Discovery of a proteolysis targeting chimera (PROTAC) as a potent regulator of FOXP3

Regulatory T cells (Tregs) play a central role in immune homeostasis. Forkhead box P3 (Foxp3), a hallmark molecule in Tregs, is a vital transcription factor for their development and function, and degradation of the Foxp3 could provide therapeutic benefits in achieving effective anti-tumor immunity. In this study, we designed three PROTAC molecules, P60-L1-VHL, P60-L2-VHL, and P60-L3-VHL, based on a 15-mer peptide inhibitor of Foxp3 (P60), and explored their potential in regulating Foxp3 expression and function. Our data show that, among these molecules, P60-L3-VHL can inhibit the expression and nuclear localization of Foxp3 in HEK 293 T and HeLa cells, respectively. Meanwhile, use of proteasome inhibitor in P60-L3-VHL treated cells revealed an increased Foxp3 expression, indicating that P60-L3-VHL mediates the inhibition of Foxp3 through its degradation in the proteasome pathway. We further substantiate that P60-L3-VHL reduces the differentiation and Foxp3 expression in the in-vitro activated Tregs. Overall, our findings suggest that P60-L3-VHL inhibits Tregs differentiation by degrading the Foxp3, and it may have potential implications in cancer immunotherapy.