RNA-binding proteins (RBPs) are emerging as crucial regulators in cancer, but the development of therapeutic strategies targeting RBPs remains limited. Here, through chemical proteomics approaches, we identify DIQ01 as a novel small-molecule inhibitor of KH-type splicing regulatory protein (KHSRP), an RBP that is aberrantly hyper-expressed in human tumors and plays an essential role in proliferation, metastasis, and tumor progression. DIQ01 specifically binds to the KH3 and KH4 domains (Phe358 as the key residue) of KHSRP, inhibiting its interaction with mRNAs. Furthermore, the binding of DIQ01 diminishes the PRMT5-mediated arginine methylation of KHSRP, a post-translational modification required for its oncogenic activity. Mechanistically, transcriptomic and proteomic profiling suggested that DIQ01 functionally inactivates KHSRP, triggering destabilization and downregulation of its target PLK1 mRNA. This leads to S-phase arrest, DNA damage, and apoptosis in HCT116 cells. Both in vitro and in vivo studies, including CRC xenograft and patient-derived organoid models, demonstrate that DIQ01 exhibits potent antitumor efficacy with minimal systemic toxicity. Our findings underscore the innovative approach of concurrently targeting the RNA-binding function of an RBP and its post-translational modification, highlighting DIQ01's unique mechanism and significant translational potential as a targeted cancer therapy.

KHSRP  /  Arginine methylation  /  DNA damage  /  PRMT5  /  PLK1  /  ABPP  /  Colon cancer  /  Small molecule inhibitor