Synthesis and evaluation of Strychnos alkaloids as MDR reversal agents for cancer cell eradication.

Natural products represent the fourth generation of multidrug resistance (MDR) reversal agents that resensitize MDR cancer cells overexpressing P-glycoprotein (Pgp) to cytotoxic agents. We have developed an effective synthetic route to prepare various Strychnos alkaloids and their derivatives. Molecular modeling of these alkaloids docked to a homology model of Pgp was employed to optimize ligand-protein interactions and design analogues with increased affinity to Pgp. Moreover, the compounds were evaluated for their (1) binding affinity to Pgp by fluorescence quenching, and (2) MDR reversal activity using a panel of in vitro and cell-based assays and compared to verapamil, a known inhibitor of Pgp activity. Compound 7 revealed the highest affinity to Pgp of all Strychnos congeners (Kd=4.4µM), the strongest inhibition of Pgp ATPase activity, and the strongest MDR reversal effect in two Pgp-expressing cell lines. Altogether, our findings suggest the clinical potential of these synthesized compounds as viable Pgp modulators justifies further investigation.

A semisynthetic taxane Yg-3-46a effectively evades P-glycoprotein and ß-III tubulin mediated tumor drug resistance in vitro.

Tumor resistance, especially that mediated by P-glycoprotein (P-gp) and ß-III tubulin, is a major obstacle to the efficacy of most microtubule-targeting anticancer drugs in clinics. A novel semisynthetic taxane, 2-debenzoyl-2-(3-azidobenzyl)-10-propionyldocetaxel (Yg-3-46a) was shown to be highly cytotoxic to breast cancer cell lines MCF-7 and MCF/ADR which overexpressed P-gp via long term culture with doxorubicin, and cervical cancer cell lines Hela and Hela/ßIII which overexpressed ßIII-tubulin via stable transfection with TUBB3 gene. siRNA transfection experiments also confirmed that Yg-3-46a can circumvent P-gp and ß-III tubulin mediated drug resistance. In addition, its cytotoxicity was lower than that of paclitaxel in the human mammary cell line HBL-100 and the human telomerase-immortalized retinal pigment epithelium cell line (hTERT-RPE1), suggesting a better safety margin for this compound in vivo. It exhibited more potent microtubule polymerization ability than paclitaxel in vitro, and also induced G2/M phase arrest in MCF-7/ADR cells. Moreover, it was found to induce apoptosis in MCF-7/ADR cells through the caspase-dependent death-receptor pathway by enhancing levels of Fas and FasL, and activating caspase-8 and 3. Yg-3-46a was found to be a poorer substrate of P-gp compared to paclitaxel, in both binding and ATPase experiments, which is likely responsible for its ability to circumvent P-gp mediated multidrug resistance (MDR). All of these results indicate that Yg-3-46a is a novel microtubule-stabilizing agent that has the potential to evade drug resistance mediated by P-gp and ß-III tubulin overexpression.

Lipid bilayer properties control membrane partitioning, binding, and transport of p-glycoprotein substrates.

The ABC protein P-glycoprotein (Pgp or ABCB1) is a multidrug efflux pump capable of transporting many structurally diverse substrates from within the lipid bilayer. Previous studies have demonstrated the importance of the membrane in modulating Pgp function, but few have quantified these effects. We employed purified Pgp reconstituted into phospholipid bilayers with defined gel to liquid-crystalline melting transitions to investigate the effect of membrane environment on the transporter and three of its substrates. Equilibrium dialysis measurements indicated that Hoechst 33342, LDS-751, and MK-571 partitioned much more readily into liquid-crystalline phase bilayers than into gel phase bilayers. However, drug binding affinities revealed that Pgp bound the three substrates more tightly when the lipid bilayer was in the gel phase. The binding affinity of the transporter for substrates within the bilayer was low, in the millimolar range, suggesting that it interacts with them weakly. Thermodynamic analysis revealed that both drug-Pgp and drug-lipid interactions contribute to binding affinity. The kinetics of LDS-751 and Hoechst 33342 transport by reconstituted Pgp was monitored using a real-time fluorescence-based assay to obtain apparent turnover frequencies. Transport rates were found to be sensitive to both drug structure and lipid environment. Arrhenius and transition state analysis of transport rates suggested that the rate of drug transport depends on both the affinity of Pgp for substrate and protein conformational changes. Transport rates did not appear to be limited exclusively by the rate of ATP hydrolysis and may be partially controlled by the rate of drug dissociation.

