Discovery of Phosphorylated Peptidomimetics Targeting Cbl-b SH2 Domain as Orthosteric Cbl-b Inhibitors by an Optimized Fluorescence Polarization Competition Assay

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, <b>Tracer 2</b>, based on the Cbl-b peptide inhibitor <b>Pep 1.</b> 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, <b>Pep 19</b>, which significantly enhanced TCR signaling and promoted IL-2 secretion in Jurkat cells. Furthermore, <i>in vivo</i> studies demonstrated that <b>Pep 19</b> 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 <b>Pep 19</b> as a lead compound for developing orthosteric Cbl-b SH2 domain small-molecule inhibitors for cancer immunotherapy.