Agents that stabilize mutated von Hippel-Lindau (VHL) protein: results of a high-throughput screen to identify compounds that modulate VHL proteostasis

J Biomol Screen. 2012 Jun;17(5):572-80. doi: 10.1177/1087057112436557. Epub 2012 Feb 21.

Abstract

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that affects multiple organs. Treatment is mainly surgical, and effective systemic therapies are needed. We developed a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point-mutated VHL protein. The 786-0 cell line was infected with full-length W117A-mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of functional readouts, including hypoxia-inducible factor 2α (HIF2α) and glucose transporter 1 (Glut1) levels, were performed. We found that bortezomib, MG132, and the Prestwick compounds 8-azaguanine, thiostrepton, and thioguanosine upregulated VHL-W117A-Venus in 786-0 cells. 8-Azaguanine downregulated HIF2α levels and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate posttranslational processing. Nuclear-cytoplasmic localization of VHL-W117A-Venus varied among the different compounds. In conclusion, a 786-0 cell line containing VHL-W117A-Venus was successfully used to identify compounds that upregulate VHL levels, with differential effect on VHL intracellular localization and posttranslational processing. Further screening efforts will broaden the number of pharmacophores available to develop therapeutic agents that will upregulate and refunctionalize mutated VHL.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Line
  • Cysteine Proteinase Inhibitors / pharmacology
  • Drug Discovery* / methods
  • High-Throughput Screening Assays*
  • Humans
  • Leupeptins / pharmacology
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Proteasome Endopeptidase Complex / metabolism
  • Proteasome Inhibitors
  • Protein Stability / drug effects
  • Reproducibility of Results
  • Small Molecule Libraries / pharmacology
  • Von Hippel-Lindau Tumor Suppressor Protein / genetics
  • Von Hippel-Lindau Tumor Suppressor Protein / metabolism*

Substances

  • Cysteine Proteinase Inhibitors
  • Leupeptins
  • Mutant Proteins
  • Proteasome Inhibitors
  • Small Molecule Libraries
  • Von Hippel-Lindau Tumor Suppressor Protein
  • Proteasome Endopeptidase Complex
  • benzyloxycarbonylleucyl-leucyl-leucine aldehyde