Development and validation of a novel assay to identify radiosensitizers that target nucleophosmin 1

Bioorg Med Chem. 2015 Jul 1;23(13):3681-6. doi: 10.1016/j.bmc.2015.04.018. Epub 2015 Apr 11.

Abstract

A series of indole analogs that are synthesized using the scaffold of a potent radiosensitizer, YTR107, were tested for their ability to alter the solubility of phosphorylated nucleophosmin 1 (pNPM1). NPM1 is critical for DNA double strand break (DSB) repair. In response to formation of DNA DSBs, phosphorylated T199 NPM1 binds to ubiquitinated chromatin, in a RNF8/RNF168-dependent manner, forming irradiation-induced foci (IRIF) that promote repair of DNA DSBs. A Western blot assay was developed using lead molecule, YTR107, for the purpose of screening newly synthesized molecules that target pNPM1 in irradiated cells. A colony formation assay was used to demonstrate the radiosensitization properties of the compounds. Compounds that enhanced the extractability of pNPM1 upon radiation treatment possessed radiosensitization properties.

Keywords: Assay development; Cancer; DNA repair; Indole analogs; Lung cancer; NPM1; Nucleophosmin 1; PNR605; Radiation; Radiosensitizers; YTR107.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Barbiturates / pharmacology*
  • Blotting, Western / methods*
  • Cell Line
  • Cell Line, Tumor
  • Cell Nucleus / chemistry
  • Cell Nucleus / metabolism
  • Cell Nucleus / radiation effects
  • Centrosome / chemistry
  • Centrosome / metabolism
  • Centrosome / radiation effects
  • Chromatin / chemistry
  • Chromatin / metabolism
  • Chromatin / radiation effects
  • DNA Breaks, Double-Stranded / radiation effects*
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Fibroblasts / radiation effects
  • Gamma Rays
  • Humans
  • Indoles / pharmacology*
  • Nuclear Proteins / genetics
  • Nuclear Proteins / isolation & purification*
  • Nuclear Proteins / metabolism
  • Nucleophosmin
  • Phosphorylation
  • Radiation-Sensitizing Agents / pharmacology*
  • Tumor Stem Cell Assay
  • Ubiquitin / genetics
  • Ubiquitin / metabolism

Substances

  • 5-((N-benzyl-1H-indol-3-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)trione
  • Barbiturates
  • Chromatin
  • Indoles
  • NPM1 protein, human
  • Nuclear Proteins
  • Radiation-Sensitizing Agents
  • Ubiquitin
  • Nucleophosmin