Pgp Modulators Research Articles

Novel pyxinol amide derivatives bearing an aliphatic heterocycle as P-glycoprotein modulators for overcoming multidrug resistance

P-glycoprotein (Pgp) modulators are promising agents for overcoming multidrug resistance (MDR) in cancer chemotherapy. In this study, via structural optimization of our lead compound S54 (nonsubstrate allosteric inhibitor of Pgp), 29 novel pyxinol amide derivatives bearing an aliphatic heterocycle were designed, synthesized, and screened for MDR reversal activity in KBV cells. Unlike S54, these active derivatives were shown to transport substrates of Pgp. The most potent derivative 4c exhibited promising MDR reversal activity (IC50 of paclitaxel = 8.80 ± 0.56 nM, reversal fold = 211.8), which was slightly better than that of third-generation Pgp modulator tariquidar (IC50 of paclitaxel = 9.02 ± 0.35 nM, reversal fold = 206.6). Moreover, the cytotoxicity of this derivative was 8-fold lower than that of tariquidar in human normal HK-2 cells. Furthermore, 4c blocked the efflux function of Pgp and displayed high selectivity for Pgp but had no effect on its expression and distribution. Molecular docking revealed that 4c bound preferentially to the drug-binding domain of Pgp. Overall, 4c is a promising lead compound for developing Pgp modulators.

Poloxamer 188-Coated Ammonium Methacrylate Copolymer Nanocarriers Enhance Loperamide Permeability across Pgp-Expressing Epithelia.

Loperamide is a μ-opioid agonist with poor gastrointestinal absorption, mainly because of its modest aqueous solubility and being a P-glycoprotein (Pgp) efflux substrate. Nevertheless, studies associated with therapeutic effects strongly suggest that loperamide holds potential pharmacological advantages over traditional μ-opioid agonists commonly used for analgesia. Thus, in this Communication, we assessed in MDCK-hMDR1 cell lines the effects over loperamide uptake and efflux ratio, when loaded into Eudragit RS (ERS) nanocarriers coated with poloxamer 188 (P188). ERS was chosen for enhancing loperamide aqueous dispersibility and P188 as a potential negative Pgp modulator. In uptake assays, it was observed that Pgp limited the accumulation of loperamide into cells and that preincubation with P188, but not coincubation, led to increasing loperamide uptake at a similar extent of Pgp pharmacological inhibition. On the other hand, the efflux ratio displayed no alterations when Pgp was pharmacologically inhibited, whereas ERS/P188 nanocarriers effectively enhanced loperamide uptake and absorptive transepithelial transport. The latter suggests that loperamide transport across cells is significantly influenced by the presence of the unstirred water layer (UWL), which could hinder the visualization of Pgp-efflux effects during transport assays. Thus, results in this work highlight that formulating loperamide into this nanocarrier enhances its uptake and transport permeability.

Imaging probes for non-invasive tumoral detection and functional monitoring of cancer multidrug resistance.

Aim: Several cationic radiotracers originally developed as myocardial perfusion agents have shown potential for both early detection of cancer and non-invasive monitoring of multiple drug resistance (MDR) by single photon emission computed tomography. We have introduced two cationic complexes, 99mTc-DMEOP [di-methoxy-tris-pyrazolyl-99mTc-(CO)3] and 99mTc-TMEOP [tri-methoxy-tris-pyrazolyl-99mTc-(CO)3], which showed excellent preclinical results as cardiac imaging probes, namely a persistent heart uptake with rapid blood and liver clearance. This study aimed at the evaluation of their usefulness for tumoral detection and functional assessment of MDR.Methods: The uptake and efflux kinetics of 99mTc-DMEOP and 99mTc-TMEOP were evaluated in human prostate, lung, and breast cancer cell lines, including drug-resistant cell lines that are known to overexpress the MDR P-glycoprotein (Pgp). The effects of MDR modulators were also studied. In vivo studies were performed in xenografted tumor models, and the MDR phenotype of the tumors was confirmed by Western blot.Results: The uptake kinetics of both complexes in human cancer cell lines is comparable with the one found for 99mTc-Sestamibi, increasing over time. The uptake of 99mTc-TMEOP is greatly reduced in cells overexpressing Pgp and increased in the presence of a Pgp modulator. In nude mice, the tumor uptake of 99mTc-TMEOP was higher in the MCF-7 xenografts compared with the MCF7 Pgp tumors.Conclusion: The uptake kinetics of both complexes in human cancer cell lines is comparable with the one found for 99mTc-Sestamibi, increasing over time. The uptake of 99mTc-TMEOP is greatly reduced in cells overexpressing Pgp, and increased in the presence of a Pgp modulator. In nude mice, the tumor uptake of 99mTc-TMEOP was higher in the MCF-7 xenografts compared with the MCF7 Pgp tumors.

