Hydrophobic Vacuum Cleaner Research Articles

In Silico Characterization of P-Glycoprotein Substrate Entrance Pathways

Due to the wide variety of substrates it extrudes, P-glycoprotein is one of the main causes of multidrug resistance. Although the chemical structure of a ligand is likely to dictate interactions within the binding site, it is equally important to understand how access to the binding site itself is controlled. Access to the binding site is via an aqueous cavity formed in the main by transmembrane helices (TMHs). These TMHs also have helical structure that extends into the cytoplasm, but the hydrophobic vacuum cleaner model suggests that access to the aqueous cavity is via gates between the membranous regions of TMHs 4 and 6 or between TMHs 10 and 12.Previously, we observed that the intra-membrane sections of these gates tend to close while cytoplasmic regions remain open. We have explored this further via the use of steered MD with substrates initially positioned in the cytoplasmic leaflet of the bilayer. Rather unexpectedly, the simulations show that entrance through the intra-membrane gate requires a significantly larger amount of work compared to the cytoplasmic entrance and with a preference for the cavity between TMH 4 and 6 compared to that between TMH 10 and 12.Once the compound has reached the aqueous cavity, it can freely diffuse towards its binding site. The binding mode was independently assessed through the use of consensus docking and appears to be similar to the so-called “R-site”. The docked conformations were then used to calculate possible unbinding pathways of amitriptyline with the Protein Energy Landscape Exploration (PELE) method.Taken together, these combined approaches suggest that a likely access route to the binding site for many compounds is through a cytoplasmic gate formed by TMHs 4 and 6.

Complex Interplay between the P-Glycoprotein Multidrug Efflux Pump and the Membrane: Its Role in Modulating Protein Function.

Multidrug resistance in cancer is linked to expression of the P-glycoprotein multidrug transporter (Pgp, ABCB1), which exports many structurally diverse compounds from cells. Substrates first partition into the bilayer and then interact with a large flexible binding pocket within the transporter’s transmembrane regions. Pgp has been described as a hydrophobic vacuum cleaner or an outwardly directed drug/lipid flippase. Recent X-ray crystal structures have shed some light on the nature of the drug-binding pocket and suggested routes by which substrates can enter it from the membrane. Detergents have profound effects on Pgp function, and several appear to be substrates. Biochemical and biophysical studies in vitro, some using purified reconstituted protein, have explored the effects of the membrane environment. They have demonstrated that Pgp is involved in a complex relationship with its lipid environment, which modulates the behavior of its substrates, as well as various functions of the protein, including ATP hydrolysis, drug binding, and drug transport. Membrane lipid composition and fluidity, phospholipid headgroup and acyl chain length all influence Pgp function. Recent studies focusing on thermodynamics and kinetics have revealed some important principles governing Pgp–lipid and substrate–lipid interactions, and how these affect drug-binding and transport. In some cells, Pgp is associated with cholesterol-rich microdomains, which may modulate its functions. The relationship between Pgp and cholesterol remains an open question; however, it clearly affects several aspects of its function in addition to substrate–membrane partitioning. The action of Pgp modulators appears to depend on their membrane permeability, and membrane fluidizers and surfactants reverse drug resistance, likely via an indirect mechanism. A detailed understanding of how the membrane affects Pgp substrates and Pgp’s catalytic cycle may lead to new strategies to combat clinical drug resistance.

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.

Shedding light on drug transport: structure and function of the P-glycoprotein multidrug transporter (ABCB1)This paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease.

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.

