ATP Acyl Phosphate Reactivity Reveals Native Conformations of Hsp90 Paralogs and Inhibitor Target Engagement

Abstract

Hsp90 is an ATP-dependent chaperone of widespread interest as a drug target. Here, using an LC-MS/MS chemoproteomics platform based on a lysine-reactive ATP acyl phosphate probe, several Hsp90 inhibitors were profiled in native cell lysates. Inhibitor specificities for all four human paralogs of Hsp90 were simultaneously monitored at their endogenous relative abundances. Equipotent inhibition of probe labeling in each paralog occurred at sites both proximal to and distal from bound ATP observed in Hsp90 cocrystal structures, suggesting that the ATP probe is assaying a native conformation not predicted by available structures. Inhibitor profiling against a comprehensive panel of protein kinases and other ATP-binding proteins detected in native cell lysates identified PMS2, a member of the GHKL ATPase superfamily as an off-target of NVP-AUY922 and radicicol. Because of the endogenously high levels of Hsp90 paralogs in typical cell lysates, the measured potency of inhibitors was weaker than published IC₅₀ values. Significant inhibition of Hsp90 required inhibitor concentrations above a threshold where off-target activity was detectable. Direct on- and off-target engagement was measured by profiling lysates derived from cells treated with Hsp90 inhibitors. These studies also assessed the downstream cellular pathway effects of Hsp90 inhibition, including the down regulation of several known Hsp90 client proteins and some previously unknown client proteins. Overall, the ATP probe-based assay methodology enabled a broad characterization of Hsp90 inhibitor activity and specificity in native cell lysates.

In Vivo Conformational Dynamics of Hsp90 and Its Interactors

“…Our results are in line with the observation that 17-AAG mimics the binding of ADP (Roe et al, 1999), and contacts between NTD and MD that are required for ATP hydrolysis (Cunningham et al, 2008). The results also agree with the observation of a recent study which found that MD residues, including and surrounding K347 of HS90B, were labeled by an ATP acyl phosphate probe (Nordin et al, 2015). Nordin et al concluded that the ATP acyl phosphate probe identified a novel, highly conserved conformational state of Hsp90, and although no model was provided, their data are in excellent agreement with our results of a compact conformation that is enriched upon 17-AAG binding.…”

The Ligand Binding Landscape of Diacylglycerol Kinases

“…Although we cannot rule out the possibility of alternative mechanisms, e.g. probe binding due to domain- (Nordin et al, 2015) or protein-interactions (Okerberg et al, 2014), we do provide evidence that the C1 domain serves as a ligand binding site for ritanserin distinct from the ATP binding region of DGKα (Figure 5A and 6A). The overlapping (DAGKa) and distinct (C1) binding sites of ritanserin compared with ATP helps explain previous kinetic findings of a mixed competitive mechanism of inhibition whereby ritanserin prefers to bind a DGKα-ATP complex (Boroda et al, 2017).…”

Identification and characterization of the antiplasmodial activity of Hsp90 inhibitors

“…Finally, it is further unlikely given the experimental validation of the geldanamycin scaffold as chaperone specific in both human and P. falciparum chaperones [24, 48], and for the lack of off-target effects against other ATP binding proteins like protein kinases [49]. Therefore, a more likely explanation for the activity of these inhibitors could be associated with the inhibition of additional members of the Hsp90 family, such as Grp94 or Trap-1 [50, 51]. The results support this hypothesis since the most active compounds against malaria parasite, 17-DMAG and NVP-AUY922, also showed the highest affinity for the two Plasmodium chaperones tested (Table 2).…”