Study of fabomotizole belonging to p-glycoprotein substrates

P-glycoprotein (Pgp) is a membrane efflux protein transporter with numerous drug-substrates. In addition, a lot of drugs alter the activity of the transporter. It can lead to drug-drug interactions during polypharmacy. Fabomotizole (afobazol) is a Russian anxiolytic drug with neuroprotective activity, applied over a wide range of indications. The drug belongs to a potential substrate of Pgp according to its chemical structure.
 Aim. The aim of the study was to assess belonging of fabomotizole to Pgp substrates.
 Materials and Methods. The work was performed on 12 male Chinchilla rabbits. The belonging of fabomotizole to Pgp substrates was evaluated by comparing pharmacokinetic parameters of the test-substance after course administration of known transporter inducers and inhibitors – rifampicin and verapamil respectively. Fabomotizole was administered orally as a single dose of 3.8 mg/kg b.w. and blood was taken from the ear vein after 5, 10, 15, 20, 30, 60, 90, 120 and 240 min followed by it's pharmacokinetic analysis by HPLC. Pharmacokinetic parameters of fabomotizole were manually calculated by a model-independent method. The animals were then divided into 2 groups of 6 rabbits each: the 1st group received verapamil at a dose 20 mg/kg b.w. 3 times a day for 14 days, the 2nd – rifampicin in a similar course and dose. After the administration of Pgp modulators the pharmacokinetics of fabomotizole were re-analyzed.
 Results. It was found that only the absorption coefficient of fabomotizole in the rifampicin series was significantly reduced by 1.27 times as compared to the parameter of intact animals (90% CI 0.66-0.94, p=0.04322). However, this change was not clinically significant, because 90% CI overlapped the range of 0.80-1.25, noted by FDA. The remaining pharmacokinetic parameters of Pgp marker substrate were not significantly changed in any series. This is evidence that fabomotizole is not a Pgp substrate. The insignificant participation of Pgp in fabomotizole pharmacokinetics testifies that the drug can be administered together with drug-modulators of transporter activity without dose correction.
 Conclusion. In vivo experiment on Chinchilla rabbits showed that fabomotizole is not a substrate of P-glycoprotein.

Deoxycholic acid derivatives as inhibitors of P-glycoprotein-mediated multidrug efflux

Deoxycholic acid derivatives were designed as P-glycoprotein (Pgp, ABCB1) inhibitors. Thus the synthesis and the biological activity of methyl deoxycholate derivatives 5–10 and their ether analogs 15–20 have been reported. The potency of these compounds to modulate Pgp-mediated MDR was evaluated through daunorubicin accumulation and potentiation of doxorubicin cytotoxicity in K562/R7 multidrug resistant cells overexpressing Pgp. In parallel, their intrinsic toxicity was appreciated on K562 sensitive cells. Methyl 12α-[(2R or 2S) tetrahydro-2H-pyran-2-yloxy]-3-oxo-5β-cholan-24-oate 9b has shown a good efficiency as a Pgp inhibitor and a low intrinsic toxicity. Therefore, this derivative constitutes a new lead compound which can be used as a starting point to improve the design of non-toxic Pgp modulators.

