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, 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.