Purification of breast cancer resistance protein ABCG2 and role of arginine-482.

Human ABCG2 was efficiently overexpressed in insect cell membranes, solubilized with 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate, and purified through N-terminal hexahistidine tag. Its functionality was assessed by high vanadate-sensitive ATPase activity, and nucleotide-binding capacity. Interestingly, the R482T point mutation increased both maximal hydrolysis rate and affinity for MgATP, and lowered sensitivity to vanadate inhibition. Direct nucleotide binding, as monitored by quenching of intrinsic fluorescence, indicated a mutation-related preference for ATP over ADP. The R482T mutation only produced a limited change, if any, on the binding of drug substrates, indicating that methotrexate, on the one hand, and rhodamine 123 or doxorubicin, on the other hand, bound similarly to wild-type and mutant transporters whether or not they were subject to cellular transport. In addition, the characteristic inhibitors GF120918 and 6-prenylchrysin, which alter mitoxantrone efflux much better for wild-type than mutant ABCG2, bound similarly to purified ABCG2, while the highly-potent Ko143 bound in the nanomolar range also effective in inhibition of drug transport. All results indicate that the role of the arginine-482 mutation on substrate drug transport and inhibitor efficiency is not mediated by changes in drug binding.

RU49953: a non-hormonal steroid derivative that potently inhibits P-glycoprotein and reverts cellular multidrug resistance.

Progesterone and the antiprogestin RU38486 have been reported as non-transported modulators of P-glycoprotein-mediated drug efflux. However, their hormonal properties limit their potential for clinical trials. The present work shows that some derivatives from either progesterone/RU38486 or estradiol, displaying differential interaction with hormone receptors, bind to P-glycoprotein and chemosensitize the growth of MDR1-transfected cells to vinblastine more strongly than does RU38486. Structure comparison of the compounds indicates that the highly hydrophobic estradiol derivative RU49953, which does not interact with any hormone receptor, inhibits P-glycoprotein-mediated drug efflux very efficiently, as monitored by flow cytometry, and prevents drug site photoaffinity labeling by azidopine. It induces a much higher chemosensitization than the well-known P-glycoprotein modulator verapamil, which is itself more efficient than RU38486. RU49953 therefore constitutes a promising new lead for steroid-type modulators of multidrug resistance.

Multidrug resistance phenotype mediated by the P-glycoprotein-like transporter in Leishmania: a search for reversal agents.

Abstract: Protozoan parasites are responsible for important diseases that threaten the lives of nearly one-quarter of the human population world-wide. Among them, leishmaniasis has become the second cause of death, mainly due to the emergence of parasite resistance to conventional drugs. P-glycoprotein (Pgp)-like transporters overexpression is a very efficient mechanism to reduce the intracellular accumulation of many drugs in cancer cells and parasitic protozoans including Plasmodium and Leishmania, thus conferring a multidrug resistance (MDR) phenotype. Therefore, there is a great clinical interest in developing inhibitors of these transporters to overcome such a resistance. Pgps are active pumps belonging to the ATP-binding cassette (ABC) superfamily of proteins, and consist of two homologous halves, each containing a transmembrane domain (TMD) involved in drug efflux, and a cytosolic nucleotide-binding domain (NBD) responsible for ATP binding and hydrolysis. Most conventional cancer MDR modulators interact with the drug-binding sites on the TMDs of Pgps, but they are also usually transported and the required concentrations for a permanent inhibition produce subsequent side-effects that hamper their clinical use. Besides, they only poorly modulate the resistance in protozoan parasites. We review here a rational strategy developed to overcome the MDR phenotype in Leishmania, consisting in: i) the selection of an MDR Leishmania tropica line that overexpresses a Pgp-like transporter; ii) the use of their cytosolic NBDs as new pharmacological targets; iii) the search of new natural compounds that revert the MDR phenotype in Leishmania by binding to the TMDs; iv) the combination of subdoses of the above selected modulators directed to both targets in the transporter, NBDs and TMDs, to accumulate their reversal effects while diminishing their toxicity. In this way, we have reverted the MDR phenotype in Leishmania, including the resistance to the most promising new antileishmania agents, the alkyl-lysophospholipids. This approach might be extrapolated to be used in other eukaryotic cells.

Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and related ABC transporters.

