ORCID Profile
0000-0001-6136-3532
Current Organisation
University of Leeds
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Publisher: Elsevier BV
Date: 2004
DOI: 10.1016/J.BCP.2003.07.016
Abstract: Platinum(II)-based anticancer drugs are associated with high reactivity and thus a poor biological stability. The platinum(IV)-complexes display potential advantages due to their greater stability and bioreductive activation, thereby allowing a greater proportion of the drug to arrive at the target intact. All compounds tested were able to produce cytotoxicity in monolayer cell cultures, however, the potencies of platinum(IV) drugs were lower than that observed for the platinum(II) compounds or established organic chemotherapeutic agents. There was no significant alteration in the potency of platinum(II) or (IV) compounds to produce cytotoxicity in multicellular tumour spheroids (MCTS) compared to monolayer cultures. All the organic and platinum-based cytotoxic agents produced, to varying degrees, either a retardation or reduction in MCTS growth. Proliferating cells were restricted to the outer two to three cellular layers in intermediate (d=350 microm) and large (d=600 microm) MCTS. Regardless of MCTS size, drug treatment produced a larger and more widely distributed proliferating cell population, consistent with the recruitment of quiescent cells to the proliferating pool following cytotoxic damage. Histology indicated that the predominant morphological change was that of apoptosis, although there was some drug-dependent effects such as the metaphase arrest produced by vinblastine and chromatin dispersal to the periphery of nuclei produced by doxorubicin. In summary, whilst the platinum(IV) derivatives were able to produce cytotoxicity via apoptosis, the introduction of a stable axial group significantly retarded the rate at which this occurred.
Publisher: Wiley
Date: 13-06-2011
DOI: 10.1111/J.1742-4658.2011.08169.X
Abstract: Mutations to members of the A subfamily of ATP binding cassette (ABC) proteins are responsible for a number of diseases typically they are associated with aberrant cellular lipid transport processes. Mutations to the ABCA4 protein are linked to a number of visual disorders including Stargardt's disease and retinitis pigmentosa. Over 500 disease-associated mutations in ABCA4 have been demonstrated however, the genotype-phenotype link has not been firmly established. This shortfall is primarily because the function of ABCA4 in the visual cycle is not yet fully understood. One hypothesis suggests that ABCA4 mediates the trans-bilayer translocation of retinal-phosphatidylethanolamine conjugates to facilitate the retinal regeneration process in the visual cycle. This review examines the evidence to support, or refute, this working hypothesis on the function of this clinically important protein.
Publisher: Elsevier BV
Date: 05-1995
DOI: 10.1016/0005-2736(95)00042-2
Abstract: It has previously been demonstrated that opiates interact directly with P-glycoprotein in drug resistant Chinese hamster ovary (CHO) cells (Callaghan, R. and Riordan, J.R. (1993) J. Biol. Chem. 268, 16059-16064). In this study we have examined the effects of several opiates on the growth of drug sensitive and resistant CHO and human MCF7 cell lines. The growth of P-glycoprotein expressing cells was inhibited by the opiates pentazocine, pethidine and naloxone to a greater extent than in drug sensitive cells. Since P-glycoprotein is localised at the plasma membrane the effects of opiates on membrane biophysical properties were investigated. The opiates caused a fluidizing effect in membranes from P-glycoprotein expressing cells and decreased the basal level of P-glycoprotein phosphorylation. In addition, they were able to increase the leakage of the membrane impermeant compound 6-carboxyfluorescein entrapped in model membrane vesicles. The ability to alter membrane biophysical properties correlated with the inhibitory effects on growth of drug resistant cells. These results suggest that the collateral sensitivity of P-glycoprotein expressing cell lines to opiates is mediated by the drugs' effects on the plasma membrane.
Publisher: Elsevier BV
Date: 10-2005
DOI: 10.1016/J.BCP.2005.07.016
Abstract: The chemotherapeutic drug cisplatin is an important treatment for many types of solid tumours, in particular non-small cell lung cancer (NSCLC). Platinum(IV) complexes offer several advantages to cisplatin due to their requirement for reduction to the active platinum(II) form to elicit cytotoxicity. This should minimise non-specific effects and facilitate higher amounts of the active complexes reaching the target DNA. Hypoxia and a quiescent cell population are features of the tumour microenvironment known to lead to resistance to many chemotherapeutic agents. It is unclear how these microenvironmental factors will impact on the efficacy of novel platinum(IV) complexes. Consequently, the cytotoxicities of several platinum drugs were determined in monolayer and tumour spheroid cultures derived from NSCLC lines. Platinum(IV) reduction potential correlated well with cytotoxicity. The complex containing a chloro axial ligand demonstrated the greatest potency and the drug with the hydroxy ligand was the least effective. Although drug cytotoxicity was not enhanced under hypoxic conditions, both cisplatin and the platinum(IV) complexes retained full potency. In addition, all of the platinum drugs retained the ability to evoke apoptosis in quiescent cells. In summary, unlike many anticancer drugs, the platinum(IV) complexes retain cytotoxic potency under resistance-inducing tumour microenvironmental conditions and warrant further investigation as more selective alternatives to current platinum-based therapy for the treatment of solid tumours.
Publisher: American Chemical Society (ACS)
Date: 29-08-2018
DOI: 10.1021/ACS.JMEDCHEM.8B00986
Abstract: A series of 5-substituted tetrahydroisoquinolines was synthesized via a 10-step linear synthesis to assess whether replacement of noscapine's southern isobenzofuranone with other moieties resulted in retained cytotoxic activity. One such molecule, 18g, bearing a para-methoxybenzyl functionality with N-ethylcarbamoyl substitution, produced cell-cycle arrest at the G2/M phase with an EC
Publisher: American Chemical Society (ACS)
Date: 06-06-2003
DOI: 10.1021/BI0341049
Abstract: Our aim is to provide molecular understanding of the mechanisms underlying the (i) interaction between the two nucleotide binding domains (NBDs) and (ii) coupling between NBDs and transmembrane domains within P-glycoprotein (Pgp) during a transport cycle. To facilitate this, we have introduced a number of unique cysteine residues at surface exposed positions (E393C, S452C, I500C, N508C, and K578C) in the N-terminal NBD of Pgp, which had previously been engineered to remove endogenous cysteines. Positions of the mutations were designed using a model based on crystallographic features of prokaryotic NBDs. The single cysteine mutants were expressed in insect cells using recombinant baculovirus and the proteins purified by metal affinity chromatography by virtue of a polyhistidine tag. None of the introduced cysteine residues perturbed the function of Pgp as judged by the characteristics of drug stimulated ATP hydrolysis. The role of residues at each of the introduced sites in the catalytic cycle of Pgp was investigated by the effect of covalent conjugation with N-ethyl-maleimide (NEM). All but one mutation (K578C) was accessible to labeling with [(3)H]-NEM. However, perturbation of ATPase activity was only observed for the derivitized N508C isoform. The principle functional manifestation was a marked inhibition of the "basal" rate of ATP hydrolysis. Neither the extent nor potency to which a range of drugs could affect the ATPase activity were altered in the NEM conjugated N508C isoform. The results imply that the accessibility of residue 508, located in the alpha-helical subdomain of NBD1 in Pgp, is altered by the conformational changes that occur during ATP hydrolysis.
Publisher: Elsevier BV
Date: 03-2003
Publisher: Wiley
Date: 09-12-2019
DOI: 10.1111/FEBS.15148
Abstract: The accumulation of amyloid-β (Aβ) peptides is a key histopathological feature of the Alzheimer's brain. Defective clearance mechanisms result in toxic levels of soluble Aβ
Publisher: The Company of Biologists
Date: 15-05-1100
DOI: 10.1242/BIO.058613
Abstract: A dramatic rise of infections with antibiotic-resistant bacterial pathogens continues to challenge the healthcare field due to the lack of effective treatment regimes. As such, there is an urgent need to develop new antimicrobial agents that can combat these multidrug-resistant superbugs. Mitochondria are central regulators of metabolism and other cellular functions, including the regulation of innate immunity pathways involved in the defense against infection. The mitochondrial unfolded protein response (UPRmt) is a stress-activated pathway that mitigates mitochondrial dysfunction through the regulation of genes that promote recovery of the organelle. In the model organism Caenorhabditis elegans, the UPRmt also mediates an antibacterial defense program that combats pathogen infection, which promotes host survival. We sought to identify and characterize antimicrobial effectors that are regulated during the UPRmt. From our search, we discovered that the antimicrobial peptide CNC-4 is upregulated during this stress response. CNC-4 belongs to the caenacin family of antimicrobial peptides, which are predominantly found in nematodes and are known to have anti-fungal properties. Here, we find that CNC-4 also possesses potent antimicrobial activity against a spectrum of bacterial species and report on its characterization.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 26-11-2018
Abstract: The antitussive agent noscapine has been shown to inhibit the proliferation of cancer cells by disruption of tubulin dynamic. However, the efficacy of several anticancer drugs that inhibit tublin dynamics (vinca alkaloids and taxanes) is reduced by the multidrug resistance phenotype. These compounds are substrates for P-glycoprotein (P-gp)-mediated extrusion from cells. Consequently, the antiproliferative activity of noscapine and a series of derivatives was measured in drug-sensitive and drug-resistant cells that overexpress P-gp. None of the noscapine derivatives displayed lower potency in cells overexpressing P-gp, thereby suggesting a lack of interaction with this pump. However, the cellular efflux of a fluorescent substrate by P-gp was potently inhibited by noscapine and most derivatives. Further investigation with purified, reconstituted P-gp demonstrated that inhibition of P-gp function was due to direct interaction of noscapine derivatives with the transporter. Moreover, coadministration of vinblastine with two of the noscapine derivatives displayed synergistic inhibition of proliferation, even in P-gp-expressing resistant cell lines. Therefore, noscapine derivatives offer a dual benefit of overcoming the significant impact of P-gp in conferring multidrug resistance and synergy with tubulin-disrupting anticancer drugs.
