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BBA - Proteins and Proteomics (v.1804, #3)

Editorial Board (pp. i).

Inhibitors of protein kinases by David Shugar (pp. 427-428).

Insights into protein kinase regulation and inhibition by large scale structural comparison by Jeyanthy Eswaran; Stefan Knapp (pp. 429-432).

Protein structure determination of soluble globular protein domains has developed into an efficient routine technology which can now be applied to generate and analyze structures of entire human protein families. In the kinase area, several kinase families still lack comprehensive structural analysis. Nevertheless, Structural Genomics (SG) efforts contributed more than 40 kinase catalytic domain structures during the past 4 years providing a rich resource of information for large scale comparisons of kinase active sites. Moreover, many of the released structures are inhibitor complexes that offer chemical starting points for development of selective and potent inhibitors. Here we discuss the currently available structural data and strategies that can be utilized for the development of highly selective inhibitors.

Keywords: Protein kinase; Structure based drug design; X-ray crystallography; Atypical kinase; Kinase regulation

Dissecting the role of mTOR: Lessons from mTOR inhibitors by Ryan J.O. Dowling; Ivan Topisirovic; Bruno D. Fonseca; Nahum Sonenberg (pp. 433-439).

Recent years have observed significant advances in our understanding of how the serine/threonine kinase target of rapamycin (TOR) controls key cellular processes such as cell survival, growth and proliferation. Consistent with its role in cell proliferation, the mTOR pathway is frequently hyperactivated in a number of human malignancies and is thus considered to be an attractive target for anti-cancer therapy. Rapamycin and its analogs (rapalogs) function as allosteric inhibitors of mTORC1 and are currently used in the treatment of advanced renal cell carcinoma. Rapamycin and its derivatives bind to the small immunophilin FKBP12 to inhibit mTORC1 signalling through a poorly understood mechanism. Rapamycin/FKBP12 efficiently inhibit some, but not all, functions of mTOR and hence much interest has been placed in the development of drugs that target the kinase activity of mTOR directly. Several novel active-site inhibitors of mTOR, which inhibit both mTORC1 and mTORC2, were developed in the last year. In this manuscript, we provide a brief outline of our current understanding of the mTOR signalling pathway and review the molecular underpinnings of the action of rapamycin and novel active-site mTOR inhibitors as well as potential advantages and caveats associated with the use of these drugs in the treatment of cancer.

Keywords: Mammalian target of rapamycin (mTOR); S6 kinases (S6Ks); eIF4E-binding proteins (4E-BPs); Eukaryotic translation initiation factor eIF4E (eIF4E); Rapamycin; Active-site mTOR inhibitor; Translation

Defining the conserved internal architecture of a protein kinase by Alexandr P. Kornev; Susan S. Taylor (pp. 440-444).

Protein kinases constitute a large protein family of important regulators in all eukaryotic cells. All of the protein kinases have a similar bilobal fold, and their key structural features have been well studied. However, the recent discovery of non-contiguous hydrophobic ensembles inside the protein kinase core shed new light on the internal organization of these molecules. Two hydrophobic “spines” traverse both lobes of the protein kinase molecule, providing a firm but flexible connection between its key elements. The spine model introduces a useful framework for analysis of intramolecular communications, molecular dynamics, and drug design.

Keywords: Protein kinase; Structure; Phosphorylation; Hydrophobic motifs

Extended kinase profile and properties of the protein kinase inhibitor nilotinib by Paul W. Manley; Peter Drueckes; Gabriele Fendrich; Pascal Furet; Janis Liebetanz; Georg Martiny-Baron; Jürgen Mestan; Jörg Trappe; Markus Wartmann; Doriano Fabbro (pp. 445-453).

As a drug used to treat imatinib-resistant and -intolerant, chronic and advanced phase chronic myelogenous leukaemia, nilotinib is well characterised as a potent inhibitor of the Abl tyrosine kinase activity of wild-type and imatinib-resistant mutant forms of BCR-Abl. Here we review the profile of nilotinib as a protein kinase inhibitor. Although an ATP-competitive inhibitor of Abl, nilotinib binds to a catalytically inactive conformation (DFG-out) of the activation loop. As a consequence of this, nilotinib exhibits time-dependent inhibition of Abl kinase in enzymatic assays, which can be extrapolated to other targets to explain differences between biochemical activity and cellular assays. Although these differences confound assessment of kinase selectivity, as assessed using a combination of protein binding and transphosphorylation assays, together with cellular autophosporylation and proliferation assays, well established kinase targets of nilotinib in rank order of inhibitory potency are DDR-1>DDR-2>BCR-Abl (Abl)>PDGFRα/β>KIT>CSF-1R. In addition nilotinib has now been found to bind to both MAPK11 (p38β) and MAPK12 (p38α), as well as with very high affinity to ZAK kinase. Although neither enzymatic nor cellular data are yet available to substantiate the drug as an inhibitor of ZAK phosphorylation, modeling predicts that it binds in an ATP-competitive fashion.

Keywords: Nilotinib; Imatinib; Abl kinase; ZAK kinase; Chronic leukaemia; Protein kinase assay

Inhibitors of the Abl kinase directed at either the ATP- or myristate-binding site by Doriano Fabbro; Paul W. Manley; Wolfgang Jahnke; Janis Liebetanz; Alexandra Szyttenholm; Gabriele Fendrich; Andre Strauss; Jianming Zhang; Nathanael S. Gray; Francisco Adrian; Markus Warmuth; Xavier Pelle; Robert Grotzfeld; Frederic Berst; Andreas Marzinzik; Sandra W. Cowan-Jacob; Pascal Furet; Jürgen Mestan (pp. 454-462).

