Mechanism of inhibition of human glucose transporter GLUT1 is conserved between cytochalasin B and phenylalanine amides.

Cancerous cells have an acutely increased demand for energy, leading to increased levels of human glucose transporter 1 (hGLUT1). This up-regulation suggests hGLUT1 as a target for therapeutic inhibitors addressing a multitude of cancer types. Here, we present three inhibitor-bound, inward-open structures of WT-hGLUT1 crystallized with three different inhibitors: cytochalasin B, a nine-membered bicyclic ring fused to a 14-membered macrocycle, which has been described extensively in the literature of hGLUTs, and two previously undescribed Phe amide-derived inhibitors. Despite very different chemical backbones, all three compounds bind in the central cavity of the inward-open state of hGLUT1, and all binding sites overlap the glucose-binding site. The inhibitory action of the compounds was determined for hGLUT family members, hGLUT1-4, using cell-based assays, and compared with homology models for these hGLUT members. This comparison uncovered a probable basis for the observed differences in inhibition between family members. We pinpoint regions of the hGLUT proteins that can be targeted to achieve isoform selectivity, and show that these same regions are used for inhibitors with very distinct structural backbones. The inhibitor cocomplex structures of hGLUT1 provide an important structural insight for the design of more selective inhibitors for hGLUTs and hGLUT1 in particular.

Revealing the fate of cell surface human P-glycoprotein (ABCB1): The lysosomal degradation pathway.

P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7±1.1 h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1±0.5 h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50 h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.

Using the BacMam Baculovirus System to Study Expression and Function of Recombinant Efflux Drug Transporters in Polarized Epithelial Cell Monolayers.

The ATP-binding cassette (ABC) transporter superfamily includes several membrane-bound proteins that are critical to drug pharmacokinetics and disposition. Pharmacologic evaluation of these proteins in vitro remains a challenge. In this study, human ABC transporters were expressed in polarized epithelial cell monolayers transduced using the BacMam baculovirus gene transfer system. The purpose of the study was to evaluate the efficacy of BacMam baculovirus to transduce cells grown in monolayers. In a porcine kidney cell line, LLC-PK1 cells, baculoviral transduction is successful only via the apical side of a polarized monolayer. We observed that recombinant ABC transporters were expressed on the cell surface with post-translational modification. Furthermore, sodium butyrate played a critical role in recombinant protein expression, and preincubation in the presence of tunicamycin or thapsigargin enhanced protein expression. Cells overexpressing human P-glycoprotein (P-gp) showed vectorial basolateral-to-apical transport of [(3)H]-paclitaxel, which could be reversed by the inhibitor tariquidar. Similarly, coexpression of human P-gp and ABCG2 in LLC-PK1 cells resulted in higher transport of mitoxantrone, which is a substrate for both transporters, than in either P-gp- or ABCG2-expressing cells alone. Taken together, our results indicate that a high level of expression of efflux transporters in a polarized cell monolayer is technically feasible with the BacMam baculovirus system.

Expression of the multidrug transporter P-glycoprotein is inversely related to that of apoptosis-associated endogenous TRAIL.

Multidrug resistance (MDR) has been associated with expression of ABC transporter genes including P-glycoprotein (Pgp, MDR1, ABCB1). However, deregulation of apoptotic pathways also renders cells resistant to chemotherapy. To discover apoptosis-related genes affected by Pgp expression, we used the HeLa MDR-off system. We found that using doxycycline to control Pgp expression has a significant advantage over tetracycline, in that doxycycline caused less endogenous gene expression modification/perturbation, and was more potent than tetracycline in suppressing Pgp expression. Cells overexpressing Pgp have lower TNFSF10 (TRAIL) expression than their parental cells. Controlled downregulation of Pgp increased endogenous TRAIL protein expression. Also, ectopic overexpression of TRAIL in Pgp-positive cells was associated with a reduction in Pgp levels. However, cells expressing a functionally defective mutant Pgp showed an increase in TRAIL expression, suggesting that Pgp function is required for TRAIL suppression. Cells in which Pgp is knocked down by upregulation of TRAIL expression are less susceptible to TRAIL ligand (sTRAIL)-induced apoptosis. Our findings reveal an inverse correlation between functional Pgp and endogenous TRAIL expression. Pgp function plays an important role in the TRAIL-mediated apoptosis pathway by regulating endogenous TRAIL expression and the TRAIL-mediated apoptosis pathway in MDR cancer cells.

