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During the past decade, genomic analyses have been introduced into cancer studies with variable success. It has become recognised, however, that genomic techniques in isolation are insufficient to study the complex pathways of carcinogenesis; this has led to the application of proteomic techniques, which allow for the reliable analysis of complex mixtures of proteins. This article reviews the basic principles of proteomics, methods currently used in proteomics including two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), and the application of proteomics in cancer research. Currently, proteomic technology has been used in two main areas of cancer research: early diagnosis and treatment (included prediction of response to treatment and targeting novel cancer agents). The initial results from both in vitro and in vivo studies are impressive. These technologies, particularly when combined with genomic analyses, will provide valuable insights into the molecular basis of carcinogenesis and the development of more effective anti-cancer therapies. [ABSTRACT FROM AUTHOR]

The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties from the IUPHAR database. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12444/full. This compilation of the major pharmacological targets is divided into seven areas of focus: G protein-coupled receptors, ligand-gated ion channels, ion channels, catalytic receptors, nuclear hormone receptors, transporters and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. A new landscape format has easy to use tables comparing related targets. It is a condensed version of material contemporary to late 2013, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in previous Guides to Receptors & Channels. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and GRAC and provides a permanent, citable, point-in-time record that will survive database updates., (Copyright © 2013 The British Pharmacological Society.)

This review will focus on two general approaches carried out at the Sandler Center, University of California, San Francisco, to address the challenge of developing new drugs for the treatment of Chagas disease. The first approach is target-based drug discovery, and two specific targets, cytochrome P450 CYP51 and cruzain (aka cruzipain), are discussed. A 'proof of concept' molecule, the vinyl sulfone inhibitor K777, is now a clinical candidate. The preclinical assessment compliance for filing as an Investigational New Drug with the United States Food and Drug Administration (FDA) is presented, and an outline of potential clinical trials is given. The second approach to identifying new drug leads is parasite phenotypic screens in culture. The development of an assay allowing high throughput screening of Trypanosoma cruzi amastigotes in skeletal muscle cells is presented. This screen has the advantage of not requiring specific strains of parasites, so it could be used with field isolates, drug resistant strains or laboratory strains. It is optimized for robotic liquid handling and has been validated through a screen of a library of FDA-approved drugs identifying 65 hits.

P-glycoprotein (P-gp) is the most intensively studied eukaryotic ATP binding cassette (ABC) transporter, due to its involvement in the multidrug resistance phenotype of a number of cancers. In common with most ABC transporters, P-gp is comprised of two transmembrane domains (TMDs) and two nucleotide binding domains (NBD), the latter coupling ATP hydrolysis with substrate transport (efflux in the case of P-gp). Biochemical investigations over the past twenty years have attempted to unlock mechanistic aspects of P-glycoprotein through scanning and site-directed mutagenesis of both the TMDs and the NBDs. Contemporaneously, crystallographers have elucidated the atomic structure of numerous ABC transporter NBDs, as well as the intact structure (i.e. NBDs and TMDs) of a distantly related ABC-exporter Sav1866. Significantly, the structure of P-gp remains unknown, and only low resolution electron microscopy data exists. Within the current manuscript we employ crystallographic data for homologous proteins, and a molecular model for P-gp, to perform a structural interpretation of the existing "mutagenesis database" for P-gp NBDs. Consequently, this will enable testable predictions to be made that will result in further in-roads into our understanding of this clinically important drug pump.

The precise mechanism of antibiotic-resistance-conferring ABC (ATP-binding-cassette) proteins (termed NBD2) remains open to debate. Currently, two hypotheses are recognized. In one, the NBD2 proteins are envisaged to act at the ribosome to impair antibiotic access to the target site on the 23 S rRNA. In the other, NBD2 proteins are believed to act as the components of ATP driven efflux pumps by associating with membrane spanning proteins capable of binding and transporting antibiotics. Pertinent data in support of these two hypotheses are discussed in this paper.

