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PubMedDate not supplied

ABC transporters of staphylococci.

Some members of the genus Staphylococcus are important human pathogens, such as Staphylococcus aureus and Staphylococcus epidermidis. ATP binding cassette (ABC) transporters of pathogenic staphylococci are often responsible for antibiotic resistance and for the uptake of essential solutes and are therefore being considered as targets for novel antistaphylococcal drugs. Other ABC transporters are involved in the biosynthesis of a special class of peptide antibiotics, the lantibiotics, and in self-protection of the producer against them.

ATP-Binding Cassette Transporters↗

PubMed2003

ABC-transporters: implications on drug resistance from microorganisms to human cancers.

Resistance to chemotherapy is a common clinical problem in patients with infectious diseases as well as in patients with cancer. During treatment of infections or malignant tumors, the drug targets of prokaryotic or eukaryotic microorganisms and neoplastic cells are often found to be refractory to a variety of drugs that have different structures and functions. This phenomenon has been termed multidrug resistance (MDR). The mechanisms leading to MDR are frequently caused by trans-membrane xenobiotic transport molecules belonging to the superfamily of ATP-binding cassette (ABC) transporters. There is an urgent need to understand the structure-function relationships of these efflux pumps that underlie their transport mechanism and drug selectivity. This knowledge may allow the rational design of new drugs that can inhibit or circumvent the activity of these MDR transport molecules. Furthermore, the development of such chemosensitizing agents would help us learn more about the physiological functions and substrates of these pump proteins. This review will discuss the current state of knowledge of the functional and structural similarities among ABC-transporters in prokaryotic and eukaryotic cells and their impact on MDR.

ATP-Binding Cassette Transporters↗

PubMed2003

Protein-mediated transbilayer movement of lipids in eukaryotes and prokaryotes: the relevance of ABC transporters.

Lipid distribution across cellular membranes is regulated by specific membrane proteins controlling transbilayer movement of lipids. Flippases facilitate flip-flop of lipids and allow them to equilibrate between the two membrane leaflets independent of ATP. Distinct P-Type-ATPases transport specific lipids unidirectionally across the membrane at the expense of ATP. A group of ATP-dependent lipid transporters, the ATP-binding cassette (ABC) transporter family, was identified in studies originally related to multidrug resistance (MDR) in cancer cells. Meanwhile, lipid transport activity has been shown for full and half size ABC proteins in eukaryotic and prokaryotic cells. This activity may not only modify the organisation of lipids in membranes, but could also be of significant consequence for cell homeostasis. The various types of lipid movement mediating proteins and their cellular localisation in eukaryotes and prokaryotes are reviewed.

ATP-Binding Cassette Transporters↗

PubMed2000

Head-head/tail-tail relative orientation of the pore-forming domains of the heterodimeric ABC transporter TAP.

BACKGROUND: The transporter associated with antigen processing (TAP) is a heterodimeric member of the large family of ABC transporters. The study of interactions between the subunits TAP1 and TAP2 can reveal the relative orientation of the transmembrane segments, which form a translocation pore for peptides. This is essential for understanding the architecture of TAP and other ABC transporters. RESULTS: The amino-terminal six transmembrane segments (TMs) of human TAP1, TAP1 (1-6), and the amino-terminal five TMs of TAP2, TAP2(1-5), are thought to constitute the pore of TAP. Two new approaches are used to define dimer interactions. We show that TM6 of TAP1 (1-6) is able to change topology post-translationally. This TM, along with a cytoplasmic tail, is translocated into the endoplasmic reticulum lumen, unless TAP2 is expressed. Coexpression of TM(4-5) of TAP2 stabilizes the topology of TAP1 (1-6), even when the TM1 of TAP1 is subsitituted with another sequence. This suggests that the carboxy-terminal TMs of the pore-forming domains TAP1 (1-6) and TAP2(1-5) interact. An alternative assay uses photobleaching in living cells using TAP1 (1-6) tagged with the green fluorescent protein (GFP). Coexpression with TAP2(1-5) results in reduced movement of the heterodimer within the endoplasmic reticulum membrane, as compared with the single TAP1 (1-6) molecule. In contrast, TAP2(1-4) has no effect on the mobility of TAP1 (1-6)-GFP, indicating the importance of TM5 of TAP2 for dimer formation. Also, TM1 of both TAP1 and TAP2 is essential for formation of a complex with low mobility. CONCLUSIONS: Dimerization of the pore-forming transmembrane domains of TAP1 (TM1-6) with its TAP2 counterpart (TM1-5) prevents the post-translational translocation of TM6 of TAP1 and results in a complex with reduced mobility within the endoplasmic reticulum membrane compared with the free subunit. These techniques are used to show that the pore-forming domains of TAP are aligned in a head-head/tail-tail orientation. This positions the following peptide-binding segments of the two TAP subunits to one side of the pore.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

ATP transport and ABC proteins.