Publisher: Informa UK Limited
Date: 03-2019
DOI: 10.2147/IJN.S189924
Publisher: Portland Press Ltd.
Date: 10-2015
DOI: 10.1042/BST20150131
Abstract: It is almost 40 years since the drug efflux pump P-glycoprotein (permeability glycoprotein or P-gp) was shown to confer multi-drug resistance in cancer cells. This protein has been one of the most extensively investigated transport proteins due to its intriguing mechanism and its affect in oncology. P-gp is known to interact with over 300 compounds and the ability to achieve this has not yet been revealed. Following the binding of substrate and nucleotide, a complex series of conformational changes in the membrane and cytosolic domains translocates substrate across the membrane. Despite over 30 years of biochemical investigation, the availability of structural data and a plethora of chemical tools to modulate its function, the molecular mechanism remains a mystery. In addition, overcoming its activity in resistant cancer cells has not been achieved in the clinic, thereby garnering some degree of pessimism in the field. This review highlights the progress that has been achieved in understanding this complex protein and the value of undertaking molecular studies.
Publisher: Wiley
Date: 03-2008
Abstract: Expression of the ABC transporter P-glycoprotein (P-gp or ABCB1) is associated with resistance to chemotherapy in cancer. However, early investigations into the regulation of ABCB1 expression revealed that the process is not a classical induction as observed for certain metabolizing enzymes. The process involves the cellular stress response pathway initiated by either inflicted (e.g., chemotherapy damage) or endogenous (e.g., hypoxia) factors. However, ABCB1 is also expressed in a number of noncancerous tissues. In particular, the protein is found at tissues providing a barrier or secretory function. The localization of ABCB1 in normal tissues will impact significantly on drug pharmacokinetics, in particular the absorption and elimination processes. This review also describes the mechanism underlying ABCB1 expression in noncancerous tissue, a process that does not involve the stress response.
Publisher: Springer Science and Business Media LLC
Date: 1998
DOI: 10.1038/34669
Abstract: Bacteria have developed many fascinating antibiotic-resistance mechanisms. A protein in Lactococcus lactis, LmrA, mediates antibiotic resistance by extruding hiphilic compounds from the inner leaflet of the cytoplasmic membrane. Unlike other known bacterial multidrug-resistance proteins, LmrA is an ATP-binding cassette (ABC) transporter. The human multidrug-resistance P-glycoprotein, encoded by the MDR1 gene, is also an ABC transporter, overexpression of which is one of the principal causes of resistance of human cancers to chemotherapy. We expressed lmrA in human lung fibroblast cells. Surprisingly, LmrA was targeted to the plasma membrane and conferred typical multidrug resistance on these human cells. The pharmacological characteristics of LmrA and P-glycoprotein-expressing lung fibroblasts were very similar, and the affinities of both proteins for vinblastine and magnesium-ATP were indistinguishable. Blockers of P-glycoprotein-mediated multidrug resistance also inhibited LmrA-dependent drug resistance. Kinetic analysis of drug dissociation from LmrA expressed in plasma membranes of insect cells revealed the presence of two allosterically linked drug-binding sites indistinguishable from those of P-glycoprotein. These findings have implications for the reversal of antibiotic resistance in pathogenic microorganisms. Taken together, they demonstrate that bacterial LmrA and human P-glycoprotein are functionally interchangeable and that this type of multidrug-resistance efflux pump is conserved from bacteria to man.
Publisher: Wiley
Date: 11-2006
Publisher: Springer Science and Business Media LLC
Date: 07-02-2008
Publisher: Elsevier BV
Date: 04-2006
DOI: 10.1016/J.BCP.2005.12.039
Abstract: Platinum complexes are widely used in cancer chemotherapy however, they are associated with toxicity, high "non-specific" reactivity and relatively poor pharmacokinetic profiles. In particular, their low cellular uptake and rapid metabolic inactivation means that the amount of "active" drug reaching the nuclear compartment is low. Our strategy to facilitate nuclear accumulation was to introduce a hydrophobic anthraquinone (1C3) moiety to the Pt-complex. Anthraquinones are known to readily intercalate into DNA strands and hence, the Pt-1C3 complex may represent an effective system for the delivery of the platinum moiety to nuclear DNA. Efficacy of the complex was determined by measuring the extent and potency of cytotoxicity in comparison to cisplatin and an anthraquinone based anticancer drug, doxorubicin. The Pt-1C3 complex generated higher levels of cytotoxicity than cisplatin, with a potency of 19 +/- 4 microM in the DLD-1 cancer cell line. However, this potency was not significantly different to that of the 1C3 moiety alone. To examine the reason for the apparent lack of platinum related cytotoxicity, the cellular distribution was characterised. Confocal fluorescence microscopy indicated that the Pt-1C3 complex was rapidly sequestered into lysosomes, in contrast to the nuclear localisation of doxorubicin. In addition, there was negligible DNA associated Pt following administration of the novel complex. Thus, the addition of a 1C3 moiety generated sequestration of the complex to lysosomes, thereby preventing localisation to the nucleus.
Publisher: American Chemical Society (ACS)
Date: 28-02-2008
DOI: 10.1021/BI7023089
Abstract: Reduced intracellular drug accumulation due to the activity of the drug efflux pump ABC (B1) is a major mechanism in the resistance of cancer cells to chemotherapy. ABC (B1) is a poly specific transporter, and the molecular mechanism of its complex translocation process remains to be elucidated. To understand the process will require information on the regions involved in drug binding and those that couple this event to nucleotide hydrolysis. The present investigation focuses on the cytosolic region of transmembrane helix 6 (TM6), which has been widely attributed with a central role in the translocation process. A series of ABC (B1) isoforms containing a unique cysteine within TM6 was constructed and the resultant proteins purified and reconstituted. Accessibility of the cysteines to covalent modification by maleimide reagents was measured for the basal, ATP bound and vanadate trapped conformations of each isoform. Residues at the two extremes of the TM6 region examined (amino acids 344 to 360) were considerably more accessible than the central segment, the latter of which also failed to undergo significant conformational changes during the catalytic cycle. Covalent modification of the cytosolic segment of TM6 did, however, attenuate drug stimulation of ATP hydrolysis and demonstrates an important role for this segment in coupling drug binding to ATP hydrolysis during translocation.
Publisher: Wiley
Date: 07-08-2008
DOI: 10.1111/J.1742-4658.2008.06578.X
Abstract: ABCG2 confers resistance to cancer cells by mediating the ATP-dependent outward efflux of chemotherapeutic compounds. Recent studies have indicated that the protein contains a number of interconnected drug binding sites. The present investigation examines the coupling of drug binding to ATP hydrolysis. Initial drug binding to the protein requires a high-affinity interaction with the drug binding site, followed by transition and reorientation to the low-affinity state to enable dissociation at the extracellular face. [3H]Daunomycin binding to the ABCG2 R482G isoform was examined in the nucleotide-bound and post-hydrolytic conformations. Binding of [3H]daunomycin was displaced by ATP analogues, indicating transition to a low-affinity conformation prior to hydrolysis. The low-affinity state was observed to be retained immediately post-hydrolysis. Therefore, the dissociation of phosphate and/or ADP is likely to be responsible for resetting of the transporter. The data indicate that, like ABCB1 and ABCC1, the 'power stroke' for translocation in ABCG2 R482G is the binding of nucleotide.
Publisher: Portland Press Ltd.
Date: 28-02-2017
DOI: 10.1042/EBC20160060
Abstract: Malarial infection continues to impart devastating health problems in the developing world. Treatment of malaria has involved chemotherapy since 168 BC, with the most prevalent and successful forms using plant alkaloids. Perhaps the greatest treatment success against malaria was by chloroquine, a synthetic derivative of the quinines found in the Cinchona tree bark. Chloroquine is able to kill parasites by interfering with haem metabolism in the parasite’s digestive vacuole. The widespread use of chloroquine predictably resulted in the development of drug-resistant malaria and the most highly implicated resistance mediators are the transporter proteins P-glycoprotein (P-gp) homologue 1 (P-gh1) and Plasmodium falciparum chloroquine-resistance transporter (PfCRT), which reside on the parasite’s digestive vacuole. The presence of PfCRT and P-gh1 on the vacuole membrane is analogous to the two-headed fictional creature known as the “Pushmi-Pullyu”. P-gh1 (Pushmi) increases influx of chloroquine into the vacuole, while PfCRT (Pullmi) causes efflux of chloroquine from the vacuole. This review describes how drug-resistant malarial parasites co-ordinate chloroquine distribution through adaptive mutations to promote their survival in the presence of this cytotoxic drug.
