep 19, that enhances immune responses and shows antitumor effects in mice.
Area of Science:
- Immunology
- Biochemistry
- Pharmacology
Background:
- Casitas B-lineage lymphoma-b (Cbl-b) negatively regulates T-cell receptor (TCR) signaling and is a target for cancer immunotherapy.
- Inhibiting Cbl-b can enhance anti-tumor immune responses.
Purpose of the Study:
- To develop a sensitive fluorescence polarization (FP) assay for screening Cbl-b inhibitors.
- To identify novel small-molecule inhibitors targeting the Cbl-b SH2 domain for cancer immunotherapy.
Main Methods:
- Construction of a fluorescent tracer (Tracer 2) based on a Cbl-b peptide inhibitor (Pep 1).
- Establishment and validation of a high-throughput FP assay for Cbl-b inhibitor screening.
- Derivation and characterization of a novel Cbl-b inhibitor (Pep 19).
Main Results:
- The FP assay demonstrated high sensitivity, discriminative capacity, and precision.
- Pep 19, a phosphorylated peptidomimetic inhibitor, significantly enhanced TCR signaling and IL-2 secretion in Jurkat cells.
- In vivo studies showed Pep 19 induced potent immune-mediated anti-tumor effects in a mouse model.
Conclusions:
- A validated FP-based screening platform for Cbl-b inhibitors was established.
- Pep 19 is a promising lead compound for developing small-molecule inhibitors targeting the Cbl-b SH2 domain.
- This work provides a new avenue for cancer immunotherapy drug discovery.
