The novel anti-cancer fluoropyrimidine NUC-3373 is a potent inhibitor of thymidylate synthase and an effective DNA-damaging agent

Cancer Chemother Pharmacol. 2023 May;91(5):401-412. doi: 10.1007/s00280-023-04528-5. Epub 2023 Mar 31.


Introduction: Fluoropyrimidines, principally 5-fluorouracil (5-FU), remain a key component of chemotherapy regimens for multiple cancer types, in particular colorectal and other gastrointestinal malignancies. To overcome key limitations and pharmacologic challenges that hinder the clinical utility of 5-FU, NUC-3373, a phosphoramidate transformation of 5-fluorodeoxyuridine, was designed to improve the efficacy and safety profile as well as the administration challenges associated with 5-FU.

Methods: Human colorectal cancer cell lines HCT116 and SW480 were treated with sub-IC50 doses of NUC-3373 or 5-FU. Intracellular activation was measured by LC-MS. Western blot was performed to determine binding of the active anti-cancer metabolite FdUMP to thymidylate synthase (TS) and DNA damage.

Results: We demonstrated that NUC-3373 generates more FdUMP than 5-FU, resulting in a more potent inhibition of TS, DNA misincorporation and subsequent cell cycle arrest and DNA damage in vitro. Unlike 5-FU, the thymineless death induced by NUC-3373 was rescued by the concurrent addition of exogenous thymidine. 5-FU cytotoxicity, however, was only reversed by supplementation with uridine, a treatment used to reduce 5-FU-induced toxicities in the clinic. This is in line with our findings that 5-FU generates FUTP which is incorporated into RNA, a mechanism known to underlie the myelosuppression and gastrointestinal inflammation associated with 5-FU.

Conclusion: Taken together, these results highlight key differences between NUC-3373 and 5-FU that are driven by the anti-cancer metabolites generated. NUC-3373 is a potent inhibitor of TS that also causes DNA-directed damage. These data support the preliminary clinical evidence that suggest NUC-3373 has a favorable safety profile in patients.

Keywords: Colorectal cancer; DNA damage; Fluoropyrimidine; NUC-3373; Thymidylate synthase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antimetabolites
  • Colorectal Neoplasms* / genetics
  • DNA
  • Enzyme Inhibitors / pharmacology
  • Enzyme Inhibitors / therapeutic use
  • Fluorodeoxyuridylate / pharmacology
  • Fluorodeoxyuridylate / therapeutic use
  • Fluorouracil / therapeutic use
  • Humans
  • Thymidylate Synthase* / genetics


  • Thymidylate Synthase
  • Fluorodeoxyuridylate
  • Fluorouracil
  • Enzyme Inhibitors
  • Antimetabolites
  • DNA