Thymidylate synthase (TS) inhibitors are an integral component of chemotherapy regimens for difficult to treat cancer subtypes. Despite initial therapeutic benefit, current inhibitors induce TS overexpression or alter folate transport metabolism feedback pathways that tumor cells exploit for drug resistance. Here we report a small molecule TS inhibitor that exhibits i) enhanced antitumor activity as compared to current fluoropyrimidines and antifolates without inducing TS overexpression, ii) is structurally distinct from classical antifolates, iii) extends survival in a pancreatic tumor mouse model, iv) and is well tolerated with equal efficacy using either intraperitoneal or oral administration. Mechanistically, we confirm the compound is a multifunctional non classical antifolate and through a series of analogues identify structural features allowing direct TS inhibition while also maintaining the ability to inhibit dihydrofolate reductase (DHFR). Collectively, this work identifies new non classical antifolate inhibitors that optimize inhibition of thymidylate biosynthesis with a favorable safety profile highlighting potential for enhanced cancer therapy.

Citation Format: Maria V. Guijarro, Patrick C. Kellish, Peter E. Dib, Nicholas G. Paciaroni, Akbar Nawab, Jacob Andring, Lidia Kulemina, Nicholas V. Borrero, Carlos Modenutti, Richard L. Bennett, Daniil Shabashvili, Jonathan D. Licht, Robert McKenna, Adrian Roitberg, Robert W. Huigens, Frederic J. Kaye, Maria Zajac-Kaye. First in class multifunctional non classical antifolates inhibits thymidylate synthase and extends survival in pancreatic cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4055.