High-throughput screen for small molecules that modulate the ATPase activity of the molecular chaperone DnaK.
DnaK is a molecular chaperone of Escherichia coli that belongs to a family of conserved 70-kDa heat shock proteins. The Hsp70 chaperones are well known for their crucial roles in regulating protein homeostasis, preventing protein aggregation, and directing subcellular traffic. Given the complexity of functions, a chemical method for controlling the activities of these chaperones might provide a useful experimental tool. However, there are only a handful of Hsp70-binding molecules known. To build this area, we developed a robust, colorimetric, high-throughput screening (HTS) method in 96-well plates that reports on the ATPase activity of DnaK. Using this approach, we screened a 204-member focused library of molecules that share a dihydropyrimidine core common to known Hsp70-binding leads and uncovered seven new inhibitors. Intriguingly, the candidates do not appear to bind the hydrophobic groove that normally interacts with peptide substrates. In sum, we have developed a reliable HTS method that will likely accelerate discovery of small molecules that modulate DnaK/Hsp70 function. Moreover, because this family of chaperones has been linked to numerous diseases, this platform might be used to generate new therapeutic leads.
Chang
Lyra
L
Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA.
Bertelsen
Eric B
EB
Wisén
Susanne
S
Larsen
Erik M
EM
Zuiderweg
Erik R P
ER
Gestwicki
Jason E
JE
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D003124:Colorimetry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism; D007700:Kinetics; D011994:Recombinant Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli