Abstract
Thymidylate synthase (TYMS) enzyme is an anti-cancer target given its role in DNA biosynthesis. TYMS inhibitors (e.g., 5-Fluorouracil) can lead to drug resistance through an autoregulatory mechanism of TYMS that causes its overexpression. Since G-quadruplexes (G4) can modulate gene expression, we searched for putative G4 forming sequences (G4FS) in the TYMS gene that could be targeted using polypurine reverse Hoogsteen hairpins (PPRH). G4 structures in the TYMS gene were detected using the quadruplex forming G-rich sequences mapper and confirmed through spectroscopic approaches such as circular dichroism and NMR using synthetic oligonucleotides. Interactions between G4FS and TYMS protein or G4FS and a PPRH targeting this sequence (HpTYMS-G4-T) were studied by EMSA and thioflavin T staining. We identified a G4FS in the 5’UTR of the TYMS gene in both DNA and RNA capable of interacting with TYMS protein. The PPRH binds to its corresponding target dsDNA, promoting G4 formation. In cancer cells, HpTYMG-G4-T decreased TYMS mRNA and protein levels, leading to cell death, and showed a synergic effect when combined with 5-fluorouracil. These results reveal the presence of a G4 motif in the TYMS gene, probably involved in the autoregulation of TYMS expression, and the therapeutic potential of a PPRH targeted to the G4FS.
Highlights
Thymidylate synthase (TYMS) has been widely studied as an anti-cancer target due to its essential role in the de novo synthesis of 2’-deoxythymidine-5’-monophosphate, a critical precursor for DNA biosynthesis
Both genomic DNA and cDNA samples originated a main product of 184 bp (Figure 1C), confirming that the identified G4 forming sequences (G4FS) was located within the 5’ untranslated region (5’UTR) of the TYMS gene and not in the promoter
We describe the effects of a polypurine reverse Hoogsteen hairpins (PPRH) targeting a polypyrimidine strand complementary to a3.nDewislcyusidsieonntified G4 motif present in the 5’UTR of the TYMS gene, whose encoded protein is a classicalInanttih-ciasncwerotrakr,gewt deuedetoscirtisbreolethine DeNffAecstsynothfesais [P2P].RSHincteairtgiestkinngowan tphoaltyGp4yrsitmruicdtiunrees sctarnand be cimompoprletamnetngteanrye rteogualanteowrylyeliedmenetniftise,dwGe 4semarochtiefdpfroerseGn4t sintruthcteur5e’Us iTnRthoef TthYeMTSYgMenSeguesnine,gwthheose QGeRnScomdaepdpperr,oatecionmispauctalatisosnicaalltaonotlif-ocar nthceerptraerdgiecttidouneotfoqiutsadrorulepilnexDfNorAmisnygntGh-ersicish[s2e]q
Summary
Thymidylate synthase (TYMS) has been widely studied as an anti-cancer target due to its essential role in the de novo synthesis of 2’-deoxythymidine-5’-monophosphate (dTMP), a critical precursor for DNA biosynthesis. 5-FU is converted to 5-fluoro-2’-deoxyuridine5’-monophosphate (FdUMP), which is an analog of the endogenous ligand dUMP and binds to the nucleotide-binding domain of TYMS. This FdUMP-TYMS-mTHF ternary complex inhibits TYMS activity and leads to the suppression of dTMP synthesis, producing deoxynucleotide pool imbalance, increased levels of deoxyuridine triphosphate (dUTP) and, DNA damage [4,5]. The effectiveness of fluoropyrimidines or other TYMS inhibitors is compromised by the development of drug resistance. Some of these have been associated with high TYMS protein levels and are thought to be related to an autoregulatory mechanism of TYMS protein that modulates its own expression [6,7]
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