Mutations in intracellular loops 1 and 3 lead to misfolding of human P-glycoprotein (ABCB1) that can be rescued by cyclosporine A, which reduces its association with chaperone Hsp70.

P-glycoprotein (P-gp) is an ATP binding cassette transporter that effluxes a variety of structurally diverse compounds including anticancer drugs. Computational models of human P-gp in the apo- and nucleotide-bound conformation show that the adenine group of ATP forms hydrogen bonds with the conserved Asp-164 and Asp-805 in intracellular loops 1 and 3, respectively, which are located at the interface between the nucleotide binding domains and transmembrane domains. We investigated the role of Asp-164 and Asp-805 residues by substituting them with cysteine in a cysteine-less background. It was observed that the D164C/D805C mutant, when expressed in HeLa cells, led to misprocessing of P-gp, which thus failed to transport the drug substrates. The misfolded protein could be rescued to the cell surface by growing the cells at a lower temperature (27 °C) or by treatment with substrates (cyclosporine A, FK506), modulators (tariquidar), or small corrector molecules. We also show that short term (4-6 h) treatment with 15 µM cyclosporine A or FK506 rescues the pre-formed immature protein trapped in the endoplasmic reticulum in an immunophilin-independent pathway. The intracellularly trapped misprocessed protein associates more with chaperone Hsp70, and the treatment with cyclosporine A reduces the association of mutant P-gp, thus allowing it to be trafficked to the cell surface. The function of rescued cell surface mutant P-gp is similar to that of wild-type protein. These data demonstrate that the Asp-164 and Asp-805 residues are not important for ATP binding, as proposed earlier, but are critical for proper folding and maturation of a functional transporter.

Insight into pleiotropic drug resistance ATP-binding cassette pump drug transport through mutagenesis of Cdr1p transmembrane domains.

The fungal ATP-binding cassette (ABC) transporter Cdr1 protein (Cdr1p), responsible for clinically significant drug resistance, is composed of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). We have probed the nature of the drug binding pocket by performing systematic mutagenesis of the primary sequences of the 12 transmembrane segments (TMSs) found in the TMDs. All mutated proteins were expressed equally well and localized properly at the plasma membrane in the heterologous host Saccharomyces cerevisiae, but some variants differed significantly in efflux activity, substrate specificity, and coupled ATPase activity. Replacement of the majority of the amino acid residues with alanine or glycine yielded neutral mutations, but about 42% of the variants lost resistance to drug efflux substrates completely or selectively. A predicted three-dimensional homology model shows that all the TMSs, apart from TMS4 and TMS10, interact directly with the drug-binding cavity in both the open and closed Cdr1p conformations. However, TMS4 and TMS10 mutations can also induce total or selective drug susceptibility. Functional data and homology modeling assisted identification of critical amino acids within a drug-binding cavity that, upon mutation, abolished resistance to all drugs tested singly or in combinations. The open and closed Cdr1p models enabled the identification of amino acid residues that bordered a drug-binding cavity dominated by hydrophobic residues. The disposition of TMD residues with differential effects on drug binding and transport are consistent with a large polyspecific drug binding pocket in this yeast multidrug transporter.

Design, synthesis, and biological evaluation of (S)-valine thiazole-derived cyclic and noncyclic peptidomimetic oligomers as modulators of human P-glycoprotein (ABCB1).

Multidrug resistance caused by ATP binding cassette transporter P-glycoprotein (P-gp) through extrusion of anticancer drugs from the cells is a major cause of failure in cancer chemotherapy. Previously, selenazole-containing cyclic peptides were reported as P-gp inhibitors and were also used for co-crystallization with mouse P-gp, which has 87 % homology to human P-gp. It has been reported that human P-gp can simultaneously accommodate two to three moderately sized molecules at the drug binding pocket. Our in silico analysis, based on the homology model of human P-gp, spurred our efforts to investigate the optimal size of (S)-valine-derived thiazole units that can be accommodated at the drug binding pocket. Towards this goal, we synthesized varying lengths of linear and cyclic derivatives of (S)-valine-derived thiazole units to investigate the optimal size, lipophilicity, and structural form (linear or cyclic) of valine-derived thiazole peptides that can be accommodated in the P-gp binding pocket and affects its activity, previously an unexplored concept. Among these oligomers, lipophilic linear (13) and cyclic trimer (17) derivatives of QZ59S-SSS were found to be the most and equally potent inhibitors of human P-gp (IC50 =1.5 µM). As the cyclic trimer and linear trimer compounds are equipotent, future studies should focus on noncyclic counterparts of cyclic peptides maintaining linear trimer length. A binding model of the linear trimer 13 within the drug binding site on the homology model of human P-gp represents an opportunity for future optimization, specifically replacing valine and thiazole groups in the noncyclic form.

