Long-term exposure to irinotecan reduces cell migration in glioma cells.

In spite of considerable research into the therapies for glioblastoma multiforme this tumour type remains very difficult to treat. As well as having a tendency to be inherently resistant to chemotherapy, glioblastoma multiforme also displays local invasion. Cell line studies have a continued and important role to play in understanding the mechanisms associated with both chemotherapy resistance and invasion. In the current study we have utilized the C6 glioma cell line to investigate the response to long-term, clinically relevant application of topoisomerase I and II inhibitors. Treatment with etoposide resulted in an increase in resistance to this topoisomerase II inhibitor. By contrast, the continuous exposure to a topoisomerase I inhibitor did not result in increased drug resistance, but was associated with a reduction in cell migration. This data supports further investigation of topoisomerase I inhibition as a means to inhibit glioma invasion without the development of parallel chemoresistance.

Two approaches to discovering and developing new drugs for Chagas disease.

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.

Two approaches to discovering and developing new drugs for Chagas disease

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.

Structure-based interpretation of the mutagenesis database for the nucleotide binding domains of P-glycoprotein.

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.

Principles of proteomics and its applications in cancer.

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.

Multiple drugbinding sites on the R482G isoform of the ABCG2 transporter.

BACKGROUND & PURPOSE: Drug-resistant cancer cells frequently display efflux pumps such as P-glycoprotein (P-gp), the multidrug resistance associated protein (MRP1) or the transporter ABCG2. These transporters are each capable of mediating the active efflux of numerous anticancer drugs and display relatively distinct substrate preferences. The last, most recently discovered member, ABCG2, plays a major role in resistance in several types of cancer and the precise pharmacology of this multidrug transporter remain unresolved as does the nature of substrate binding. EXPERIMENTAL APPROACH: Plasma membranes from insect cells expressing ABCG2 were used to characterise binding of [3H]daunomycin to the multidrug transporter. The kinetics of association and dissociation for this substrate and several other compounds were also determined in this experimental system. KEY RESULTS: The dissociation constant for [3H]daunomycin binding was 564 +/- 57 nM and a Hill slope of 1.4 suggested cooperative binding. Doxorubicin, prazosin and daunomycin completely displaced the binding of radioligand, while mitoxantrone and Hoechst 33342 produced only a partial displacement. Analysis of the dissociation rates revealed that [3H]daunomycin and doxorubicin bind to multiple sites on the transporter. CONCLUSIONS: Both kinetic and equilibrium data support the presence of at least two symmetric drug binding sites on ABCG2, which is distinct from the asymmetry observed for P-gp. The data provide the first molecular details underlying the mechanism by which this transporter is capable of interacting with multiple substrates.

ABC proteins and antibiotic drug resistance: is it all about transport?

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.

A structural perspective on the enzymes that convert dTDP-d-glucose into dTDP-l-rhamnose.

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.

Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle.

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.

Overexpression, purification, crystallization and data collection of a single-stranded DNA-binding protein from Sulfolobus solfataricus.

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.

Simulation studies on bacteriorhodopsin bundle of transmembrane alpha segments.

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.

Protein-water-ion interactions in a model of the pore domain of a potassium channel: a simulation study.

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.

The pore-lining region of shaker voltage-gated potassium channels: comparison of beta-barrel and alpha-helix bundle models.

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 influenza A virus M2 channel: a molecular modeling and simulation study.

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 - modelling via restrained molecular dynamics simulations.

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.

Ion channels formed by HIV-1 Vpu: a modelling and simulation study.

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.

Ferrocenoyl derivatives of alamethicin: redox-sensitive ion channels.

The synthesis and single-channel characterization of two redox-active C-terminal derivatives of alamethicin are herein described. The reduced [Fe(II)] forms of ferrocenoyl-alamethicin (Fc-ALM) and 1'-carboxyferrocenoyl-alamethicin (cFc-ALM) are shown to form voltage-dependent ion channels at cis positive potentials in planar lipid bilayers (PLB) with conductance properties similar to those of alamethicin. In situ oxidation of Fc-ALM [to Fe(III)] in the PLB apparatus causes a time-dependent elimination of channel openings, which can be restored by an increase in the transbilayer potential. In contrast, oxidation of cFc-ALM leads to the formation of shorter-lived channels. Pretreatment of the ferrocenoyl peptides with oxidizing agent alters their single-channel properties in a qualitatively similar manner, establishing that the changes in channel properties in the presence of oxidizing agents are due specifically to ferrocenoyl oxidation. We suggest that the redox sensitivity of these ferrocene-containing ion channels may be governed by a combination of the following factors: (1) changes in hydrophobicity; (2) alteration of peptide molecular dipole; and (3) alterations in tendencies toward self-association. However, oxidation induced changes in peptide conformation cannot be ruled out. Our results provide evidence that it is possible to engineer channel-forming peptides that respond to specific changes in the chemical environment.