The P-glycoprotein multidrug transporter.

Pgp (P-glycoprotein) (ABCB1) is an ATP-powered efflux pump which can transport hundreds of structurally unrelated hydrophobic amphipathic compounds, including therapeutic drugs, peptides and lipid-like compounds. This 170 kDa polypeptide plays a crucial physiological role in protecting tissues from toxic xenobiotics and endogenous metabolites, and also affects the uptake and distribution of many clinically important drugs. It forms a major component of the blood-brain barrier and restricts the uptake of drugs from the intestine. The protein is also expressed in many human cancers, where it probably contributes to resistance to chemotherapy treatment. Many chemical modulators have been identified that block the action of Pgp, and may have clinical applications in improving drug delivery and treating cancer. Pgp substrates are generally lipid-soluble, and partition into the membrane before the transporter expels them into the aqueous phase, much like a 'hydrophobic vacuum cleaner'. The transporter may also act as a 'flippase', moving its substrates from the inner to the outer membrane leaflet. An X-ray crystal structure shows that drugs interact with Pgp within the transmembrane regions by fitting into a large flexible binding pocket, which can accommodate several substrate molecules simultaneously. The nucleotide-binding domains of Pgp appear to hydrolyse ATP in an alternating manner; however, it is still not clear whether transport is driven by ATP hydrolysis or ATP binding. Details of the steps involved in the drug-transport process, and how it is coupled to ATP hydrolysis, remain the object of intensive study.

Effects of C7 substitutions in a high affinity microtubule-binding taxane on antitumor activity and drug transport.

Some C-7 modified analogs of 3, a taxane with high affinity for binding to microtubules, were prepared through multistep transformations. Most of the analogs, bearing less lipophilic C-7 substituents than propionyl in 3, exhibited comparable binding affinities to microtubules but less cytotoxicity against drug-sensitive as well as multidrug-resistant tumor cells overexpressing P-glycoprotein. In addition, these C7 modifications increased P-glycoprotein-mediated drug transport in both directions in a Caco-2 cell assay.

Flipping and flopping--lipids on the move.

The rapid movement of polar lipids from one membrane leaflet to the other is facilitated by lipid flippases or translocases. Although their activity was first observed over 30 years ago, the structures, physiological roles, and molecular mechanisms of this group of proteins remain enigmatic. Lipid flippases maintain membrane lipid asymmetry, and in eukaryotes they are also intimately involved in membrane budding and vesicle trafficking. The ATP-dependent flippases are members of well-characterized protein families, whose other members transport nonlipid substrates across cell membranes. The P(4)-type ATPases carry out the inward translocation of phospholipids, and various ABC transporters are involved in outward lipid movement. The ATP-independent flippases move lipid substrates in both directions between membrane leaflets. With only a few exceptions, the molecular identity of these proteins is still unknown, despite their involvement in key biosynthetic pathways in both bacteria and eukaryotes. This review provides an overview of the different classes of flippases, and summarizes recent progress in their identification and functional characterization. The possible mechanisms of action of lipid flippases are discussed, and future directions explored.

Fluorescence studies of drug binding and translocation by membrane transporters.

Resistance to multiple drugs is a serious limitation to chemotherapy treatment of human cancers. In addition, many clinically useful drugs show limited uptake in the intestine and cannot gain access to the brain. Three multidrug efflux pumps of the ABC superfamily (P-glycoprotein/ABCB1, MRP1/ABCC1, and BCRP/ABGG2) are responsible for most drug transport out of mammalian cells. P-glycoprotein is the best characterized of the ABC drug transporters. However, the lipophilic nature of its substrates has made it difficult to directly quantitate drug binding to the protein by classical biochemical methods, and the measurement of drug transport rates has also proved challenging. In recent years, fluorescence spectroscopic approaches have proved very useful in overcoming these problems. This chapter focuses on the use of fluorescence tools to quantitate the affinity of binding of various drugs to purified P-glycoprotein and to measure its drug transport activity in reconstituted proteoliposomes in real time. The ability of various drugs to inhibit P-glycoprotein mediated transport can also be assessed using this approach.