Bacteria have a rich collection of biochemical pathways for the synthesis of complex metabolites. These conversions often involve chemical reactions that are hard to reproduce in the laboratory. An area of considerable interest is in the manipulation and synthesis of carbohydrates. In contrast with amino acids, carbohydrates are densely functionalized (each carbon atom is attached to at least one heteroatom) and this holds out the prospect of discovering novel enzyme mechanisms. The results from the study of the biosynthetic dTDP-L-rhamnose pathway are discussed. dTDP-L-rhamnose is a key intermediate in many pathogenic bacteria, as it is the donor for L-rhamnose, which is found in the cell wall of important human pathogens, such as Mycobacteria tuberculosis and Salmonella typhimurium. All four enzymes have been structurally characterized; in particular, the acquisition of structural data on substrate complexes was extremely useful. The structural data have guided site-directed-mutagenesis studies that have been used to test mechanistic hypotheses. The results shed light on three classes of enzyme mechanism: nucleotide condensation, short-chain dehydrogenase activity and epimerization.

P-glycoprotein (P-gp) is an ABC (ATP-binding cassette) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells. P-gp consists of two transmembrane domains (TMDs) that span the membrane multiple times, and two cytoplasmic nucleotide-binding domains (NBDs). We have determined projection structures of P-gp trapped at different steps of the transport cycle and correlated these structures with function. In the absence of nucleotide, an approximately 10 A resolution structure was determined by electron cryo-microscopy of two-dimensional crystals. The TMDs form a chamber within the membrane that appears to be open to the extracellular milieu, and may also be accessible from the lipid phase at the interfaces between the two TMDs. Nucleotide binding causes a repacking of the TMDs and reduction in drug binding affinity. Thus, ATP binding, not hydrolysis, drives the major conformational change associated with solute translocation. A third distinct conformation of the protein was observed in the post-hydrolytic transition state prior to release of ADP/P(i). Biochemical data suggest that these rearrangements may involve rotation of transmembrane alpha-helices. A mechanism for transport is suggested.

Single-stranded DNA-binding proteins are recruited when single-stranded DNA is exposed by disruption of the duplex. Many important biological processes such as DNA replication can only occur when the two strands of the duplex are separated. A defining trait of these proteins is the presence of the so-called OB fold. The single-stranded DNA-binding protein of the crenarchaeote Sulfolobus solfataricus has a number of interesting differences and similarities to both the eubacterial and eukaryotic homologues. It has an extended C-terminal tail with significant sequence identity to a similar region in the eubacterial protein. However, the sequence of the OB fold is much more like the eukaryotic and euryarchaeal proteins. The S. solfataricus protein remains a monomer in the absence of DNA but rapidly polymerizes upon binding - a behaviour not seen in the Escherichia coli protein. The protein has been overexpressed, purified and crystallized. The protein crystallizes in two related forms, both having space group P6(1) (or P6(5)) with approximate unit-cell parameters a = b = 75, c = 69 A, but the crystals are distinguished by their size and morphology. The larger crystals are hexagonal bipyramids and are merohedrally twinned, diffracting to 1.34 A with diffraction observed to 1.2 A. Smaller needle-like crystals diffract to about 2.0 A but are not twinned. Molecular-replacement attempts have failed owing to low identity with available search models. The structure will be determined by multiple-wavelength methods.

Bacteriorhodopsin (BR) is a membrane protein which pumps protons through the plasma membrane. Seven transmembrane BR helical segments are subjected to simulation studies in order to investigate the packing process of transmembrane helices. A Monte Carlo simulated annealing protocol is employed to optimize the helix bundle system. Helix packing is optimized according to a semi-empirical potential mainly composed of six components: a bilayer potential, a crossing angle potential, a helix dipole potential, a helix-helix distance potential, a helix orientation potential and a helix-helix distance restraint potential (a loop potential). Necessary parameters are derived from theoretical studies and statistical analysis of experimentally determined protein structures. The structures from the simulations are compared with the experimentally determined structures in terms of geometry. The structures generated show similar shapes to the experimentally suggested structure even without the helix-helix distance restraint potential. However, the relative locations of individual helices were reproduced only when the helix-helix distance restraint potential was used with restraint conditions. Our results suggest that transmembrane helix bundles resembling those observed experimentally may be generated by simulations using simple potentials.

A model of the selectivity filter of a voltage-gated K+ (Kv) channel formed by an eight-stranded beta-barrel is compared with physiological properties of the channel. Continuum electrostatic calculations suggest that only two of the eight Asp sidechains at the extracellular mouth of the pore will ionise. A ring of four Tyr sidechains forms the narrowest region of the pore. Molecular dynamic simulations of the potential energy of a K+ ion as translated along the model pore indicate that the two ionised Asp sidechains and the hydroxyl groups of the Tyr sidechains stabilise the partially desolvated ion as it passes through the narrowest region., (Copyright 1998 Elsevier Science B.V.)