ATP can be exported into the extracellular space, where it has important biological effects. Recent evidence shows that direct ATP export across the plasma membrane is associated with the presence of ABC proteins. Do the ABC proteins pump ATP as well as their other substrates, and if so, why?

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

Monitoring the kinetics and performance of yeast membrane ABC transporters by diS-C3(3) fluorescence.

Kinetic features (initial start-up phase, drug pumping velocity and efficiency as dependent on drug concentration and growth phase) of yeast plasma membrane multidrug resistance ABC pumps were studied by monitoring the uptake of the fluorescent potentiometric dye diS-C3(3), which has been found to be expelled from the cells by these pumps. The monitoring was done with Saccharomyces cerevisiae mutants AD1-8 and AD1-3 deleted in different ABC pumps, and in their pump-competent parent strain US50-18C overexpressing transcriptional activators Pdr1p and Pdr3p. On addition to the cells, diS-C3(3) is expelled by the Pdr5p, Yor1p and Snq2p pumps with overlapping substrate specificity. The pump action can be assessed as a difference between the dye uptake curve for pump-competent and pump-deleted cells. The pump-mediated dye efflux, which shows an initial lag of various lengths, maintains a certain residual intracellular dye level. In the absence of external glucose the dye efflux ability of the pumps depends on the growth phase; late exponential and stationary cells can maintain the export for tens of minutes, whereas exponential cells keep up the pump action for limited time periods. This may reflect an insufficient number of pump molecules in the membrane or an effect of insufficient pump energization from endogenous sources. This effect is not mediated by changes in membrane potential because lowered membrane potential caused by inhibition of the plasma membrane H+-ATPase does not affect the pump action.

ATP-Binding Cassette Transporters↗

PubMed2000

Three ways to learn the ABCs.

The ABC model of flower development represents a milestone in explaining how the fate of emerging floral organ primordia is specified. This model states that organ identity is specified by different combinations of the activities of the A, B and C class homeotic genes. In spite of the remarkable simplicity of this model, the complex regulatory interactions that establish the initial pattern of A, B and C gene activity have yet to be fully explained. It has been shown that the LEAFY gene functions early to promote flower meristem identity, and that it is subsequently required for the normal expression of the ABC genes. Recently, LEAFY has been identified as an immediate upstream regulator of the floral homeotic genes, thus opening up an avenue to examine the transcriptional interactions that underlie floral patterning.

AGAMOUS Protein, Arabidopsis↗

PubMed2000

Disruption of the ABC transporter genes PDR5, YOR1, and SNQ2, and their participation in improved fermentative activity of a sake yeast mutant showing pleiotropic drug resistance.

Clotrimazole-resistant mutants from sake yeasts show improved fermentative activity in sake mash and pleiotropic drug resistance (PDR). The PDR mechanism is interpreted by overexpression of ATP-binding cassette (ABC) transporters, which extrude various kinds of drugs out of a cell. In a clotrimazole-resistant mutant, CTZ21, isolated from the haploid sake yeast HL69, the levels of mRNA for three major ABC transporter genes, PDR5, SNQ2, and YOR1, markedly increased. These three genes of CTZ21 were disrupted to investigate which participated in the improved fermentative activity of CTZ21. The fermentative activities of deltapdr5 and deltasnq2 strains of CTZ21 were reduced to that of HL69 in the initial and middle stages of fermentation. In the last stage, however, the sake meter [(1/gravity - 1) x 1443] of the deltapdr5 and deltasnq2 strains rose faster than that of HL69. On the other hand, a deltayor1 strain of CTZ21 fermented sake mash in a manner nearly identical to that of CTZ21 until the last stage of fermentation. But in the last stage, fermentation of the deltayor1 slowed down compared with that of CTZ21. A deltayor1 strain of HL69 also exhibited much reduced fermentative activity in the middle and last fermentation stages. The YOR1 gene seems necessary for sake fermentation to be completed efficiently. The ATP content in sake mash brewed with CTZ21 was drastically decreased throughout the whole fermentation period. This low ATP level was restored to a medium level in the cases of both the deltapdr5 and deltasnq2 strains of CTZ21. In contrast, the deltayor1 of CTZ21 exhibited a low ATP level in sake mash in the same manner as CTZ21. These results suggest that the low ATP level of CTZ21 contributes to a certain extent its improved fermentative activity in the initial and middle stages of sake fermentation.

