Abstract P025: NUC-3373 is a more potent inhibitor of thymidylate synthase than 5-FU and reduces generation of toxic metabolites
Authors: Bré Jennifer , Dickson Alison L. , Read Oliver J. , Zhang Ying , Mullen Peter , Czekster Clarissa M. , Harrison David J. 
Published: December 2021

Abstract
Background NUC-3373 is a novel anti-cancer agent designed to replace 5-FU, one of the most widely used therapies across a broad range of tumors, including colorectal cancer (CRC). 5-FU exerts its main anti-cancer activity via the active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), which binds and inhibits thymidylate synthase (TS), preventing the conversion of dUMP into dTMP and disrupting DNA synthesis and repair. NUC-3373, a phosphoramidate transformation of FUDR-MP, is designed to bypass 5-FU resistance mechanisms associated with transport, activation and breakdown, and reduce generation of toxic metabolites including FUTP, which is incorporated into RNA causing myelosuppression and gastrointestinal toxicity, and FBAL which causes hand-foot syndrome. Thymidine supplementation rescues cells from NUC-3373 induced cytotoxicity, supporting a DNA targeted mode of action. The aim of this study was to further evaluate the differences between NUC-3373 and 5-FU. Methods CRC cell lines HCT116 and SW480 were exposed to sub-IC50 doses of NUC-3373 or 5-FU (0.1-25 μM) for 6h. At specific time-points, cells were harvested and analyzed as follows: TS inhibition by western blot, metabolite levels by mass spectrometry (LC-MS & LC-MS/MS) and cell cycle by flow cytometry. In rescue experiments, NUC-3373 and 5-FU were supplemented with thymidine (8 μg/mL) for 24h and cell survival assessed at 96h post-treatment. Results In both cell lines, NUC-3373 was a more potent inhibitor of TS than 5-FU with a higher proportion of TS protein bound to FUDR-MP at low concentrations. At 24h, 10 µM 5-FU was required to achieve the same level of TS binding as 0.1 µM NUC-3373 in HTC116 cells and as 0.5 µM NUC-3373 in SW480 cells. TS inhibition by NUC-3373 was almost maximal by 6 hours and was sustained for at least 48 hours. NUC-3373 generated significantly higher levels (50x) of free FUDR-MP compared to 5-FU, resulting in a more pronounced increase in dUMP levels (5x compared to 5-FU). At 48h, NUC-3373 treated cells remained arrested in S phase, while 5-FU treated cells had reverted to a normal cell cycle. FUTP was present in cells exposed to low doses of 5-FU (0.5 μM) but was not detectable in NUC-3373 treated cells. Finally, thymidine supplementation did not alter cell sensitivity to 5-FU but rescued cells treated with NUC-3373. Conclusion NUC-3373 generates higher intracellular levels of FUDR-MP and is a more potent inhibitor of TS than 5-FU, leading to more pronounced effects on cell cycle arrest and perturbation of the nucleotide pool that can result in misincorporation of uracil into DNA. Furthermore, NUC-3373 did not generate FUTP, consistent with the observation that patients treated with NUC-3373 in clinical studies have experienced much lower rates of FUTP-related toxicities. NUC-3373 is a potent TS inhibitor with a favorable safety profile.
Citation Format: Jennifer Bré, Alison L. Dickson, Oliver J. Read, Ying Zhang, Peter Mullen, Clarissa M. Czekster, David J. Harrison. NUC-3373 is a more potent inhibitor of thymidylate synthase than 5-FU and reduces generation of toxic metabolites [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P025.