Identification of small molecules that modify the protein folding activity of heat shock protein 70.
Molecular chaperones, such as heat shock protein 70 (Hsp70) and its bacterial ortholog DnaK, play numerous important roles in protein folding. In vitro, this activity can be observed by incubating purified chaperones with denatured substrates and measuring the recovery of properly folded protein. In an effort to rapidly identify small molecules that modify this folding activity, we modified an existing method for use in 96-well plates. In this assay, denatured firefly luciferase was treated with a mixture of DnaK and prospective chemical modulators. The luminescence of refolded luciferase was used to follow the reaction progress, and counterscreens excluded compounds that target luciferase; thus, hits from these screens modify protein folding via their effects on the function of the chaperone machine. Using this platform, we screened a pilot chemical library and found five new inhibitors of DnaK and one compound that promoted folding. These chemical probes may be useful in studies aimed at understanding the many varied roles of chaperones in cellular protein folding. Moreover, this assay provides the opportunity to rapidly screen for additional compounds that might regulate the folding activity of Hsp70.
Wisén
Susanne
S
Departments of Pathology and Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA.
Gestwicki
Jason E
JE
D000255:Adenosine Triphosphate / Q000378:metabolism; D002623:Chemistry Techniques, Analytical / Q000295:instrumentation; D004353:Drug Evaluation, Preclinical; D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D049409:Luciferases, Firefly / Q000378:metabolism; D017510:Protein Folding; D020673:Protein Renaturation / Q000187:drug effects; D012680:Sensitivity and Specificity; D054852:Small Molecule Libraries / Q000494:pharmacology*
D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D054852:Small Molecule Libraries; D000255:Adenosine Triphosphate; D049409:Luciferases, Firefly