ATP Acyl Phosphate Reactivity Reveals Native Conformations
of Hsp90 Paralogs and Inhibitor Target Engagement
- Publication date
- Publisher
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 IC<sub>50</sub> 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