Targeting a Regulatory Element in Human Thymidylate Synthase mRNA

Abstract

Thymidylate synthase (TS) is a key enzyme in the biosynthesis of thymidine. TS inhibitors, which are used in cancer chemotherapy, suffer from resistance development in tumors through upregulation of TS expression. Autoregulatory translation control has been implicated with TS overexpression. TS binding at its own mRNA, which leads to sequestration of the start codon, is abolished when the enzyme forms an inhibitor complex, thereby relieving translation suppression. We have used the protein binding site from the TS mRNA in the context of a bicistronic expression system to validate targeting the regulatory motif with stabilizing ligands that prevent ribosomal initiation. Stabilization of the RNA by mutations, which were studied as surrogates of ligand binding, suppresses translation of the TS protein. Compounds that stabilize the TS binding RNA motif and thereby inhibit ribosomal initiation might be used in combination with existing TS enzyme-targeting drugs to overcome resistance development during chemotherapy.

Functional conservation despite structural divergence in ligand-responsive RNA switches

“…FRET folding and compound screening experiments were performed as described earlier (29). The in vitro transcription-translation assay was performed using HCV bicistronic luciferase constructs, as previously reported (40). HCV replicon testing followed procedures outlined earlier (23,41).…”

Screening for inhibitors of the hepatitis C virus internal ribosome entry site RNA

“…4b). 25 The benzoxazole 9 selectively inhibited IRES-initiated translation (30% inhibition at 100 mM compound concentration) while not affecting cap dependent translation (Fig. 4c).…”

“…4b) as previously described. 25 Compound stock solution in DMSO was added to the assay mixture to a final concentration of 10μM or 100μM, respectively, and adjusted to a final DMSO concentration of 1vol%. Detection of firefly and Renilla luciferase levels was done using the Dual-Glo Luciferase Assay System (Promega) according to manufactures recommendations.…”

“…A cap-driven firefly reporter gene is followed by Renilla luciferase reporter under the control of the internal ribosome entry site (IRES) from hepatitis C virus (HCV). 25 (c) Translation inhibition by compound 9 which at a concentration of 100μM selectively inhibits the IRES-dependent process while not affecting cap-driven translation.…”

Analysis of mRNA recognition by human thymidylate synthase

“…TS protein binding to the regulatory mRNA site 1 motif likely stabilizes the hairpin loop that renders the start codon unavailable for ribosomal recognition. In a previous investigation we had demonstrated that the TS site 1 hairpin constitutes an autonomous regulatory RNA motif that maintains its function when transplanted into heterologous reporter systems [ 19 ]. From mutational and mechanistic studies of the TS site 1 motif we concluded that secondary structure of the RNA by itself provides only a marginally stable roadblock to ribosomal initiation, whereas binding of the TS protein reduces translation initiation by sequestration of the start codon.…”