Publisher: Springer Science and Business Media LLC
Date: 14-09-2013
DOI: 10.1007/S12013-013-9757-7
Abstract: Solid tumours undergo considerable alterations in their metabolism of nutrients in order to generate sufficient energy and biomass for sustained growth and proliferation. During growth, the tumour microenvironment exerts a number of influences (e.g. hypoxia and acidity) that affect cellular biology and the flux or utilisation of fuels including glucose. The tumour spheroid model was used to characterise the utilisation of glucose and describe alterations to the activity and expression of key glycolytic enzymes during the tissue growth curve. Glucose was avidly consumed and associated with the production of lactate and an acidified medium, confirming the reliance on glycolytic pathways and a diminution of oxidative phosphorylation. The expression levels and activities of hexokinase, phosphofructokinase-1, pyruvate kinase and lactate dehydrogenase in the glycolytic pathway were measured to assess glycolytic capacity. Similar measurements were made for glucose-6-phosphate dehydrogenase, the entry point and regulatory step of the pentose-phosphate pathway (PPP) and for cytosolic malate dehydrogenase, a key link to TCA cycle intermediates. The parameters for these key enzymes were shown to undergo considerable variation during the growth curve of tumour spheroids. In addition, they revealed that the dynamic alterations were influenced by both transcriptional and posttranslational mechanisms.
Publisher: American Chemical Society (ACS)
Date: 03-05-2011
DOI: 10.1021/AC1031594
Publisher: Elsevier BV
Date: 11-2012
DOI: 10.1016/J.BIORTECH.2012.07.073
Abstract: Bio-oil from pyrolysis of mallee (Eucalyptus loxophleba ssp. gratiae) leaves differs from that obtained with wood by its content of cyclic ethers, terpenoids and N-containing organic compounds. Upgrading of the leaf bio-oil in methanol with a solid acid catalyst was investigated and it was found that the N-containing organics in the bio-oil lead to deactivation of the catalyst in the initial stage of exposure and have to be removed via employing high catalyst loading to allow the occurrence of other acid-catalysed reactions. Eucalyptol, the main cyclic ether in the bio-oil, could be converted into the aromatic hydrocarbon, p-cymene, through a series of intermediates including α-terpineol, terpinolene, and α-terpinene. Various steps such as ring-opening, dehydration, isomerisation, and aromatization were involved in the conversion of eucalyptol. The terpenoids in bio-oil could also be converted into aromatic hydrocarbons that can serve as starting materials for the synthesis of fine chemicals, via the similar processes.
Publisher: Elsevier BV
Date: 10-2004
Publisher: Wiley
Date: 10-1997
Publisher: Wiley
Date: 22-11-2019
Publisher: Humana Press
Date: 05-10-2010
Publisher: Elsevier BV
Date: 09-2010
Publisher: Springer Science and Business Media LLC
Date: 10-1998
DOI: 10.1038/BJC.1998.597
Abstract: XR9051 (N-(4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydro-2-isoquinolyl)ethyl)phe nyl)-3-((3Z,6Z)-6-benzylidene-1-methyl-2,5-dioxo-3-pipera zinylidene) methylbenzamide) was identified as a potent modulator of P-glycoprotein-mediated multidrug resistance (MDR) following a synthetic chemistry programme based on a natural product lead compound. The activity of XR9051 was determined using a panel of human and murine drug-resistant cell lines (H69/LX4, 2780AD, EMT6/AR 1.0, MC26 and P388/DX Johnson). XR9051 was able to reverse resistance to a variety of cytotoxic drugs, including doxorubicin, etoposide and vincristine, which are associated with classical MDR. At a concentration of 0.3-0.5 microM, XR9051 was able to fully sensitize resistant cells to cytotoxics, whereas little or no effect was observed on the corresponding parental cell lines. No effect of XR9051 was observed on the response of cells to non-MDR cytotoxics such as methotrexate and 5-fluorouracil. XR9051 was consistently more potent than cyclosporin A (CsA) and verapamil (Vpm) in all assays used. XR9051 inhibited the efflux of [3H]daunorubicin from preloaded cells and, unlike CsA and Vpm, remained active for several hours after removal of resistance-modifying agent. In photoaffinity labelling experiments employing [3H]azidopine, XR9051 was able to displace binding to P-glycoprotein. In binding studies using [3H]vinblastine, XR9051 was shown to be a potent inhibitor of the binding of the cytotoxic to P-glycoprotein (EC50 = 1.4 +/- 0.5 nM). Taken together, the results indicate that XR9051 reverses the MDR phenotype through direct interaction with P-glycoprotein.
Publisher: MDPI AG
Date: 03-05-2020
Abstract: Investigative systems for purified membrane transporters are almost exclusively reliant on the use of phospholipid vesicles or liposomes. Liposomes provide an environment to support protein function however, they also have numerous drawbacks and should not be considered as a “one-size fits all” system. The use of artificial vesicles comprising block co-polymers (polymersomes) offers considerable advantages in terms of structural stability provision of sufficient lateral pressure and low passive permeability, which is a particular issue for transport assays using hydrophobic compounds. The present investigation demonstrates strategies to reconstitute ATP binding cassette (ABC) transporters into hybrid vesicles combining phospholipids and the block co-polymer poly (butadiene)-poly (ethylene oxide). Two efflux pumps were chosen namely the Novosphingobium aromaticivorans Atm1 protein and human P-glycoprotein (Pgp). Polymersomes were generated with one of two lipid partners, either purified palmitoyl-oleoyl-phosphatidylcholine, or a mixture of crude E. coli lipid extract and cholesterol. Hybrid polymersomes were characterised for size, structural homogeneity, stability to detergents, and permeability. Two transporters, NaAtm1 and P-gp, were successfully reconstituted into pre-formed and surfactant-destabilised hybrid polymersomes using a detergent adsorption strategy. Reconstitution of both proteins was confirmed by density gradient centrifugation and the hybrid polymersomes supported substrate dependent ATPase activity of both transporters. The hybrid polymersomes also displayed low passive permeability to a fluorescent probe (calcein acetomethoxyl-ester (C-AM)) and offer the potential for quantitative measurements of transport activity for hydrophobic compounds.
Publisher: Elsevier BV
Date: 09-2018
DOI: 10.1016/J.YMETH.2018.02.012
Abstract: Membrane proteins are notoriously difficult to investigate in isolation. The focus of this chapter is the key step following extraction and purification of membrane proteins namely reconstitution. The process of reconstitution re-inserts proteins into a lipid bilayer that partly resembles their native environment. This native environment is vital to the stability of membrane proteins, ensuring that they undergo vital conformational transitions and maintain optimal interaction with their substrates. Reconstitution may take many forms and these have been classified into two broad categories. Symmetric systems enable unfettered access to both sides of a bilayer. Compartment containing systems contain a lumen and are ideally suited to measurement of transport processes. The investigator is encouraged to ascertain what aspects of protein function will be undertaken and to apply the most advantageous reconstitution system or systems. It is important to note that the process of reconstitution is not subject to defined protocols and requires empirical optimisation to specific targets.
Publisher: Wiley
Date: 09-1999
Publisher: Elsevier BV
Date: 07-2017
Publisher: Elsevier BV
Date: 09-2006
DOI: 10.1016/J.EJCA.2006.05.020
Abstract: The distribution of chemotherapeutics in solid tumours is poorly understood and the contribution it makes to treatment failure is unknown. Novel approaches are required to understand how the three-dimensional organisation of cancer cells in solid tumours affects drug availability. Since convective drug transport is limited by increased interstitial pressure in poorly vascularised cancers, the aim of this study was to measure the diffusive hindrance exerted by solid tumour tissue. Multicell layer tumour models comprising DLD1 colon cancer cells were characterised and fluxes were determined for [3H]-vinblastine and [14C]-sucrose. The mathematical models provided the diffusion coefficients for both compounds and predicted higher exposure of cells in the vicinity of vessels. The diffusion of vinblastine was three times slower than that of sucrose. Although slow diffusion delays vinblastine penetration into the avascular regions of tumours, the proliferating cells are generally in the marginal area of tumours. The mathematical model that we have developed enabled accurate quantification of drug pharmacokinetic behaviour, in particular, the diffusivity of vinblastine within solid tissue. This mathematical model may be adapted readily to incorporate the influence of factors mediating pharmacokinetic drug resistance.
Publisher: Elsevier BV
Date: 09-1997
DOI: 10.1016/S0005-2736(97)00079-5
Abstract: P-Glycoprotein (P-gp) is a 180-kDa membrane-bound transporter which can confer the multi-drug resistance phenotype on tumor cells. We have examined the factors required to preserve activity of P-gp during its purification. The starting material for purification was plasma membranes from Chinese hamster ovary (CHrB30) cells, overexpressing P-glycoprotein. These membranes displayed drug stimulated ATPase activity (Vm = 897 +/- 55 nmol min(-1) mg(-1) Km = 1.8 +/- 0.4 mM) and high affinity binding of [3H]vinblastine (Kd = 36 +/- 5 nM Bm = 161 +/- 11 pmol/mg). Several non-ionic detergents which readily solubilized P-glycoprotein significantly inhibited ATPase activity and drug binding at concentrations well below their respective CMC values. This inactivation was prevented by excess crude lipid mixtures, with the greatest protection afforded against dodecyl-maltoside. Furthermore, the significantly reduced binding affinity and capacity of solubilized P-gp was partly reversed by the addition of lipids. A combination of anion-exchange and hydroxyapatite chromatography were used to purify P-gp with high yield to greater than 90%. The purified, reconstituted P-gp displayed high ATPase activity (Vm = 2137 +/- 309 Km = 2.9 +/- 0.9 mM) which was stimulated by verapamil (EC50 = 3.8 +/- 0.6 microM) and inhibited by orthovanadate (3.1 +/- 0.8 microM). Pure P-gp also displayed high affinity vinblastine binding (Kd = 64 +/- 9 nM) with a capacity of 2320 +/- 192 pmol/mg. This purification scheme yields the highest P-gp activity reported to date, and indicates a dependence of function on maintaining a lipid-protein interface.
