The proposed research is intended to discover novel anti-cancer agents that inhibit expression of thymidylate synthase (TS), the sole intracellular de novo source of thymidylate for DNA biosynthesis. Specifically, we want to synthesize and identify compounds that selectively bind and stabilize a structured regulatory element in the 5'nontranslated region (NTR) of the TS mRNA which contains the translation start codon sequestered in an RNA hairpin element. By preventing access of the cellular translation machinery to the initiation site, TS mRNA-binding compounds would mimic the action of TS protein which acts as a ligand and repressor of its own mRNA. In preliminary experiments we have validated the TS mRNA as a target for translation inhibition via stabilization of the mRNA secondary structure. In the proposed research we will establish an iterative process aiming at the discovery of lead compounds for the development of novel cancer therapeutics for use in combination with existing anti-cancer drugs that target the enzyme but suffer from resistance development via upregulation of TS expression.
The specific aims of this project are to: 1) design and synthesize novel RNA-biased ligands based on two classes of heterocycles privileged for RNA binding;2) design and validate model oligonucleotides representing the regulatory RNA element in the 5'-NTR of TS mRNA for ligand binding studies;3) determine the three- dimensional structure of the regulatory RNA element in the TS 5'-NTR by X-ray crystallography;4) develop RNA affinity and target specificity assays for the TS mRNA and identify small molecule binders among the newly synthesized RNA-biased ligands and known RNA-binding natural products;5) evaluate TS mRNA binders for specific target recognition in binding and in vitro translation assays;6) test TS-specific translation inhibitors in human cell lines to determine membrane permeability, interference with TS expression and antiproliferative activity;7) determine the three-dimensional structure of TS mRNA-ligand complexes by X-ray crystallography;8) design modified ligands with potentially improved binding affinity by using biological activity data and structural information (structure activity relationships, SAR).

Public Health Relevance

This research is aimed at the discovery of novel anti-cancer therapeutics which will greatly increase the efficacy of existing drugs for the treatment of resistant breast and colorectal cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA132753-05
Application #
8462455
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Fu, Yali
Project Start
2009-06-01
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
5
Fiscal Year
2013
Total Cost
$296,612
Indirect Cost
$98,069
Name
University of California San Diego
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Brunn, Nicholas D; Dibrov, Sergey M; Kao, Melody B et al. (2014) Analysis of mRNA recognition by human thymidylate synthase. Biosci Rep 34:e00168
Ding, Kejia; Dibrov, Sergey M; Hermann, Thomas (2013) Synthesis and crystal structure of a phenazine N-oxide. J Chem Crystallogr 43:550-553
Cruz, José Almeida; Blanchet, Marc-Frédérick; Boniecki, Michal et al. (2012) RNA-Puzzles: a CASP-like evaluation of RNA three-dimensional structure prediction. RNA 18:610-25
Brunn, Nicholas D; Garcia Sega, Emily; Kao, Melody B et al. (2012) Targeting a regulatory element in human thymidylate synthase mRNA. Chembiochem 13:2738-44
Dibrov, Sergey; McLean, Jaime; Hermann, Thomas (2011) Structure of an RNA dimer of a regulatory element from human thymidylate synthase mRNA. Acta Crystallogr D Biol Crystallogr 67:97-104
Carnevali, Maia; Parsons, Jerod; Wyles, David L et al. (2010) A modular approach to synthetic RNA binders of the hepatitis C virus internal ribosome entry site. Chembiochem 11:1364-7
Dutta, Sanjay; Higginson, Cody J; Ho, Bao T et al. (2010) 1,3-Diazepanes of natural product-like complexity from cyanamide-induced rearrangement of epoxy-delta-lactams. Org Lett 12:360-3