The Role of Efflux Pumps in Schistosoma mansoni Praziquantel Resistant Phenotype

BackgroundSchistosomiasis is a neglected disease caused by a trematode of the genus Schistosoma that is second only to malaria in public health significance in Africa, South America, and Asia. Praziquantel (PZQ) is the drug of choice to treat this disease due to its high cure rates and no significant side effects. However, in the last years increasingly cases of tolerance to PZQ have been reported, which has caused growing concerns regarding the emergency of resistance to this drug.Methodology/Principal FindingsHere we describe the selection of a parasitic strain that has a stable resistance phenotype to PZQ. It has been reported that drug resistance in helminths might involve efflux pumps such as members of ATP-binding cassette transport proteins, including P-glycoprotein and multidrug resistance-associated protein families. Here we evaluate the role of efflux pumps in Schistosoma mansoni resistance to PZQ, by comparing the efflux pumps activity in susceptible and resistant strains. The evaluation of the efflux activity was performed by an ethidium bromide accumulation assay in presence and absence of Verapamil. The role of efflux pumps in resistance to PZQ was further investigated comparing the response of susceptible and resistant parasites in the absence and presence of different doses of Verapamil, in an ex vivo assay, and these results were further reinforced through the comparison of the expression levels of SmMDR2 RNA by RT-PCR.Conclusions/SignificanceThis work strongly suggests the involvement of Pgp-like transporters SMDR2 in Praziquantel drug resistance in S. mansoni. Low doses of Verapamil successfully reverted drug resistance. Our results might give an indication that a combination therapy with PZQ and natural or synthetic Pgp modulators can be an effective strategy for the treatment of confirmed cases of resistance to PZQ in S. mansoni.

Diterpenic compounds with selective antiproliferative activity in multidrug resistant cancer cells

The failure of chemotherapeutics in cancer is mainly imputed to multidrug resistance (MDR). Among several proposed approaches to overcome MDR, the modulation of P-glycoprotein (Pgp) is the most highlighted [1]. Another valuable strategy is to take advantage of the collateral sensitivity effect, where some compounds may selectively exert a more pronounced cytotoxicity on cells with MDR phenotype than in the parental ones [1]. Macrocyclic diterpenes isolated from Euphorbia species, have been recognized as potent Pgp modulators and as having a collateral sensitivity effect on MDR cell variants [2]. Our main goal is to explore these two approaches, since tackling more than one MDR mechanism simultaneously might be a solution to control MDR in its complexity. The fractionation of the methanol extract of the aerial parts of E. piscatoria led to the isolation of a macrocyclic diterpene that allowed to build a small library of derivatives [3]. In this work, eleven compounds were evaluated for their potential collateral sensitivity effect on nine cell lines, gastric, pancreatic and colon cancer cell models (drug sensitive and drug resistant sublines), using the SRB assay. Most of the derivatives exhibited significant collateral sensitivity effect towards the MDR gastric cell line (with Pgp overexpression) and the MDR colon cells. The most significant results were obtained with a benzoyl derivative that demonstrated ability to target different cellular contexts with concomitant high antiproliferative activity. These compounds were previously assessed as Pgp modulators, at non-cytotoxic doses, on MDR1-mouse lymphoma cells [3]. A regression analysis between the antiproliferative activity presented herein and the previously assessed Pgp modulatory effect showed a strong relation between the compounds that presented both high Pgp modulation and cytotoxicity. Acknowledgements: FCT, Portugal (PTDC/QUI-QUI/099815/2008; PTDC/QEQ-MED/0905/2012; SFRH/BD/72915/2010).

Improving the MDR reversal activity of 6,17-epoxylathyrane diterpenes

Aiming to optimize macrocyclic lathyrane-type diterpenes as effective Pgp modulators, the phytochemical study of the methanolic extract of Euphorbia boetica aerial parts was carried out. Two new macrocyclic 6,17-epoxylathyrane-type diterpenes, named epoxyboetiranes A (1) and B (2), along with three known analogues (3–5) were isolated. Epoxyboetirane A (1), a triacetate isolated in large amounts, was hydrolyzed to give epoxylathyrol (6). In order to study the effect of the substitution pattern of the macrocyclic scaffold on MDR reversal, 6 was acylated with aroyl, phenylacetyl, cinnamoyl and alkanoyl chlorides/anhydrides, yielding eight new esters, epoxyboetiranes C–J (7–14). The ability of compounds 1–14 as P-glycoprotein (Pgp, ABCB1) modulators was evaluated through combination of transport and chemosensitivity assays, using L5178Y mouse T lymphoma cell line transfected with the human MDR1 gene. In the transport assay, excepting 1, 3 and 6, the compounds, at non-cytotoxic concentrations, displayed strong MDR reversing activity in a dose-dependent mode, exhibiting all the new acyl derivatives (7–14) a many fold increase in the activity when compared with 1. Apart from 11 and 12, all compounds exhibited remarkable synergistic effects in combination with doxorubicin. An ATPase assay, using membrane vesicles from mammalian cells overexpressing Pgp, was also performed with two representatives of the modulators (4 and 5). The results suggest that both compounds compete with substrates for the Pgp drug-binding sites.