Characterization of an asymmetric occluded state of P-glycoprotein with two bound nucleotides: implications for catalysis.

P-glycoprotein (ABCB1), a member of the ABC superfamily, functions as an ATP-driven multidrug efflux pump. The catalytic cycle of ABC proteins is believed to involve formation of a sandwich dimer in which two ATP molecules are bound at the interface of the nucleotide binding domains (NBDs). However, such dimers have only been observed in isolated NBD subunits and catalytically arrested mutants, and it is still not understood how ATP hydrolysis is coordinated between the two NBDs. We report for the first time the characterization of an asymmetric state of catalytically active native P-glycoprotein with two bound molecules of adenosine 5'-(gamma-thio)triphosphate (ATPgammaS), one of low affinity (K(d) 0.74 mm), and one "occluded" nucleotide of 120-fold higher affinity (K(d) 6 microm). ATPgammaS also interacts with P-glycoprotein with high affinity as assessed by inhibition of ATP hydrolysis and protection from covalent labeling of a Walker A Cys residue, whereas other non-hydrolyzable ATP analogues do not. Binding of ATPgammaS (but not ATP) causes Trp residue heterogeneity, as indicated by collisional quenching, suggesting that it may induce conformational asymmetry. Asymmetric ATPgammaS-bound P-glycoprotein does not display reduced binding affinity for drugs, implying that transport is not driven by ATP binding and likely takes place at a later stage of the catalytic cycle. We propose that this asymmetric state with two bound nucleotides represents the next intermediate on the path toward ATP hydrolysis after nucleotide binding, and an alternating sites mode of action is achieved by simultaneous switching of the two active sites between high and low affinity states.

Differential interactions between statins and P-glycoprotein: implications for exploiting statins as anticancer agents.

Statins, prescribed for decades to control cholesterol, have more recently been shown to have promising anticancer activity. Statins induce tumor-selective apoptosis by inhibiting the mevalonate (MVA) pathway. In addition, we have recently demonstrated that lovastatin modulates drug accumulation in a MVA-independent manner in multidrug-resistant (MDR) tumor cells overexpressing the P-glycoprotein (P-gp) multidrug transporter. P-gp-mediated drug efflux can contribute to chemotherapy failure. However, direct statin-mediated inhibition of P-gp in human MDR tumor cells at clinically achievable concentrations remains unexplored. An understanding of these interactions is crucial, both to appreciate differences in the anticancer potential of different statins and to safely and effectively integrate statins into traditional chemotherapy regimens that include P-gp substrates. Here we evaluate interactions between 4 statins (lovastatin, atorvastatin, fluvastatin and rosuvastatin) and P-gp, at both the molecular level using purified P-gp and at the cellular level using human MDR tumor cells. Lovastatin bound directly to purified P-gp with high affinity and increased doxorubicin accumulation in MDR tumor cells, potentiating DNA damage, growth arrest and apoptosis. By contrast, while atorvastatin inhibited substrate transport by purified P-gp in proteoliposomes, it had no effect on doxorubicin transport in MDR tumor cells. Finally, fluvastatin and rosuvastatin only interacted with P-gp in vitro at high concentrations and did not inhibit doxorubicin transport in MDR cells. These differential interactions should be considered when combining statins with traditional chemotherapeutic drugs.

Interaction of the P-glycoprotein multidrug efflux pump with cholesterol: effects on ATPase activity, drug binding and transport.

Resistance to a broad spectrum of structurally diverse chemotherapeutic drugs (multidrug resistance; MDR) is a major impediment to the treatment of cancer. One cause of MDR is the expression at the tumor cell surface of P-glycoprotein (Pgp), which functions as an ATP-powered multidrug efflux pump. Since Pgp interacts with its substrates after they partition into the lipid bilayer, changes in membrane physicochemical properties may have substantial effects on its functional activity. Various interactions between cholesterol and Pgp have been suggested, including a role for the protein in transbilayer movement of cholesterol. We have characterized several aspects of Pgp-cholesterol interactions, and found that some of the previously reported effects of cholesterol result from inhibition of Pgp ATPase activity by the cholesterol-extracting reagent, methyl-beta-cyclodextrin. The presence of cholesterol in the bilayer modulated the basal and drug-stimulated ATPase activity of reconstituted Pgp in a modest fashion. Both the ability of drugs to bind to the protein and the drug transport and phospholipid flippase functions of Pgp were also affected by cholesterol. The effects of cholesterol on drug binding affinity were unrelated to the size of the compound. Increasing cholesterol content greatly altered the partitioning of hydrophobic drug substrates into the membrane, which may account for some of the observed effects of cholesterol on Pgp-mediated drug transport. Pgp does not appear to mediate the flip-flop of a fluorescent cholesterol analogue across the bilayer. Cholesterol likely modulates Pgp function via effects on drug-membrane partitioning and changes in the local lipid environment of the protein.