Several analogues of the channel-forming peptaibol alamethicin have been demonstrated to exhibit faster switching between channel substates than does unmodified alamethicin. Molecular modelling studies are used to explore the possible molecular basis of these differences. Models of channels formed by alamethicin analogues were generated by restrained molecular dynamics in vacuo and refined by short molecular dynamics simulations with water molecules within and at either mouth of the channel. A decrease in backbone solvation was found to correlate with a decrease in open channel stability between alamethicin and an analogue in which all alpha-amino-isobutyric acid residues of alamethicin were replaced by leucine. A decrease in the extent of hydrogen-bonding at residue 7 correlates with lower open channel stabilities of analogues in which the glutamine at position 7 was replaced by smaller polar sidechains. These two observations indicate the importance of alamethicin/water H-bonds in stabilizing the open channel.

Although there is a large body of site-directed mutagenesis data that identify the pore-lining sequence of the voltage-gated potassium channel, the structure of this region remains unknown. We have interpreted the available biochemical data as a set of topological and orientational restraints and employed these restraints to produce molecular models of the potassium channel pore region, H5. The H5 sequence has been modeled either as a tetramer of membrane-spanning beta-hairpins, thus producing an eight-stranded beta-barrel, or as a tetramer of incompletely membrane-spanning alpha-helical hairpins, thus producing an eight-staved alpha-helix bundle. In total, restraints-directed modeling has produced 40 different configurations of the beta-barrel model, each configuration comprising an ensemble of 20 structures, and 24 different configurations of the alpha-helix bundle model, each comprising an ensemble of 24 structures. Thus, over 1300 model structures for H5 have been generated. Configurations have been ranked on the basis of their predicted pore properties and on the extent of their agreement with the biochemical data. This ranking is employed to identify particular configurations of H5 that may be explored further as models of the pore-lining region of the voltage-gated potassium channel pore.

The M2 protein of influenza virus forms ion channels activated by low pH which are proton permeable and play a key role in the life cycle of the virus. M2 is a 97-residue integral membrane protein containing a single transmembrane (TM) helix. M2 is present as disulfide-linked homotetramers. The TM domain of M2 has been modeled as a bundle of four parallel M2 helices. The helix bundle forms a left-handed supercoil surrounding a central pore. Residue H37 has been implicated in the mechanism of low-pH activation of the channel. Models generated with H37 in a fully deprotonated state exhibit a pore occluded by a ring of H37 side chains oriented toward the lumen of the pore. Models with H37 in a fully protonated state no longer exhibit such occlusion of the pore, as the H37 side chains adopt a more interfacial location. Extended molecular dynamics simulations with water molecules within and at the mouths of the pores support this distinction between the H37-deprotonated and H37-protonated models. These simulations suggest that only in the H37-protonated model is there a continuous column of water extending the entire length of the central pore. A mechanism for activation of M2 by low pH is presented in which the H37-deprotonated model corresponds to the "closed" form of the channel, while the H37-protonated model corresponds to the "open" form. A switch from the closed to the open form of the channel occurs if H37 is protonated midway through a simulation. The open channel is suggested to contain a wire of H-bonded water molecules which enables proton permeability.

Alamethicin channels have been modelled as approximately parallel bundles of transbilayer helices containing between N = 4 and 8 helices per bundle. Initial models were generated by in vacuo restrained molecular dynamics (MD) simulations, and were refined by 60 ps MD simulations with water molecules present within and at the mouths of the central pore. The helix bundles were stabilized by networks of H-bonds between intra-pore water molecules and Gln-7 side-chains. Channel conductances were predicted on the basis of pore radius profiles, and suggested that the N = 4 bundle formed an occluded pore, whereas pores with N > or = 5 helices per bundle were open. Continuum electrostatics calculations suggested that the N = 6 pore is cation-selective, whereas pores with N > or = 7 helices per bundle were predicted to be somewhat less ion-selective.

Vpu is an oligomeric integral membrane protein encoded by HIV-1 which forms ion channels, each subunit of which contains a single transmembrane helix. Models of Vpu channels formed by bundles of N = 4, 5 or 6 transmembrane helices have been developed by restrained molecular dynamics and refined by 100 ps simulations with water molecules within the pore. Pore radius profiles and conductance predictions suggest that the N = 5 model corresponds to the predominant channel conductance level of the channel. Potential energy profiles for translation of Na+ or Cl- ions along the Vpu N = 5 pore are consistent with the weak cation selectivity of Vpu channels.