Journal Article↗

PubMed2003

Macrophage apolipoprotein A-I expression protects against atherosclerosis in ApoE-deficient mice and up-regulates ABC transporters.

The antiatherogenic effect of high-density lipoprotein (HDL) and its major protein component apolipoprotein A-I (apoA-I) has been largely attributed to their key roles in reverse cholesterol transport (RCT) and cellular cholesterol efflux. Substantial evidence shows that overexpression of human apoA-I reduces atherosclerosis in animal models. However, it is uncertain whether this protection is due to an increase in plasma HDL level or to a local effect in the artery wall. To test the hypothesis that expression of human apoA-I in macrophages can promote RCT in the artery wall, we used a retroviral construct expressing human apoA-I cDNA (MFG-HAI) to transduce ApoE(-/-) bone marrow cells and then transplanted these cells into ApoE(-/-) mice with preexisting atherosclerosis. ApoE(-/-) mice reconstituted with MFG-HAI marrow had a significant reduction (30%) in atherosclerotic lesions in the proximal aorta compared to control mice that received marrow expressing MFG parental virus. Peritoneal macrophages isolated from MFG-HAI mice showed a four- to fivefold increase in mRNA expression levels of both ATP-binding cassette (ABC) A1 and ABCG1 compared to controls. Our data demonstrate that gene transfer-mediated expression of human apoA-I in macrophages can compensate in part for apoE deficiency and delay the progression of atherosclerotic lesions by stimulating ABC-dependent cholesterol efflux and RCT.

ATP-Binding Cassette Transporters↗

PubMedDate not supplied

pH-responsive three-layered PEGylated polyplex micelle based on a lactosylated ABC triblock copolymer as a targetable and endosome-disruptive nonviral gene vector.

Nonviral vectors for gene therapy have recently received an increased impetus because of the inherent safety problems of the viral vectors, while their transfection efficiency is generally low compared to the viral vectors. The lack of the ability to escape from the endosomal compartments is believed to be one of the critical barriers to the intracellular delivery of noviral gene vectors. This study was devoted to the design and preparation of a novel ABC triblock copolymer for constructing a pH-responsive and targetable nonviral gene vector. The copolymer, lactosylated poly(ethylene glycol)-block-poly(silamine)-block-poly[2-(N,N-dimethylamino)ethyl methacrylate] (Lac-PEG-PSAO-PAMA), consists of lactosylated poly(ethylene glycol) (A-segment), a pH-responsive polyamine segment (B-segment), and a DNA-condensing polyamine segment (C-segment). The Lac-PEG-PSAO-PAMA spontaneously associated with plasmid DNA (pDNA) to form three-layered polyplex micelles with a PAMA/pDNA polyion complex (PIC) core, an uncomplexed PSAO inner shell, and a lactosylated PEG outer shell, as confirmed by 1H NMR spectroscopy. Under physiological conditions, the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles prepared at an N/P (number of amino groups in the copolymer/number of phosphate groups in pDNA) ratio above 3 were found to be able to condense pDNA, thus adopting a relatively small size (< 150 nm) and an almost neutral surface charge (zeta approximately +5 mV). The micelle underwent a pH-induced size variation (pH = 7.4, 132.6 nm --> pH = 4.0, 181.8 nm) presumably due to the conformational changes (globule-rod transition) of the uncomplexed PSAO chain in response to pH, leading to swelling of the free PSAO inner shell at lowered pH while retaining the condensed pDNA in the PAMA/pDNA PIC core. Furthermore, the micelles exhibited a specific cellular uptake into HuH-7 cells (hepatocytes) through asialoglycoprotein (ASGP) receptor-mediated endocytosis and achieved a far more efficient transfection ability of a reporter gene compared to the Lac-PEG-PSAO/pDNA and Lac-PEG-PAMA/pDNA polyplex micelles composed of the diblock copolymers and pDNA. The effect of hydroxychloroquine as an endosomolytic agent on the transfection efficiency was not observed for the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles, whereas the nigericin treatment of the cell as an inhibitor for the endosomal acidification induced a substantial decrease in the transfection efficiency, suggesting that the protonation of the free PSAO inner shell in response to a pH decrease in the endosome might lead to the disruption of the endosome through buffering of the endosomal cavity. Therefore, the polyplex micelle composed of ABC (ligand-PEG/pH-responsive segment/DNA-condensing segment) triblock copolymer would be a promising approach to a targetable and endosome disruptive nonviral gene vector.