Localization of Multidrug Transporter Substrates within Model Membranes

Active extrusion of drugs from the cell interior by primary and secondary efflux pumps is an essential mechanism underlying the phenomenon of multidrug resistance. The first discovered and best characterized primary efflux pump found in humans is the ABC transporter P-glycoprotein (PGP), which shows very broad substrate specificity. Many of these molecules are lipophilic, and binding most likely takes place within the membrane. PGP could either translocate them from the inner to the outer leaflet (flippase) or extrude them from the membrane into the extracellular environment (hydrophobic vacuum cleaner). Recognition and binding of such a diverse set of substrates must be associated with a preferred membrane location, determined by molecular properties and lipid interactions. Therefore, a systematic study of the interaction among seven PGP substrates (phenazine, doxorubicin, cephalexin, ampicillin, chloramphenicol, penicillin G, and quercetin) and two modulators (quinidine and nicardipine) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) model membranes is reported here. The location profile of these molecules across the membrane was determined by (1)H NOESY MAS NMR based on (1)H-(1)H cross-peaks between their aromatic fingerprint region and lipid resonances. Although structurally rather diverse, all tested substances are found to have their highest concentration between the phosphate of the lipid headgroup and the upper segments of the lipid hydrocarbon chains. Our findings are consistent with PGP substrate and modulator binding from the membrane interface region.

Cyclosporins: structure-activity relationships for the inhibition of the human MDR1 P-glycoprotein ABC transporter.

Cyclic undecapeptide cyclo-[MeBmt(1)-Abu(2)-MeGly(3)-MeLeu(4)-Val(5)-MeLeu(6)-Ala(7)-D-Ala(8)-MeLeu(9)-MeLeu(10)-MeVal(11)], the immunosuppressive and antifungal antibiotic cyclosporin A (CsA), was reported to interfere with the MDR1 P-glycoprotein (Pgp), a transmembranous adenosine 5'-triphosphate binding cassette (ABC) transporter with phospholipid flippase or "hydrophobic vacuum cleaner" properties that mediate multidrug resistance (MDR) of cancer cells. By use of photoaffinity-labeled cyclosporins and membranes from Pgp-expressing cells, it was recently shown that in vitro, Pgp molecules could bind a large cyclosporin domain involving residues 4-9 as well as the side chain of residue 1. Tumor cell MDR can also be reversed by a product more distantly related to cyclosporin with the structure [Thr(2), Leu(5), D-Hiv(8), Leu(10)]-CsA (SDZ 214-103). In a standardized assay that measures Pgp function in vivo (on intact live cells) by the Pgp-mediated efflux of the calcein-AM Pgp substrate and uses human lymphoblastoid MDR-CEM (VBL(100)) cells as highly resistant Pgp-expressing cells, SDZ 214-103 was found to be one of the most active Pgp inhibitors among naturally occurring cyclosporins, with an IC(50) of 1.6 microM in an assay where CsA gives an IC(50) of 3.4 microM. Using the in vivo assay, 60, mostly natural, cyclosporin analogues were analyzed to establish structure-activity relationships (SAR). Our SAR are compatible with the in vitro-defined Pgp binding domain model and further disclose that in vivo Pgp inhibition is favored by larger hydrophobic side chains on cyclosporin residues 1, 4, 6, and 8 and a smaller one on residue 7, although with no effect on the residue 5 side chain; moreover, larger hydrophobic side chains on other residues 2, 3, 10, and 11 (outside the in vitro-defined Pgp binding domain) also favor the eventual inhibition of Pgp function. The N-desmethylation of any of the seven N-methylated amides, as naturally occurring in numerous cyclosporins, regularly leads to a decreased Pgp inhibitory activity (Pgp-InhA), up to its abrogation if it occurs at residues 4 and 9. Nevertheless, despite unfavorable use of [Thr(2)] and [Leu(10)] residues, all [D-Hiv(8)] analogues whose lead is SDZ 214-103 show a large Pgp-InhA. The SAR for Pgp inhibition by cyclosporins are thus very complex. Because CsA and SDZ 214-103 show largely different conformations when free in solution, but remarkably similar ones when bound to the cytosolic cyclophilins, SAR for Pgp inhibition must similarly include requirements for occurrence of suitable conformers for insertion in the cell membrane, sufficient conformational plasticity for gaining access to Pgp binding sites, and an adequate conformer structure there to achieve such binding with a high enough affinity and possibly escape from sequestration on cyclophilins.