The ATP-competitive inhibitors dasatinib and nilotinib, which bind to catalytically different conformations of the Abl kinase domain, have recently been approved for the treatment of imatinib-resistant CML. These two new drugs, albeit very efficient against most of the imatinib-resistant mutants of Bcr–Abl, fail to effectively suppress the Bcr–Abl activity of the T315I (or gatekeeper) mutation. Generating new ATP site-binding drugs that target the T315I in Abl has been hampered, amongst others, by target selectivity, which is frequently an issue when developing ATP-competitive inhibitors. Recently, using an unbiased cellular screening approach, GNF-2, a non-ATP-competitive inhibitor, has been identified that demonstrates cellular activity against Bcr–Abl transformed cells. The exquisite selectivity of GNF-2 is due to the finding that it targets the myristate binding site located near the C-terminus of the Abl kinase domain, as demonstrated by genetic approaches, solution NMR and X-ray crystallography. GNF-2, like myristate, is able to induce and/or stabilize the clamped inactive conformation of Abl analogous to the SH2-Y527 interaction of Src. The molecular mechanism for allosteric inhibition by the GNF-2 inhibitor class, and the combined effects with ATP-competitive inhibitors such as nilotinib and imatinib on wild-type Abl and imatinib-resistant mutants, in particular the T315I gatekeeper mutant, are reviewed.

Keywords: Abl; Imatinib resistance; Myristate binders; Kinase inhibitors; Abl structure

c-Jun N-terminal kinase (JNK) signaling: Recent advances and challenges by Marie A. Bogoyevitch; Kevin R.W. Ngoei; Teresa T. Zhao; Yvonne Y.C. Yeap; Dominic C.H. Ng (pp. 463-475).

c-Jun N-terminal kinases (JNKs), first characterized as stress-activated members of the mitogen-activated protein kinase (MAPK) family, have become a focus of inhibitor screening strategies following studies that have shown their critical roles in the development of a number of diseases, such as diabetes, neurodegeneration and liver disease. We discuss recent advances in the discovery and development of ATP-competitive and ATP-noncompetitive JNK inhibitors. Because understanding the modes of actions of these inhibitors and improving their properties will rely on a better understanding of JNK structure, JNK catalytic mechanisms and substrates, recent advances in these areas of JNK biochemistry are also considered. In addition, the use of JNK gene knockout animals is continuing to reveal in vivo functions for these kinases, with tissue-specific roles now being dissected with tissue-specific knockouts. These latest advances highlight the many challenges now faced, particularly in the directed targeting of the JNK isoforms in specific tissues.

Keywords: Abbreviations; Ago; Argonaute; DELFIA; Dissociation Enhanced Lathanide Fluoro-Immuno Assay; D-JNKI-1; cell-permeable JIP1-derived JNK inhibitory peptide-1 (D-amino acids); ELISA; enzyme-linked immunosorbent assays; ERK; extracellular signal-regulated kinase (ERK); FITC; fluorescein-isothiocyanate; FRET; Fluorescence Resonance Energy Transfer; GSK; glycogen synthase kinase; JNKs; c-Jun N-terminal kinases; JIP1; JNK-interacting protein-1; L-JNKI-1; cell-permeable JIP1-derived JNK inhibitory peptide-1 (L-amino acids); MAPK; mitogen-activated protein kinase; miRNA; microRNA; MKK; MAPK kinase; Myt1; membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase; NMR; nuclear magnetic resonance; PDB; Protein DataBase; PP2C; protein phosphatase 2C; RISC; RNA-induced silencing complex; SAPK; stress-activated protein kinase; Smac; second mitochondria-derived activator of caspases; TAT-TIJP; shorter version of the L-JNKI-1 TAT-linked JIP-based inhibitory peptide; XIAP; X-linked inhibitor of apoptosisc-Jun N-terminal kinase (JNK); ATP-competitive inhibitor; ATP non-competitive inhibitor; JNK structure; JNK substrate; Autophagy; microRNA; JNK knockout mouse

Deregulated signalling networks in human brain tumours by Michal Grzmil; Brian A. Hemmings (pp. 476-483).

Despite the variety of modern therapies against human brain cancer, in its most aggressive form of glioblastoma multiforme (GBM) it is a still deadly disease with a median survival of approximately 1 year. Over the past 2 decades, molecular profiling of low- and high-grade malignant brain tumours has led to the identification and molecular characterisation of mechanisms leading to brain cancer development, maintenance and progression. Genetic alterations occurring during gliomagenesis lead to uncontrolled tumour growth stimulated by deregulated signal transduction pathways. The characterisation of hyperactivated signalling pathways has identified many potential molecular targets for therapeutic interference in human gliomas. Overexpressed or mutated and constitutively active kinases are attractive targets for low-molecular-weight inhibitors. Although the first attempts with mono-therapy using a single targeted kinase inhibitor were not satisfactory, recent studies based on the simultaneous targeting of several core hyperactivated pathways show great promise for the development of novel therapeutic approaches. This review focuses on genetic alterations leading to the activation of key deregulated pathways in human gliomas.

