scholarly journals Linear and cyclic peptides as substrates and modulators of P-glycoprotein: peptide binding and effects on drug transport and accumulation

1998 ◽  
Vol 333 (3) ◽  
pp. 621-630 ◽  
Author(s):  
Frances J. SHAROM ◽  
Peihua LU ◽  
Ronghua LIU ◽  
Xiaohong YU
Keyword(s):  
Cyclic Peptides ◽  
Drug Transport ◽  
Membrane Vesicles ◽  
Drug Uptake ◽  
Multidrug Transporter ◽  
Chemotherapeutic Drugs ◽  
Plasma Membrane Vesicles ◽  
Hydrophobic Peptides ◽  
P Glycoprotein ◽  
Highly Correlated

One cause of multidrug resistance (MDR) in human cancers is the overexpression of the P-glycoprotein multidrug transporter, a member of the ABC superfamily of membrane proteins. Natural products and chemotherapeutic drugs are pumped out of the cell by P-glycoprotein in an ATP-dependent fashion. There is growing evidence that many hydrophobic peptides are also P-glycoprotein substrates. With the use of a fluorescence-quenching assay, we have shown that some linear and cyclic hydrophobic peptides interact with P-glycoprotein, whereas others do not. The measured values of the quenching constant, Kq, for interaction of peptides with P-glycoprotein ranged from 200 nM for cyclosporine A to 138 µM for the tripeptide N-acetyl-leucyl-leucyl-norleucinal. Peptides that interacted with P-glycoprotein in the fluorescence assay also blocked colchicine transport into plasma membrane vesicles from MDR cells. The values of Dm, the peptide concentration causing 50% inhibition of drug uptake, were highly correlated with the values of Kq, over three orders of magnitude. The P-glycoprotein ATPase stimulation/inhibition profile of the peptides was not helpful in making a quantitative assessment of the ability of a peptide to interact with P-glycoprotein or to block drug transport. Some hydrophobic peptides were able to restore accumulation in MDR cells of the chemotherapeutic drug daunorubicin and the fluorescent dye rhodamine 123 to the levels observed in the drug-sensitive parent. Peptides that interacted with P-glycoprotein also displayed a relatively low overall toxicity to intact MDR cells, and inhibited drug transport at concentrations below the toxic range. Hydrophobic peptides should be given serious consideration for development as clinical chemosensitizing agents.

scholarly journals Synthetic hydrophobic peptides are substrates for P-glycoprotein and stimulate drug transport

1996 ◽  
Vol 320 (2) ◽  
pp. 421-428 ◽  
Author(s):  
Frances J. SHAROM ◽  
Xiaohong YU ◽  
Giulio DiDIODATO ◽  
Joseph W. K. CHU
Keyword(s):  
Drug Transport ◽  
Efflux Pump ◽  
Membrane Vesicles ◽  
Multidrug Resistant ◽  
Drug Uptake ◽  
Peptide Transport ◽  
Active Efflux ◽  
Hydrophobic Peptides ◽  
P Glycoprotein ◽  
Drug Studies

P-Glycoprotein functions as an ATP-driven active efflux pump for many natural products and chemotherapeutic drugs. Hydrophobic peptides have been shown to block drug uptake by P-glycoprotein, indicating that they might be transport substrates. The present study examines the interaction of the synthetic peptide series NAc-LnY-amide with the multidrug transporter. Several peptides in this series caused up to 3.5-fold enhancement of colchicine accumulation in membrane vesicles from multidrug resistant (MDR) cells, which suggests the existence of novel interactions between the binding sites for peptides and drug. Peptides did not stimulate vinblastine transport, which was inhibited as expected for competing substrates. These peptides displayed modest stimulatory effects on the ATPase activity of P-glycoprotein. None blocked azidopine photoaffinity labelling, showing that they probably occupy a binding site separate from that for the drug. Studies with 125I-labelled NAc-LLY-amide showed that it was transported by P-glycoprotein in both membrane vesicles and reconstituted proteoliposomes. Uptake of the peptide was rapid, saturable, osmotically sensitive and occurred against a concentration gradient. The enhancing effect of NAc-LLY-amide on colchicine transport was reciprocated, i.e. colchicine greatly increased the transport of labelled peptide by P-glycoprotein. Peptide transport was also modulated, both positively and negatively, by other MDR spectrum drugs. It is concluded that linear hydrophobic peptides are indeed transported by P-glycoprotein, and some have interactions with drug substrates that result in mutual stimulation of transport.