Cell Line, Tumor↗

PubMed2003

Purification and characterization of the N-terminal nucleotide binding domain of an ABC drug transporter of Candida albicans: uncommon cysteine 193 of Walker A is critical for ATP hydrolysis.

The Candida drug resistance protein Cdr1p (approximately 170 kDa) is a member of ATP binding cassette (ABC) superfamily of drug transporters, characterized by the presence of 2 nucleotide binding domains (NBD) and 12 transmembrane segments (TMS). NBDs of these transporters are the hub of ATP hydrolysis activity, and their sequence contains a conserved Walker A motif (GxxGxGKS/T). Mutations of the lysine residue within this motif abrogate the ability of NBDs to hydrolyze ATP. Interestingly, the sequence alignments of Cdr1p NBDs with other bacterial and eukaryotic transporters reveal that its N-terminal NBD contains an unusual Walker A sequence (GRPGAGCST), as the invariant lysine is replaced by a cysteine. In an attempt to understand the significance of this uncommon positioning of cysteine within the Walker A motif, we for the first time have purified and characterized the N-terminal NBD (encompassing first N-terminal 512 amino acids) of Cdr1p as well as its C193A mutant protein. The purified NBD-512 protein could exist as an independent functional general ribonucleoside triphosphatase with strong divalent cation dependence. It exhibited ATPase activity with an apparent K(m) in the 0.8-1.0 mM range and V(max) in the range of 147-160 nmol min(-)(1) (mg of protein)(-)(1). NBD-512-associated ATPase activity was also sensitive to inhibitors such as vanadate, azide, and NEM. The Mut-NBD-512 protein (C193A) showed a severe impairment in its ability to hydrolyze ATP (95%); however, no significant effect on ATP (TNP-ATP) binding was observed. Our results show that C193 is critical for N-terminal NBD-mediated ATP hydrolysis and represents a unique feature distinguishing the ATP-dependent functionality of the ABC transporters of fungi from those found in bacteria and other eukaryotes.

ATP-Binding Cassette Transporters↗

PubMed2005

Functional characterization of N-terminal nucleotide binding domain (NBD-1) of a major ABC drug transporter Cdr1p of Candida albicans: uncommon but conserved Trp326 of Walker B is important for ATP binding.

Using purified N-terminal NBD (NBD-512) domain of Cdr1p, a major multidrug extrusion pump of human pathogenic yeast Candida albicans, we show the relevance of the unique positioning of an atypical Trp326 residue. Similar to Cys193 in Walker A, Trp326 in the Walker B motif of Cdr1p is also a conserved feature of other fungal ATP Binding Cassette (ABC) transporters. By employing fluorescence spectroscopy, chemical modification, and site-directed mutagenesis, we demonstrate that of the five Trp residues in the NBD-512 domain, Trp326 alone is important for nucleotide binding and subsequent conformational changes within the domain. Furthermore, mutation of Trp326 to Ala results in an increased K(M) without appreciably affecting V(max) of ATPase activity. Thus, Trp326 in NBD-512 appears to be important for nucleotide binding and not for its hydrolysis. Additionally, the role of Trp326 in ATP binding is independent of the presence of the adjacent well-conserved Asp327 residue which, like Cys193, has a catalytic role in ATP hydrolysis. Considering that Trp326 of Cdr1p is a typical feature of fungal transporters alone, our study suggests that these ABC transporters may reflect mechanistic differences with regard to nucleotide binding and hydrolysis as compared to their counterparts of non-fungal origin.

ATP-Binding Cassette Transporters↗

PubMed2005

Direct evidence for Sphingomonas sp. A1 periplasmic proteins as macromolecule-binding proteins associated with the ABC transporter: molecular insights into alginate transport in the periplasm.