Keywords: Abbreviations; GBM; glioblastoma multiforme; RTK; receptor tyrosine kinase; EGF(R); epidermal growth factor (receptor); PDGF(R); platelet-derived growth factor (receptor); VEGF(R); vascular endothelial growth factor (receptor); ERBB2; v-erb-b2 erythroblastic leukemia viral oncogene homolog 2; MET; met proto-oncogene (hepatocyte growth factor receptor); PI3K; phosphoinositide-3-kinase; AKT; v-akt murine thymoma viral oncogene; TP53; tumour protein p53; RB1; retinoblastoma 1; CDK 4 (6); cyclin-dependent kinase 4 (6); CDKN2A (B) (C); cyclin-dependent kinase inhibitor 2A, (B) (C); CCND2; cyclin D2; MDM2; Mdm2 p53-binding protein homolog (mouse); MAPK; mitogen-activated protein kinase; RAF1; v-raf-1 murine leukemia viral oncogene homolog 1; ERK; mitogen-activated protein kinase 1; MEK; mitogen-activated protein kinase kinase; FOXO1; forkhead box O1; PTEN; phosphatase and tensin homolog; NF1; neurofibromin 1; MGMT; O-6-methylguanine-DNA methyltransferase; mTOR; mechanistic target of rapamycin; NFk-β; nuclear factor of kappa light polypeptide gene enhancer in B-cells 1; BAD; BCL2-associated agonist of cell death; BAX; BCL2-associated; ×; protein; BCL2L12; BCL2-like 12 (proline rich); TGF-α; transforming growth factor, alpha; MCL1; myeloid cell leukemia sequence 1; S6K1; 40S ribosomal protein S6 kinase 1; eIF4EBP1; eukaryotic translation initiation factor 4E; -; binding protein 1; HIF1; hypoxia-inducible factor 1; IDH1; isocitrate dehydrogenase 1Glioblastoma; Signalling pathways; Kinase inhibitor

Conformational plasticity of the catalytic subunit of protein kinase CK2 and its consequences for regulation and drug design by Karsten Niefind; Olaf-Georg Issinger (pp. 484-492).

At the first glance CK2α, the catalytic subunit of protein kinase CK2, is a rigid molecule: in contrast to many eukaryotic protein kinases in CK2α the canonical regulatory key elements like the activation segment occur exclusively in their typical active conformations. This observation fits well to the constitutive activity of the enzyme, meaning, its independence from phosphorylation or other characteristic control factors. Most CK2α structures are based on the enzyme from Zea mays, supplemented by an increasing number of human CK2α structures. In the latter a surprising plasticity of important ATP-binding elements – the interdomain hinge region and the glycine-rich loop – was discovered. In fully active CK2α the hinge region is open and does not anchor the ATP ribose, but alternatively it can adopt a closed conformation, form hydrogen bonds to the ribose moiety and thus retract the γ-phospho group from its functional position. In addition to this partially inactive state human CK2α was recently found in a fully inactive conformation. It is incompatible with ATP-binding due to a combination of a closed hinge and a collapse of the glycine-rich loop into the ATP cavity. These conformational transitions are apparently correlated with the occupation state of a remote docking site located at the interface to the non-catalytic subunit CK2β: if CK2β blocks this site, the fully active conformation of CK2α is stabilized, while the binding of certain small molecule seems to favour the partially and fully inactive states. This observation may be exploited to design effective and selective CK2 inhibitors.

Keywords: Protein kinase CK2; Casein kinase 2; CK2α/CK2β interaction; Conformational plasticity at the interdomain hinge region and the glycin-rich loop; Protein kinase inhibitors; CK2β antagonists binding at a remote docking site of CK2α

Structure-based discovery of small molecules targeting different surfaces of protein-kinase CK2 by Renaud Prudent; Céline F. Sautel; Claude Cochet (pp. 493-498).

Protein kinase CK2 is an unfavorable pronostic marker in several cancers and has consequently emerged as a relevant therapeutic target. Several classes of ATP-competitive inhibitors have been identified, showing variable effectiveness. The molecular architecture of this multisubunit enzyme could offer alternative strategies to develop small molecule inhibitors targeting different surfaces of the kinase. Polyoxometalates were identified as original CK2 inhibitors targeting key structural elements located outside the active site. In addition, the CK2 subunit interface represents an exosite distinct from the catalytic cavity that can be targeted by peptides or small molecules to achieve functional effects.

Keywords: Abbreviations; POMs; polyoxometalates; V; vanadium; Mo; molybdenum; W; tungsten; DRB; D-ribofuranosylbenzimidazoleProtein kinase CK2; Non-competitive inhibitor; Allosteric inhibition; Protein–protein interaction

Addiction to protein kinase CK2: A common denominator of diverse cancer cells? by Maria Ruzzene; Lorenzo A. Pinna (pp. 499-504).

At variance with most oncogenic protein kinases whose malignancy is generally due to genetic alterations conferring constitutive activity, CK2 is a highly pleiotropic Ser/Thr protein kinase naturally endowed with constitutive activity and lacking gain-of-function mutants. Nonetheless CK2 is abnormally elevated in a wide variety of tumors and there is strong evidence that it operates as a cancer driver by creating a cellular environment favorable to neoplasia: notably, CK2 plays a global role as an anti-apoptotic and pro-survival agent, it enhances the multi-drug resistance (MDR) phenotype, it assists the chaperone machinery which protects the “onco-kinome” and it promotes neo-angiogenesis. Based on this scenario we propose that the implication of CK2 in neoplasia is an example of “non oncogene addiction”, i.e. over reliance of the perturbed cellular signaling network on high CK2 level for its own maintenance. Consistent with this, an ample spectrum of diverse types of cancer cells have been already shown to rely on high CK2 level for their survival, as judged from their response to specific CK2 inhibitors and silencing of endogenous CK2 catalytic subunits. Remarkably, among these are cells whose cancer phenotype arises from the genetic alteration of onco-kinases (e.g. Abl and Alk) different from CK2 and insensitive to the CK2 inhibitors used in those experiments. Based on these premises, CK2 could represent a “multi-purpose” target for the treatment of different kinds of tumors.