Cancer cell resistance to chemotherapy is often mediated by overexpression of P-glycoprotein, a plasma membrane ABC (ATP-binding cassette) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. P-glycoprotein (ABCB1, according to the human gene nomenclature committee) consists of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. Homologous ABC multidrug transporters, from the same ABCB family, are found in many species such as Plasmodiumfalciparum and Leishmania spp. protozoa, where they induce resistance to antiparasitic drugs. In yeasts, some ABC transporters involved in resistance to fungicides, such as Saccharomyces cerevisiae Pdr5p and Snq2p, display a different (NBD-TMD)2 domain topology and are classified in another family, ABCG. Much effort has been spent to modulate multidrug resistance in the different species by using specific inhibitors, but generally with little success due to additional cellular targets and/or extrusion of the potential inhibitors. This review shows that due to similarities in function and maybe in three-dimensional organization of the different transporters, common potential modulators have been found. An in vitro 'rational screening' was performed among the large flavonoid family using a four-step procedure: (i) direct binding to purified recombinant cytosolic NBD and/or full-length transporter, (ii) inhibition of ATP hydrolysis and energy-dependent drug interaction with transporter-enriched membranes, (iii) inhibition of cell transporter activity monitored by flow cytometry and (iv) chemosensitization of cell growth. The results indicate that prenylated flavonoids bind with high affinity, and strongly inhibit drug interaction and nucleotide hydrolysis. As such, they constitute promising potential modulators of multidrug resistance.

Alkyl-lysophospholipid resistance in multidrug-resistant Leishmania tropica and chemosensitization by a novel P-glycoprotein-like transporter modulator.

Drug resistance has emerged as a major impediment in the treatment of leishmaniasis. Alkyl-lysophospholipids (ALP), originally developed as anticancer drugs, are considered to be the most promising antileishmanial agents. In order to anticipate probable clinical failure in the near future, we have investigated possible mechanisms of resistance to these drugs in Leishmania spp. The results presented here support the involvement of a member of the ATP-binding cassette (ABC) superfamily, the Leishmania P-glycoprotein-like transporter, in the resistance to ALP. (i) First, a multidrug resistance (MDR) Leishmania tropica line overexpressing a P-glycoprotein-like transporter displays significant cross-resistance to the ALP miltefosine and edelfosine, with resistant indices of 9.2- and 7.1-fold, respectively. (ii) Reduced expression of P-glycoprotein in the MDR line correlates with a significant decrease in ALP resistance. (iii) The ALP were able to modulate the P-glycoprotein-mediated resistance to daunomycin in the MDR line. (iv) We have found a new inhibitor of this transporter, the sesquiterpene C-3, that completely sensitizes MDR parasites to ALP. (v) Finally, the MDR line exhibits a lower accumulation than the wild-type line of bodipy-C(5)-PC, a fluorescent analogue of phosphatidylcholine that has a structure resembling that of edelfosine. Also, C-3 significantly increases the accumulation of the fluorescent analogue to levels similar to those of wild-type parasites. The involvement of the Leishmania P-glycoprotein-like transporter in resistance to drugs used in the treatment of leishmaniasis also supports the importance of developing new specific inhibitors of this ABC transporter.

Protein kinase C effectors bind to multidrug ABC transporters and inhibit their activity.

P-Glycoprotein and homologous multidrug transporters contain a phosphorylatable linker sequence that was proposed to control drug efflux on the basis that it was indeed phosphorylated in vitro and in vivo, and that inhibitors of protein kinase C (PKC) inhibited both P-glycoprotein phosphorylation and activity. However, site-directed mutagenesis of all phosphorylatable residues did not alter the drug resistance. The present work shows that PKC effectors are able to bind directly to multidrug transporters, from either cancer cells (mouse P-glycoprotein), yeast (Saccharomyces cerevisiae Pdr5p), or protozoan parasite (Leishmania tropica ltmdr1), and to inhibit their energy-dependent drug-efflux activity. The binding of staurosporine and derivatives such as CGP 41251 is prevented by preincubation with ATP, suggesting at least partial interaction at the ATP-binding site. In contrast, more hydrophobic compounds such as calphostin C and CGP 42700 bind outside the ATP-binding site and strongly interfere with drug interaction. A direct correlation is obtained between the efficiencies of PKC effectors to inhibit energy-dependent interaction of rhodamine 6G with yeast Pdr5p, to promote intracellular drug accumulation in various multidrug resistant cells, and to chemosensitize growth of resistant cells. The noncompetitive inhibition by PKC effectors of rhodamine 6G interaction with Pdr5p suggests that the binding might interfere with signal transduction between nucleotide hydrolysis and drug interaction. The overall results indicate that the multidrug transporters from different species display common features for interaction with PKC inhibitors. The hydrophobic derivative of staurosporine, CGP 42700, constitutes a potentially powerful modulator of P-glycoprotein-mediated multidrug resistance.

High-affinity binding of silybin derivatives to the nucleotide-binding domain of a Leishmania tropica P-glycoprotein-like transporter and chemosensitization of a multidrug-resistant parasite to daunomycin.