Exploring the structure and function of the P-glycoprotein multidrug transporter using fluorescence spectroscopic tools

P-glycoprotein is an ABC protein that functions as an efflux pump for multiple drugs, natural products and peptides. It is proposed to operate as a hydrophobic vacuum cleaner, expelling non-polar compounds from the membrane bilayer to the exterior, driven by the energy of ATP hydrolysis. The nucleotide-binding domains of P-glycoprotein appear to operate by an alternating sites mechanism to power drug transport. In recent years, purification and functional reconstitution of the protein has allowed the application of fluorescence spectroscopic techniques. This approach has led to insights into the structural architecture of the P-glycoprotein molecule, and a more detailed understanding of the way in which it interacts with nucleotides and drugs.

Drug transport by reconstituted P‐glycoprotein in proteoliposomes

The P‐glycoprotein multidrug transporter (Pgp) is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Pgp is envisaged as a ‘hydrophobic vacuum cleaner’, and drugs are believed to gain access to the substrate binding sites from within the membrane, rather than from the aqueous phase. The intimate association of both Pgp and its substrates with the membrane suggests that its function may be regulated by the biophysical properties of the lipid bilayer. Using the high affinity fluorescent substrate tetramethylrosamine (TMR), we have monitored, in real time, transport in proteoliposomes containing reconstituted Pgp. The TMR concentration gradient generated by Pgp was collapsed by the addition of either the ATPase inhibitor, vanadate, or Pgp modulators. TMR transport by Pgp obeyed Michaelis–Menten kinetics with respect to both of its substrates. The Km for ATP was 0.48 mm, close to the Km for ATP hydrolysis, and the Km for TMR was 0.3 µm. TMR transport was inhibited in a concentration‐dependent fashion by verapamil and cyclosporin A, and activated (probably by a positive allosteric effect) by the transport substrate colchicine. TMR transport by Pgp reconstituted into proteoliposomes composed of two synthetic phosphatidylcholines showed a highly unusual biphasic temperature dependence. The rate of TMR transport was relatively high in the rigid gel phase, reached a maximum at the melting temperature of the bilayer, and then decreased in the fluid liquid crystalline phase. This pattern of temperature dependence suggests that the rate of drug transport by Pgp may be dominated by partitioning of drug into the bilayer.

Drug transport by reconstituted P-glycoprotein in proteoliposomes Effect of substrates and modulators, and dependence on bilayer phase state

The P-glycoprotein multidrug transporter (Pgp) is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Pgp is envisaged as a 'hydrophobic vacuum cleaner', and drugs are believed to gain access to the substrate binding sites from within the membrane, rather than from the aqueous phase. The intimate association of both Pgp and its substrates with the membrane suggests that its function may be regulated by the biophysical properties of the lipid bilayer. Using the high affinity fluorescent substrate tetramethylrosamine (TMR), we have monitored, in real time, transport in proteoliposomes containing reconstituted Pgp. The TMR concentration gradient generated by Pgp was collapsed by the addition of either the ATPase inhibitor, vanadate, or Pgp modulators. TMR transport by Pgp obeyed Michaelis--Menten kinetics with respect to both of its substrates. The Km for ATP was 0.48 mM, close to the K(m) for ATP hydrolysis, and the K(m) for TMR was 0.3 microM. TMR transport was inhibited in a concentration-dependent fashion by verapamil and cyclosporin A, and activated (probably by a positive allosteric effect) by the transport substrate colchicine. TMR transport by Pgp reconstituted into proteoliposomes composed of two synthetic phosphatidylcholines showed a highly unusual biphasic temperature dependence. The rate of TMR transport was relatively high in the rigid gel phase, reached a maximum at the melting temperature of the bilayer, and then decreased in the fluid liquid crystalline phase. This pattern of temperature dependence suggests that the rate of drug transport by Pgp may be dominated by partitioning of drug into the bilayer.

