The interaction of a drug with its target is critical to achieve drug efficacy. In cases where cellular environment influences target engagement, differences between individuals and cell types ...present a challenge for
prediction of drug efficacy. As such, characterization of environments conducive to achieving the desired pharmacologic outcome is warranted. We recently reported that the clinical CDK4/6 inhibitor palbociclib displays cell type-specific target engagement: Palbociclib engaged CDK4 in cells biologically sensitive to the drug, but not in biologically insensitive cells. Here, we report a molecular explanation for this phenomenon. Palbociclib target engagement is determined by the interaction of CDK4 with CDKN2A, a physiologically relevant protein inhibitor of CDK4. Because both the drug and CDKN2A prevent CDK4 kinase activity, discrimination between these modes of inhibition is not possible by traditional kinase assays. Here, we describe a chemo-proteomics approach that demonstrates high CDK4 target engagement by palbociclib in cells without functional CDKN2A and attenuated target engagement when CDKN2A (or related CDKN2/INK4 family proteins) is abundant. Analysis of biological sensitivity in engineered isogenic cells with low or absent CDKN2A and of a panel of previously characterized cell lines indicates that high levels of CDKN2A predict insensitivity to palbociclib, whereas low levels do not correlate with sensitivity. Therefore, high CDKN2A may provide a useful biomarker to exclude patients from CDK4/6 inhibitor therapy. This work exemplifies modulation of kinase target engagement by endogenous proteinaceous regulators and highlights the importance of cellular context in predicting inhibitor efficacy.
The central role of protein kinases in signal transduction pathways has generated intense interest in targeting these enzymes for a wide range of therapeutic indications. Here we report a method for ...identifying and quantifying protein kinases in any biological sample or tissue from any species. The procedure relies on acyl phosphate-containing nucleotides, prepared from a biotin derivative and ATP or ADP. The acyl phosphate probes react selectively and covalently at the ATP binding sites of at least 75% of the known human protein kinases. Biotinylated peptide fragments from labeled proteomes are captured and then sequenced and identified using a mass spectrometry-based analysis platform to determine the kinases present and their relative levels. Further, direct competition between the probes and inhibitors can be assessed to determine inhibitor potency and selectivity against native protein kinases, as well as hundreds of other ATPases. The ability to broadly profile kinase activities in native proteomes offers an exciting prospect for both target discovery and inhibitor selectivity profiling.
We describe the identification of a novel, tumor-specific missense mutation in the active site of casein kinase 1α (CSNK1A1) using activity-based proteomics. Matched normal and tumor colon samples ...were analyzed using an ATP acyl phosphate probe in a kinase-targeted LC-MS2 platform. An anomaly in the active-site peptide from CSNK1A1 was observed in a tumor sample that was consistent with an altered catalytic aspartic acid. Expression and analysis of the suspected mutant verified the presence of asparagine in the probe-labeled, active-site peptide for CSNK1A1. Genomic sequencing of the colon tumor samples confirmed the presence of a missense mutation in the catalytic aspartic acid of CSNK1A1 (GAC→AAC). To our knowledge, the D163N mutation in CSNK1A1 is a newly defined mutation to the conserved, catalytic aspartic acid of a protein kinase and the first missense mutation identified using activity-based proteomics. The tumorigenic potential of this mutation remains to be determined.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Protein kinases are intensely studied mediators of cellular signaling, yet important questions remain regarding their regulation and in vivo properties. Here, we use a probe-based chemoprotemics ...platform to profile several well studied kinase inhibitors against >200 kinases in native cell proteomes and reveal biological targets for some of these inhibitors. Several striking differences were identified between native and recombinant kinase inhibitory profiles, in particular, for the Raf kinases. The native kinase binding profiles presented here closely mirror the cellular activity of these inhibitors, even when the inhibition profiles differ dramatically from recombinant assay results. Additionally, Raf activation events could be detected on live cell treatment with inhibitors. These studies highlight the complexities of protein kinase behavior in the cellular context and demonstrate that profiling with only recombinant/purified enzymes can be misleading.
