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)

Early clinical PET imaging results with the novel PHF-tau radioligand [F18]-T808.

Aggregates of hyperphosphorylated tau (PHF-tau), such as neurofibrillary tangles, are linked to the degree of cognitive impairment in Alzheimer's disease. We have recently reported early clinical results of a novel PHF-tau targeting PET imaging agent, [F18]-T807. Since then, we have investigated a second novel PHF-tau targeting PET imaging agent, [F18]-T808, with different pharmacokinetic characteristics, which may be favorable for imaging Alzheimer's disease and other tauopathies. Here, we describe the first human brain images with [F18]-T808

Early clinical PET imaging results with the novel PHF-tau radioligand [F18]-T808.

Aggregates of hyperphosphorylated tau (PHF-tau), such as neurofibrillary tangles, are linked to the degree of cognitive impairment in Alzheimer's disease. We have recently reported early clinical results of a novel PHF-tau targeting PET imaging agent, [F18]-T807. Since then, we have investigated a second novel PHF-tau targeting PET imaging agent, [F18]-T808, with different pharmacokinetic characteristics, which may be favorable for imaging Alzheimer's disease and other tauopathies. Here, we describe the first human brain images with [F18]-T808