Keywords: Abbreviations; ALCL; anaplastic large cell lymphoma; ALL; acute lymphoblastic leukemia; AML; acute myeloid leukemia; CML; chronic myeloid leukemia; MDR; multi-drug resistance; MM; multiple myeloma; NF-kB; nuclear factor-kB; PDK1; 3′-phosphoinositide-dependent kinase-1; PI3K; phosphatidylinositol-3 kinase; PIP2; phosphatidylinositide 4,5-diphosphate; PIP3; phosphatidylinositol 3,4,5-triphosphate; PML; promyelocytic leukemia gene; PTEN; phosphatase and tensin homolog deleted on chromosome 10; PTK; protein tyrosine kinase; STT; signal transduction therapyCK2, casein kinase 2 (II); Signal transduction therapy; Addiction; Protein kinase inhibitors; Cancer

Regulation of cell proliferation and survival: Convergence of protein kinases and caspases by James S. Duncan; Jacob P. Turowec; Greg Vilk; Shawn S.C. Li; Gregory B. Gloor; David W. Litchfield (pp. 505-510).

Intricate networks of protein kinases are intimately involved in the regulation of cellular events related to cell proliferation and survival. In addition to protein kinases, cells also contain networks of proteases including aspartic-acid directed caspases organized in cascades that play a major role in the regulation of cell survival through their involvement in the initiation and execution phases of apoptosis. Perturbations in regulatory protein kinase and caspase networks induce alterations in cell survival and frequently accompany transformation and tumorigenesis. Furthermore, recent studies have documented that caspases or their substrates are subject to phosphorylation in cells illustrating a potential convergence of protein kinase and caspase signaling pathways. Interestingly, a number of caspase substrates are protected from cleavage when they are phosphorylated at sites that are adjacent to caspase cleavage sites. While it is theoretically possible that many distinct protein kinases could protect proteins from caspase-mediated cleavage, protein kinase CK2 is of particular interest because acidic amino acids, including aspartic acid residues that are recognized by caspases, are its dominant specificity determinants.

Keywords: Protein kinase; Caspase; Apoptosis; Protein kinase CK2; Phosphorylation-regulated cleavage

Recent developments in cyclin-dependent kinase biochemical and structural studies by Aude Echalier; Jane A. Endicott; Martin E.M. Noble (pp. 511-519).

The cyclin-dependent kinases (CDKs) have been intensely studied because of their involvement in regulating essential cellular activities that include proliferation and transcription. A series of CDK2-containing structures have informed a general model for the molecular details of CDK activation and regulation. Recent structural studies of other members of the CDK family have lead to a re-appraisal of this model. In this review, we describe alternative CDK-cyclin assemblies taking the recently characterised CDK/cyclin complexes, CDK9/cyclinT1 and CDK4/cyclinD as examples. The differential effects of CDK phosphorylation on CDK activation state and substrate specificity are examined in the light of recent data on CDK2/cyclinA, CDK9/cyclinT, CDK4/cyclinD and Pho85/Pho80. We also present an overview of factors that affect CDK substrate specificity, and, in particular, the contributions that are made by the cyclin subunit. Finally, we review recent results that have helped to unravel the molecular mechanisms underlying the conflicting roles of the Cip/Kip CDK inhibitor family in CDK regulation.

Keywords: Abbreviations; CDK; cyclin-dependent kinase; CKI; cyclin-dependent kinase inhibitor; T160pCDK2/cyclinA; CDK2 phosphorylated on Thr160 bound to cyclinA; Y15pT160pCDK2/cyclinA; CDK2 phosphorylated on Tyr15 and on Thr160 bound to cyclinA; CBF; cyclin box foldCDK; Cyclin; Cell cycle regulation; X-ray crystallography

Regulatory mechanism of the initiation step of DNA replication by CDK in budding yeast by Hiroyuki Araki (pp. 520-523).

Cyclin-dependent kinases (CDKs) regulate the progression of the cell cycle in eukaryotes. At the onset of chromosomal DNA replication, CDKs phosphorylate two replication proteins, Sld2 and Sld3, in budding yeast. Phosphorylated Sld2 and Sld3 enhance the formation of complexes with the BRCT (BRCA1 C-terminal)-containing replication protein Dpb11. The formation of these complexes is essential and sufficient for the CDK-dependent activation of the initiation of chromosomal DNA replication. Multiple phosphorylation of Sld2 by CDKs fine-tunes the process of complex formation. Here, we discussed the regulation of the initiation step of chromosomal DNA replication via CDK-dependent phosphorylation.

Keywords: CDK; DNA-replication; Eukaryotes; Yeast; Multiple phosphorylation

(d)-Amino acid analogues of DT-2 as highly selective and superior inhibitors of cGMP-dependent protein kinase Iα by Christian K. Nickl; Shiv Kumar Raidas; Hong Zhao; Matthias Sausbier; Peter Ruth; Werner Tegge; Joseph E. Brayden; Wolfgang R. Dostmann (pp. 524-532).