Aureobasidins: Structure−Activity Relationships for the Inhibition of the Human MDR1 P-Glycoprotein ABC-Transporter

Cyclic depsipeptide cyclo-[D-Hmp(1)-L-MeVal(2)-L-Phe(3)-L-MePhe(4)-L-Pro(5)-L-aIle+ ++(6)-L-MeVal(7)-L-Leu(8)-L-betaHOMeVal(9)], the antifungal antibiotic aureobasidin A (AbA), was reported to interfere with ATP-binding cassette (ABC) transporters in yeast and mammalian cells, particularly the MDR1 P-glycoprotein (Pgp), a transmembrane phospholipid flippase or "hydrophobic vacuum cleaner" that mediates multidrug resistance (MDR) of cancer cells. In a standardized assay that measures Pgp function by the Pgp-mediated efflux of the calcein-AM Pgp substrate and uses human lymphoblastoid MDR-CEM (VBL(100)) cells as highly resistant Pgp-expressing cells and the cyclic undecapeptide cyclosporin A (CsA) as a reference MDR-reversing agent (IC(50) of 3.4 microM), AbA was found to be a more active Pgp inhibitor (IC(50) of 2.3 microM). Out of seven natural analogues and 18 chemical derivatives of AbA, several were shown to display even more potent Pgp-inhibitory activity. The Pgp-inhibitory activity was increased about 2-fold by some minor modifications such as those found in the naturally occurring aureobasidins AbB ([D-Hiv(1)]-AbA), AbC ([Val(6)]-AbA), and AbD [gammaHOMeVal(9)]-AbA). The replacement of the [Phe(3)-MePhe(4)-Pro(5)] tripeptide by an 8-aminocaprylic acid or the N(7)()-desmethylation of MeVal(7) led to only a 3.3-fold decreased capacity to inhibit Pgp function, suggesting that the Pgp inhibitory potential of aureobasidins, though favored by the establishment of an antiparallel beta-sheet between the [D-Hmp(1)-L-MeVal(2)-L-Phe(3)] and [L-aIle(6)-L-MeVal(7)-L-Leu(8)-] tripeptides, does not critically depend on the occurrence of the [L-Phe(3)-L-MePhe(4)-L-Pro(5)-L-aIle(6)] type II' beta-turn secondary structure. In contrast, the most potent Pgp inhibitors were found among AbA analogues with [betaHO-MeVal(9)] residue alterations, with some data suggesting a negative impact of the [L-Leu(8)-L-betaHOMeVal(9)-D-Hmp(1)] gamma-turn secondary structure on Pgp inhibitory potential. The [2,3-dehydro-MeVal(9)]-AbA was the most potent Pgp inhibitory aureobasidin, being 13-fold more potent than AbA and 19-fold more potent (on a molar basis) than CsA. Finally, there was no correlation between the SAR for the human MDR1 Pgp inhibition and the SAR for Saccharomyces cerevisiae antifungal activity, which is mediated by an inositol phosphoceramide synthase activity.

P-glycoprotein-mediated resistance to chemotherapy in cancer cells: using recombinant cytosolic domains to establish structure-function relationships.

Resistance to chemotherapy in cancer cells is mainly mediated by overexpression of P-glycoprotein (Pgp), a plasma membrane ATP-binding cassette (ABC) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. Pgp consists of two homologous halves each containing a transmembrane domain and a cytosolic nucleotide-binding domain (NBD) which contains two consensus Walker motifs, A and B, involved in ATP binding and hydrolysis. The protein also contains an S signature characteristic of ABC transporters. The molecular mechanism of Pgp-mediated drug transport is not known. Since the transporter has an extraordinarily broad substrate specificity, its cellular function has been described as a "hydrophobic vacuum cleaner". The limited knowledge about the mechanism of Pgp, partly due to the lack of a high-resolution structure, is well reflected in the failure to efficiently inhibit its activity in cancer cells and thus to reverse multidrug resistance (MDR). In contrast to the difficulties encountered when studying the full-length Pgp, the recombinant NBDs can be obtained in large amounts as soluble proteins. The biochemical and biophysical characterization of recombinant NBDs is shown here to provide a suitable alternative route to establish structure-function relationships. NBDs were shown to bind ATP and analogues as well as potent modulators of MDR, such as hydrophobic steroids, at a region close to the ATP site. Interestingly, flavonoids also bind to NBDs with high affinity. Their binding site partly overlaps both the ATP-binding site and the steroid-interacting region. Therefore flavonoids constitute a new promising class of bifunctional modulators of Pgp.