► Chemoproteomics method allows quantitative profiling of >150 kinases per cell type ► Reports binding constants for ATP, Staurosporine, and five advanced kinase inhibitors ► Identifies MAP2K5 as a novel target of dasatinib and verifies this result in cellular assays ► Native kinase profiling capable of detecting inhibitor induced kinase activation ► Differences were observed between recombinant and native Raf kinase inhibition ► Native Raf kinase binding correlates well with in vivo activity of Raf inhibitors
Abstract
GTPases constitute an important class of regulatory proteins that participate as signal transducers in the capacity of molecular switches. The GTPase superfamily consists of 250 members in ...the human genome. These fall into several families, including the small GTPases (Ras Rho, Arf, Rab and Ran), heterotrimeric GTPases, elongation and initiation factors, and dynamins. In order to profile the distribution of GTPases in various cell lines and tissues, we have developed a chemical probe comprised of GTP linked to desthiobiotin through an acyl-phosphate bond. By treating complex proteomes with the probe, we are able to isolate probe-labeled peptides with streptavidin, and subsequently, identify the probe-labeled peptides by mass spectrometry. GTPases were identified from almost all known GTPase families indicating that this approach can be used to globally profile GTPases in various proteomes. The most common labeling site occurs at the conserved lysine residue in the phosphate binding loop (GXXXXGK) of the GTP-binding site. Significantly, the probe-labeled peptide for H-,K- and N-RAS includes amino acid positions 12 and 13, which are frequently mutated in cancers. Thus, the GTP probe can be used to directly quantify mutant RAS isoforms at the protein level.
Citation Format: Tyzoon K. Nomanbhoy, Ann Shih, Arwin Aban. Profiling GTPases in native proteomes with a GTP acyl phosphate probe. abstract. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5167. doi:10.1158/1538-7445.AM2013-5167
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.
Acyl phosphates of ATP (ATPAc) and related nucleotides have proven to be useful for the interrogation of known nucleotide binding sites via specific acylation of conserved lysines (K). In addition, ...occasional K acylations are identified in proteins without such known sites. Here we present a robust and specific acylation of procaspase-6 by ATPAc at K133 in Jurkat cell lysates. The K133 acylation is dependent on π–π stacking interactions between the adenine moiety of ATPAc and a conserved Y198–Y198 site formed at the homodimeric interface of procaspase-6. Significantly, the Y198A mutation in procaspase-6 abolishes K133 acylation but has no effect on the proteolytic activity of the mature, active caspase-6 Y198A variant. Additional in vitro studies show that ATP can inhibit the autoproteolytic activation of procaspase-6. These observations suggest that ATP, and possibly other nucleotides, may serve as the endogenous ligands for the allosteric site at the procaspase-6 dimer interface, a site that has persisted in its “orphan” status for more than a decade.
Abstract
Palbociclib (PD0332991) is a cyclin dependent kinase 4 and 6 inhibitor being developed for treatment of breast cancer. In order to evaluate the kinome-wide impacts of Palbociclib in an ...in-vivo setting, nude mice inoculated with COLO-205 (human colon carcinoma) cells were administered Palbociclib at different doses (12.5, 75 and 150mg/kg once daily for 3 days. Tumors were collected at various time points after the final dose (1, 3, 6, 12, 24 & 48 hours), lysed and subjected to in-situ kinase profiling using the KinativTM technology. Tumor lysates were probed with an ATP acylphosphate desthiobiotin probe which covalently modifies lysine residues in the kinase active site. Samples are then denatured, trypsinized, and the probe-labeled peptides captured with streptavidin, eluted, and quantitated by targeted mass spectrometry. The presence of an inhibitor bound to the active site of a kinase prevents the probe binding, resulting in the loss of MS signal for peptides from that kinase. In addition, the upregulation or downregulation of kinase proteins can also result in changes in the MS signals in palbociclib versus vehicle-treated samples. A total of 180 kinases were quantified. As expected, direct targets of palbociclib were observed to be inhibited in this study (i.e., kinases inhibited by Palbociclib in Colo205 lysate), including CDK4 &6 as well as ERK5, JNK, CDK16 &17, PIK3C3 and PIP4K2C. The observed inhibition of these targets was both dose and time dependent. Significantly, the MS signals for additional kinases not observed to directly bind Palbociclib in Colo205 lysate were also strongly downregulated in this study. These include CDC2, AurA, AurB, PLK1 and MASTL; kinases critical for cell cycle progression. We hypothesize that the downregulation of these kinases was due to cell cycle arrest in G1 phase. Consistent with this hypothesis, the downregulation of these kinases corresponded well with the inhibition of phosphorylated (Ser780; pRb) as demonstrated by Western Blots of a part of tumor tissue used for the Kinome profiling. Taken together, these results highlight the utility of kinome wide profiling in tumor samples collected from xenograft mice to better understand both pharmacodynamics of target engagement as well as pathway effects elicited by compound treatment.
Citation Format: Geeta Sharma, Subha Vogeti, Wendy Grant, Shuzhen Wu, Christa Dias, Tyzoon Nomanbhoy, Jiangyue Wu, Arwin Aban. In-situ Kinome wide profiling of Palbociclib treated COLO-205 human tumor xenograft samples. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3092. doi:10.1158/1538-7445.AM2015-3092
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