Kinetics of the multidrug transporter (P-glycoprotein) and its reversal

1997 ◽  
Vol 77 (2) ◽  
pp. 545-590 ◽  
Author(s):  
W. D. Stein
Keyword(s):  
Final Section ◽  
Transport Kinetics ◽  
Multidrug Transporter ◽  
Chemotherapeutic Drugs ◽  
Multidrug Resistance Proteins ◽  
Binding Properties ◽  
Membrane Pump ◽  
Resistance Proteins ◽  
P Glycoprotein ◽  
Kinetics Of

Most cancer deaths result from the cancer's either being intrinsically resistant to chemotherapeutic drugs or becoming resistant after being initially sensitive. Often, in cells grown in cell culture, drug resistance correlates with the presence of one or more of the so-called P-glycoproteins or multidrug resistance proteins, products of the mdr family of genes. This review is largely concerned with the transport kinetics of the P-glycoproteins. We first present a brief overview of the P-glycoproteins, their properties, and their clinical significance. Later sections of the review expand on this material with special emphasis on the substrates of P-glycoprotein and how they cross the cell membrane, on the transport kinetics of the P-glycoprotein, on reversers of its action, and on its activity as an ATPase. In a final section, we consider the mechanism of action of P-glycoprotein as an actively transporting membrane pump. The characteristic of P-glycoprotein considered the most difficult to explain is its very broad specificity (or lack of specificity), but there are precedents for this property in well-known proteins such as serum albumin, which binds a range of molecular types, including substrates and reversers of P-glycoprotein, seemingly as broad as does P-glycoprotein. Pointing out this analogy does not provide a molecular explanation for the substrate-binding properties of P-glycoprotein but does make those properties more assimilable.

scholarly journals Reversing the direction of drug transport mediated by the human multidrug transporter P-glycoprotein

2020 ◽  
Vol 117 (47) ◽  
pp. 29609-29617
Author(s):  
Andaleeb Sajid ◽  
Sabrina Lusvarghi ◽  
Megumi Murakami ◽  
Eduardo E. Chufan ◽  
Biebele Abel ◽  
...  
Keyword(s):  
Drug Transport ◽  
Atp Hydrolysis ◽  
Binding Pocket ◽  
Drug Binding ◽  
Drug Uptake ◽  
Cancer Drug ◽  
Binding Domains ◽  
Cancer Drug Resistance ◽  
P Glycoprotein ◽  
Cell Transforming

P-glycoprotein (P-gp), also known as ABCB1, is a cell membrane transporter that mediates the efflux of chemically dissimilar amphipathic drugs and confers resistance to chemotherapy in most cancers. Homologous transmembrane helices (TMHs) 6 and 12 of human P-gp connect the transmembrane domains with its nucleotide-binding domains, and several residues in these TMHs contribute to the drug-binding pocket. To investigate the role of these helices in the transport function of P-gp, we substituted a group of 14 conserved residues (seven in both TMHs 6 and 12) with alanine and generated a mutant termed 14A. Although the 14A mutant lost the ability to pump most of the substrates tested out of cancer cells, surprisingly, it acquired a new function. It was able to import four substrates, including rhodamine 123 (Rh123) and the taxol derivative flutax-1. Similar to the efflux function of wild-type P-gp, we found that uptake by the 14A mutant is ATP hydrolysis-, substrate concentration-, and time-dependent. Consistent with the uptake function, the mutant P-gp also hypersensitizes HeLa cells to Rh123 by 2- to 2.5-fold. Further mutagenesis identified residues from both TMHs 6 and 12 that synergistically form a switch in the central region of the two helices that governs whether a given substrate is pumped out of or into the cell. Transforming P-gp or an ABC drug exporter from an efflux transporter into a drug uptake pump would constitute a paradigm shift in efforts to overcome cancer drug resistance.

scholarly journals Hop Resistance in the Beer Spoilage Bacterium Lactobacillus brevis Is Mediated by the ATP-Binding Cassette Multidrug Transporter HorA