Publisher: Elsevier BV
Date: 03-2004
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 03-02-2014
Publisher: Elsevier BV
Date: 02-2007
DOI: 10.1016/J.PHARMTHERA.2006.10.003
Abstract: This year marks the 30th anniversary of the discovery of the multidrug resistance (MDR) ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp). Since then a considerable research effort has attempted to provide a greater understanding of the biological enigma of "multidrug" efflux. Moreover, the growing correlation between P-gp expression and a negative prognosis or poor outcome for chemotherapy has sparked significant interest in the generation of inhibitors. How close are we to overcoming the unwanted actions of P-gp in resistant cancer following 30 years of research? The initial inhibitors were pre-existing clinically used compounds and exploited the broad specificity of P-gp. Unfortunately, the concentrations required to inhibit P-gp meant that these compounds generated considerable toxicity. Pharmacological investigations progressed to rational design using the 1st generation compounds as a template structure. Inherent toxicity of the drugs was reduced however, pharmacokinetic interactions with the anticancer drugs were unsustainable. Generation of the most recent of inhibitors employed combinatorial chemistry to produce a handful of potent and selective P-gp inhibitors. Some of these drugs have progressed to clinical trials with poor results or in some cases, undisclosed progress. There remains a clear need for the generation of P-gp inhibitors and this review describes the potential for a structure-based design to facilitate this undertaking. In particular, the plethora of functional data can provide important regions on the protein that could conceivably be exploited as inhibitor targets.
Publisher: Oxford University Press (OUP)
Date: 09-07-2008
Publisher: Springer Science and Business Media LLC
Date: 15-03-2011
DOI: 10.1007/S00280-011-1598-8
Abstract: The intra-tumour distribution of anticancer drugs remains an important, but often under-estimated, influence on drug efficacy. Tumour acidity and the presence of efflux pumps were examined for their influence on the distribution of doxorubicin in a solid tumour model. Anticancer drug distribution and overall accumulation was measured in tumour spheroids (TS) of varying sizes. The distribution profiles were examined in normoxic and hypoxic TS, the latter generating metabolic acidosis. Finally, the drug distribution profiles were related to efficacy using radial outgrowth assays. In large tumour spheroids (TS) (d ~500 μm), intracellular accumulation of doxorubicin was restricted to cells in the outermost layers and failed to accumulate within the viable cells in the 'intermediate' hypoxic zone. A similar profile was obtained for another protonatable amine, 7-AAD. In contrast, the distribution of the non-ionisable drug (at physiological pH) BODIPY-Taxol was uniform throughout the TS. In order to independently model the hypoxic and normoxic zones of TS, we compared drug accumulation in small entirely normoxic TS (d ~200 μm) with equivalent sized ones exposed to hypoxia in an anaerobic chamber. Exposure of TS to hypoxia caused a considerable reduction in the pH of the bathing medium and lower tissue accumulation of doxorubicin. Interstitial acidity reduces the proportion of doxorubicin in the non-ionised form. In TS, the accumulation and distribution of doxorubicin was influenced by both the expression of P-glycoprotein and hypoxia-induced acidity. Therefore, optimisation of doxorubicin chemotherapy for hypoxic tumours will require circumvention of both of these crucial pharmacokinetic determinants.
Publisher: Springer Science and Business Media LLC
Date: 10-2003
Publisher: Elsevier BV
Date: 11-2017
DOI: 10.1016/J.EJPHAR.2017.09.001
Abstract: ZSTK474 is a potent phosphoinositide 3-kinase (PI3K) inhibitor that reduces cell proliferation via G
Publisher: Elsevier BV
Date: 11-2006
DOI: 10.1016/J.STR.2006.08.014
Abstract: ABCG2 is a multidrug efflux pump associated with resistance of cancer cells to a plethora of unrelated drugs. ABCG2 is a "half-transporter," and previous studies have indicated that it forms homodimers and higher oligomeric species. In this manuscript, electron microscopic structural analysis directly addressed this issue. An N-terminal hexahistidine-tagged ABCG2(R482G) isoform was expressed to high levels in insect cells. An extensive detergent screen was employed to effect extraction of ABCG2(R482G) from membranes and identified only the fos-choline detergents as efficient. Soluble protein was purified to >95% homogeneity by a three-step procedure while retaining the ability to bind substrates. Cryonegative stain electron microscopy of purified ABCG2(R482G) provided 3D structural data at a resolution of approximately 18 A. Single-particle analysis revealed that the complex forms a tetrameric complex ( approximately 180 A in diameter x approximately 140 A high) with an aqueous central region. We interpret the tetrameric structure as comprising four homodimeric ABCG2(R482G) complexes.
Publisher: S. Karger AG
Date: 2008
DOI: 10.1159/000115967
Abstract: Inherent and acquired resistance pathways account for the high rate of failure in cancer chemotherapy. The mechanisms or pathways mediating resistance may be classified as pharmacokinetic (i.e. alter intratumour drug exposue) or pharmacodynamic (i.e. failure to elicit cytotoxicity). More often than not, the resistant phenotype is characterised by alterations in multiple pathways. Consequently, the pathways may act synergistically or generate a broad spectrum of resistance to anticancer drugs. There has been a great deal of systematic characterisation of drug resistance in vitro. However, translating this greater understanding into clinical efficacy has rarely been achieved. This review explores the phenomenon of drug resistance in cancer and highlights the gap between in vitro and in vivo observations. This gap presents a major obstacle in overcoming drug resistance and restoring sensitivity to chemotherapy.
Publisher: Wiley
Date: 2006
DOI: 10.1002/JGM.947
Abstract: Our current understanding of how the unique tumour microenvironment influences the efficacy of gene delivery is limited. The current investigation systematically examines the efficiency of several non-viral gene transfer agents to transfect multicellular tumour spheroids (MCTS), an in vitro model that displays a faithful three-dimensional (3D) representation of solid tumour tissue. Using a luciferase reporter assay, gene transfer to MCTS was optimised for 22 kDa linear and 25 kDa branched polyethyleneimine (PEI), the cationic lipids Lipofectamine(trade mark) and DCChol : DOPE, and the physical approach of tissue electroporation. Confocal microscopy was used to take optical tissue slices to identify the tissue localisation of green fluorescent protein (GFP) reporter gene expression and the distribution of fluorescently labelled complexes. A MCTS model of quiescent tumour regions was used to establish the influence of cellular proliferation status on gene transfer efficiency. Of the polyplexes tested, 22 kDa linear PEI provided optimal gene delivery, with gene expression peaking at 46 h. Despite being the optimal vector tested, PEI-mediated transfection was limited to cells at the MCTS periphery. Using fluorescent PEI, it was found that complexes could only penetrate the outer 3-5 proliferating cell layers of the MCTS, sparing the deeper quiescent cells. Gene delivery in an MCTS model comprised entirely of quiescent cells demonstrated that in addition to being inaccessible to the vector, quiescent tumour regions are inherently less susceptible to PEI-mediated transfection than proliferating regions. This 'resistance' to transfection observed in quiescent cells was overcome through the use of electroporation. Despite the improved efficacy of electroporation in quiescent tissue, the gene expression was still confined to the outer regions of MCTS. The results suggest that limited access to central regions of an MCTS remain a significant barrier to gene delivery. This data provides new insights into tumour-specific factors affecting non-viral gene transfer and highlights the difficulties in delivering genes to avascular tumour regions. The MCTS model is a useful system for the initial screening of future gene therapy strategies for solid tumours.
Publisher: Elsevier BV
Date: 04-2021
Publisher: Wiley
Date: 12-11-2019
Abstract: Noscapine displays weak anticancer efficacy and numerous research efforts have attempted to generate more potent noscapine analogues. These modifications included the replacement of the N-methyl group in the 6'-position with a range of substituents, where N-ethylcarbamoyl substitution was observed to possess enhanced anticancer activity. Herein, we describe advances in this area, namely the synthesis and pharmacological evaluation of a series of N-sulfonyl and N-sulfamoyl noscapine derivatives. A number of these sulfonyl-containing noscapinoids demonstrated improved activities compared to noscapine. ((R)-5-((S)-4,5-Dimethoxy-1,3-dihydroisobenzofuran-1-yl)-4-methoxy-6-((1-methyl-1H-imidazol-4-yl)sulfonyl)-5,6,7,8-tetrahydro[1,3]dioxolo[4,5-g]isoquinoline) (14 q) displayed sub-micromolar activities of 560, 980, 271 and 443 nM against MCF-7, PANC-1, MDA-MB-435 and SK-MEL-5 cells, respectively. This antiproliferative effect was also maintained against drug-resistant NCI/Adr
Publisher: Wiley
Date: 11-12-2014
Abstract: Noscapine, a phthalideisoquinoline alkaloid derived from Papaver somniferum, is a well-known antitussive drug that has a relatively safe in vitro toxicity profile. Noscapine is also known to possess weak anticancer efficacy, and since its discovery, efforts have been made to design derivatives with improved potency. Herein, the synthesis of a series of noscapine analogues, which have been modified in the 6', 9', 1 and 7-positions, is described. In a previous study, replacement of the naturally occurring N-methyl group in the 6'-position with an N-ethylaminocarbonyl was shown to promote cell-cycle arrest and cytotoxicity against three cancer cell lines. Here, this modification has been combined with other structural changes that have previously been shown to improve anticancer activity, namely halo substitution in the 9'-position, regioselective O-demethylation to reveal a free phenol in the 7-position, and reduction of the lactone to the corresponding cyclic ether in the 1-position. The incorporation of new aryl substituents in the 9'-position was also investigated. The study identified interesting new compounds able to induce G2/M cell-cycle arrest and that possess cytotoxic activity against the human prostate carcinoma cell line PC3, the human breast adenocarcinoma cell line MCF-7, and the human pancreatic epithelioid carcinoma cell line PANC-1. In particular, the ethyl urea cyclic ether noscapinoids and a compound containing a 6'-ethylaminocarbonyl along with 9'-chloro, 7-hydroxy and lactone moieties exhibited the most promising biological activities, with EC50 values in the low micromolar range against all three cancer cell lines, and these derivatives warrant further investigation.
