title: An electrophoretic mobility shift assay identifies a mechanistically unique inhibitor of protein sumoylation.
abstract: 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.
authors.affiliation: Chemical Biology Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: Yeong Sang
authors.initials: YS
authors.lastname: Kim
authors.forename: Katelyn
authors.initials: K
authors.lastname: Nagy
authors.forename: Samantha
authors.initials: S
authors.lastname: Keyser
authors.forename: John S
authors.initials: JS
authors.lastname: Schneekloth
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D025842:SUMO-1 Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000494:pharmacology; D013329:Structure-Activity Relationship; D058207:Sumoylation / Q000187:drug effects
chemical_list: D011994:Recombinant Proteins; D025842:SUMO-1 Protein; D054852:Small Molecule Libraries