2001 ◽  
Vol 183 (18) ◽  
pp. 5371-5375 ◽  
Author(s):  
Kanta Sakamoto ◽  
Abelardo Margolles ◽  
Hendrik W. van Veen ◽  
Wil N. Konings
Keyword(s):  
Drug Transport ◽  
Expression System ◽  
Membrane Vesicles ◽  
Lactobacillus Brevis ◽  
Atp Binding ◽  
Multidrug Transporter ◽  
Transport Studies ◽  
Beer Spoilage ◽  
Spoilage Bacterium ◽  
Atp Binding Cassette

ABSTRACT Lactobacillus brevis is a major contaminant of spoiled beer. The organism can grow in beer in spite of the presence of antibacterial hop compounds that give the beer a bitter taste. The hop resistance in L. brevis is, at least in part, dependent on the expression of the horA gene. The deduced amino acid sequence of HorA is 53% identical to that of LmrA, an ATP-binding cassette multidrug transporter in Lactococcus lactis. To study the role of HorA in hop resistance, HorA was functionally expressed in L. lactis as a hexa-histidine-tagged protein using the nisin-controlled gene expression system. HorA expression increased the resistance of L. lactis to hop compounds and cytotoxic drugs. Drug transport studies with L. lactiscells and membrane vesicles and with proteoliposomes containing purified HorA protein identified HorA as a new member of the ABC family of multidrug transporters.

scholarly journals Modulation of P-glycoprotein-mediated drug transport by alterations in lipid fluidity of rat liver canalicular membrane vesicles.

1992 ◽  
Vol 267 (35) ◽  
pp. 24995-25002
Author(s):  
F.A. Sinicrope ◽  
P.K. Dudeja ◽  
B.M. Bissonnette ◽  
A.R. Safa ◽  
T.A. Brasitus
Keyword(s):  
Rat Liver ◽  
Drug Transport ◽  
Membrane Vesicles ◽  
Canalicular Membrane ◽  
Lipid Fluidity ◽  
P Glycoprotein

scholarly journals Effects of steroids and verapamil on P-glycoprotein ATPase activity: progesterone, desoxycorticosterone, corticosterone and verapamil are mutually non-exclusive modulators

1996 ◽  
Vol 317 (2) ◽  
pp. 515-522 ◽  
Author(s):  
Stéphane ORLOWSKI ◽  
Lluis M. MIR ◽  
Jean BELEHRADEK ◽  
Manuel GARRIGOS
Keyword(s):  
Atpase Activity ◽  
Binding Sites ◽  
Drug Transport ◽  
Competitive Inhibition ◽  
Membrane Vesicles ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Glycoprotein P ◽  
P Glycoprotein ◽  
Synergistic Modulation

P-glycoprotein (P-gp) is a membranous ATPase responsible for the multidrug resistance (MDR) phenotype. Using membrane vesicles prepared from the highly resistant cell line DC-3F/ADX we studied the influence on P-gp ATPase activity of four progesterone derivatives which specifically bind to P-gp and reverse MDR. Progesterone and desoxycorticosterone stimulate P-gp ATPase activity with, respectively, apparent concentrations giving half-maximal activation of 20–25 μM and 40–50 μM, and activation factors of 2.3 (at 100 μM progesterone) and 1.8 (at 170 μM desoxycorticosterone). Hydrocortisone above 100 μM stimulates P-gp ATPase activity while corticosterone has no apparent stimulating effect. Our data are consistent with the location of the binding sites for the progesterone derivatives on the P-gp membranous domain. The effects of these steroids on verapamil-stimulated P-gp ATPase activity support a non-competitive mechanism, i.e. the binding sites for verapamil and steroids are mutually non-exclusive for P-gp ATPase modulation. A similar non-competitive inhibition of progesterone-stimulated P-gp ATPase activity by desoxycorticosterone or by corticosterone leads to the conclusion that these steroids, although sharing related structures, have distinct modulating sites on P-gp. As expected from their mutually non-exclusive interactions on P-gp, progesterone and verapamil when mixed induce a synergistic modulation of P-gp ATPase activity. Since drug transport by P-gp is believed to be coupled to its ATPase activity, a corresponding synergistic effect of these two modulators for the inhibition of P-gp-mediated drug resistance can be expected.

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