Functional characterization of Escherichia coli MsbA: interaction with nucleotides and substrates.

The Escherichia coli MsbA protein is a 65-kDa member of the ATP-binding cassette superfamily. It is thought to function as an ATP-dependent lipid translocase that transports lipid A from the inner to the outer leaflet of the cytoplasmic membrane. MsbA with high ATPase activity was isolated and found to be homodimeric in detergent solution. The protein ATPase activity was inhibited by vanadate and showed variable patterns of stimulation and inhibition by lipid A and other compounds. The intrinsic tryptophan fluorescence of the protein was characterized, and dynamic quenching using acrylamide showed that a conformational change took place on binding of lipid A. Fluorescence quenching was used to characterize the interactions of MsbA with nucleotides and various putative substrates, including lipids, lipid-like compounds, and drugs. MsbA had an apparent binding affinity for ATP of approximately 2 mm and also bound nonhydrolyzable ATP analogs and fluorescent ATP derivatives. The putative substrate lipid A interacted with the protein with an affinity of 6.4 microm. Drugs that are known to be substrates for ABC multidrug transporters also interacted with MsbA with affinities in the range 0.25-50 microm. This study represents the first use of fluorescence approaches to estimate MsbA binding affinities for nucleotides and putative transport substrates.

ABC multidrug transporters: structure, function and role in chemoresistance.

Three ATP-binding cassette (ABC)-superfamily multidrug efflux pumps are known to be responsible for chemoresistance; P-glycoprotein (ABCB1), MRP1 (ABCC1) and ABCG2 (BCRP). These transporters play an important role in normal physiology by protecting tissues from toxic xenobiotics and endogenous metabolites. Hydrophobic amphipathic compounds, including many clinically used drugs, interact with the substrate-binding pocket of these proteins via flexible hydrophobic and H-bonding interactions. These efflux pumps are expressed in many human tumors, where they likely contribute to resistance to chemotherapy treatment. However, the use of efflux-pump modulators in clinical cancer treatment has proved disappointing. Single nucleotide polymorphisms in ABC drug-efflux pumps may play a role in responses to drug therapy and disease susceptibility. The effect of various genotypes and haplotypes on the expression and function of these proteins is not yet clear, and their true impact remains controversial.

Interaction of insecticides with mammalian P-glycoprotein and their effect on its transport function.

We studied the effects of four commonly used insecticides (methylparathion, endosulfan, cypermethrin and fenvalerate) on P-glycoprotein isolated from multidrug-resistant cells. All the pesticides stimulated P-glycoprotein ATPase activity, with maximum stimulation of up to 213% in a detergent-solubilized preparation, and up to 227% in reconstituted liposomes. The ATPase stimulation profiles were biphasic, displaying lower stimulation, and in the case of methylparathion, inhibition of activity, at higher insecticide concentrations. Quenching of the intrinsic Trp fluorescence of purified P-glycoprotein was used to quantitate insecticide binding; the estimated K(d) values fell in the range 4-6 microM. Transport of the fluorescent substrate tetramethylrosamine (TMR) into proteoliposomes containing P-glycoprotein was monitored in real time. The TMR concentration gradient generated by the transporter was collapsed by the addition of insecticides, and prior addition of these compounds prevented its formation. The rate of TMR transport was inhibited in a saturable fashion by all the compounds, indicating that they compete with the substrate for membrane translocation. Taken together, these data suggest that the insecticides bind to Pgp with high affinity and effectively block drug transport. Inhibition of Pgp by pesticides may compromise its ability to clear xenobiotics from the body, leading to a higher risk of toxicity.