A Gram-negative bacterium, Sphingomonas sp. A1, has a macromolecule (alginate) import system consisting of a pit on the cell surface and an alginate-specific ATP-binding cassette importer in the inner membrane. Transport of alginate from the pit to the ABC importer is probably mediated by two periplasmic binding protein homologues (AlgQ1 and AlgQ2). Here we describe characteristics of binding of AlgQ1 and AlgQ2 to alginate and its oligosaccharides through surface plasmon resonance biosensor analysis, UV absorption difference spectroscopy, and X-ray crystallography. Both AlgQ1 and AlgQ2 were inducibly expressed in the periplasm of alginate-grown cells of strain A1. Biosensor analysis indicated that both proteins specifically bind alginate with a high degree of polymerization (>100) and that dissociation constants for alginate with an average molecular mass of 26 kDa are 2.3 x 10(-)(7) M for AlgQ1 and 1.5 x 10(-)(7) M for AlgQ2. An in vitro ATPase assay using the membrane complex, including the alginate ABC importer, suggested that both alginate-bound forms of AlgQ1 and AlgQ2 are closely associated with the importer. X-ray crystallography showed that AlgQ1 consisted of two domains separated by a deep cleft that binds alginate oligosaccharides through a conformational change in the two domains. These results directly show that alginate-binding proteins play an important role in the efficient transport of alginate macromolecules with different degrees of polymerization in the periplasm.

ATP-Binding Cassette Transporters↗

PubMed2005

Functional characterization and ATP-induced dimerization of the isolated ABC-domain of the haemolysin B transporter.

Nucleotide-binding domains (NBD) are highly conserved constituents of ATP-binding cassette (ABC) transporters. Members of this family couple ATP hydrolysis to the transfer of various molecules across cell membranes. The NBD of the HlyB transporter, HlyB-NBD, was characterized with respect to its uncoupled ATPase activity, oligomeric state, and stability in solution. Experimental data showed that both the nature and pH of an assay buffer influenced the level of protein activity. Comparative analysis of protein stability and ATPase activity in various buffers suggests an inverse relationship between the two. The highest ATPase activity was detected in HEPES, pH 7.0. A kinetic analysis of the ATPase activity in this buffer revealed an enzyme concentration dependence and ATP-induced protein oligomerization. Assuming that the dimer is the active form of enzyme, at least half of the purified HlyB-NBD was estimated to be a dimer at 1.2 microM under the most optimal conditions for ATP hydrolysis. This is about 2 orders of magnitude lower than reported for other canonical ABC-ATPases. The maximum reaction velocity of 0.6 micromol/mg x min at 22 degrees C and the apparent kinetic constant K(app)(0.5) of 0.26 mM for ATP were determined for the dimerized HlyB-NBD. Gel filtration experiments with the wild-type protein and HlyB-NBD mutated in a key catalytic residue, H662A, provided further evidence for ATP-induced protein dimerization. ATPase activity experiments with protein mixtures composed of wild-type and the ATPase-deficient H662A mutant demonstrated that one intact NBD within a dimer is sufficient for ATP hydrolysis. This single site turnover might suggest a sequential mechanism of ATP hydrolysis in the intact HlyB transporter.

ATP-Binding Cassette Transporters↗

PubMed2005

In vitro functional characterization of BtuCD-F, the Escherichia coli ABC transporter for vitamin B12 uptake.

BtuCD is an ATP binding cassette (ABC) transporter that facilitates uptake of vitamin B(12) into the cytoplasm of Escherichia coli. The crystal structures of BtuCD and its cognate periplasmic binding protein BtuF have been recently determined. We have now explored BtuCD-F function in vitro, both in proteoliposomes and in various detergents. BtuCD reconstituted into proteoliposomes has a significant basal ATP hydrolysis rate that is stimulated by addition of BtuF and inhibited by sodium ortho-vanadate. When using different detergents to solubilize BtuCD, the basal ATP hydrolysis rate, the ability of BtuF to stimulate hydrolysis, and the extent to which sodium ortho-vanadate inhibits ATP hydrolysis all vary significantly. Reconstituted BtuCD can mediate transport of vitamin B(12) against a concentration gradient when coupled to ATP hydrolysis by BtuD in the liposome lumen and BtuF outside the liposomes. These in vitro studies establish the functional competence of the BtuCD and BtuF preparations used in the crystallographic analyses for both ATPase and transport activities. Furthermore, the tight binding of BtuF to BtuCD under the conditions studied suggests that the binding protein may not dissociate from the transporter during the catalytic cycle, which may be relevant to the mechanisms of other ABC transporter systems.

