New mechanism-based inhibitors of aspartate transcarbamoylase for anticancer drug development

Aspartate transcarbamoylase (ATCase) is a key enzyme which regulates and catalyzes the second step of pyrimidine synthesis in all organisms. ATCase is a prototypic enzyme regulated by both product feedback and substrate cooperativity, whereas human ATCase is a potential anticancer target. Through structural and biochemical analyses, we revealed that R167/130’s loop region in ATCase serves as a gatekeeper for the active site, playing a new and unappreciated role in feedback regulation. Based on virtual compound screening simultaneously targeting the new regulatory region and active site of human ATCase, two compounds were identified to exhibit strong inhibition of ATCase activity, proliferation of multiple cancer cell lines, and growth of xenograft tumors. Our work has not only revealed a previously unknown regulatory region of ATCase that helps explain feedback regulation, but also successfully guided the identification of new ATCase inhibitors for anticancer drug development using a dual-targeting strategy.