Kinetic Analysis of the Mechanism of Action of the Multidrug Transporter

To clarify the mechanistic role of PGP (P-glycoprotein) in multidrug transport, we constructed a kinetic model composed of four compartments: (1) the extracellular space; (2) the space in the membrane; (3) the intracellular space; and (4) the pore-like space in the PGP molecule. The kinetics of the concentration of ADM (adriamycin) in each compartment were formulated based on the assumptions that (a) the movement of ADM between two compartments by diffusion is dependent on a dynamic distribution coefficient introduced here, (b) the uptake of ADM into the pore-like structure by the pump mechanism activated by ATP is described by enzyme kinetics, (c) the movement of ADM out of the pore-like structure to the extracellular medium through a valve-like mechanism is also expressed by enzyme kinetics. The mathematical analysis of the exact solution can explain the distinct effects of verapamil and vanadate on the accumulation and release of ADM, where verapamil inhibits the efflux by the valve-like mechanism and vanadate blocks the influx by the pump mechanism. We also performed a numerical calculation with this model for a quantitative explanation and found the valid parameter values to fit the experimental data. These results support the modified hydrophobic vacuum cleaner model.

Energetics and Mechanism of Drug Transport Mediated by the Lactococcal Multidrug Transporter LmrP

The gene encoding the secondary multidrug transporter LmrP of Lactococcus lactis was heterologously expressed in Escherichia coli. The energetics and mechanism of drug extrusion mediated by LmrP were studied in membrane vesicles of E. coli. LmrP-mediated extrusion of tetraphenyl phosphonium (TPP+) from right-side-out membrane vesicles and uptake of the fluorescent membrane probe 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) into inside-out membrane vesicles are driven by the membrane potential (Deltapsi) and the transmembrane proton gradient (DeltapH), pointing to an electrogenic drug/proton antiport mechanism. Ethidium bromide, a substrate for LmrP, inhibited the LmrP-mediated TPP+ extrusion from right-side-out membrane vesicles, showing that LmrP is capable of transporting structurally unrelated drugs. Kinetic analysis of LmrP-mediated TMA-DPH transport revealed a direct relation between the transport rate and the amount of TMA-DPH associated with the cytoplasmic leaflet of the lipid bilayer. This observation indicates that drugs are extruded from the inner leaflet of the cytoplasmic membrane into the external medium. This is the first report that shows that drug extrusion by a secondary multidrug resistance (MDR) transporter occurs by a "hydrophobic vacuum cleaner" mechanism in a similar way as was proposed for the primary lactococcal MDR transporter, LmrA.

Multidrug resistance in Lactococcus lactis: evidence for ATP-dependent drug extrusion from the inner leaflet of the cytoplasmic membrane.

Lactococcus lactis possesses an ATP-dependent drug extrusion system which shares functional properties with the mammalian multidrug resistance (MDR) transporter P-glycoprotein. One of the intriguing aspects of both transporters is their ability to interact with a broad range of structurally unrelated amphiphilic compounds. It has been suggested that P-glycoprotein removes drugs directly from the membrane. Evidence is presented that this model is correct for the lactococcal multidrug transporter through studies of the extrusion mechanism of BCECF-AM and cationic diphenylhexatriene (DPH) derivatives from the membrane. The non-fluorescent probe BCECF-AM can be converted intracellularly into its fluorescent derivative, BCECF, by non-specific esterase activities. The development of fluorescence was decreased upon energization of the cells. These and kinetic studies showed that BCECF-AM is actively extruded from the membrane before it can be hydrolysed intracellularly. The increase in fluorescence intensity due to the distribution of TMA-DPH into the phospholipid bilayer is a biphasic process. This behaviour reflects the fast entry of TMA-DPH into the outer leaflet followed by a slower transbilayer movement to the inner leaflet of the membrane. The initial rate of TMA-DPH extrusion correlates with the amount of probe associated with the inner leaflet. Taken together, these results demonstrate that the lactococcal MDR transporter functions as a 'hydrophobic vacuum cleaner', expelling drugs from the inner leaflet of the lipid bilayer. Thus, the ability of amphiphilic substrates to partition in the inner leaflet of the membrane is a prerequisite for recognition by multidrug transporters.