Background A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. Better understanding of the molecular mechanisms leading to platelet activation is of importance for the development of improved therapies. Recently, protein tyrosine phosphatases (PTPs) have emerged as critical regulators of platelet function. Methods and Results This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated through the collagen receptor glycoprotein VI (GPVI) and the C-type lectin-like receptor 2 (CLEC-2). DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism, compared to wild-type mice, and showed severely impaired thrombus formation upon ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of PLCγ2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen and CLEC-2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. Conclusions DUSP3 plays a selective and essential role in collagen- and CLEC-2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a PTP, implicated in platelet signaling, has been targeted with a small-molecule drug.

We have studied the in vitro elongation and termination properties of several yeast RNA polymerase III (pol III) mutant enzymes that have altered in vivo termination behavior (S. A. Shaaban, B. M. Krupp, and B. D. Hall, Mol. Cell. Biol. 15:1467-1478, 1995). The pattern of completed-transcript release was also characterized for three of the mutant enzymes. The mutations studied occupy amino acid regions 300 to 325, 455 to 521, and 1061 to 1082 of the RET1 protein (P. James, S. Whelen, and B. D. Hall, J. Biol. Chem. 266:5616-5624, 1991), the second largest subunit of yeast RNA pol III. In general, mutant enzymes which have increased termination require a longer time to traverse a template gene than does wild-type pol III; the converse holds true for most decreased-termination mutants. One increased-termination mutant (K310T I324K) was faster and two reduced termination mutants (K512N and T455I E478K) were slower than the wild-type enzyme. In most cases, these changes in overall elongation kinetics can be accounted for by a correspondingly longer or shorter dwell time at pause sites within the SUP4 tRNA Tyr gene. Of the three mutants analyzed for RNA release, one (T455I) was similar to the wild type while the two others (T455I E478K and E478K) bound the completed SUP4 pre-tRNA more avidly. The results of this study support the view that termination is a multistep pathway in which several different regions of the RET1 protein are actively involved. Region 300 to 325 likely affects a step involved in RNA release, while the Rif homology region, amino acids 455 to 521, interacts with the nascent RNA 3 end. The dual effects of several mutations on both elongation kinetics and RNA release suggest that the protein motifs affected by them have multiple roles in the steps leading to transcription termination.The two largest subunits of the DNA-dependent RNA polymerases from eubacteria, archaebacteria, and the eukaryotic nucleus are structurally similar (3, 49). Functional roles in RNA synthesis have been assigned to these subunits on the basis of affinity labeling with nucleotide substrates, DNA templates, and nascent RNA products. The binding pocket for the nucleoside triphosphate (NTP) substrate spans both subunits (18,19,40,44,55). Both subunits also seem to make up the protein surfaces that contact the DNA template (5, 16) and the nascent RNA product (6,14,20,32,45). Binding interactions between these two subunits and the DNA and RNA components of the transcription complex most likely contribute to the extreme stability and remarkable processivity of the ternary complex. At certain sequences, known as intrinsic termination sites, this stability is markedly diminished, with the result that RNA is released from the complex and RNA polymerase dissociates from the DNA. The most studied intrinsic termination sequences for Escherichia coli RNA polymerase give rise to short hairpin loops in the transcript followed by short oligo(U) sequences at the 3Ј RNA terminus (15).To terminate transcription by RNA polymerase III (pol III)...

Highlights d We identified an allosteric inhibitory site for an activating enzyme d The lead compound inhibits the ATP-dependent step of SUMO E1 catalysis d The compound has specificity to 1 out of 18 non-disulfide bonded Cys residues d The compound increased miR-34b and reduced c-Myc in cellular and xenograft models

Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.PDK1 (phosphoinositide-dependent kinase-1) was first identified as a protein serine/threonine kinase that linked phosphatidylinositol 3-kinase (PI3K) to AKT (protein kinase B) activation in response to growth factor receptor signaling (1, 2). Growth factor binding to receptor tyrosine kinases (RTKs) 3 results in activated PI3K, which phosphorylates the 3Ј-position of the inositol ring in phosphatidylinositol 4,5-bisphosphate to produce the second messenger phosphatidylinositol 3,4,5-trisphosphate (3). Membrane-bound phosphatidylinositol 3,4,5-trisphosphate recruits AKT to the plasma membrane, where it co-localizes with PDK1 in a pleckstrin homology domain-dependent manner (4 -6). The binding of phosphatidylinositol 3,4,5-trisphosphate to AKT induces a conformational shift that alleviates AKT autoinhibition (7) and allows for PDK1-mediated phosphorylation of AKT Thr-308, an event that is absent in both PDK1 null mouse embryonic stem (ES) cells (8) and tissue-specific PDK1 knock-out mice (9). In parallel with the elucidation of the above PI3K/ PDK1/AKT signaling cascade, PDK1 has been shown to phosphorylate the conserved threonine/serine residue in the activation loop (T-loop) of about 20 related protein kinases (10). Because this phosphorylation event is a prerequisite for full catalytic activity...