The cGMP-dependent protein kinase type I (PKG I) is an essential regulator of cellular function in blood vessels throughout the body. DT-2, a peptidic inhibitor of PKG, has played a central role in determining the molecular mechanisms of vascular control involving PKG and its signaling partners. Here, we report the development of (d)-amino acid DT-2 derivatives, namely the retro-inverso ri-(d)-DT-2 and the all (d)-amino acid analog, (d)-DT-2. Both peptide analogs were potent PKG Iα inhibitors with K_i values of 5.5 nM (ri-(d)-DT-2) and 0.8 nM ((d)-DT-2) as determined using a hyperbolic mixed-type inhibition model. Also, both analogs were proteolytically stable in vivo, showed elevated selectivity, and displayed enhanced membrane translocation properties. Studies on isolated arteries from the resistance vasculature demonstrated that intraluminally perfused (d)-DT-2 significantly inhibited vasodilation induced by 8-Br-cGMP. Furthermore, in vivo application of (d)-DT-2 established a uniform translocation pattern in the resistance vasculature, with exception of the brain. Thus, (d)-DT-2 caused significant increases in mean arterial blood pressure in unrestrained, awake mice. Further, mesenteric arteries isolated from (d)-DT-2 treated animals showed a markedly reduced dilator response to 8-Br-cGMP in vitro. Our results clearly demonstrate that (d)-DT-2 is a superior inhibitor of PKG Iα and its application in vivo leads to sustained inhibition of PKG in vascular smooth muscle cells. The discovery of (d)-DT-2 may help our understanding of how blood vessels constrict and dilate and may also aid the development of new strategies and therapeutic agents targeted to the prevention and treatment of vascular disorders such as hypertension, stroke and coronary artery disease.

Keywords: Abbreviations; cGMP; cyclic guanosine 3′,5′-mono-phosphate; i.p.; intraperitoneal; i.v.; intravenous; K; _i; inhibition constant; MAP; mean arterial blood pressure; K; _m; Michaelis–Menten constant; MPP; membrane-permeable peptides; PKA; cAMP-dependent protein kinase; PKG; cGMP-dependent protein kinase; V; _max; maximal (enzyme) velocitycGMP-dependent protein kinase; Protein kinase inhibitors; DT-2; (; d; )-DT-2; Smooth muscle

Somatic mutations in PI3Kα: Structural basis for enzyme activation and drug design by Sandra B. Gabelli; Diana Mandelker; Oleg Schmidt-Kittler; Bert Vogelstein; L. Mario Amzel (pp. 533-540).

The PI3K pathway is a communication hub coordinating critical cell functions including cell survival, cell growth, proliferation, motility and metabolism. Because PI3Kα harbors recurrent somatic mutations resulting in gains of function in human cancers, it has emerged as an important drug target for many types of solid tumors. Various PI3K isoforms are also being evaluated as potential therapeutic targets for inflammation, heart disease, and hematological malignancies. Structural biology is providing insights into the flexibility of the PI3Ks, and providing basis for understanding the effects of mutations, drug resistance and specificity.

Keywords: PI3K; p110/p85; Somatic mutation; Gain of function; H1047R mutant

Bisubstrate fluorescent probes and biosensors in binding assays for HTS of protein kinase inhibitors by Asko Uri; Marje Lust; Angela Vaasa; Darja Lavogina; Kaido Viht; Erki Enkvist (pp. 541-546).

Conjugates of adenosine mimics andd-arginine-rich peptides (ARCs) are potent inhibitors of protein kinases (PKs) from the AGC group. Labeling ARCs with fluorescent dyes or immobilizing on chip surfaces gives fluorescent probes (ARC-Photo) and biosensors that can be used for high-throughput screening (HTS) of inhibitors of protein kinases. The bisubstrate character (simultaneous association with both binding sites of the kinase) and high affinity of ARCs allow ARC-based probes and sensors to be used for characterization of inhibitors targeted to either binding site of the kinase with affinities in whole nanomolar to micromolar range. The ability to penetrate cell plasma membrane and bind to the target kinase fused with a fluorescent protein leads to the possibility to use ARC-Photo probes for high content screening (HCS) of inhibitors in cellular milieu with detection of intensity of Förster resonance energy transfer (FRET) between two fluorophores.

Keywords: Abbreviations; ARC; adenosine analogue–oligoarginine conjugate; CHO; Chinese Hamster Ovary; EFC; enzyme fragment complementation technology; FA; fluorescence anisotropy; FITC; Fluorescein isothiocyanate; FRET; Förster-resonance energy transfer; GST; glutathione; S; -transferase; H1152P; (; S; )-(+)-4-methyl-5-(2-methyl-[1,4]diazepane-1-sulfonyl)-isoquinoline; H89; N; -[2-((; p; -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; HCS; high content screening; HTS; high-throughput screening; ITC; isothermal titration calorimetry; K; _d; displacement constant; K; _D; dissociation constant; K; _i; inhibition constant; MS; mass spectrometry; MSK1; mitogen- and stress-activated protein kinase 1; NMR; nuclear magnetic resonance spectroscopy; PK; protein kinase; PKA; cAMP-dependent protein kinase; PKAc; cAMP-dependent protein kinase catalytic subunit type α; PKC; protein kinase C; PKIα; heat-stable protein kinase inhibitor α-isoform; qPCR; quantitative polymerase chain reaction; RIα; cAMP-dependent protein kinase regulatory subunit isoform Iα; RIIα; cAMP-dependent protein kinase regulatory subunit isoform IIα; ROCK-II; Rho-dependent protein kinase type II; SPR; surface plasmon resonance; TAMRA; carboxytetramethylrhodamine; TR-FRET; time-resolved Förster-resonance energy transfer; Y-27623; (; R; )-(+)-trans-; N; -(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide; YFP; yellow fluorescent proteinProtein kinase; Bisubstrate inhibitor; Fluorescent probe; Biosensor; ARC; Binding assay; Fluorescence anisotropy; Fluorescence polarization; FRET; SPR; HTS

Light-mediated remote control of signaling pathways by Melanie A. Priestman; David S. Lawrence (pp. 547-558).

