An electrophoretic mobility shift assay identifies a mechanistically unique inhibitor of protein sumoylation

Chem Biol. 2013 Apr 18;20(4):604-13. doi: 10.1016/j.chembiol.2013.04.001.


The dynamic, posttranslational modification of proteins with a small ubiquitin-like modifier (SUMO) tag has been recognized as an important cellular regulatory mechanism relevant to a number of cancers as well as normal embryonic development. As part of a program aimed toward the identification of inhibitors of SUMO-conjugating enzymes, we developed a microfluidic electrophoretic mobility shift assay to monitor sumoylation events in real time. We disclose herein the use of this assay to identify a cell-permeable compound capable of blocking the transfer of SUMO-1 from the E2 enzyme Ubc9 to the substrate. We screened a small collection of compounds and identified an oxygenated flavonoid derivative that inhibits sumoylation in vitro. Next, we carried out an in-depth mechanistic analysis that ruled out many common false-positive mechanisms such as aggregation or alkylation. Furthermore, we report that this flavonoid inhibits a single step in the sumoylation cascade: the transfer of SUMO from the E2 enzyme (Ubc9) thioester conjugate to the substrate. In addition to having a unique mechanism of action, this inhibitor has a discrete structure-activity relationship uncharacteristic of a promiscuous inhibitor. Cell-based studies showed that the flavonoid inhibits the sumoylation of topoisomerase-I in response to camptothecin treatment in two different breast cancer cell lines, while isomeric analogs are inactive. Importantly, this compound blocks sumoylation while not affecting ubiquitylation in cells. This work identifies a point of entry for pharmacologic inhibition of the sumoylation cascade and may serve as the basis for continued study of additional pharmacophores that modulate SUMO-conjugating enzymes such as Ubc9.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Cell Line, Tumor
  • Electrophoretic Mobility Shift Assay*
  • Humans
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • SUMO-1 Protein / antagonists & inhibitors
  • SUMO-1 Protein / genetics
  • SUMO-1 Protein / metabolism*
  • Small Molecule Libraries / chemistry*
  • Small Molecule Libraries / pharmacology
  • Structure-Activity Relationship
  • Sumoylation / drug effects


  • Recombinant Proteins
  • SUMO-1 Protein
  • Small Molecule Libraries