Conserved Walker A cysteines 431 and 1074 in human P-glycoprotein are accessible to thiol-specific agents in the apo and ADP-vanadate trapped conformations.

P-Glycoprotein (P-gp) is an ATP-binding cassette efflux transporter involved in the development of multidrug resistance in cancer cells. Although the mechanism of P-gp efflux has been extensively studied, aspects of its catalytic and transport cycle are still unclear. In this study, we used conserved C431 and C1074 in the Walker A motif of nucleotide-binding domains (NBDs) as reporter sites to interrogate the interaction between the two NBDs during the catalytic cycle. Disulfide cross-linking of the C431 and C1074 residues in a Cys-less background can be observed in the presence of M14M and M17M cross-linkers, which have spacer arm lengths of 20 and 25 Å, respectively. However, cross-linking with both cross-linkers was prevented in the ADP-vanadate trapped (closed) conformation. Both C431 and C1074 alone or together (double mutant) in the apo and closed conformations were found to be accessible to fluorescein 5-maleimide (FM) and methanethiosulfonate derivatives of rhodamine and verapamil. In addition, C1074 showed 1.4- and 2-fold higher degrees of FM labeling than C431 in the apo and closed conformations, respectively, demonstrating that C1074 is more accessible than C431 in both conformations. In the presence of P-gp substrates, cross-linking with M17M is still observed, suggesting that binding of substrate in the transmembrane domains does not change the accessibility of the cysteines in the NBDs. In summary, the cysteines in the Walker A motifs of NBDs of human P-gp are differentially accessible to thiol-specific agents in the apo and closed conformations.

A key structural domain of the Candida albicans Mdr1 protein.

A major multidrug transporter, MDR1 (multidrug resistance 1), a member of the MFS (major facilitator superfamily), invariably contributes to an increased efflux of commonly used azoles and thus corroborates their direct involvement in MDR in Candida albicans. The Mdr1 protein has two transmembrane domains, each comprising six transmembrane helices, interconnected with extracellular loops and ICLs (intracellular loops). The introduction of deletions and insertions through mutagenesis was used to address the role of the largest interdomain ICL3 of the MDR1 protein. Most of the progressive deletants, when overexpressed, eliminated the drug resistance. Notably, restoration of the length of the ICL3 by insertional mutagenesis did not restore the functionality of the protein. Interestingly, most of the insertion and deletion variants of ICL3 became amenable to trypsinization, yielding peptide fragments. The homology model of the Mdr1 protein showed that the molecular surface-charge distribution was perturbed in most of the ICL3 mutant variants. Taken together, these results provide the first evidence that the CCL (central cytoplasmic loop) of the fungal MFS transporter of the DHA1 (drug/proton antiporter) family is critical for the function of MDR. Unlike other homologous proteins, ICL3 has no apparent role in imparting substrate specificity or in the recruitment of the transporter protein.

Alanine scanning of all cysteines and construction of a functional cysteine-less Cdr1p, a multidrug ABC transporter of Candida albicans.

Herein, we discuss the role of the native cysteines present in a major multidrug ABC transporter of Candida albicans, Cdr1p, and describe the construction of this transporter's functional cysteine-less (cysless) protein version for cross-linking studies. In the experiments in which all 23 cysteines were replaced individually, we observed that most of the cysteine replacements were tolerated by the protein, but the replacement of C1056, C1091, C1106, C1294 or C1336 resulted in an enhanced drug susceptibility together with an abrogated drug efflux. Notably, the ATPase activity was uncoupled, which largely remained unaffected in these variants. The substitution of the critical cysteines with serines restored the normal expression and functionality of Cdr1p because serine can effectively mimic the hydrogen bonding properties of cysteine. Finally, we constructed a functional cysless His-tagged Cdr1p in which all the cysteines of the native protein were replaced with alanines and the critical cysteines were replaced with serines. Notably, cysless GFP-tagged variant of Cdr1p was non-functional. The cysless His-tagged variant of Cdr1p is the first example of a cysless ABC transporter in yeast, and it will lead to a greater understanding of the architecture of this important protein and provide insight into the nature of drug binding and interdomain communication.