Cell signaling networks display an extraordinary range of temporal and spatial plasticity. Our programmatic approach focuses on the construction of intracellular probes, including sensors, inhibitors, and functionally unique proteins that can be temporally and spatially controlled by the investigator even after they have entered the cell. We have designed and evaluated protein kinase sensors that furnish a fluorescent readout upon phosphorylation. In addition, since the sensors are inert (i.e., cannot be phosphorylated) until activated by light, they can be carried through the various stages of any given cell-based behavior without being consumed. Using this strategy, we have shown that PKCβ is essential for nuclear envelope breakdown and thus the transition from prophase to metaphase in actively dividing cells. Photoactivatable proteins furnish the means to initiate cellular signaling pathways with a high degree of spatial and temporal control. We have used this approach to demonstrate that cofilin serves as a component of the steering apparatus of the cell. Finally, inhibitors are commonly used to assess the participation of specific enzymes in signaling pathways that control cellular behavior. We have constructed a photo- deactivatable inhibitor, an inhibitory species that can be switched off with light. In the absence of light, the target enzyme is inactive due to the presence of the potent inhibitory molecule. Upon photolysis, the inhibitory molecule is destroyed and enzymatic activity is released.

Keywords: Signal transduction; Photoactivation; Caged protein; Kinase sensor; Inhibitor

Structural and mechanistic underpinnings of the differential drug sensitivity of EGFR mutations in non-small cell lung cancer by Michael J. Eck; Cai-Hong Yun (pp. 559-566).

EGFR and other ErbB-family tyrosine kinases are overexpressed in many human tumors, and their aberrant expression and mutational activation is associated with the development, progression and aggressiveness of a number of malignancies. Thus the EGFR kinase has long been recognized as a potential drug target in oncology, and small-molecule inhibitors have been under development for more than two decades. As a result of their effectiveness in treating non-small cell lung cancers (NSCLCs) driven by somatic mutations in the EGFR kinase, gefitinib and erlotinib were the first EGFR tyrosine kinase inhibitors (TKIs) approved for clinical use. Ironically, these drugs found their target against mutant forms of the EGFR kinase, which have altered enzyme active sites, and not against the wild type (WT) kinase against which their potency and selectivity was carefully honed. Here we review recent structural and enzymological studies that explore the exquisite sensitivity of a subset of these lung cancer mutants to gefitinib and erlotinib. We discuss available structural evidence for the mechanisms of activation of the EGFR kinase by these mutants, and compare it to physiologic activation of the kinase by ligand-induced dimerization. Finally, we consider the mechanisms by which the secondary T790M “gatekeeper” mutation confers resistance to gefitinib and erlotinib.

Keywords: Tyrosine kinase; Epidermal growth factor receptor; X-ray crystallography; Gefitinib; Lung cancer; Drug resistance

Structure–function analysis of VEGF receptor activation and the role of coreceptors in angiogenic signaling by Felix S. Grünewald; Andrea E. Prota; Alexandra Giese; Kurt Ballmer-Hofer (pp. 567-580).

Vascular endothelial growth factors (VEGFs) constitute a family of six polypeptides, VEGF-A, -B, -C, -D, -E and PlGF, that regulate blood and lymphatic vessel development. VEGFs specifically bind to three type V receptor tyrosine kinases (RTKs), VEGFR-1, -2 and -3, and to coreceptors such as neuropilins and heparan sulfate proteoglycans (HSPG). VEGFRs are activated upon ligand-induced dimerization mediated by the extracellular domain (ECD). A study using receptor constructs carrying artificial dimerization-promoting transmembrane domains (TMDs) showed that receptor dimerization is necessary, but not sufficient, for receptor activation and demonstrates that distinct orientation of receptor monomers is required to instigate transmembrane signaling. Angiogenic signaling by VEGF receptors also depends on cooperation with specific coreceptors such as neuropilins and HSPG. A number of VEGF isoforms differ in binding to coreceptors, and ligand-specific signal output is apparently the result of the specific coreceptor complex assembled by a particular VEGF isoform. Here we discuss the structural features of VEGF family ligands and their receptors in relation to their distinct signal output and angiogenic potential.

Keywords: VEGF; Angiogenesis; Neuropilin; Receptor tyrosine kinase

Development of protein kinase activators: AMPK as a target in metabolic disorders and cancer by S. Fogarty; D.G. Hardie (pp. 581-591).

AMP-activated protein kinase (AMPK) is a cellular energy sensor activated by metabolic stresses that either inhibit ATP synthesis or accelerate ATP consumption. Activation of AMPK in response to an increase in the cellular AMP:ATP ratio results in inhibition of ATP-consuming processes such as gluconeogenesis and fatty acid synthesis, while stimulating ATP-generating processes, including fatty acid oxidation. These alterations in lipid and glucose metabolism would be expected to ameliorate the pathogenesis of obesity, type 2 diabetes and other metabolic disorders. Recently, AMPK has also been identified as a potential target for cancer prevention and/or treatment. Cell growth and proliferation are energetically demanding, and AMPK may act as an “energy checkpoint” that permits growth and proliferation only when energy reserves are sufficient. Thus, activators of AMPK could have potential as novel therapeutics both for metabolic disorders and for cancer, which together constitute two of the most prevalent groups of diseases worldwide.

Keywords: AMPK; Activator; Metabolic Syndrome; Obesity; Diabetes; Cancer

Fine tuning T lymphocytes: A role for the lipid phosphatase SHIP-1 by Richard V. Parry; Stephanie J. Harris; Stephen G. Ward (pp. 592-597).