In the middle of the S6 helix in voltage-gated potassium channels there is a highly conserved Pro-Val-Pro motif, while the equivalent M2 helix of inward rectifier potassium channels contains a conserved glycine residue in a comparable position. The structural implications of these conserved motifs are of interest given the evidence that S6 and M2 are components of the lining of their respective pores. Multiple sequence alignment and TM helix prediction methods were used to define consensus regions for S6 and M2. Ensembles of 50 structures for each helix were generated by simulated annealing and restrained molecular dynamics. Time-dependent fluctuations of S6 and M2 were investigated by long time scale molecular dynamics simulations on representative members of each ensemble carried out in vacuo in the presence and absence of a hydrophobic potential that mimics a lipid bilayer. The results are discussed in terms of the structural basis of the kink in S6 and M2 and of a putative functional role for flexible helices as "molecular swivels."

Phosphatidylcholine-specific phospholipase D (PLD) enzymes catalyze hydrolysis of phospholipid phosphodiester bonds, and also transphosphatidylation of phospholipids to acceptor alcohols. Bacterial and plant PLD enzymes have not been shown previously to be homologues or to be homologous to any other protein. Here we show, using sequence analysis methods, that bacterial and plant PLDs show significant sequence similarities both to each other, and to two other classes of phospholipid-specific enzymes, bacterial cardiolipin synthases, and eukaryotic and bacterial phosphatidylserine synthases, indicating that these enzymes form an homologous family. This family is suggested also to include two Poxviridae proteins of unknown function (p37K and protein K4), a bacterial endonuclease (nuc), an Escherichia coli putative protein (o338) containing an N-terminal domain showing similarities with helicase motifs V and VI, and a Synechocystis sp. putative protein with a C-terminal domain likely to possess a DNA-binding function. Surprisingly, four regions of sequence similarity that occur once in nuc and o338, appear twice in all other homologues, indicating that the latter molecules are bi-lobed, having evolved from an ancestor or ancestors that underwent a gene duplication and fusion event. It is suggested that, for each of these enzymes, conserved histidine, lysine, aspartic acid, and/or asparagine residues may be involved in a two-step ping pong mechanism involving an enzyme-substrate intermediate.

The transbilayer pores formed by ion channel proteins contain extended columns of water molecules. The dynamic properties of such waters have been suggested to differ from those of water in its bulk state. Molecular dynamics simulations of ion channel models solvated within and at the mouths of their pores are used to investigate the dynamics and structure of intra-pore water. Three classes of channel model are investigated: a) parallel bundles of hydrophobic (Ala20) alpha-helices; b) eight-stranded hydrophobic (Ala10) antiparallel beta-barrels; and c) parallel bundles of amphipathic alpha-helices (namely, delta-toxin, alamethicin, and nicotinic acetylcholine receptor M2 helix). The self-diffusion coefficients of water molecules within the pores are reduced significantly relative to bulk water in all of the models. Water rotational reorientation rates are also reduced within the pores, particularly in those pores formed by alpha-helix bundles. In the narrowest pore (that of the Ala20 pentameric helix bundle) self-diffusion coefficients and reorientation rates of intra-pore waters are reduced by approximately an order of magnitude relative to bulk solvent. In Ala20 helix bundles the water dipoles orient antiparallel to the helix dipoles. Such dipole/dipole interaction between water and pore may explain how water-filled ion channels may be formed by hydrophobic helices. In the bundles of amphipathic helices the orientation of water dipoles is modulated by the presence of charged side chains. No preferential orientation of water dipoles relative to the pore axis is observed in the hydrophobic beta-barrel models.

