Monitoring Native p38α:MK2/3 Complexes via Trans Delivery of an ATP Acyl Phosphate Probe

Here we describe a chemical proteomics strategy using ATP acyl phosphates to measure the formation of a protein:protein complex between p38α and mapkap kinases 2 and/or 3. Formation of the protein:protein complex results in a new probe labeling site on p38α that can be used to quantify the extent of interaction in cell lysates and the equilibrium binding constant for the interaction in vitro. We demonstrate through RNA interference that the labeling site is dependent on formation of the protein:protein complex in cells. Further, we identify that active-site-directed, small-molecule inhibitors of MK2/3 selectively inhibit the heterodimer-dependent probe labeling, whereas p38α inhibitors do not. These findings afford a new method to evaluate p38α and MK2/3 inhibitors within native biological systems and a new tool for improved understanding of p38α signaling pathways

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 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

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

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

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

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

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