Identification of the target ANTXR1 and covalent acylation mechanism of 8-esterified cycloberberines against cancer.

An ester group can serve as a covalent warhead to acylate amino acid residues around the active cavity of the target protein, thereby silencing its biological function. In this study, taking 8-(benzoyloxy) cycloberberine (1) as the lead, twenty-seven 8-esterified cycloberberine (CBBR) derivatives were continuously synthesized and evaluated for their anti-tumor activities. Among them, compound 8a exhibited an appealing potency against a variety of tumor cells with IC50 values ranging 1.91-5.09 μM, significantly outperforming the lead 1. Cell cycle analysis showed 8a-induced G2/M phase arrest, which suggested ANTXR1 as a candidate target via thermal proteome profiling (TPP) assay. Further cellular thermal shift assay (CETSA) and surface plasmon resonance (SPR) analysis identified ANTXR1 as a direct target of 8a (KD = 2.88 μM). LC-MS/MS, molecule docking and molecular dynamics simulations (MD) demonstrated that the 8-adamantane acetyl in 8a could undergo an acylation reaction with Ser229 of ANTXR1, thereby inhibiting tumor cell proliferation. Therefore, compound 8a is a covalent inhibitor of ANTXR1, and introducing an ester group as the chemical warhead represents a highly effective structural modification strategy in medicinal chemistry.