Use of baculovirus BacMam vectors for expression of ABC drug transporters in mammalian cells.

ATP-binding cassette (ABC) drug transporters ABCB1 [P-glycoprotein (Pgp)] and ABCG2 are expressed in many tissues including those of the intestines, the liver, the kidney and the brain and are known to influence the pharmacokinetics and toxicity of therapeutic drugs. In vitro studies involving their functional characteristics provide important information that allows improvements in drug delivery or drug design. In this study, we report use of the BacMam (baculovirus-based expression in mammalian cells) expression system to express and characterize the function of Pgp and ABCG2 in mammalian cell lines. BacMam-Pgp and BacMam-ABCG2 baculovirus-transduced cell lines showed similar cell surface expression (as detected by monoclonal antibodies with an external epitope) and transport function of these transporters compared to drug-resistant cell lines that overexpress the two transporters. Transient expression of Pgp was maintained in HeLa cells for up to 72 h after transduction (48 h after removal of the BacMam virus). These BacMam-baculovirus-transduced mammalian cells expressing Pgp or ABCG2 were used for assessing the functional activity of these transporters. Crude membranes isolated from these cells were further used to study the activity of these transporters by biochemical techniques such as photo-cross-linking with transport substrate and adenosine triphosphatase assays. In addition, we show that the BacMam expression system can be exploited to coexpress both Pgp and ABCG2 in mammalian cells to determine their contribution to the transport of a common anticancer drug substrate. Collectively, these data demonstrate that the BacMam-baculovirus-based expression system can be used to simultaneously study the transport function and biochemical properties of ABC transporters.

Rational mutational analysis of a multidrug MFS transporter CaMdr1p of Candida albicans by employing a membrane environment based computational approach.

CaMdr1p is a multidrug MFS transporter of pathogenic Candida albicans. An over-expression of the gene encoding this protein is linked to clinically encountered azole resistance. In-depth knowledge of the structure and function of CaMdr1p is necessary for an effective design of modulators or inhibitors of this efflux transporter. Towards this goal, in this study, we have employed a membrane environment based computational approach to predict the functionally critical residues of CaMdr1p. For this, information theoretic scores which are variants of Relative Entropy (Modified Relative Entropy RE(M)) were calculated from Multiple Sequence Alignment (MSA) by separately considering distinct physico-chemical properties of transmembrane (TM) and inter-TM regions. The residues of CaMdr1p with high RE(M) which were predicted to be significantly important were subjected to site-directed mutational analysis. Interestingly, heterologous host Saccharomyces cerevisiae, over-expressing these mutant variants of CaMdr1p wherein these high RE(M) residues were replaced by either alanine or leucine, demonstrated increased susceptibility to tested drugs. The hypersensitivity to drugs was supported by abrogated substrate efflux mediated by mutant variant proteins and was not attributed to their poor expression or surface localization. Additionally, by employing a distance plot from a 3D deduced model of CaMdr1p, we could also predict the role of these functionally critical residues in maintaining apparent inter-helical interactions to provide the desired fold for the proper functioning of CaMdr1p. Residues predicted to be critical for function across the family were also found to be vital from other previously published studies, implying its wider application to other membrane protein families.

Multidrug resistance in yeast Candida.

The opportunistic human pathogens Candida albicans and other non-albicans species have acquired considerable significance in the recent past due to the enhanced susceptibility of immunocompromised patients. These pathogenic species of Candida derive their importance not only from the severity of their infections but also from their ability to develop resistance against antifungals. Widespread and prolonged use of azoles has led to the rapid development of the phenomenon of multidrug resistance (MDR), which poses a major hurdle in antifungal therapy. Various mechanisms that contribute to the development of MDR have been implicated in Candida as well as in other human fungal pathogens, and some of these include overexpression of or mutations in the target enzyme of azoles, lanosterol 14 alpha-demethylase, and transcriptional activation of genes encoding drug efflux pump proteins belonging to ATP-binding cassette (ABC) as well as to major facilitator superfamilies (MFS) of transporters. The ABC transporters, CDR1, CDR2, and an MFS pump CaMDR1, play a key role in azole resistance as deduced from their high level of expression found in several azole-resistant clinical isolates.