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.

Synthesis and evaluation of Strychnos alkaloids as MDR reversal agents for cancer cell eradication

Natural products represent the fourth generation of multidrug resistance (MDR) reversal agents that resensitize MDR cancer cells overexpressing P-glycoprotein (Pgp) to cytotoxic agents. We have developed an effective synthetic route to prepare various Strychnos alkaloids and their derivatives. Molecular modeling of these alkaloids docked to a homology model of Pgp was employed to optimize ligand–protein interactions and design analogues with increased affinity to Pgp. Moreover, the compounds were evaluated for their (1) binding affinity to Pgp by fluorescence quenching, and (2) MDR reversal activity using a panel of in vitro and cell-based assays and compared to verapamil, a known inhibitor of Pgp activity. Compound 7 revealed the highest affinity to Pgp of all Strychnos congeners (Kd=4.4μM), the strongest inhibition of Pgp ATPase activity, and the strongest MDR reversal effect in two Pgp-expressing cell lines. Altogether, our findings suggest the clinical potential of these synthesized compounds as viable Pgp modulators justifies further investigation.

P-glycoprotein expression and pharmacological modulation in larval stages of Echinococcus granulosus

P-glycoprotein (Pgp) is an ATP-dependent transporter involved in the efflux of a wide variety of lipophilic substrates, such as toxins and xenobiotics, out of cells. Pgp expression level is associated with the ineffective therapeutic treatment of cancer cells and microbial pathogens which gives it high clinical importance. Research on these transporters in helminths is limited. This work describes for the first time the Echinococcus granulosus Pgp (Eg-Pgp) expression, in a model cestode parasite and an important human pathogen. Based on calcein efflux assays in the presence of common Pgp modulators, we demonstrated the occurrence of active Eg-Pgp in protoscoleces and metacestodes. Eg-Pgp, which showed a molecular mass of ~130kDa in western blots, is localized in the suckers and the tegument of control protoscoleces as well as in the subtegument or all parenchymatous cells of protoscoleces treated with Pgp-interfering agents. We also identified five genes encoding Pgp which are constitutively expressed in protoscoleces and metacestodes. We showed that the Eg-pgp1 and Eg-pgp2 transcripts were up-regulated in response to in vitro drug treatment with amiodarone and loperamide, in agreement with the increased polypeptide levels. Finally, in vitro treatment of protoscoleces and metacestodes with trifluoperazine and loperamide was lethal to the parasites. This indicates that both drugs as well as cyclosporine A negatively modulate the E. granulosus Pgp efflux activity, favoring the retention of these drugs in the larval tissue. These events could be associated with the reduction in protoscolex and metacestode viability.

P-glycoprotein-dependent resistance of cancer cells toward the extrinsic TRAIL apoptosis signaling pathway

The TNF-related apoptosis-inducing ligand (TRAIL or Apo2L) preferentially cause apoptosis of malignant cells in vitro and in vivo without severe toxicity. Therefore, TRAIL or agonist antibodies to the TRAIL DR4 and DR5 receptors are used in cancer therapy. However, many malignant cells are intrinsically resistant or acquire resistance to TRAIL. It has been previously proposed that the multidrug transporter P-glycoprotein (Pgp) might play a role in resistance of cells to intrinsic apoptotic pathways by interfering with components of ceramide metabolism or by modulating the electrochemical gradient across the plasma membrane. In this study we investigated whether Pgp also confers resistance toward extrinsic death ligands of the TNF family. To this end we focused our study on HeLa cells carrying a tetracycline-repressible plasmid system which shuts down Pgp expression in the presence of tetracycline. Our findings demonstrate that expression of Pgp is a significant factor conferring resistance to TRAIL administration, but not to other death ligands such as TNF-α and Fas ligand. Moreover, blocking Pgp transport activity sensitizes the malignant cells toward TRAIL. Therefore, Pgp transport function is required to confer resistance to TRAIL. Although the resistance to TRAIL-induced apoptosis is Pgp specific, TRAIL itself is not a direct substrate of Pgp. Pgp expression has no effect on the level of the TRAIL receptors DR4 and DR5. These findings might have clinical implications since the combination of TRAIL therapy with administration of Pgp modulators might sensitize TRAIL resistant tumors.

