Richard Callaghan

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 ∼10 Å 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/Pi. Biochemical data suggest that these rearrangements may involve rotation of transmembrane α‐helices. A mechanism for transport is suggested.

The molecular interaction of the high affinity reversal agent XR9576 with P‐glycoprotein

The kinetics and nature of equilibrium binding were used to characterize the molecular interaction of the anthranilic acid derivative [3H]‐XR9576 with the multidrug resistance P‐glycoprotein (P‐gp). XR9576 displayed specific high‐affinity binding to P‐gp (Bmax=275 pmol mg−1, Kd=5.1 nM). The transport substrates [3H]‐vinblastine and [3H]‐paclitaxel displayed 4 fold and 20 fold lower affinity respectively for P‐gp. The duration of action of XR9576 with P‐gp was increased in comparison to that of vinblastine which displayed a slower rate of association and a faster dissociation rate. The relative affinities of several modulators and transport substrates to interact with P‐gp were determined from displacement drug equilibrium binding assays. Vinblastine and paclitaxel could only fractionally displace [3H]‐XR9576 binding, displaying Ki values significantly different from their measured Kd values. This suggests a non‐competitive interaction between XR9576 and the P‐gp substrates vinblastine and paclitaxel. XR9576 was shown to be a potent modulator of P‐gp mediated [3H]‐vinblastine and [3H]‐paclitaxel transport as it increased the steady‐state accumulation of these cytotoxics in CHrB30 cells to levels observed in non‐P‐gp‐expressing AuxB1 cells (EC50=487±50 nM). This inhibition of drug transport is not mediated through competition for transport since [3H]‐XR9576 accumulation was not influenced by P‐gp expression or function. These results demonstrate that the P‐gp modulator XR9576 exhibits greater selectivity, duration of inhibition and potency of interaction with this transporter than any other reported modulators. Several lines of evidence suggest that XR9576 inhibits P‐gp function by binding at a site which is distinct from the site of interaction of transport substrates. The two sites may be classified as serving modulatory or transport functions.

Collateral sensitivity as a strategy against cancer multidrug resistance

While chemotherapy remains the most effective treatment for disseminated tumors, acquired or intrinsic drug resistance accounts for approximately 90% of treatment failure. Multidrug resistance (MDR), the simultaneous resistance to drugs that differ both structurally and mechanistically, often results from drug efflux pumps in the cell membrane that reduce intracellular drug levels to less than therapeutic concentrations. Expression of the MDR transporter P-glycoprotein (P-gp, MDR1, ABCB1) has been shown to correlate with overall poor chemotherapy response and prognosis. This review will focus on collateral sensitivity (CS), the ability of compounds to kill MDR cells selectively over the parental cells from which they were derived. Insights into CS may offer an alternative strategy for the clinical resolution of MDR, as highly selective and potent CS agents may lead to drugs that are effective at MDR cell killing and tumor resensitization. Four main mechanistic hypotheses for CS will be reviewed, followed by a discussion on quantitative and experimental evaluation of CS.

Resistance to Chemotherapy in Cancer: A Complex and Integrated Cellular Response

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.

Protein kinase C-mediated phosphorylation does not regulate drug transport by the human multidrug resistance P-glycoprotein.

P-glycoprotein (P-gp) is an active transporter that can confer multidrug resistance by pumping cytotoxic drugs out of cells and tumors. P-gp is phosphorylated at several sites in the “linker” region, which separates the two halves of the molecule. To examine the role of phosphorylation in drug transport, we mutated P-gp such that it could no longer be phosphorylated by protein kinase C (PKC). When expressed in yeast, the ability of the mutant proteins to confer drug resistance, or to mediate [3H]vinblastine accumulation in secretory vesicles, was indistinguishable from that of wild type P-gp. A matched pair of mammalian cell lines were generated expressing wild type P-gp and a non-phosphorylatable mutant protein. Mutation of the phosphorylation sites did not alter P-gp expression or its subcellular localization. The transport properties of the mutant and wild type proteins were indistinguishable. Thus, phosphorylation of the linker of P-gp by PKC does not affect the rate of drug transport. In light of these data, the use of agents that alter PKC activity to reverse multidrug resistance in the clinic should be considered with caution.

Inhibition of the multidrug resistance P-glycoprotein; time for a change of strategy?

P-glycoprotein (P-gp) is a key player in the multidrug-resistant phenotype in cancer. The protein confers resistance by mediating the ATP-dependent efflux of an astonishing array of anticancer drugs. Its broad specificity has been the subject of numerous attempts to inhibit the protein and restore the efficacy of anticancer drugs. The general strategy has been to develop compounds that either compete with anticancer drugs for transport or act as direct inhibitors of P-gp. Despite considerable in vitro success, there are no compounds currently available to “block” P-gp–mediated resistance in the clinic. The failure may be attributed to toxicity, adverse drug interaction, and numerous pharmacokinetic issues. This review provides a description of several alternative approaches to overcome the activity of P-gp in drug-resistant cells. These include 1) drugs that specifically target resistant cells, 2) novel nanotechnologies to provide high-dose, targeted delivery of anticancer drugs, 3) compounds that interfere with nongenomic transfer of resistance, and 4) approaches to reduce the expression of P-gp within tumors. Such approaches have been developed through the pursuit of greater understanding of resistance mediators such as P-gp, and they show considerable potential for further application.

P‐glycoprotein: So Many Ways to Turn It On

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 (eg, chemotherapy damage) or endogenous (eg, 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.

A model of quiescent tumour microregions for evaluating multicellular resistance to chemotherapeutic drugs

The quiescent cell population of tumours poses a barrier to the success of many cancer therapies. Most chemotherapeutic drugs target proliferating cells, but the growth fraction of many tumours is low. Based on the multicellular tumour spheroid model, a system was developed using human colon adenocarcinoma (DLD-1) cells to mimic the microenvironment of quiescent microregions of solid tumours. The quiescent tumour spheroids (TSQ) showed decreased expression of the proliferation marker Ki-67 and increased expression of the quiescence marker p27kip1 compared to proliferating spheroids (TSP). The quiescent status of the TSQ was confirmed by long-term growth assessment. The quiescence was completely reversible demonstrating that the TSQ retained the ability to proliferate and morphological assessment by light microscopy confirmed the absence of significant apoptosis. When the efficacy of widely used chemotherapeutic drugs was determined, vinblastine, doxorubicin, cisplatin and 5-fluorouracil (5-FU) all produced significant cell death in the TSP. However, while still effective, the potencies of doxorubicin and cisplatin were significantly reduced in TSQ. In contrast, 5-FU and vinblastine did not produce cell death in the TSQ. In summary, TSQ show considerable resistance to a panel of established chemotherapeutic agents and represent a useful model for evaluating the efficacy of drugs and other cancer therapies in quiescent tumours.

THE FUNCTIONAL PURIFICATION OF P-GLYCOPROTEIN IS DEPENDENT ON MAINTENANCE OF A LIPID-PROTEIN INTERFACE

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.

The translocation mechanism of P-glycoprotein.

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.