Polo-like kinases (PLKs) play critical roles throughout mitosis. Here, we report that wortmannin, which was previously thought to be a highly selective inhibitor of phosphoinositide (PI) 3-kinases, is a potent inhibitor of mammalian PLK1. Observation of the wortmannin-PLK1 interaction was enabled by a tetramethylrhodamine-wortmannin conjugate (AX7503) that permits rapid detection of PLK1 activity and expression in complex proteomes. Importantly, we show that wortmannin inhibits PLK1 activity in an in vitro kinase assay with an IC(50) of 24 nM and when incubated with intact cells. Taken together, our results indicate that, at the concentrations of wortmannin commonly used to inhibit PI 3-kinases, PLK1 is also significantly inhibited.

Characterization and functional annotation of the large number of proteins predicted from genome sequencing projects poses a major scientific challenge. Whereas several proteomics techniques have been developed to quantify the abundance of proteins, these methods provide little information regarding protein function. Here, we present a gel-free platform that permits ultrasensitive, quantitative, and high-resolution analyses of protein activities in proteomes, including highly problematic samples such as undiluted plasma. We demonstrate the value of this platform for the discovery of both disease-related enzyme activities and specific inhibitors that target these proteins.capillary electrophoresis ͉ fluorescence ͉ MS ͉ protease T he completion of several major genome sequencing projects has both accelerated the pace and broadened the scale of modern biology. For example, it is estimated that a complete molecular understanding of the human organism will require the characterization of at least 100,000 proteins (1). Several innovations in the field of proteomics have advanced to meet the demands of postgenome biology. Particularly, improvements in the resolving capacity and reproducibility of 2D gel electrophoresis (2) and the increased sensitivity and accuracy of modern MS methods (3) have enabled the global characterization of protein expression levels in complex biological systems. Nonetheless, the large dynamic range of protein expression in biological tissues and fluids renders these methods ineffective at detecting and͞or quantitating low-abundance proteins in the absence of labor-intensive prefractionation or enrichment procedures (2, 4). Moreover, abundance-based proteomic strategies fail to provide direct information regarding protein function or activity.To address these limitations, a chemical proteomic technology called activity-based protein profiling (ABPP) (5) has emerged that utilizes active site-directed probes to enable the parallel measurement of the activity of many enzymes in complex proteomic mixtures (5-8). Activity-based probes (ABPs) enable the detection of changes in enzyme activity independent of alterations in protein abundance, and provide a specific enrichment strategy for low-copy-number enzymes without interference from more abundant proteins. To date, ABPP has relied on gel-based methods for proteome analysis that are difficult to automate and often fail to resolve highly related protein species (5, 6, 9, 10). Here, we describe a gel-free platform for ABPP, termed Xsite, that permits the rapid and systematic quantification and identification of enzyme activities in complex protein mixtures. Materials and MethodsGeneral Materials and Methods. Fluorophosphonate probes were synthesized as described (5). Synthesis of the cathepsin probe, AX6429, is detailed in supplemental materials (Fig. 3, which is published as supporting information on the PNAS web site). Rat FAAH was expressed in Escherichia coli and purified as described (11). Porcine trypsin and equine butyrylcholinesterase were p...

are reported. X-ray diffraction studies of the dihydroxylated and decaalkyl derivatives 2 and 3 reveal that the diprotonated forms of sapphyrin are capable of stabilizing 1:2 inner-sphere complexes with phosphate-derived monoanions, such as diphenyl phosphate and monobasic phenyl phosphate. Similar analyses reveal that the diprotonated form of dihydroxysapphyrin 2 is capable of forming a 1:1 chelate complex in the solid state with either mono-or dibasic phosphoric acid. Solution-phase studies, involving 1 H and 31 P NMR spectroscopy, confirm that these same sapphyrins are capable of binding phosphate anions in organic solution, a conclusion that is supported by qualitative fast atom bombardment mass spectrometric (FAB MS) and extractive partition studies. In the case of phenylphosphonic acid and sapphyrin 2, extraction studies were consistent with 2:1 and 1:1 phosphate-to-sapphyrin binding stochiometries at pH 1.68 and 5.6, respectively. Similar studies using NMR and visible spectroscopy carried out with the water-soluble tetrahydroxy sapphyrin derivative, 1, and 2 indicate that these species bind phosphate anion in both methanolic and aqueous solution. Calculated association constants are on the order of 10 4 M -1 in methanol and 10 2 M -1 in 10 mM aqueous bis-Tris, pH 6.1.

A model for the interaction of the water-soluble sapphyrin derivative 1 with a variety of nucleic acid species is presented. Three modes of interaction are described: The first mode, seen with all the nucleic acid species, is that of "phosphate chelation". This mode is exemplified by a solid state structure of the complex formed between the monobasic form of cAMP and the sapphyrin species [2H‚2] 2+ . It involves the specific chelation of the oxyanion of a phosphorylated nucleotide or nucleic acid species with the protonated core of sapphyrin Via Coulombic interactions that include H-bonding interactions. Spectroscopically, this interaction is characterized by a visible absorption at 422 nm and corresponds to complexes formed between the dimeric form of 1 and phosphorylated nucleotides. In the case of double-stranded DNA, this mode of binding shows a preference for the more flexible copolymer [poly-(dA-dT)] 2 over [poly(dG-dC)] 2 . The second mode involves a hydrophobic interaction with the nucleobases present in both monomeric and single-stranded polymeric nucleotides. Spectroscopically, this nucleotide-dependent interaction is characterized by the absorption of the monomeric species of 1 at ca. 450 nm. The third mode involves the highly ordered aggregation of 1 on the surface of certain double-stranded, helical nucleic acids at low phosphate ester to sapphyrin (P/S) ratios and is templated by the higher order structure of these nucleic acid polymers. Spectroscopically, this mode is characterized by a visible absorption at ca. 400 nm and a large, conservative induced CD signal for 1.

No abstract