Ion channels contain narrow columns of water molecules. It is of interest to compare the structure and dynamics of such intrapore water with those of the bulk solvent. Molecular dynamics simulations of modified TIP3P water molecules confined within channel-like cavities have been performed and the orientation and dynamics of the water molecules analyzed. Channels were modeled as cylindrical cavities with lengths ranging from 15 to 60 A and radii from 3 to 12 A. At the end of the molecular dynamics simulations water molecules were observed to be ordered into approximately concentric cylindrical shells. The waters of the outermost shell were oriented such that their dipoles were on average perpendicular to the normal of the wall of the cavity. Water dynamics were analyzed in terms of self-diffusion coefficients and rotational reorientation rates. For cavities of radii 3 and 6 A, water mobility was reduced relative to that of simulated bulk water. For 9- and 12-A radii confined water molecules exhibited mobilities comparable with that of the bulk solvent. If water molecules were confined within an hourglass-shaped cavity (with a central radius of 3 A increasing to 12 A at either end) a gradient of water mobility was observed along the cavity axis. Thus, water within simple models of transbilayer channels exhibits perturbations of structure and dynamics relative to bulk water. In particular the reduction of rotational reorientation rate is expected to alter the local dielectric constant within a transbilayer pore.

Delta-Toxin is a 26-residue channel-forming peptide from Staphylococcus aureus which forms an amphipathic alpha-helix in a membrane environment. Channel formation in planar bilayers suggests that an average of six delta-toxin helices self-assemble to form transbilayer pores. Molecular models for channels formed by delta-toxin and by a synthetic analogue have been generated using a simulated annealing protocol applied via restrained molecular dynamics. These models are analysed in terms of the predicted geometric and energetic properties of the transbilayer pores. Pore radius calculations of the models demonstrate that rings of channel-lining residues contribute a series of constrictions along the pore. Electrostatic properties of the pores are determined both by pore-lining charged side chains and by the aligned helix dipoles of the parallel helix bundle. Molecular dynamics simulations (100 ps) of delta-toxin models containing intra-pore water were performed. Analysis of the resultant dynamics trajectories further supports the proposal that alternative conformations of pore-constricting side chains may be responsible for the observed conductance heterogeneity of delta-toxin ion channels.

Transmembrane beta-barrels, first observed in bacterial porins, are possible models for a number of membrane channels. Restrained molecular dynamics simulations based on idealized C alpha beta templates have been used to generate models of such beta-barrels. Model beta-barrels have been analyzed in terms of their conformational, energetic, and pore properties. Model beta-barrels formed by N = 4, 8, 12 and 16 anti-parallel Ala10 strands have been developed. For each N, beta-barrels with shear numbers S = N to 2N have been modeled. In all beta-barrel models the constituent beta-strands adopt a pronounced right-handed twist. Interstrand interactions are of approximately equal stability for all models with N > or = 8, whereas such interactions are weaker for the N = 4 beta-barrels. In N = 4 beta-barrels the pore is too narrow (minimum radius approximately 0.6 A) to allow ion permeation. For N > or = 8, the pore radius depends on both N and S; for a given value of N an increase in S from N to 2N is predicted to result in an approximately threefold increase in pore conductance. Calculated maximal conductances for the beta-barrel models are compared with experimental values for porins and for K+ channels.

Here we present a procedure for modelling membrane proteins which employs molecular dynamics simulations incorporating target restraints derived from low-resolution structures alongside distance restraints derived from mutagenesis data. The application of the modelling procedure to the closed conformation of the pore domain of the nicotinic acetylcholine receptor is described. This domain is formed by a parallel bundle of five M2 helices. Each M2 helix is kinked due to cumulative distortions of backbone (phi, psi) values. The central region of M2 may adopt a more distorted conformation. This would enable a ring of conserved leucine residues (one from each M2 helix) to pack together, occluding the central pore and thus preventing ion permeation. Molecular dynamics simulations on isolated helices that kink formation is not an inherent property of M2.

Ion channel formation by three analogues of staphylococcal delta-toxin, an amphipathic and alpha-helical channel-forming peptide, has been evaluated by measurement of ionic currents across planar lipid bilayers. Replacement of beta-branched, hydrophobic residues by leucine and movement of a tryptophan residue from the hydrophilic to the hydrophobic face of the helix does not significantly alter ion channel activity. Removal of the N-terminal blocking group combined with the substitution of glycine-10 by leucine changes the single channel properties of delta-toxin, without altering macroscopic conductance/voltage behaviour. Truncation of the N-terminus by three residues results in complete loss of channel-forming activity. These changes in channel-forming properties upon altering the peptide sequence do not mirror changes in haemolytic activity. The results lend support to the proposal that channel formation and haemolysis are distinct events. Channel properties are discussed in the context of a model in which the pore is formed by a bundle of approximately parallel transbilayer helices.