In Situ Kinase Profiling Reveals Functionally Relevant Properties of Native Kinases

SummaryProtein 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

High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity

Expression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. Human ASNS is therefore emerging as a bona fide drug target for cancer therapy. Here we show that a slow-onset, tight binding inhibitor, which exhibits nanomolar affinity for human ASNS in vitro, exhibits excellent selectivity at 10 μM concentration in HCT-116 cell lysates with almost no off-target binding. The high-resolution (1.85 Å) crystal structure of human ASNS has enabled us to identify a cluster of negatively charged side chains in the synthetase domain that plays a key role in inhibitor binding. Comparing this structure with those of evolutionarily related AMP-forming enzymes provides insights into intermolecular interactions that give rise to the observed binding selectivity. Our findings demonstrate the feasibility of developing second generation human ASNS inhibitors as lead compounds for the discovery of drugs against metastasis

Functional Interrogation of the Kinome Using Nucleotide Acyl Phosphates

ABSTRACT: 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. Protein kinases represent the single largest mammalian enzyme family with more than 500 members in the human proteome. These enzymes have been implicated in a wide array of complex cellular functions and pathways, ranging from metabolic regulation to tumorigenesis. Assessing kinase function in vivo is complicated by a high degree of posttranslational regulation, generally low expression levels, and overlapping substrate selectivity. Thus, despite intense efforts, the physiological function of the majority of protein kinases remains unknown. Considerable effort in the pharmaceutical industry is currently directed at the generation of novel protein kinase inhibitors (1). Several kinase inhibitors have been approved for clinical indications, including the anticancer drugs Gleevec (2) and Iressa (3). These drugs, as well as the majority of other kinase inhibitors in development, are designed to bind to the kinase ATP-binding site, thereby preventing substrate phosphorylation. Since the ATP-binding sites of protein kinases are highly conserved, the identification of inhibitors that are both potent and selective is paramount. At present, even the most comprehensive kinase selectivity screens cover less than 30% of the predicted protein kinases, leaving the majority of kinases unexplored. Recent reports have demonstrated that unexpected activities of protein kinase inhibitors can be found in kinase families structurally remote from the primary target (4, 5), highlighting the need for comprehensive screening. Additionally, due to the high degree of post-translational regulation of kinase activity, it is not clear whether assays of recombinant enzymes address the physiologically relevant state of each kinase. These observations, and the fact that many other families of ATP binding proteins exist with potential affinity for protein kinase inhibitors, highlight the need for improved methods for the comprehensive screening of protein kinases and other ATP binding proteins. Here we describe a probe-based technology (6, 7) that is uniquely capable of profiling the selectivity of kinase inhibitors against a broad range of protein kinases and other nucleotide binding proteins directly in native proteomes. This method also enables researchers to identify protein kinases and other ATPases with altered expression or activity in human disease or cell models. The method is based on novel biotinylated acyl phosphates of ATP or ADP that irreversibly react with protein kinases on conserved lysine residues in the ATP binding pocket. To date, more than 400 different protein kinases (>80% of the predicted kinome) have been identified and, in most cases, functionally assayed in various mammalian tissues and cell lines using this method. MATERIALS AND METHODS Synthesis of (+)-Biotin-Hex-Acyl-ATP (BHAcATP). 1 To a stirred suspension of N-(+)-biotinyl-6-aminohexanoic acid (30 mg, 0.085 mmol) in 3 mL of a dioxane/DMF/DMSO mixture (1:1:1) were added triethylamine (47 µL, 0.34 mmol) and isobutyl chloroformate (33 µL, 0.255 mmol) at 0°C. The cloudy mixture was kept at that temperature for 20 min, allowed to warm up to room temperature, and then stirred for an additional 1.5 h. A solution of ATP triethylammonium salt (69 mg, 0.085 mmol) in anhydrous DMSO (1 mL) was added to the mixture described above to give a clear solution. The reaction was monitored by 31 P NMR and MALDI. After 18 h, the reaction was quenched with water (4 mL) and the solution quickly extracted with ethyl acetate (3 × 4 mL)

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