Overcoming tumor drug resistance with high-affinity taxanes: a SAR study of C2-modified 7-acyl-10-deacetyl cephalomannines.

A series of C2-modified 10-deacetyl-7-propionyl cephalomannine derivatives was designed, prepared, and biologically evaluated. Some C2 meta-substituted benzoate analogues showed potent activity against both drug-sensitive and drug-resistant tumor cells in which resistance is mediated through either P-gp overexpression or beta-tubulin mutation mechanisms. The taxoid 15 b and related compounds are of particular interest, as they are much more cytotoxic than paclitaxel, especially against drug-resistant tumor cells; they are able to kill both drug-resistant and drug-sensitive cells (low R/S ratio), and they have high affinity for beta-tubulin. Our research results led to an important hypothesis, that is, a taxane with very high binding affinity for beta-tubulin is able to counteract drug resistance, which may assist in future taxane-based drug-discovery efforts.

Conformational and functional characterization of trapped complexes of the P-glycoprotein multidrug transporter.

The Pgp (P-glycoprotein) multidrug transporter couples ATP hydrolysis at two cytoplasmic NBDs (nucleotide-binding domains) to the transport of hydrophobic compounds. Orthovanadate (V(i)) and fluoroaluminate (AlF(x)) trap nucleotide in one NBD by forming stable catalytically inactive complexes (Pgp-M2+-ADP-X), which are proposed to resemble the catalytic transition state, whereas the complex formed by beryllium fluoride (BeF(x)) is proposed to resemble the ground state. We studied the trapped complexes formed via incubation of Pgp with ATP (catalytically forward) or ADP (reverse) and V(i), BeF(x) or AlF(x) using Mg2+ or Co2+ as the bivalent cation. Quenching of intrinsic Pgp tryptophan fluorescence by acrylamide, iodide and caesium indicated that conformational changes took place upon formation of the trapped complexes. Trapping with V(i) and ATP led to a 6-fold increase in the acrylamide quenching constant, K(SV), suggesting that large conformational changes take place in the Pgp transmembrane regions on trapping in the forward direction. Trapping with V(i) and ADP gave only a small change in quenching, indicating that the forward- and reverse-trapped complexes are different. TNP (trinitrophenyl)-ATP/TNP-ADP interacted with all of the trapped complexes, however, the fluorescence enhancement differed for the trapped states, suggesting a change in polarity in the nucleotide-binding sites. The nucleotide-binding site of the BeF(x)-trapped complex was much more polar than that of the V(i) and AlF(x) complexes. Functionally, all the trapped complexes were able to bind drugs and TNP-nucleotides with unchanged affinity compared with native Pgp.

Combined chemical and enzymatic stable isotope labeling for quantitative profiling of detergent-insoluble membrane proteins isolated using Triton X-100 and Brij-96.

Effective quantitative profiling of detergent-insoluble membrane proteins using high-throughput mass spectrometry (MS)-based proteomics would allow a better understanding of physiological and pathological processes that take place at the cell surface. To increase the coverage of proteins present in detergent-resistant membrane microdomains (DRMMs), a combination of 16O/18O and isotope coded affinity tags (ICAT) labeling was used in a comparative analysis of detergent-insoluble membrane proteins isolated from rat basophilic leukemia cells (RBL-2H3), with either Triton X-100 or Brij-96. The analysis resulted in the quantification of 738 unique proteins from Triton X-100 and Brij-96 isolated DRMMs, significantly exceeding the number of proteins quantified from either single labeling technique. Twenty-five noncysteine-containing proteins were quantified, as well as 32 cysteine-containing proteins that would have been missed if either 16O/18O or ICAT labeling had been used exclusively, which illustrate better proteome coverage and enhanced ability to quantitate. The comparative analysis revealed that proteins were more readily extracted using Triton X-100 than Brij-96; however, Triton X-100 also extracted larger quantities of non-DRMMs-associated proteins. This result confirms previous, targeted studies suggesting that DRMMs isolated using Triton X-100 and Brij-96 differ in their protein content.

Shedding light on drug transport: structure and function of the P-glycoprotein multidrug transporter (ABCB1).