Publisher: Elsevier BV
Date: 10-2022
DOI: 10.1016/J.BBAMEM.2022.184005
Abstract: A mechanistic understanding of how P-glycoprotein (Pgp) is able to bind and transport its astonishing range of substrates remains elusive. Pharmacological data demonstrated the presence of at least four distinct binding sites, but their locations have not been fully elucidated. The combination of biochemical and structural data suggests that initial binding may occur in the central cavity or at the lipid-protein interface. Our objective was to define the binding sites for two transported substrates of Pgp the anticancer drug vinblastine and the fluorescent probe rhodamine 123. A series of mutations was generated in positions proximal to previously defined drug-interacting residues on Pgp. The protein was purified and reconstituted into styrene-maleic acid lipid particles (SMALPs) to measure the apparent drug binding constant or into liposomes for assessment of drug-stimulated ATP hydrolysis. The biochemical data were reconciled with structural models of Pgp using molecular docking. The data indicated that the binding of rhodamine 123 occurred predominantly within the central cavity of Pgp. In contrast, the significantly more hydrophobic vinblastine bound to both the lipid-protein interface and within the central cavity. The data suggest that the initial interaction of vinca alkaloids with Pgp occurs at the lipid interface followed by internalisation into the central cavity, which also provides the transport conduit. This model is supported by recent structural observations with Pgp and early biophysical and cross-linking approaches. Moreover, the proposed model illustrates that the broad substrate profile for Pgp is underpinned by a combination of multiple initial interaction sites and an accommodating transport conduit.
Publisher: Wiley
Date: 03-12-2009
DOI: 10.1111/J.1742-4658.2009.07484.X
Abstract: The role of the ATP-binding cassette ABCB1 in mediating the resistance to chemotherapy in many forms of cancer has been well established. The protein is also endogenously expressed in numerous barrier and excretory tissues, thereby regulating or impacting on drug pharmacokinetic profiles. Given these prominent roles in health and disease, a great deal of biochemical, structural and pharmacological research has been directed towards modulating its activity. Despite the effort, only a small handful of compounds have reached the later stages of clinical trials. What is responsible for this poor return on the heavy research investment? Perhaps the most significant factor is the lack of information on the location, physical features and chemical properties of the drug-binding site(s) in ABCB1. This minireview outlines the various strategies and outcomes of research efforts to pin-point the sites of interaction. The data may be assimilated into two working hypotheses to describe drug binding to ABCB1 (a) the central cavity and the (b) domain interface models.
Publisher: Springer Science and Business Media LLC
Date: 26-06-2003
DOI: 10.1007/S00249-003-0327-6
Abstract: Heterologous expression of domains of eukaryotic proteins is frequently associated with formation of inclusion bodies, consisting of aggregated mis-folded protein. This phenomenon has proved a significant barrier to the characterization of domains of eukaryotic ATP binding cassette (ABC) transporters. We hypothesized that the solubility of heterologously expressed nucleotide binding domains (NBDs) of ABC transporters is dependent on the definition of the domain boundaries. In this paper we have defined a core NBD, and tested the effect of extensions to and deletions of this core domain on protein expression. Of 10 NBDs constructed, only one was expressed as a soluble protein in Escherichia coli, with expression of the remaining NBDs being associated with inclusion body formation. The soluble NBD protein we have obtained corresponds to residues 386-632 of P-glycoprotein and represents an optimally defined domain. The NBD has been isolated and purified to 95% homogeneity by a two-step purification protocol, involving affinity chromatography and gel filtration. Although showing no detectable ATP hydrolysis, the protein retains specific ATP binding and has a secondary structure compatible with X-ray crystallographic data on bacterial NBDs. We have interpreted our results in terms of homology models, which suggest that the N-terminal NBD of P-glycoprotein can be produced as a stable, correctly folded, isolate domain with judicious design of the expression construct.
Publisher: Elsevier BV
Date: 08-2004
Publisher: Springer Science and Business Media LLC
Date: 16-06-2008
Publisher: Wiley
Date: 04-2003
DOI: 10.1046/J.1432-1033.2003.03514.X
Abstract: The two nucleotide-binding domains (NBDs) of a number of ATP-binding cassette (ABC) transporters have been shown to be functionally dissimilar, playing different roles in the transport process. A high degree of co-operativity has been determined for the NBDs of the human multidrug transporter, P-glycoprotein. However, the issue of functional symmetry in P-glycoprotein remains contentious. To address this, the NBDs of P-glycoprotein were expressed and purified to 95% homogeneity, as fusions to maltose-binding protein. The NBDs were engineered to contain a single cysteine residue in the Walker-A homology motif. Reactivity of this cysteine residue was demonstrated by specific, time-dependent, covalent labelling with N-ethylmaleimide. No differences in the rates of labelling of the two NBDs were observed. The relative affinity of binding to each NBD was determined for a number of nucleotides by measuring their ability to effect a reduction in N-ethylmaleimide labelling. In general, nucleotides bound identically to the two NBDs, suggesting that there is little asymmetry in the initial step of the transport cycle, namely the recognition and binding of nucleotide. Any observed functional asymmetry in the intact transporter presumably reflects different rates of hydrolysis at the two NBDs or interdomain communications.
Publisher: Elsevier BV
Date: 09-2004
Publisher: Elsevier BV
Date: 04-2011
DOI: 10.1016/J.BIOS.2011.02.019
Abstract: This paper describes the first immunosensing system reported for the detection of bacteria combining immunomagnetic capture and erometric detection in a one-step sandwich format, and in a microfluidic environment. Detection is based on the electrochemical monitoring of the activity of horseradish peroxidase (HRP), an enzyme label, through its catalysis of hydrogen peroxide (H(2)O(2)) in the presence of the mediator hydroquinone (HQ). The enzymatic reaction takes place in an incubation micro-chamber where the magnetic particles (MPs) are confined, upstream from the working electrode. The enzyme product is then pumped along a microchannel, where it is erometrically detected by a set of microelectrodes. This design avoids direct contact of the biocomponents with the electrode, which lowers the risk of electrode fouling. The whole assay can be completed in 1h. The experiments performed with Escherichia coli evidenced a linear response for concentrations ranging 10(2)-10(8) cell ml(-1), with a limit of detection of 55 cells ml(-1) in PBS, without pre-enrichment steps. Furthermore, 100 cells ml(-1) could be detected in milk, and with negligible interference by non-target bacteria such as Pseudomonas.
Publisher: Elsevier BV
Date: 2005
Publisher: Elsevier BV
Date: 05-2012
DOI: 10.1016/J.CANLET.2011.12.002
Abstract: Survivin is an important member of the Inhibitor of Apoptosis Proteins (IAPs) family and has essential roles in apoptosis and cell cycle progression. This gene is commonly upregulated in human cancer and provides an exciting diagnostic and therapeutic target. Survivin is expressed as several isoforms that are generated by alternative splicing, and some of these present antagonistic activities. Currently, information regarding the regulation of these isoforms is lacking. In this study, we sought to analyze survivin Delta Ex3 expression in a three-dimensional model of avascular tumors and its overexpression effects in processes such as proliferation, clonogenicity and apoptosis. We found a positive correlation between spheroid growth and survivin Delta Ex3 expression during the exponential phase. We demonstrated that this isoform not only decreased apoptosis but also inhibited tumor spheroid formation by decreasing proliferation and clonogenic survival. These results point toward a dual and antagonistic effect of this spliced survivin isoform in cancer development.
Publisher: Springer Science and Business Media LLC
Date: 19-06-2007
Publisher: Springer International Publishing
Date: 17-12-2015
Publisher: Springer Science and Business Media LLC
Date: 02-1995
DOI: 10.1038/BJC.1995.59
Abstract: Tamoxifen is an anti-oestrogen which is currently being assessed as a prophylactic for women at high risk of breast cancer. Taxoxifen has also been shown to reverse multidrug resistance in P-glycoprotein (P-gp)-expressing cells, although the mechanism of action is unknown. In this study we demonstrate that tamoxifen interacts directly with P-gp. Plasma membranes from P-gp-expressing cells bound [3H]tamoxifen in a specific and saturable fashion. A 180 kDa membrane protein in these membranes, labelled by the affinity analogue tamoxifen aziridine and azidopine, was shown to be P-gp. Tamoxifen reduced the binding of vinblastine and azidopine to P-gp, and tamoxifen increased [3H]vinblastine accumulation in P-gp-expressing cells to levels approaching those in non-P-gp-expressing cells. However, the cellular accumulation of [3H]tamoxifen itself was not influenced by the presence of P-gp. Thus, tamoxifen appears to reverse multidrug resistance by binding to P-gp and inhibiting the transport of cytotoxic drugs, but does not itself appear to be transported by the protein.