In order to overcome the multidrug resistance mediated by P-glycoprotein-like transporters in Leishmania spp., we have studied the effects produced by derivatives of the flavanolignan silybin and related compounds lacking the monolignol unit on (i) the affinity of binding to a recombinant C-terminal nucleotide-binding domain of the L. tropica P-glycoprotein-like transporter and (ii) the sensitization to daunomycin on promastigote forms of a multidrug-resistant L. tropica line overexpressing the transporter. Oxidation of the flavanonol silybin to the corresponding flavonol dehydrosilybin, the presence of the monolignol unit, and the addition of a hydrophobic substituent such as dimethylallyl, especially at position 8 of ring A, considerably increased the binding affinity. The in vitro binding affinity of these compounds for the recombinant cytosolic domain correlated with their modulation of drug resistance phenotype. In particular, 8-(3,3-dimethylallyl)-dehydrosilybin effectively sensitized multidrug-resistant Leishmania spp. to daunomycin. The cytosolic domains are therefore attractive targets for the rational design of inhibitors against P-glycoprotein-like transporters.

ABC transporters in the protozoan parasite Leishmania.

ATP-binding cassette (ABC) transporters constitute one of the biggest and most conserved protein families in the evolutionary scale. Many of them are of enormous clinical relevance, due to their relationship with genetic diseases and drug resistance during the treatment of cancer and infectious diseases. Leishmaniasis is a major and globally widespread group of parasitic diseases, whose treatment has been complicated by the expansion of resistance to conventional drugs. Here, we review the current knowledge about ABC transporters in Leishmania spp, with special attention to their relationship with the drug-resistance phenotype.

New natural sesquiterpenes as modulators of daunomycin resistance in a multidrug-resistant Leishmania tropica line.

The effects produced by nine dihydro-beta-agarofuran sesquiterpenes isolated from Crossopetalum tonduzii (1-8) and Maytenus macrocarpa (9) (Celastraceae) on the reversion of the resistant phenotype on a multidrug-resistant Leishmania line and their binding to recombinant C-terminal nucleotide-binding domain of Leishmania P-glycoprotein-like transporter were studied. The structures of the new compounds (1-5) were elucidated by spectroscopic methods, including (1)H-(13)C heteronuclear correlation (HMQC), long-range correlation spectra with inversal detection (HMBC), ROESY experiments, and chemical correlations. The absolute configuration of one of them (1) was determined by CD studies. The structure-activity relationship is discussed.

Involvement of thiol metabolism in resistance to glucantime in Leishmania tropica.

Clinical resistance to pentavalent antimonials, in the form of pentostam (sodium stibogluconate) or glucantime (N-methylglucamine antimoniate), has long been recognized as a problem in Leishmaniasis. However, the mechanisms of resistance are unclear. We selected in vitro a Leishmania tropica line resistant to 1.2 mg/mL of Sb(V) of glucantime (GLU-R10). The cell line has a stable phenotype for at least 6 months and a resistance index of 1400-fold. The resistant line has no cross-resistance to pentostam or to SbCl3 and SbCl5. The resistance to glucantime was reverted by buthionine sulfoximine (BSO) and chlorambucil (CLB); however, thiol analyses by HPLC of wild-type and GLU-R10 cell lines, in the presence or absence of the drug, showed no differences between these two cell lines. The resistant line had a DNA amplification shown as a circular extrachromosomal element (G-circle) of approximately 22 kb. However, the specific probes for gamma-glutamyl cysteine synthetase, ornithine decarboxylase and trypanothione reductase did not recognize the G-circle amplified in the GLU-R10. The G-circle did not arise from the H region and was not related with P-glycoprotein Pgp-MDR- or Pgp-MRP-like genes. Northern blot analysis of the G-circle showed that a single transcript of approximately 6 kb was overexpressed in the resistant line. Molecular characterization of the G-circle would lead to the determination of the gene(s) involved in resistance to glucantime in Leishmania.

Altered drug membrane permeability in a multidrug-resistant Leishmania tropica line.

We selected a Leishmania tropica cell line resistant to daunomycin (DNM) that presents a multidrug-resistant (MDR) phenotype characterized by overexpression of a P-glycoprotein of 150 kDa. The resistant line overexpressed an MDR-like gene, called ltrmdr1, located in an extrachromosomal circular DNA. DNM uptake experiments using laser flow cytometry showed a significant reduction in drug accumulation in the resistant parasites. The initial stages of the interaction of DNM with membranes from wild-type and DNM-resistant parasites were defined by a rapid kinetic stopped-flow procedure which can be described by two kinetic components. On the basis of a previous similar kinetic study with tumor cells, we ascribed the fast component to rapid interaction of DNM with membrane surface components and the slow component to passive diffusion of the drug across the membranes. The results reported here indicate that entrance of DNM into wild-type parasites was facilitated in respect to the resistant ones. We propose that resistance to DNM in L. tropica is a multifactorial event involving at least two complementary mechanisms. an altered drug membrane permeability and the overexpression of a protein related to P-glycoprotein that regulates drug efflux.