P-glycoprotein (Pgp; ABCB1), a member of the ATP-binding cassette (ABC) superfamily, exports structurally diverse hydrophobic compounds from the cell, driven by ATP hydrolysis. Pgp expression has been linked to the efflux of chemotherapeutic drugs in human cancers, leading to multidrug resistance (MDR). The protein also plays an important physiological role in limiting drug uptake in the gut and entry into the brain. Substrates partition into the lipid bilayer before interacting with Pgp, which has been proposed to function as a hydrophobic vacuum cleaner. Low- and medium-resolution structural models of Pgp suggest that the 2 nucleotide-binding domains are closely associated to form a nucleotide sandwich dimer. Pgp is an outwardly directed flippase for fluorescent phospholipid and glycosphingolipid derivatives, which suggests that it may also translocate drug molecules from the inner to the outer membrane leaflet. The ATPase catalytic cycle of the protein is thought to proceed via an alternating site mechanism, although the details are not understood. The lipid bilayer plays an important role in Pgp function, and may regulate both the binding and transport of drugs. This review focuses on the structure and function of Pgp, and highlights the importance of fluorescence spectroscopic techniques in exploring the molecular details of this enigmatic transporter.

P-glycoprotein (ABCB1) interacts directly with lipid-based anti-cancer drugs and platelet-activating factors.

The P-glycoprotein multidrug transporter (Pgp; ABCB1) is an ATP-binding cassette (ABC) protein that has been implicated in the multidrug resistance of human cancers. Pgp couples ATP hydrolysis to active extrusion from the cell of a broad array of amphipathic compounds via an ill-defined mechanism. Substrates are believed to interact with Pgp within the membrane. Reconstituted Pgp functions as an ATP-dependent flippase for a variety of fluorescently labelled membrane lipids. The protein may also function as a drug 'flippase', moving its substrates from the inner to the outer leaflet of the bilayer. We show that lipid-based anti-cancer drugs, such as miltefosine, and signaling molecules, such as platelet-activating factors, bind saturably to Pgp with Kd values in the low micromolar range, and modulate its ATPase activity. These compounds also inhibit Pgp-mediated flipping of fluorescent lipids and transport of Hoechst 33342 and tetramethylrosamine, which occupy different subsites in the drug-binding pocket. Bacterial lipid A modulates Pgp ATPase activity, and glycolipid flipping is inhibited by unlabelled glucosylceramide, suggesting that these lipids also interact with the transporter. These results indicate that Pgp treats a variety of lipid-based molecules as substrates, and likely interacts with lipids and drugs in the same manner.

P-Glycoprotein is localized in intermediate-density membrane microdomains distinct from classical lipid rafts and caveolar domains.

P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) superfamily responsible for the ATP-driven extrusion of diverse hydrophobic molecules from cells, is a cause of multidrug resistance in human tumours. Pgp can also operate as a phospholipid and glycosphingolipid flippase, and has been functionally linked to cholesterol, suggesting that it might be associated with sphingolipid-cholesterol microdomains in cell membranes. We have used nonionic detergent extraction and density gradient centrifugation of extracts from the multidrug-resistant Chinese hamster ovary cell line, CH(R)B30, to address this question. Our data indicate that Pgp is localized in intermediate-density membrane microdomains different from classical lipid rafts enriched in Src-family kinases. We demonstrate that Brij-96 can selectively isolate the Pgp domains, separating them from the caveolar and classical lipid rafts. Pgp was found entirely in the Brij-96-insoluble domains, and only partially in the Triton X-100-insoluble membrane microdomains. We studied the sensitivity of these domains to cholesterol removal, as well as their relationship to GM(1) ganglioside- and caveolin-1-enriched caveolar domains. We found that the buoyant density of the Brij-96-based Pgp-containing microdomains was sensitive to cholesterol removal by methyl-beta-cyclodextrin. The Brij-96 domains retained their structural integrity after cholesterol depletion while, in contrast, the Triton X-100-based caveolin-1/GM(1) microdomains did not. Using confocal fluorescence microscopy, we determined that caveolin-1 and GM(1) colocalized, while Pgp and caveolin-1, or Pgp and GM(1), did not. Our results suggest that Pgp does not interact directly with caveolin-1, and is localized in intermediate-density domains, distinct from classical lipid rafts and caveolae, which can be isolated using Brij-96.