Publisher: American Chemical Society (ACS)
Date: 05-09-2008
DOI: 10.1021/AC800643K
Publisher: Wiley
Date: 25-07-2008
DOI: 10.1016/J.FEBSLET.2008.07.022
Abstract: P-glycoprotein (ABCB1) is an ATP-binding cassette protein that is associated with the acquisition of multi-drug resistance in cancer and the failure of chemotherapy in humans. Structural insights into this protein are described using a combination of small angle X-ray scattering data and cryo-electron crystallography data. We have compared the structures with bacterial homologues, and discuss the development of homology models for P-glycoprotein based on the bacterial Sav1866 structure.
Publisher: Wiley
Date: 02-02-2016
DOI: 10.1002/JCB.25488
Abstract: Solid tumors contend with, and adapt to, a hostile micro-environment that includes limited availability of nutrient fuels and oxygen. The presence of hypoxia (O2 <5%) stabilizes the transcription factor Hif1 and results in numerous cellular adaptations including increased flux of glucose through glycolysis. Increasingly, more sophisticated analysis of tumor oxygenation has revealed large gradients of oxygen tension and significant regions under severe hypoxia (O2 ∼0.1%). The present investigation has demonstrated a significant increase in the glycolytic flux rate when tumor spheroids were exposed to 0.1% O2 . The severe hypoxia was associated with uniform pimonidazole adduct formation and elevated levels of Hif1α and c-Myc. This resulted in elevated expression of GLUT and MCT transporters, in addition to increased activity of PFK1 in comparison to that observed in normoxia. However, the protein expression and enzymatic capacity of HK2, G6PDH, PK, and LDH were all reduced by severe hypoxia. Clearly, the effects of exposure to severe hypoxia lead to a significantly abridged Hif1 response, yet one still able to elevate glycolytic flux and prevent loss of intermediates to anabolism. J. Cell. Biochem. 117: 1890-1901, 2016. © 2016 Wiley Periodicals, Inc.
Publisher: Elsevier BV
Date: 07-2008
Publisher: Spandidos Publications
Date: 14-06-2018
DOI: 10.3892/OR.2018.6492
Abstract: In the present study, a lipid-polymer hybrid drug carrier system was developed to encapsulate psoralen (PSO), a multidrug resistance reversal agent and traditional Chinese medicine. Emphasis was focused the parameters that influence physicochemical characteristics, and then the drug release profile, stability, cytotoxicity and drug resistance reversal effect of the lipid-polymer hybrid nanoparticles (LPNs) were investigated. It was found that various formulation parameters affected NP size, drug loading (DL) and release characteristics. Hydrophilic 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy(polyethylene glycol)2000 increased the ζ potential and thus the stability of the NPs, but also enlarged their diameter. The amount of PSO influenced their DL and encapsulation efficiency, but did not show any effect on drug release kinetics. Next, the stability of the LPNs in different media and their storage characteristics were assessed. Finally, the cytotoxicity and multidrug resistance reversal effect was studied in the K562 and HepG2 cell lines. The analysis of half maximal inhibitory concentration values demonstrated that combination therapy with doxorubicin (DOX) and PSO-loaded LPNs (P-LPNs) was 14- and 23-fold more effective than a single-dose DOX treatment in resistant K562 and HepG2 cells, respectively, and 2.2- and 2.1-fold more effective than a single-dose combination regimen of DOX and PSO in solution, respectively. These data indicate that the LPNs have superior properties compared with a combination therapy in solution.
Publisher: Elsevier BV
Date: 02-2012
Publisher: Wiley
Date: 19-12-2005
DOI: 10.1016/J.FEBSLET.2005.11.083
Abstract: Multidrug transporters are involved in mediating the failure of chemotherapy in treating several serious diseases. The archetypal multidrug transporter P-glycoprotein (P-gp) confers resistance to a large number of chemically and functionally unrelated anti-cancer drugs by mediating efflux from cancer cells. The ability to efflux such a large number of drugs remains a biological enigma and the lack of mechanistic understanding of the translocation pathway used by P-gp prevents rational design of compounds to inhibit its function. The translocation pathway is critically dependent on ATP hydrolysis and drug interaction with P-gp is possible at one of a multitude of allosterically linked binding sites. However, aspects such as coupling stoichiometry, molecular properties of binding sites and the nature of conformational changes remain unresolved or the centre of considerable controversy. The present review attempts to utilise the available data to generate a detailed sequence of events in the translocation pathway for this dexterous protein.
Publisher: American Chemical Society (ACS)
Date: 12-10-2007
DOI: 10.1021/JA076281T
Publisher: Wiley
Date: 12-2001
Publisher: Elsevier BV
Date: 1994
DOI: 10.1016/S0168-8278(05)80144-8
Abstract: The factors affecting drug elimination following acute administration of carbon tetrachloride (CCl4) were investigated using a perfused rat liver system. Morphine and pethidine were used as markers of hepatic glucuronidation and oxidation, respectively. Hepatoxicity of CCl4 was indicated by widespread cellular necrosis and raised serum asparatate aminotransferase levels. At a perfusion rate of 10 ml/min, the extraction ratio of morphine in the normal liver was 0.67 +/- 0.18 and fell to 0.48 +/- 0.03 (p < 0.001) in the acutely damaged livers. The hepatic clearance of morphine fell from 6.7 +/- 0.2 ml/min in controls to 4.7 +/- 0.3 (p < 0.005) in the treated livers. Similar changes were seen at perfusion rates of 7 and 12 ml/min. Intrinsic clearances calculated according to both the venous equilibrium and the undistributed sinusoidal models were independent of perfusion rate and were also lower in the damaged livers. Perfusion rate was a dominant factor in determining morphine elimination in control livers. However, in the damaged livers, the fall in intrinsic clearance resulted in the elimination of morphine being mixed, i.e. both capacity and flow limited. At a perfusion rate of 10 ml/min, the extraction ratio of pethidine was 0.97 +/- 0.01 in control livers and was reduced to 0.91 +/- 0.02 (p < 0.005) in damaged livers. The hepatic clearance also fell at each perfusion rate in the damaged livers. As for morphine, the intrinsic clearances of pethidine calculated by both venous equilibrium and undistributed sinusoidal models gave qualitatively similar results and were lower in the damaged livers than controls.(ABSTRACT TRUNCATED AT 250 WORDS)
Publisher: Elsevier BV
Date: 2017
DOI: 10.1016/J.BCP.2016.10.002
Abstract: The multidrug resistance P-glycoprotein (P-gp) is characterised by the ability to bind and/or transport an astonishing array of drugs. This poly-specificity is imparted by at least four pharmacologically distinct binding sites within the transmembrane domain. Whether or not these sites are spatially distinct has remained unclear. Biochemical and structural investigations have implicated a central cavity as the likely location for the binding sites. In the present investigation, a number of contact residues that are involved in drug binding were identified through biochemical assays using purified, reconstituted P-gp. Drugs were selected to represent each of the four pharmacologically distinct sites. Contact residues important in rhodamine123 binding were identified in the central cavity of P-gp. However, contact residues for the binding of vinblastine, paclitaxel and nicardipine were located at the lipid-protein interface rather than the central cavity. A key residue (F978) within the central cavity is believed to be involved in coupling drug binding to nucleotide hydrolysis. Data observed in this investigation suggest the presence of spatially distinct drug binding sites connecting through to a single translocation pore in the central cavity.
Publisher: Elsevier BV
Date: 07-2020
Publisher: American Chemical Society (ACS)
Date: 04-06-2009
DOI: 10.1021/BI900373X
Publisher: American Chemical Society (ACS)
Date: 07-09-2000
DOI: 10.1021/BI000559B
Abstract: P-glycoprotein (P-gp) confers multiple drug resistance on cancer cells by acting as a plasma membrane localized ATP-dependent drug efflux pump. Currently, there is little information on the nature of the communication between the energy-providing nucleotide binding domains (NBDs) and the drug binding sites of P-gp to generate transport of substrate. Many substrates and modulators cause alterations in ATP hydrolysis, but what effect do the various stages of the catalytic cycle have on drug interaction with P-gp? Vanadate trapping of Mg.ADP caused a reversible decrease in the binding capacity of the transported substrate [(3)H]-vinblastine and the nontransported modulator [(3)H]XR9576 to P-gp in CH(r)B30 cell membranes. The non-hydrolyzable nucleotide analogue ATP-gamma-S also caused a reduction in the binding capacity of [(3)H]-vinblastine but not for the modulator [(3)H]XR9576. This indicates that signaling to the NBDs following binding of a nontransported modulator is different to that transmitted upon interaction of a transported substrate. Second, it appears that the binding of nucleotide, rather than its hydrolysis, causes the initial conformational shift in the drug-binding site during a transport cycle.
