Rations used the compound did not reveal 1805787-93-2 binding to a specific site within Hsp70 but instead interacted with Hsp70 in an undefined, nonsaturable and non-stoichiometric manner. For this interaction the SBD of Hsp70 was necessary. How this interaction is able to inhibit the chaperone activity of Hsp70 is not clear. Based on the observation that deletion of the disordered C-terminal tail of the Escherichia coli Hsp70 homolog DnaK reduces slightly chaperone activity and cell viability under sever stress conditions it was proposed that the disordered C terminus of Hsp70s contains a weak substrate binding site. This site was not excluded as potential binding site for PES in our study. However, Hsp70 with a deleted C-terminal tail is pulled down with similar efficiency by order 2222-07-3 biotinylated PES/avidin beads as wild type Hsp70, excluding such a possibility. In contrast, single amino acid replacement variants of Hsp70 were shown recently to be resistant to pull-down by biotinylated PES/avidin beads. These data suggest an interaction of PES with the helical lid. Interestingly, it was shown earlier that deletion of the helical lid in E. coli DnaK abrogates its ability to refold denatured firefly luciferase and compromises complementation of dnaK-deletion in vivo. It is therefore possible that the helical lid contains additional low affinity substrate binding sites that are important for refolding. Unfortunately, such binding sites for substrate proteins have not been demonstrated directly so far and, to our knowledge, there is currently no assay available to test whether PES prevents such binding. Nevertheless, the existence of several such sites would explain the non-saturable low-affinity binding of PES detected in our study by surface plasmon resonance spectroscopy and by differential scanning calorimetry. Whether such a binding mode of PES is specific for Hsp70 and whether this is the mechanism by which PES acts in vivo remains to be shown, for example by rescuing PES-induced apoptosis and autophagy through expression of mutant but not wild type Hsp70. Several other Hsp70 inhibitors of different classes have been described. Derivatives of spergualin, which are supposed to interact with the C-terminal EEVD motive, were reported to increase or decrease Hsp70 activity bringing the whole chaperone system out of balance. Although the specificity and mode of action remain elusive, clinical trials against different cancer types were undertaken but without any result. The search for different scaffolds resulted in the identification of a series of dihydropyrimidines, which modulate Hsp40-mediated ATPase activity. Although they exhibit weak activity and selectivity needs to be shown, some have anti-proliferative activity against cancer cell lines. A completely different c