Drug-protein hydrogen bonds govern the inhibition of the ATP hydrolysis of the multidrug transporter P-glycoprotein.

P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter superfamily. This multidrug transporter utilizes energy from ATP hydrolysis for the efflux of a variety of hydrophobic and amphipathic compounds including anticancer drugs. Most of the substrates and modulators of P-gp stimulate its basal ATPase activity, although some inhibit it. The molecular mechanisms that are in play in either case are unknown. In this report, mutagenesis and molecular modeling studies of P-gp led to the identification of a pair of phenylalanine-tyrosine structural motifs in the transmembrane region that mediate the inhibition of ATP hydrolysis by certain drugs (zosuquidar, elacridar and tariquidar), with high affinity (IC50's ranging from 10 to 30nM). Upon mutation of any of these residues, drugs that inhibit the ATPase activity of P-gp switch to stimulation of the activity. Molecular modeling revealed that the phenylalanine residues F978 and F728 interact with tyrosine residues Y953 and Y310, respectively, in an edge-to-face conformation, which orients the tyrosines in such a way that they establish hydrogen-bond contacts with the inhibitor. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport function. Upon mutation, they bind at lower affinity sites, stimulating ATP hydrolysis and only poorly inhibiting transport. These results also reveal that screening chemical compounds for their ability to inhibit the basal ATP hydrolysis can be a reliable tool to identify modulators with high affinity for P-gp.

Multiple transport-active binding sites are available for a single substrate on human P-glycoprotein (ABCB1).

P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [(125)I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each substrate.

Employing information theoretic measures and mutagenesis to identify residues critical for drug-proton antiport function in Mdr1p of Candida albicans.

By employing information theoretic measures, this study presents a structure and functional analysis of a multidrug-proton antiporter Mdr1p of Candida albicans. Since CaMdr1p belongs to drug-proton antiporter (DHA1) family of Major Facilitator Superfamily (MFS) of transporters, we contrasted DHA1 (antiporters) with Sugar Porter family (symporters). Cumulative Relative Entropy (CRE) calculated for these two sets of alignments enabled us to selectively identify conserved residues of not only CaMdr1p but for the entire DHA1 family. Based on CRE, the highest scoring thirty positions were selected and predicted to impart functional specificity to CaMdr1p as well as to other drug-proton antiporters. Nineteen positions wherein the CaMdr1p residue matched with the most frequent amino acid at a particular alignment position of DHA1 members were subjected to site-directed mutagenesis and were replaced with either alanine or leucine. All these mutant variants, except one, displayed either complete or selective sensitivity to the tested drugs. The enhanced susceptibility of these mutant variants was corroborated with the simultaneously abrogated efflux of substrates. Taken together, based on scaled CRE between two MFS sub-families, we could accurately predict the functionally relevant residues of CaMdr1p. An extrapolation of these predictions to the entire DHA1 family members as validated from previously published data shows that these residues are functionally critical in other members of the DHA1 family also.

Phospholipidome of Candida: each species of Candida has distinctive phospholipid molecular species.

By employing electrospray ionization tandem mass spectrometry (ESI-MS/MS), the phospholipidomes of eight hemiascomycetous human pathogenic Candida species have been characterized. Over 200 phospholipid molecular species were identified and quantified. There were no large differences among Candida species in phosphoglyceride class composition; however, differences in phosphoglycerides components (i.e., fatty acyl chains) were identified. In contrast, differences in sphingolipid class composition as well as in molecular species were quite evident. The phospholipid compositions of C. albicans, C. glabrata, C. parapsilosis, C. kefyr, C. tropicalis, C. dubliniensis, C. krusei, and C. utilis could be further discriminated by principal component analysis. Notwithstanding that a single strain of each species was analyzed, our data do point to a typical molecular species imprint of Candida strains.