Even more strikingly, we observed a slowdown 
of the association of fluorescently labeled nucleotide to Hsp70 by two orders of magnitude in the presence of VER-155008. As a functional consequence of this inhibition, the rates of ATPinduced opening of the SBD and acceleration of substrate release are reduced and thus refolding of the model substrate firefly luciferase is impaired. VER-155008 by itself did not trigger transmission of a signal to the SBD and we did not observe any influence of the compound on substrate binding. Recently, PES, originally described as an inhibitor of p53mediated apoptosis, was reported to promote cancer cell death by specifically inhibiting the heat-inducible Hsp70 and its interactions with co-chaperones without affecting the constitutively expressed Hsc70. In pull down VE-822 experiments it was observed that the SBD of Hsp70 is required to detect an interaction between the chaperone and PES. Due to the lower sequence conservation of the SBD as compared to the NBD an inhibitory mechanism involving this domain could explain the proposed isoform specificity. As such an isoform specific inhibitor can help understanding the different roles of the two isoforms within the background of a living cell and can act as a specialized drug, we were eager to elucidate its mode of action. To our surprise PES inhibited, yet only slightly, the refolding of heat-denatured luciferase by both Hsp70 and Hsc70, which is consistent with a more recent study, which detected also an interaction of biotinylated PES with Hsc70. As the interaction is supposed to be mediated via the SBD we put great efforts into analyzing substrate affinity and binding dynamics in the presence and absence of PES in detail. Despite these efforts we were not able to detect any direct influence of PES on the interaction of Hsp70 with a peptide substrate. We also did not observe any influence of PES on the ATPase cycle of Hsp70. Finally, under our experimental conditions and with the concentrations used the compound did not reveal 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 PF-04217903 moa 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 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.