ATP-Binding Cassette Transporters↗

PubMed2006

Interaction of the nucleotide binding domains and regulation of the ATPase activity of the human retina specific ABC transporter, ABCR.

We report here a novel regulation of the ATPase activity of the human retina specific ATP binding cassette transporter (ABC), ABCR, by nucleotide binding domain interactions. We also present evidence that recombinant nucleotide binding domains of ABCR interact in vitro in the complete absence of transmembrane domains (TMDs). Although similar domain-domain interactions have been described in other ABC transporters, the roles of such interactions on the enzymatic mechanisms of these transporters have not been demonstrated experimentally. A quantitative analysis of the in vitro interactions as a function of the nucleotide-bound state demonstrated that the interaction takes place in the absence of nucleotide as well as in the presence of ATP and that it only attenuates in the ADP-bound state. Analysis of the ATPase activities of these proteins in free and complex states indicated that the NBD1-NBD2 interaction significantly influences the ATPase activity. Further investigation, using site-specific mutants, showed that mutations in NBD2 but not NBD1 led to the alteration of the ATPase activity of the NBD1.NBD2 complex and residue Arg 2038 is critical to this regulation. These data indicate that changes in the oligomeric state of the nucleotide binding domains of ABCR are coupled to ATP hydrolysis and might represent a possible signal for the TMDs of ABCR to export the bound substrate. Furthermore, the data support a mechanistic model in which, upon binding of NBD2, NBD1 binds ATP but does not hydrolyze it or does so with a significantly reduced rate.

ATP-Binding Cassette Transporters↗

PubMed2006

Molecular basis for differential nucleotide binding of the nucleotide-binding domain of ABC-transporter CvaB.

The cytoplasmic membrane protein CvaB, involved in colicin V secretion in Escherichia coli, belongs to the ABC-transporter family in which ATP hydrolysis is typically the driving force for substrate transport. However, our previous studies indicated that the nucleotide-binding domain of CvaB could also bind and hydrolyze GTP and, indeed, highly preferred GTP over ATP at low temperatures. In this study, we have examined the molecular basis of this preference. Sequence alignment and homology modeling of the CvaB nucleotide-binding domain predicted that the aromatic stacking region of CvaB (Y501DSQ loop) had a role in the differential binding of nucleotides, and Ser503 and Gln504 provided potential hydrogen bonds to GTP but not to ATP. Site-directed mutagenesis of the Y501DSQ loop, mutations S503A, Q504L, and double mutation S503A/Q504L, was made to test the predicted hydrogen bonds with GTP. The double mutation S503A/Q504L increased the affinity for ATP by 6-fold, whereas the affinity for GTP was reduced slightly: the ATP/GTP-binding ratio increased about 10-fold. The temperature effect assays on nucleotide binding and hydrolysis further indicated that the double mutant protein had largely eliminated the difference for substrates ATP and GTP, and behaved more similarly to the NBD of typical ABC-transporter HlyB. Therefore, we conclude that Ser503 and Gln504 in aromatic stacking region of CvaB block the ATP binding and are important for the GTP-binding preference.

ATP-Binding Cassette Transporters↗

PubMed2005

Transmembrane inhibitors of P-glycoprotein, an ABC transporter.

Drug resistance mediated by ABC transporters such as P-glycoprotein (P-gp) continues to be a major impediment to effective cancer chemotherapy. We have developed a panel of highly specific peptide inhibitors of P-gp based on the structure of the transmembrane domains of the transporter. These peptides are thought to exert their inhibitory action by disrupting the proper assembly of P-gp. A novel 96-well-plate assay based on the efflux of fluorescent P-gp substrate DiOC2 (3-ethyl-2-[3-(3-ethyl-2(3H)-benzoxazolylidene)-1-propenyl]benzoxazolium iodide) was developed and used for structure-functional characterization of transporter inhibitors. The studies strongly suggest that potent and selective inhibitors of ABC transporters can now be developed solely on the basis of the primary structures of the target proteins. The inhibition of P-gp with transmembrane peptides was shown to be chirality-independent. A 25-residue long retroinverso D-analogue of transmembrane domain 5 inhibited the efflux of the fluorescent P-gp substrate with an IC50 of 500 nM. Transmembrane peptides effectively sensitized resistant cancer cells to doxorubicin in vitro without demonstrating any cell toxicity of their own. The newly synthesized P-gp antagonists appear to be promising nontoxic drug resistance inhibitors that merit further development.

ATP Binding Cassette Transporter, Subfamily B, Mem↗