High-throughput screen for small molecules that modulate the ATPase activity of the molecular chaperone DnaK

• Published in: Analytical biochemistry 2008-01, Vol.372 (2), p.167-176
• Main Authors: Chang, Lyra, Bertelsen, Eric B., Wisén, Susanne, Larsen, Erik M., Zuiderweg, Erik R.P., Gestwicki, Jason E.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; Bacterial Proteins - metabolism; Colorimetry; Dihydropyrimidine; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Heat shock protein; Heat-Shock Proteins - metabolism; Hsp70; HSP70 Heat-Shock Proteins - genetics; HSP70 Heat-Shock Proteins - metabolism; Kinetics; Malachite green; Recombinant Proteins - metabolism; Stress response
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2007.08.020

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.