Thymidylate synthase inhibition is a critical mechanism in cancer treatment, as it disrupts DNA synthesis by preventing the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), leading to cell death. This approach targets rapidly dividing cancer cells and is a cornerstone of chemotherapeutic regimens involving drugs like 5-fluorouracil and pemetrexed.