Publisher: Elsevier BV
Date: 08-2023
Publisher: Springer Science and Business Media LLC
Date: 11-1992
DOI: 10.1038/BJC.1992.360
Abstract: Cell lines selected (CHRC5) and transfected (LR-73-1A) for multi-drug resistance have total lipid compositions which are indistinguishable between resistant and parental cells. Lipid composition was evaluated by 1H NMR and the total fatty acid content by GLC. No change in surface hydrophobicity, as measured with the fluorescent probe dansyl-PE, was observed as a result of transfection of CHO cells with the mdr1 gene. However, the selected cell line, CHRC5, showed a decreased surface hydrophobicity. This decreased surface hydrophobicity was indicated by an 8 nm increase in the fluorescence emission of dansyl-PE in the CHRC5 cell line compared with the AB1. Both resistant cell lines showed an increase in the polarisation of the fluorescent probe, TMA-DPH in the plasma membranes corresponding to a 14% and a 24% change in fluorescence polarisation for the selected and transfected cell lines, respectively. This is indicative of reduced mobility of the acyl chains in the resistant cell lines. Both the CHRC5 and the transfected cell lines showed almost a 2-fold increase in the initial rate of membrane cycling. The membrane cycling could be inhibited by a known bilayer stabiliser, the N-carbobenzoxy-D-Phe-L-Phe-Gly. These results indicate that the properties of the plasma membrane from resistant cells are altered compared with their parental cell line.
Publisher: Elsevier BV
Date: 05-2006
Publisher: American Chemical Society (ACS)
Date: 28-06-2007
DOI: 10.1021/JM070280U
Publisher: Wiley
Date: 20-10-2020
Publisher: Wiley
Date: 20-08-2010
DOI: 10.1111/J.1742-4658.2010.07789.X
Abstract: Describing the molecular details of the multidrug efflux process of ABCB1, in particular the interdomain communication associated with bioenergetic coupling, continues to prove difficult. A number of investigations to date have implicated transmembrane helix 12 (TM12) in mediating communication between the transmembrane domains and nucleotide-binding domains (NBDs) of ABCB1. The present investigation further addressed the role of TM12 in ABCB1 by characterizing its topography during the multidrug efflux process with the use of cysteine-directed mutagenesis. Cysteines were introduced at various positions along TM12 and assessed for their ability to covalently bind thiol-reactive fluorescent probes with differing physiochemical properties. By analysing each isoform in the basal, ATP-bound and posthydrolytic states, it was possible to determine how the local environment of TM12 alters during the catalytic cycle. Labelling with hydrophobic CM and zwitterionic BM was extensive throughout the helix in the basal, prehydrolytic and posthydrolytic states, suggesting that TM12 is in a predominantly hydrophobic environment. Overall, the carboxy region (intracellular half) of TM12 appeared to be more responsive to changes in the catalytic state of the protein than the amino region (extracellular half). Thus, the carboxy region of TM12 is suggested to be responsive to nucleotide binding and hydrolysis at the NBDs and therefore directly involved in interdomain communication. This data can be reconciled with an atomic-scale model of human ABCB1. Taken together, these results indicate that TM12 plays a key role in the progression of the ATP hydrolytic cycle in ABCB1 and, in particular, in coordinating conformational changes between the NBDs and transmembrane domains.
Publisher: Elsevier BV
Date: 2009
DOI: 10.1016/J.ADDR.2008.07.004
Abstract: ABCG2 is best known as a multidrug transporter capable of conferring resistance to cancer cells. However, the protein is also inherently expressed in numerous barrier tissues and intriguingly within hematopoietic stem cells. Unlike its partners ABCB1 and ABCC1, there is considerably less information available on the molecular mechanism of ABCG2. The transporter has a distinct topology and is presumed to function as a homodimer. However, a number of biochemical studies have presented data to suggest that the protein adopts higher order oligomers. This review focuses on this controversial issue with particular reference to findings from low resolution structural data. In addition, a number of molecular models of ABCG2 based on high resolution structures of bacterial ABC transporters have recently become available and are critically assessed. ABCG2 is a structurally distinct member of the triumvirate of human multidrug transporters and continues to evade description of a unifying molecular mechanism.
Publisher: Wiley
Date: 03-12-2009
Publisher: Elsevier BV
Date: 2021
Publisher: Wiley
Date: 04-2014
DOI: 10.1111/FEBS.12773
Publisher: Elsevier BV
Date: 11-2008
DOI: 10.1016/J.EJPHAR.2008.09.014
Abstract: Approximately one-third of patients with epilepsy display an inherent resistance to pharmacological therapy, manifest as continuing seizures despite maximal tolerated doses of anti-epileptic drugs. One hypothesis for the underlying mechanism of anti-epileptic drug pharmacoresistance is lower drug entry to the epileptic neurones due to the activity of multidrug efflux pumps from the ATP Binding Cassette (ABC) superfamily at the blood-brain barrier. There has been a steady accumulation of animal and human data supporting this theory, particularly for ABC(B1) (P-glycoprotein). However, much of this evidence is indirect. In the present study, several anti-epileptic drugs (carbamazepine, valproic acid, phenytoin, lamotrigine and primidone) were examined for their ability to interact with three ABC transporters that have been implicated pharmacoresistance of anti-epileptic drugs - ABC(B1), ABC(C1) and ABC(G2). Interaction of anti-epileptic drugs with the transporters was assessed by determining whether they could reverse the ability of multidrug ABC transporters to confer a drug resistance phenotype on cancer cell lines. None of these compounds was able to affect the phenotype, suggesting an absence of any interaction with the multidrug transporters. This finding was further investigated by examination of transporter activity namely the ability to reduce steady-state intracellular [(3)H]-radiolabelled drug accumulation. None of the anti-epileptic drugs affected labelled drug accumulation by any of the triumvirate of multidrug transporters examined, indicating that they are unlikely to be substrates. The lack of direct modulation by anti-epileptic drugs of ABC transporter function suggests that these proteins do not contribute significantly to resistance in epilepsy.
Publisher: Springer Science and Business Media LLC
Date: 15-10-1999
Publisher: American Chemical Society (ACS)
Date: 12-2001
DOI: 10.1021/BI011211Z
Abstract: Conceptually one may envisage that substrate binding sites on the ABC transporter P-gp cycle between high- and low-affinity conformations in response to signals arising from nucleotide hydrolysis to effect active transport. A radioligand binding assay was used to characterize the interaction of [3H]vinblastine with P-gp and determine how drug binding site parameters are altered during a catalytic cycle of P-gp. In the absence of nucleotide, we show that [3H]vinblastine interacts with a single class of binding site with high affinity (K(d) = 80 +/- 18 nM). In the presence of the nonhydrolyzable ATP analogue AMP-PNP, the drug binding site was in a low-affinity conformation, manifest by a 9-fold increase in K(d) (K(d) = 731 +/- 20 nM). There was no alteration in the binding capacity, reflecting a complete shift in the high-affinity site to a low-affinity form. The posthydrolytic (Mg-ADP-V(i) bound) form of P-gp also exhibited low-affinity substrate binding (K(d) = 446 +/- 57 nM). Restoration of the high-affinity drug binding site conformation (K(d) = 131 +/- 32 nM) did not occur until release of phosphate from the posthydrolysis P-gp-Mg-ADP-P(i). complex. Our results suggest that alteration of the affinity of the vinblastine binding site involves only one nucleotide binding domain per transport cycle. The binding of ATP provides the signal to instigate this change, while release of phosphate post-ATP hydrolysis returns the transporter to its original state to complete the cycle.
Publisher: MDPI AG
Date: 29-12-2020
DOI: 10.3390/IJMS22010246
Abstract: Defective clearance mechanisms lead to the accumulation of amyloid-beta (Aβ) peptides in the Alzheimer’s brain. Though predominantly generated in neurons, little is known about how these hydrophobic, aggregation-prone, and tightly membrane-associated peptides exit into the extracellular space where they deposit and propagate neurotoxicity. The ability for P-glycoprotein (P-gp), an ATP-binding cassette (ABC) transporter, to export Aβ across the blood-brain barrier (BBB) has previously been reported. However, controversies surrounding the P-gp–Aβ interaction persist. Here, molecular data affirm that both Aβ40 and Aβ42 peptide isoforms directly interact with and are substrates of P-gp. This was reinforced ex vivo by the inhibition of Aβ42 transport in brain capillaries from P-gp-knockout mice. Moreover, we explored whether P-gp could exert the same role in neurons. Comparison between non-neuronal CHO-APP and human neuroblastoma SK-N-SH cells revealed that P-gp is expressed and active in both cell types. Inhibiting P-gp activity using verapamil and nicardipine impaired Aβ40 and Aβ42 secretion from both cell types, as determined by ELISA. Collectively, these findings implicate P-gp in Aβ export from neurons, as well as across the BBB endothelium, and suggest that restoring or enhancing P-gp function could be a viable therapeutic approach for removing excess Aβ out of the brain in Alzheimer’s disease.