Effects of β-cyclodextrin on the intestinal absorption of berberine hydrochloride, a P-glycoprotein substrate

The major objective of this work is to investigate the enhancing effect of β-cyclodextrin on the intestinal absorption of berberine hydrochloride, a P-glycoprotein (Pgp) substrate. The inclusion complexation behavior of BBH with β-CD was investigated by phase-solubility diagram, Fourier transform infrared spectroscopy, differential scanning calorimetry, X-ray powder diffractometry, NMR spectroscopy, and molecular modeling studies. Results indicated that the 1,3-benzodioxole of BBH was included into the cavity of β-CD to form an inclusion complex which exhibited higher dissolution rate than BBH in vitro. The intestinal absorption of the inclusion complex in rats was significantly higher than the free drug due to its solubilizing effect and Pgp modulatory activity. The mechanisms of β-CD on Pgp modulation were demonstrated by modifying the Pgp ATPase activity, the Pgp mRNA level and the Pgp expression.

P-glycoprotein efflux pump plays an important role in Trypanosoma cruzi drug resistance.

Drug resistance in protozoan parasites has been associated with the P-glycoprotein (Pgp), an energy-dependent efflux pump that transports substances across the membrane. Interestingly, the genes TcPGP1 and TcPGP2 have been described in Trypanosoma cruzi, although the function of these genes has not been fully elucidated. The main goal of this work was to investigate Pgp efflux pump activity and expression in T. cruzi lines submitted to in vitro induced resistance to the compounds 4-N-(2-methoxy styryl)-thiosemicarbazone (2-Meotio) and benznidazole (Bz) and to verify the stability of the resistant phenotypes during the parasite life cycle. We observed that the EC50 values for the treatment of epimastigotes with 2-Meotio or Bz were increased at least 4.7-fold in resistant lines, and this phenotype was maintained in metacyclic trypomastigotes, cell-derived trypomastigotes, and intracellular amastigotes. However, in epimastigotes, 2-Meotio resistance is reversible, but Bz resistance is irreversible. When compared with the parental line, the resistant lines exhibited higher Pgp efflux activity, reversion of the resistant phenotypes in the presence of Pgp inhibitors, cross-resistance with Pgp modulators, higher basal Pgp ATPase activity, and overexpression of the genes TcPGP1 and TcPGP2. In conclusion, the resistance induced in T. cruzi by the compounds 2-Meotio and Bz is maintained during the entire parasite life cycle. Furthermore, our data suggest the participation of the Pgp efflux pump in T. cruzi drug resistance.

Keratinocyte growth factor-2 stimulates P-glycoprotein expression and function in intestinal epithelial cells

Intestinal P-glycoprotein (Pgp/multidrug resistance 1), encoded by the ATP-binding cassette B1 gene, is primarily involved in the transepithelial efflux of toxic metabolites and xenobiotics from the mucosa into the gut lumen. Reduced Pgp function and expression has been shown to be associated with intestinal inflammatory disorders. Keratinocyte growth factor-2 (KGF2) has emerged as a potential target for modulation of intestinal inflammation and maintenance of gut mucosal integrity. Whether KGF2 directly regulates Pgp in the human intestine is not known. Therefore, the present studies were undertaken to determine the modulation of Pgp by KGF2 using Caco-2 cells. Short-term treatment of Caco-2 cells with KGF2 (10 ng/ml, 1 h) increased Pgp activity (~2-fold, P < 0.05) as measured by verapamil-sensitive [(3)H]digoxin flux. This increase in Pgp function was associated with an increase in surface Pgp levels. The specific fibroblast growth factor receptor (FGFR) antagonist PD-161570 blocked the KGF2-mediated increase in Pgp activity. Inhibition of the mitogen-activated protein kinase (MAPK) pathway by PD-98059 attenuated the stimulatory effects of KGF2 on Pgp activity. Small-interfering RNA knockdown of Erk1/2 MAPK blocked the increase in surface Pgp levels by KGF2. Long-term treatment with KGF2 (10 ng/ml, 24 h) also significantly increased PgP activity, mRNA, protein expression, and promoter activity. The long-term effects of KGF2 on Pgp promoter activity were also blocked by the FGFR antagonist and mediated by the Erk1/2 MAPK pathway. In conclusion, our findings define the posttranslational and transcriptional mechanisms underlying stimulation of Pgp function and expression by KGF2 that may contribute to the beneficial effects of KGF2 in intestinal inflammatory disorders.