The phosphoinositide 3-kinase/AKT signaling pathway plays a key role in cancer cell growth, survival, and angiogenesis. Phosphoinositide-dependent protein kinase-1 (PDK1) acts at a focal point in this pathway immediately downstream of phosphoinositide 3-kinase and PTEN, where it phosphorylates numerous AGC kinases. The PDK1 kinase domain has at least three ligand-binding sites: the ATP-binding pocket, the peptide substrate-binding site, and a groove in the N-terminal lobe that binds the C-terminal hydrophobic motif of its kinase substrates. Based on the unique PDK1 substrate recognition system, ultrahigh throughput TR-FRET and Alphascreen screening assays were developed using a biotinylated version of the PDK1-tide substrate containing the activation loop of AKT fused to a pseudo-activated hydrophobic motif peptide. Using full-length PDK1, K m values were determined as 5.6 M for ATP and 40 nM for the fusion peptide, revealing 50-fold higher affinity compared with the classical AKT(Thr-308)-tide. Kinetic and biophysical studies confirmed the PDK1 catalytic mechanism as a rapid equilibrium random bireactant reaction. Following an ultrahigh throughput screen of a large library, 2,000 compounds were selected from the reconfirmed hits by computational analysis with a focus on novel scaffolds. ATP-competitive hits were deconvoluted by dose-response studies at 1؋ and 10؋ K m concentrations of ATP, and specificity of binding was assessed in thermal shift assay. Inhibition studies using fusion PDK1-tide1 substrate versus AKT(Thr-308)-tide and kinase selectivity profiling revealed a novel selective alkaloid scaffold that evidently binds to the PDK1-interacting fragment pocket. Molecular modeling suggests a structural paradigm for the design of inhibitory versus activating allosteric ligands of PDK1.Protein kinases are the second largest group of drug targets after G-protein-coupled receptors. They account for an estimated 20 -30% of drug development pipelines and comprise the largest enzyme family, with more than 500 members encoded in the human genome (1, 2). It is estimated that protein kinases catalyze the reversible phosphorylation of more than 10,000 substrate proteins at greater than 100,000 sites (3, 4). Consequently, malfunctioning of kinases can have profound effects on human health, and the discovery of selective kinase inhibitors is critical in helping to delineate the role of these enzymes in disease processes.In cancer, oncogenic transformations are frequently associated with increased activity of protein-serine/threonine kinases, many of which are key signaling molecules in the phosphoinositide 3-kinase and mitogen-activated protein kinase (MAPK) 2 pathways. Recent clinical studies have demonstrated that activating mutations in the MAPK pathway (i.e. KRAS and BRAF) confer resistance to anti-epidermal growth factor receptor antibody therapy (5, 6), suggesting that co-targeting of receptor tyrosine kinases and downstream serine/threonine kinases may be an attractive therapeutic strategy.The serine/threonine pr...

Using yeast RNA polymerase III ternary complexes stalled at various positions on the template, we have analyzed the cleavage products that are retained and released by the transcription complexes. The retained 5 products result from cleavage at uridine residues during retraction, whereas the yield of mononucleotides and dinucleotides released indicates that multiple cuts occur near the 3 end. Comparison of the cleavage patterns of uridine-containing and 5-bromouridine-containing transcripts suggests that RNA within an RNA-DNA hybrid duplex is the substrate for the 3-5 exonuclease. During transcription of the SUP4 tRNA Tyr gene, RNA polymerase III produces not only full-length pre-tRNA Tyr but also short oligonucleotides, indicating that exonuclease digestion and transcription are concurrent processes. To explore the possibility that these oligonucleotides are released by the action of the RNA polymerase III nuclease at previously observed uridinerich pause sites, we tested modified templates lacking the arrest sites present in the SUP4 tRNA Tyr gene. Comparative studies of cleavage during transcription for these templates show a direct correlation between the number of natural pause sites and the yield of 3 products made. At the natural arrest sites and the terminator, RNA polymerase III carries out multiple cleavage resynthesis steps, producing short oligoribonucleotides with uridine residues at the 3 terminus.A hydrolytic activity that effectively reverses the course of gene transcription has been found for Escherichia coli RNA polymerase, eukaryotic RNA polymerases I, II, and III, and the vaccinia virus RNA polymerase (1-9). In the first three cases a separate protein co-factor serves to activate the nuclease, whereas for RNA polymerase III (Pol III) 1 and the vaccinia polymerase no such co-factor is known. In attributing a functional role to the transcription factor IIS-stimulated nuclease activity of Pol II, major emphasis has been placed upon the role that nucleolytic retraction plays in overcoming the arrest of elongation at certain template positions (10 -12) that are rich in adenosine residues in the template DNA strand (13-16). In the only previous report of an intrinsic nuclease in RNA polymerase III, Whitehall, Bardeleben, and Kassavetis (8) noted the excision of dinucleotide cleavage products when transcription was halted within the sequence 5Ј-UCUC in a yeast tRNA Tyr gene. In this paper we have extended the study of pol III nuclease activity to elongation complexes stalled within a number of different sequence contexts. For several of these we characterized both products of nuclease cutting, the released 3Ј oligonucleotides as well as the shortened 5Ј transcript. An examination of the respective 3Ј product sizes relative to that of the RNA transcript underscores a surprising dilemma. More than one oligonucleotide fragment must have been produced per surviving 5Ј fragment to satisfy conservation of mass. That is, during conversion of the initial RNA into the first large product observed, the nuclea...