Publisher: Elsevier BV
Date: 2014
DOI: 10.1016/J.BBAMEM.2013.09.001
Abstract: ATP Binding Cassette (ABC) transporters play prominent roles in numerous cellular processes and many have been implicated in human diseases. Unfortunately, detailed mechanistic information on the majority of ABC transporters has not yet been elucidated. The slow rate of progress of molecular and high resolution structural studies may be attributed to the difficulty in the investigation of integral membrane proteins. These difficulties include the expression of functional, non-aggregated protein in heterologous systems. Furthermore, the extraction of membrane proteins from source material remains a major bottle-neck in the process since there are relatively few guidelines for selection of an appropriate detergent to achieve optimal extraction. Whilst affinity tag strategies have simplified the purification of membrane proteins many challenges remain. For ex le, the chromatographic process and associated steps can rapidly lead to functional inactivation, random aggregation, or even precipitation of the target protein. Furthermore, optimisation of high yield and purity, does not guarantee successful structure determination. Based on this series of potential issues, any investigation into structure-function of membrane proteins requires a systematic evaluation of preparation quality. In particular, the evaluation should focus on function, homogeneity and mono-dispersity. The present investigation provides a detailed assessment of the quality of purified ATP Binding Cassette (ABC) transporters namely ABCB1 (P-gp) and ABCA4 (ABCR). A number of suggestions are provided to facilitate the production of functional, homogeneous and mono-disperse preparations using the insect cell expression system. Finally, the ABCA4 s les have been used to provide structural insights into this essential photo-receptor cell protein.
Publisher: Wiley
Date: 15-10-2001
Publisher: Wiley
Date: 28-06-2005
DOI: 10.1016/J.FEBSLET.2005.06.030
Abstract: The transmembrane (TM) domains in P-glycoprotein (P-gp) contain the drug binding sites and undergo conformational changes driven by nucleotide catalysis to effect translocation. However, our understanding of exactly which regions are involved in such events remains unclear. A site-directed labelling approach was used to attach thiol-reactive probes to cysteines introduced into transmembrane segment 6 (TM6) in order to perturb function and infer involvement of specific residues in drug binding and/or interdomain communication. Covalent attachment of coumarin-maleimide at residue 339C within TM6 resulted in impaired ATP hydrolysis by P-gp. The nature of the effect was to reduce the characteristic modulation of basal activity caused by transported substrates, modulators and the potent inhibitor XR9576. Photoaffinity labelling of P-gp with [(3)H]-azidopine indicated that residue 339C does not alter drug binding per se. However, covalent modification of this residue appears to prevent conformational changes that lead to drug stimulation of ATP hydrolysis.
Publisher: Springer Science and Business Media LLC
Date: 09-09-2015
DOI: 10.1007/S00280-015-2858-9
Abstract: Multidrug efflux pumps such as ABCG2 confer drug resistance to a number of cancer types, leading to poor prognosis and outcome. To date, the strategy of directly inhibiting multidrug efflux pumps in order to overcome drug resistance in cancer has been unsuccessful. An alternative strategy is to target proteins involved in the regulation of multidrug efflux pump activity or expression. Pim1 kinase has been demonstrated to phosphorylate ABCG2, promote its oligomerisation and contribute to its ability to confer drug resistance. In the present manuscript, imidazo-pyridazine-based inhibitors of Pim1 were examined for their ability to overcome ABCG2-mediated drug resistance. Drug efficacy was measured as a cytotoxic response or an effect on transport by ABCG2. Protein expression patterns were assessed using western immuno-blotting. The two Pim1 inhibitors increased the potency of flavopiridol, mitoxantrone, topotecan and doxorubicin, specifically in ABCG2-expressing cells. This effect was associated with an increase in the cellular accumulation of [(3)H]-mitoxantrone, suggesting direct impairment of the transporter. However, prolonged pre-incubation with the studied inhibitors greatly enhanced the effect on mitoxantrone accumulation. The inhibitors caused a significant time-dependent reduction in the expression of ABCG2 in the resistant cells, an effect that would improve drug efficacy. Consequently, it appears that the Pim1 inhibitors display a dual-mode effect on ABCG2-expressing cancer cells. This may provide a powerful new strategy in overcoming drug resistance by targeting proteins that regulate expression of efflux pumps.
Publisher: Springer Science and Business Media LLC
Date: 19-07-2005
Publisher: Elsevier BV
Date: 06-1997
Abstract: In order to elucidate the mechanism by which the multidrug resistance P-glycoprotein extrudes cytotoxic drugs from the cell, and particularly the number and nature of the drug binding site(s), knowledge of the structure of P-gp is essential. A considerable body of genetic and biochemical data has accrued which gives insights into P-gp structure and function. These data are critically reviewed, particularly in relation to the low resolution structure of P-gp which has recently been determined by electron microscopy. P-gp is one of the best characterised of the ABC transporters and these structure-function studies may have more general implications.
Publisher: Elsevier BV
Date: 02-1993
DOI: 10.1016/0167-4889(93)90217-D
Abstract: Growth of CHRC5 multidrug resistant cells in media enriched in a saturated C-17 fatty acid, heptadecanoic acid, resulted in these cells accumulating vinblastine at a rate and to an extent comparable to that of the parental cell line AB1. The fatty acid-enriched growth media had no effect on the ability of AB1 cells to take up vinblastine. The action of hiphiles on the uptake of rhodamine dyes by CHRC5 cells was compared with the increased dye accumulation affected by verapamil. Membrane rigidifying agents, such as the saturated fatty acid stearic acid, or the cholesterol derivatives, cholesteryl hemisuccinate and cholesteryl phosphorylcholine, as well as a membrane fluidizing unsaturated fatty acid, linoleic acid, could significantly increase dye uptake, although not as well as verapamil. These results taken in conjunction with other reports in the literature, demonstrate that multidrug resistance is sensitive to alterations of membrane properties. They suggest that perturbation of the membrane to either increased or to decreased membrane fluidity can lower the level of resistance.
Publisher: Springer Science and Business Media LLC
Date: 10-2001
Abstract: In tumour cell lines that display multidrug resistance, expression of P-glycoprotein (P-gp) alters many aspects of biomembrane organization in addition to its well-characterized drug transport activity. We have developed a reconstitution system to directly investigate the effect of purified P-gp on the biophysical properties of lipid bilayers. Using a mixed detergent system it was possible to efficiently reconstitute P-gp at lipid:protein ratios as low as 2.5 (w/w) by removal of detergent using adsorption to SM-2 BioBeads. P-gp was able to alter many biophysical parameters associated with lipid organization within bilayers. For ex le, the changes in overall fluidity and excimer formation by lipid analogues indicate modified packing organization of bilayer constituents. Surprisingly, given its role in conferring drug resistance, P-gp insertion into bilayers also caused significantly increased permeability to aqueous compounds, also reflecting a modified phospholipid environment. Translocation of various phospholipid species between leaflets of the bilayer was increased in the presence of P-gp however, the effect was not dependent on ATP hydrolysis by the protein. Physiological concentrations of cholesterol modified P-gp function and the degree to which it perturbed bilayer organization. The basal ATPase activity of P-gp was increased in a dose-dependent fashion by the incorporation of cholesterol in PC:PE liposomes. In addition, the degree to which the modulator verapamil was able to stimulate this basal ATPase activity was reduced by the presence of cholesterol in proteoliposomes. However, the potency of verapamil was unaltered, suggesting a specific effect, not simply caused by lower drug penetration into the cholesterol containing bilayers. In summary, P-gp is able to cause perturbation in the organization of bilayer constituents. Cholesterol imparted "stability" to this perturbation of bilayer organization by P-gp and moreover this led to altered protein function.
Publisher: Public Library of Science (PLoS)
Date: 14-05-2010
Publisher: American Chemical Society (ACS)
Date: 14-08-2007
DOI: 10.1021/BI700447P
Abstract: Multidrug transporters such as P-glycoprotein require considerable inter-domain communication to couple energy utilization with substrate translocation. Elucidation of the regions or residues involved in these communication pathways is a key step in the eventual molecular description of multidrug transport. We used cysteine-scanning mutagenesis to probe the functional involvement of residues along the cytoplasmic half of transmembrane segment 6 (TM6) and its extension toward the nucleotide binding domain. The mutation of one residue (G346C) in this segment adversely affected drug transport in cells. Further investigation using purified protein revealed that the underlying biochemical effect was a reduction in basal ATP hydrolysis. This G346C mutation also affected the stimulation of ATPase activity in a drug dependent manner but had no effect on drug binding, ATP binding, or ADP release. Homology modeling of P-glycoprotein indicated that the G346C mutation caused a steric interaction between TM5 and TM6, thereby precluding a helical movement required to support ATP hydrolysis.
Publisher: Elsevier BV
Date: 2006
DOI: 10.1016/J.EJPHAR.2005.11.038
Abstract: Recently developed glucosylceramide synthase inhibitors with enhanced hydrophobicity display increased bioavailability in the central nervous system (CNS). Have these improvements come at a potential risk given that the improved glucosylceramide synthase inhibitors bear the hallmarks of P-glycoprotein substrates? This question warrants attention given the potential to induce adverse drug interactions or toxicity, if glucosylceramide synthase inhibitors are administered with other P-glycoprotein substrates. The aim of this study was to determine if glucosylceramide synthase inhibitors are substrates for the multidrug transporter P-glycoprotein. Direct measurements of glucosylceramide synthase inhibitors binding to P-glycoprotein were examined, as was their ability to modulate transport by the protein. The more hydrophobic glucosylceramide synthase inhibitors caused a reduction in drug binding to P-glycoprotein. However, the compounds did not achieve this by direct interaction with the protein, but through a general membrane perturbation. Furthermore, the alterations in drug-P-glycoprotein interaction did not manifest as altered cellular accumulation of glucosylceramide synthase inhibitors or altered efficacy to reduce cellular glycolipid levels. Consequently, P-glycoprotein expression will not contribute significantly to the pharmacokinetic profile of the iminosugar glucosylceramide synthase inhibitors.
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Richard Callaghan.