Design, synthesis and biological evaluation of novel TMPRSS2-PROTACs with florosubstituted 4-guanidino-N-phenylbenzamide derivative ligands

Abstract

Transmembrane Serine Protease 2 (TMPRSS2) plays a critical role in tumorigenesis and progression, making its degradation a promising therapeutic strategy. In this study, we designed and synthesized TMPRSS2-PROTACs, including VPOT64 and VPOT76, based on camostat. Both compounds exhibited superior inhibitory effects on HT-29 colorectal and Calu-3 lung cancer cells compared to paclitaxel. Notably, VPOT76 effectively degraded TMPRSS2, significantly inhibiting the proliferation of TMPRSS2-positive HT-29 cells and inducing apoptosis with an IC₅₀ of 0.39 ± 0.01 μM. Flow cytometry analysis demonstrated that VPOT76 increased early apoptotic cells in a dose-dependent manner and caused G2 phase arrest at 0.8 μM. Colony formation assays showed that VPOT76 inhibited HT-29 colony formation, even at low concentrations, further confirming its anti-proliferative effect. Additionally, wound healing assays indicated that VPOT76 reduced the migration of HT-29 cells after 48 h, suggesting its potential to impair tumor cell invasion and metastasis. These findings highlight the multifaceted anticancer activities of VPOT76, including apoptosis induction, cell cycle arrest, colony formation inhibition, and migration suppression. Overall, this study establishes VPOT76 as a potent TMPRSS2-degrading PROTAC with strong therapeutic potential, laying the groundwork for further development of TMPRSS2-targeting treatments for colorectal and other cancers.