PDK1 activates AKT suggesting that PDK1 inhibition might suppress tumor development. However, while PDK1 has been investigated intensively as an oncology target, selective inhibitors suitable for in vivo studies have remained elusive. In this study we present the results of in vivo PDK1 inhibition through a universally applicable RNAi approach for functional drug target validation in oncogenic pathway contexts. This approach, which relies on doxycycline-inducible shRNA expression from the Rosa26 locus, is ideal for functional studies of genes like PDK1 where constitutive mouse models lead to strong developmental phenotypes or embryonic lethality. We achieved more than 90% PDK1 knockdown in vivo, a level sufficient to impact physiological functions resulting in hyperinsulinemia and hyperglycemia. This phenotype was reversible on PDK1 reexpression. Unexpectedly, longterm PDK1 knockdown revealed a lack of potent antitumor efficacy in 3 different mouse models of PTENdeficient cancer. Thus, despite efficient PDK1 knockdown, inhibition of the PI3K pathway was marginal suggesting that PDK1 was not a rate limiting factor. Ex vivo analysis of pharmacological inhibitors revealed that AKT and mTOR inhibitors undergoing clinical development are more effective than PDK1 inhibitors at blocking activated PI3K pathway signaling. Taken together our findings weaken the widely held expectation that PDK1 represents an appealing oncology target. Cancer Res; 71(8); 3052-65. Ó2011 AACR.

Kinetic analysis of ribosomal peptidyltransferase activity in a methanolic puromycin reaction with wild type and drug-resistant 23 S RNA mutants was used to probe the structural basis of catalysis and mechanism of resistance to antibiotics. 23 S RNA mutants G2032A and G2447A are resistant to oxazolidinones both in vitro and in vivo with the latter displaying a 5-fold increase in the value of K m for initiator tRNA and a 100-fold decrease in V max in puromycin reaction. Comparison of the K i values for oxazolidinones, chloramphenicol, and sparsomycin revealed partial cross-resistance between oxazolidinones and chloramphenicol; no cross-resistance was observed with sparsomycin, a known inhibitor of the peptidyltransferase A-site. Inhibition of the mutants using a truncated CCA-Phe-X-Biotin fragment as a P-site substrate is similar to that observed with the intact initiator tRNA, indicating that the inhibition is substrateindependent and that the peptidyltransferase itself is the oxazolidinone target. Mapping of all known mutations that confer resistance to these drugs onto the spatial structure of the 50 S ribosomal subunit allows for docking of an oxazolidinone into a proposed binding pocket. The model suggests that oxazolidinones bind between the P-and A-loops, partially overlapping with the peptidyltransferase P-site. Thus, kinetic, mutagenesis, and structural data suggest that oxazolidinones interfere with initiator fMet-tRNA binding to the P-site of the ribosomal peptidyltransferase center.Oxazolidinones, the only novel class of antibiotics identified in the last two decades, are the focus of intensive discovery efforts (1-9). Linezolid, an oxazolidinone, is approved for treatment of infections caused by Gram-positive bacteria that are resistant to other antibiotics. Emerging resistance to all known drugs, including the "last resort" vancomycin family, poses a serious threat to the public health worldwide. Understanding the mechanism of action of oxazolidinones at the molecular level, therefore, has a great importance for the development of the next generation of these novel antibiotics and, ultimately, for the outcome of the ongoing battle against drug-resistant pathogens.Oxazolidinones impose their action at the initiation stage of translation (4, 5), apparently via inhibition of preinitiation complex formation (9). Our recent finding that oxazolidinones interfere with binding of initiator tRNA to the ribosomal P-site (1), thus inhibiting formation of the first peptide bond, prompted a search for similarities between oxazolidinones and known inhibitors of peptidyltransferase. To address this and other mechanistic questions, we have studied catalytic properties of oxazolidinone-resistant ribosomes and compared the mechanism of oxazolidinone inhibition with the action of known peptidyltransferase inhibitors, such as chloramphenicol and sparsomycin. To further define the oxazolidinone binding site, we have mapped resistant mutations onto the three-dimensional structure of the ribosomal 50 S subunit to reveal a...