The phosphoinositide 3-kinase signaling pathway regulates a range of T lymphocyte cellular functions including growth, proliferation, cytokine secretion and survival. Aberrant regulation of phosphoinositide 3-kinase-dependent signaling in T lymphocytes has been implicated in inflammatory and autoimmune diseases. In common with much of the immune system, several mechanisms exist to ensure the pathway is tightly regulated to elicit appropriate responses. One level of control involves the Src homology 2 domain-containing inositol-5-phosphatase-1 (SHIP-1) that modulates phosphoinositide 3-kinase signaling by degrading the key signaling lipid PI(3,4,5)P₃ to PI(3,4)P₂, but also serves as a key scaffolding molecule in the formation of multi-protein complexes. Here we discuss the role of SHIP-1 in regulating T lymphocyte and immune function, as well as its potential as a therapeutic target.

Keywords: T Lymphocyte; Lipid phosphatase; PI3K; Chemotaxis; miRNA; T; _Reg

Substrate Competitive GSK-3 Inhibitors strategy and Implications by Hagit Eldar-Finkelman; Avital Licht-Murava; Shmuel Pietrokovski; Miriam Eisenstein (pp. 598-603).

Glycogen synthase kinase-3 (GSK-3) is a highly conserved protein serine/threonine kinase ubiquitously distributed in eukaryotes as a constitutively active enzyme. Abnormally high GSK-3 activity has been implicated in several pathological disorders, including diabetes and neuron degenerative and affective disorders. This led to the hypothesis that inhibition of GSK-3 may have therapeutic benefit. Most GSK-3 inhibitors developed so far compete with ATP and often show limited specificity. Our goal is to develop inhibitors that compete with GSK-3 substrates, as this type of inhibitor is more specific and may be useful for clinical applications. We have employed computational, biochemical, and molecular analyses to gain in-depth understanding of GSK-3's substrate recognition. Here we argue that GSK-3 is a promising drug discovery target and describe the strategy and practice for developing specific substrate-competitive inhibitors of GSK-3.

Keywords: Protein kinase; Phosphorylation; GSK-3; Inhibitors; Substrate recognition

Malaria: Targeting parasite and host cell kinomes by Christian Doerig; Abdirahman Abdi; Nicholas Bland; Sylvain Eschenlauer; Dominique Dorin-Semblat; Clare Fennell; Jean Halbert; Zoe Holland; Marie-Paule Nivez; Jean-Philippe Semblat; Audrey Sicard; Luc Reininger (pp. 604-612).

Malaria still remains one of the deadliest infectious diseases, and has a tremendous morbidity and mortality impact in the developing world. The propensity of the parasites to develop drug resistance, and the relative reluctance of the pharmaceutical industry to invest massively in the developments of drugs that would offer only limited marketing prospects, are major issues in antimalarial drug discovery. Protein kinases (PKs) have become a major family of targets for drug discovery research in a number of disease contexts, which has generated considerable resources such as kinase-directed libraries and high throughput kinase inhibition assays. The phylogenetic distance between malaria parasites and their human host translates into important divergences in their respective kinomes, and most Plasmodium kinases display atypical properties (as compared to mammalian PKs) that can be exploited towards selective inhibition. Here, we discuss the taxon-specific kinases possessed by malaria parasites, and give an overview of target PKs that have been validated by reverse genetics, either in the human malaria parasite Plasmodium falciparum or in the rodent model Plasmodium berghei. We also briefly allude to the possibility of attacking Plasmodium through the inhibition of human PKs that are required for survival of this obligatory intracellular parasite, and which are targets for other human diseases.

Keywords: Malaria; Protein kinase; Target validation; Plasmodium falciparum; Plasmodium berghei; Transgenesis; Transfection

The two faces of PTP1B in cancer by Laurent Lessard; Matthew Stuible; Michel L. Tremblay (pp. 613-619).

PTP1B is a classical non-transmembrane protein tyrosine phosphatase that plays a key role in metabolic signaling and is a promising drug target for type 2 diabetes and obesity. Accumulating evidence also indicates that PTP1B is involved in cancer, but contrasting findings suggest that it can exert both tumor suppressing and tumor promoting effects depending on the substrate involved and the cellular context. In this review, we will discuss the diverse mechanisms by which PTP1B may influence tumorigenesis as well as recent in vivo data on the impact of PTP1B deficiency in murine cancer models. Together, these results highlight not only the great potential of PTP1B inhibitors in cancer therapy but also the need for a better understanding of PTP1B function prior to use of these compounds in human patients.

Keywords: Abbreviations; CSF1R; macrophage colony stimulating factor 1 receptor; EGFR; epidermal growth factor receptor; ER; endoplasmic reticulum; FAK; focal adhesion kinase; IL; interleukin; IR; insulin receptor; JAK; janus kinase; MAPK; mitogen activated protein kinase; MMTV; mouse mammary tumor virus; PDGFR; platelet-derived growth factor receptor; PI3K; phosphatidylinositol 3-kinase; PLC; phospholipase C; PTK; protein tyrosine kinase; PTP; protein tyrosine phosphatase; PTP1B; protein tyrosine phosphatase 1B; QTL; quantitative trait locus; ROS; reactive oxygen species; RTK; receptor tyrosine kinase; STAT; signal transducer and activator of transcription; SUMO; small ubiquitin-like modifier; SYK; spleen tyrosine kinase; TC-PTP; T-cell protein tyrosine phosphatase; VEGFR; vascular endothelial growth factor receptorPTP1B; Protein tyrosine phosphatase; Cancer; Tumor promoter; Tumor suppressor

Protein kinase and phosphatase signaling in Mycobacterium tuberculosis physiology and pathogenesis by Joseph Chao; Dennis Wong; Xingji Zheng; Valerie Poirier; Horacio Bach; Zakaria Hmama; Yossef Av-Gay (pp. 620-627).

