PDL1 degrader-3

PDL1 degrader-3 (compound e24) is an antitumor immunomodulator that targets and binds to CSN5 with a Kd of 4.53 μM. It inhibits the enzymatic activity of CSN5, enhances the ubiquitination of PD-L1, and induces its degradation through the ubiquitin-proteasome pathway, reducing PD-L1 expression on tumor cell membranes. By blocking the PD-1/PD-L1 interaction, PDL1 degrader-3 activates the tumor immune microenvironment, enhances the immune function of tumor-infiltrating T cells, and inhibits the activation of immunosuppressive MDSCs and Tregs. It demonstrates antitumor effects in mouse tumor models and is applicable for research in colorectal and lung cancers.

PDL1 degrader-3 (compound e24) at 20 μM for 24 hours significantly reduces PD-L1 protein expression in human colorectal cancer RKO cells with effectiveness superior to JQ-1 and with lower cytotoxicity. The compound (1-20 μM; 3-24 h) decreases PD-L1 protein levels in RKO and human lung cancer H1975 cells in a dose- and time-dependent manner; significant reduction is observed at concentrations ≥5 μM in RKO cells and ≥10 μM in H1975 cells after 24 hours, with notable downregulation appearing as early as 3 hours at 20 μM. Similarly, PDL1 degrader-3 (5-20 μM; 3-24 h) reduces cell membrane PD-L1 expression; at 10 μM and 20 μM concentrations after 24 hours, significant downregulation is noted, and at 20 μM, this effect is apparent from 6 hours. Via immunofluorescence, PDL1 degrader-3 (5-20 μM; 24 h) also exhibits a dose-dependent decrease in PD-L1 protein in both cell types. Noteworthy, at concentrations up to 20 μM for 24 hours, no significant reduction in cell viability is detected. At 20 μM for 24 hours, it markedly inhibits PD-L1/PD-1 interaction in both RKO and H1975 cells. Additionally, PDL1 degrader-3 (5-20 μM; 24 h) enhances T cell-mediated cytotoxicity in a dose-dependent manner against both RKO and H1975 cells. No significant effect on PD-L1 mRNA expression in RKO cells is noted, indicating protein-level downregulation. When combined with 60 μg/mL CHX for up to 12 hours, PDL1 degrader-3 (20 μM) substantially shortens the PD-L1 protein half-life in RKO cells. It promotes PD-L1 degradation via the ubiquitin-proteasome pathway, as 10 μM MG132 blocks this effect, unlike lysosome or autophagy inhibitors. Moreover, PDL1 degrader-3 (20 μM; 6 h) enhances polyubiquitination of PD-L1 in RKO cells, confirming degradation via this pathway. The compound's ability to induce PD-L1 downregulation in RKO cells depends on CSN5; overexpression hinders, while knockdown enhances the effect. Direct binding to CSN5 in RKO cells is displayed by increased thermal stability, with a dissociation constant (K d) of 4.53 μM mediated by Asp151 and Lys210 residues.

PDL1 degrader-3 (also known as compound e24) administered via intraperitoneal injection daily for 16 consecutive days at doses ranging from 2.5 to 10 mg/kg effectively suppresses the growth of subcutaneous MC38 colorectal cancer in C57BL/6J mice, achieving a maximum tumor growth inhibition rate of 78.88% at 10 mg/kg. This compound reduces immunosuppressive Treg and MDSC in a dose-dependent manner and activates tumor-infiltrating CD8+ T cells. Furthermore, when administered at 2.5-5 mg/kg doses in C57BL/6J mice with subcutaneous Lewis lung cancer, it reaches an 88.56% tumor growth inhibition rate at 5 mg/kg, while modulating the tumor immune microenvironment to enhance antitumor immune activity. However, in T cell-deficient nude mice, PDL1 degrader-3 at 10 mg/kg (daily for 16 days) does not inhibit subcutaneous MC38 colorectal cancer growth, nor does it suppress subcutaneous Lewis lung cancer growth at 5 mg/kg, indicating its reliance on functional T cells for antitumor efficacy. Additionally, when combined with an anti-CTLA-4 antibody (10 mg/kg; intraperitoneal; daily for 18 days), PDL1 degrader-3 enhances antitumor immunity and more effectively inhibits subcutaneous MC38 colorectal cancer growth in C57BL/6J mice compared to either monotherapy.