7 research outputs found
ATP Acyl Phosphate Reactivity Reveals Native Conformations of Hsp90 Paralogs and Inhibitor Target Engagement
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
Chemoproteomic Evaluation of Target Engagement by the Cyclin-Dependent Kinase 4 and 6 Inhibitor Palbociclib Correlates with Cancer Cell Response
Palbociclib
is a cyclin-dependent kinase (CDK) 4/CDK6 inhibitor
approved for breast cancer that is estrogen receptor (ER)-positive
and human epidermal growth factor receptor 2 (HER2)-negative. We profiled
palbociclib in cells either sensitive or resistant to the drug using
an ATP/ADP probe-based chemoproteomics platform. Palbociclib only
engaged CDK4 or CDK6 in sensitive cells. In resistant cells, no inhibition
of CDK4 or CDK6 was observed, although the off-target profiles were
similar in both cell types. Prolonged incubation of sensitive cells
with the compound (24 h) resulted in the downregulation of additional
kinases, including kinases critical for cell cycle progression. This
downregulation is consistent with cell cycle arrest caused by palbociclib
treatment. Both the direct and indirect targets were also observed
in a human tumor xenograft study using the COLO-205 cell line in which
phosphorylation of the retinoblastoma protein was tracked as the pharmacodyanamic
marker. Together, these results suggest that this probe-based approach
could be an important strategy toward predicting patient responsiveness
to palbociclib
The structure of an ATP acyl phosphate probe containing a desthiobiotin affinity tag and a schematic mechanism for covalent labeling of conserved lysines in the kinase active site.
<p>The structure of an ATP acyl phosphate probe containing a desthiobiotin affinity tag and a schematic mechanism for covalent labeling of conserved lysines in the kinase active site.</p
Sequence analysis of cloned PCR products from the 815zp tumor sample.
<p>A) A representative clone showing the mutated nucleotide (*). B) Alignment of all 41 sequences. Differences between pseudogene and CSNK1A1 are indicated by a plus symbol.</p
Immunoblot of recombinant CSNK1A1 samples.
<p>HEK293 cells were transfected with either no DNA (Mock), <b>CSNK1A1</b> wild type or D136N plasmids (CK1-WT and CK1-M, respectively).</p
Genomic DNA sequencing of CSNK1A1.
<p>A) PCR sequencing results for control using intronic primer. B) PCR sequencing results for patient 815zp using an intronic primer. In both cases, nucleotide of interest is highlighted with an asterisk.</p
Analysis of a CSNK1A1 active site peptide from wild-type and mutant CSNK1A1 expressed in HEK293 cells.
<p>A) Amino acid sequence of the mutant peptide. B) LC-MS/MS spectra for wild-type and mutant protein. The mutant sample is off-set by 50 units for clarity and the region of interest is highlighted. C) Region of interest in the LC-MS/MS spectra for colon tumor and matched control samples. Each spectrum is normalized to the highest intensity ion. D) Extracted ion chromatograms from HEK293 lysates transfected with either wild-type or mutant CSNK1A1. The wild-type sample is normalized to the peak eluting at 63.0 minutes and the mutant sample is normalized to the peak eluting at 59.8 minutes.</p