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), evades the antimicrobial defenses of the host and survives within the infected individual through a complex set of strategies. These include active prevention of host cellular killing processes as well as overwhelming adaptive gene expression. In the past decade, we have gained an increased understanding of how mycobacteria not only have the ability to adapt to a changing host environment but also actively interfere with the signaling machinery within the host cell to counteract or inhibit parts of the killing apparatus employed by the macrophage. Mtb is able to sense its environment via a set of phospho-signaling proteins which mediate its response and interaction with the host in a coordinated manner. In this review, we summarize the current knowledge about selected Mtb serine, threonine, and tyrosine kinase and phosphatase signaling proteins, focusing on the protein kinases, PknG and PtkA, and the protein phosphatase, PtpA.

Keywords: Mycobacterium tuberculosis; Ser/Thr Tyr phosphorylation; Kinase substrates; PknG; PtpA; PtkA

Bacterial tyrosine-kinases: Structure–function analysis and therapeutic potential by Christophe Grangeasse; Raphaël Terreux; Sylvie Nessler (pp. 628-634).

Since the characterization of genes encoding Ser/Thr-kinases and Tyr-kinases in bacteria, in 1991 and 1997, respectively, a growing body of evidence has been reported showing the important role of these enzymes in the regulation of bacterial physiology. While most Ser/Thr-kinases share structural similarity with their eukaryotic counterparts, it seems that bacteria have developed their own Tyr-kinases to catalyze protein phosphorylation on tyrosine. Different types of Tyr-kinases have been identified in bacteria and a large number of them are similar to ATP-binding proteins with Walker motifs. These enzymes have been grouped in the same family (BY-kinases) and the crystal structures of two of them have been recently characterized. Phosphoproteome analysis suggest that BY-kinases are involved in several cellular processes and to date, the best-characterized role of BY-kinases concerns the control of extracellular polysaccharide synthesis. Knowing the role of these compounds in the virulence of bacterial pathogens, BY-kinases can be considered as promising targets to combat some diseases. Here, we review the current knowledge on BY-kinases and discuss their potential for the development of new antibiotics.

Keywords: BY-kinase; Tyrosine phosphorylation; Signalisation; Virulence; Kinase inhibitor; Antibiotic

A generalized numerical approach to steady-state enzyme kinetics: Applications to protein kinase inhibition by Petr Kuzmič (pp. 635-641).

A generalized numerical treatment of steady-state enzyme kinetics is presented. This new approach relies on automatic computer derivation of the underlying mathematical model (a system of simultaneous nonlinear algebraic equations) from a symbolic representation of the reaction mechanism (a system of biochemical equations) provided by the researcher. The method allows experimental biochemists to analyze initial-rate enzyme kinetic data, under the steady-state approximation, without having to use any mathematical equations. An illustrative example is based on the inhibition kinetics of p56^lck kinase by an ATP competitive inhibitor. A computer implementation of the new method, in the modified software package DYNAFIT [Kuzmič, P. (1996) Anal. Biochem. 237, 260–273], is freely available to all academic researchers.

Keywords: DYNAFIT; Protein Kinase; Inhibition; p56lck tyrosine kinase; PKA; Bisubstrate analog; Enzyme kinetics; Theory; Steady state approximation

Structure-guided expansion of kinase fragment libraries driven by support vector machine models by Jon A. Erickson; Mary M. Mader; Ian A. Watson; Yue W. Webster; Richard E. Higgs; Michael A. Bell; Michal Vieth (pp. 642-652).

This work outlines a new de novo design process for the creation of novel kinase inhibitor libraries. It relies on a profiling paradigm that generates a substantial amount of kinase inhibitor data from which highly predictive QSAR models can be constructed. In addition, a broad diversity of X-ray structure information is needed for binding mode prediction. This is important for scaffold and substituent site selection. Borrowing from FBDD, the process involves fragmentation of known actives, proposition of binding mode hypotheses for the fragments, and model-driven recombination using a pharmacophore derived from known kinase inhibitor structures. The support vector machine method, using Merck atom pair derived fingerprint descriptors, was used to build models from activity from 6 kinase assays. These models were qualified prospectively by selecting and testing compounds from the internal compound collection. Overall hit and enrichment rates of 82% and 2.5%, respectively, qualified the models for use in library design. Using the process, 7 novel libraries were designed, synthesized and tested against these same 6 kinases. The results showed excellent results, yielding a 92% hit rate for the 179 compounds that made up the 7 libraries. The results of one library designed to include known literature compounds, as well as an analysis of overall substituent frequency, are discussed.

Keywords: Abbreviations; FBDD; fragment-based drug design; SBDD; structure-based drug design; QSAR; quantitative structure activity relationship; SVM-FP; support vector machine—fingerprints; PPV; positive predicted value; LE; ligand efficiency; HAC; heavy atom count; AMW; atomic molecular weight; PDB; protein databank; ATP; Adenosine triphosphate; ABL1; c-abl oncogene 1, receptor tyrosine kinase; CHK2; CHK2 checkpoint kinase; FLT3; fms-related tyrosine kinase 3; MET; met proto-oncogene (hepatocyte growth factor receptor); P70S6K; RPS6KB1 ribosomal protein S6 kinase; ROCK2; Rho-associated, coiled-coil containing protein kinase 2De novo design; Kinase inhibitor library; Fragment-based drug design; Structure-based drug design; QSAR model; Binding mode prediction

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BBA - Proteins and Proteomics (v.1804, #3)