Novel Approaches for Targeting Thymidylate Synthase to Overcome the Resistance and Toxicity of Anticancer Drugs

The antifolate drug, methotrexate, was introduced to the clinic as an anticancer agent in the early 1950s.1 Subsequently, its mechanism of action was elucidated and it was found to bind in mono- and poly-glutamated forms to dihydrofolate reductase (DHFR),2,3 thymidylate synthase (TS)4 and amino-imidazolecarboxamide-ribonucleotide transformylase (AICARTF).5 A fluoropyrimidine, 5-fluorouracil (5FU), was conceived in 19576 following the observation that uracil was utilised preferentially in malignant over non-malignant cells,7 and has since been a first line drug in cancer chemotherapy. Subsequently, 5-fluoro-2'-deoxyuridine-5’-monophosphate (5FdUMP), an active metabolite of 5FU, was found to inhibit TS by forming a covalent ternary complex with the enzyme and 5,10-methylenetetrahydrofolate (mTHF).8 These discoveries marked the dawn of exploiting TS as an anticancer target.TS (EC 2.1.1.45), which is encoded by the TYMS gene in humans, catalyses the conversion of 2’-deoxyuridine-5’-monophosphate (dUMP) to 2’-deoxythymidine-5’-monophosphate (dTMP) by using mTHF as a cosubstrate. dTMP is then phosphorylated by thymidylate kinase to 2’-deoxythymidine diphosphate (dTDP) and then to 2’-deoxythymidine triphosphate (dTTP) by nucleoside-diphosphate kinase for use in the synthesis of new DNA. Thus, in human cells, TS plays a key role in the biosynthetic pathway that provides the sole de novo source of thymidylate, an essential precursor required for DNA replication and repair.9 In addition to its catalytic function, TS acts as a regulator of translation for some mRNAs. One of these is its own mRNA,10 and others include p53,11 which is a tumour suppressor, and c-myc,12 which is oncogenic. Binding of the TS protein to its own mRNA leads to the formation of an autoregulatory feedback loop for repression of the translation of TS mRNA (Fig 1). A 36 nucleotide sequence (75-110, Site I),13 encompassing the start codon, and a 70 nucleotide sequence (480-550, Site II)14 within the coding region have been identified as the most essential regions in the TS mRNA for binding to the TS protein. On the p53 mRNA, the nucleotide sequence from 531-1020 in the protein coding region,15 and for the c-myc mRNA, the C terminal coding region covering nucleotide positions 1625-1790,12 have been identified to be important for binding to the TS protein. Based on the observation that overexpression of TS sets the cells into a neoplastic phenotype, oncogenic behaviour is a novel role that has been attributed to TS recently.16