Synthesis of 5,7-disubstituted-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-amines as microtubule inhibitors

Compounds 1–4 were previously reported as potent antimitotic and antitumor agents with Pgp modulatory effects. Compounds 5–18 have been synthesized in an attempt to optimize the various activities of 1–4. Compounds 5–10 explored the influence of methoxy substitutions on the 7-benzyl moiety in 1, while 11–18 investigated the influence of incorporation of a sulfur linker at C5 compared to 1–3. Compounds 5–10 demonstrated potent single-digit micromolar tumor cell cytotoxicity, Pgp modulation and microtubule inhibition. Compound 7 of this series was the most potent and showed GI50 values in the nanomolar range against several human tumor cell lines in the standard NCI preclinical in vitro screen. Antitumor activity and Pgp modulatory effects were found to decrease for the 5-phenylthio compounds 11–14 compared to their 5-phenylethyl analogs 2–4 and the standard compound Taxol. Incorporation of methoxy substitutions on the 7-benzyl moiety improved antitumor activity for the 5-phenylthio compounds 16 and 17. Compounds 16 and 17 demonstrated single to two-digit micromolar inhibition of tumor cells.

Evidence for Modulatory Sites at the Lipid–Protein Interface of the Human Multidrug Transporter P-Glycoprotein

The human multidrug transporter P-glycoprotein (Pgp or ABCB1) sets up pharmacological barriers to many clinically important drugs, a therapeutic remedy for which has yet to be formulated. For the rational design of mechanism-based inhibitors (or modulators), it is necessary to map the potential sites for modulator interaction and understand their modes of communication with the other functional domains of Pgp. In this study, combining directed mutagenesis with homology modeling, we provide evidence of two modulator-specific sites at the lipid protein interface of Pgp. Targeting 21 variant positions in the COOH-terminal transmembrane (TM) regions, we find residues M948 (in TM11) and F983, M986, V988, and Q990 (all four in TM12) critically involved in substrate-site modulation by a thioxanthene-based allosteric modulator cis-(Z)-flupentixol. Interestingly, for ATP-site modulation by the same modulator, only two (M948 and Q990) of those four residues appear indispensable, together with two additional residues, T837 and I864 in TM9 and TM10, respectively, suggesting independent modes of communication linking the allosteric site with the substrate binding and ATPase domains. None of the seven residues identified prove to be critical for modulation of the substrate or ATP sites by Pgp modulators that are transported by the pump, such as cyclosporin A or verapamil, indicating their specificity for cis-(Z)-flupentixol. On the other hand, ATP-site modulation by verapamil proves to be highly sensitive to replacement at positions F716 (in TM7) and I765 (in TM8), and to a more moderate extent at I764 and L772 (both in TM8). Homology modeling based on the known crystal structures of the bacterial multidrug transporter SAV1866 and the mouse Pgp homologue maps the identified residues primarily at the lipid-protein interface of Pgp, in two spatially distinct modulator-specific clusters. The two modulatory sites demonstrate negative synergism in influencing ATP hydrolysis, consolidating their spatial distinctness. Because Pgp is known to recruit drug molecules directly from the lipid bilayer, identification of modulatory sites at the lipid-protein interface and at the same time outside the conventional central drug binding cavity is mechanistically revealing.

Three Decades of P-gp Inhibitors: Skimming Through Several Generations and Scaffolds

Many tumor cells become resistant to commonly used cytotoxic drugs due to the overexpression of ATP-binding cassette (ABC) transporters, namely P-glycoprotein (P-gp). The discovery of the reversal of multidrug resistance (MDR) by verapamil occured in 1981, and in 1968 MDR Chinese hamster cell lines were isolated for the first time. Since then, P-gp inhibitors have been intensively studied as potential MDR reversers. Initially, drugs to reverse MDR were not specifically developed for inhibiting P-gp; in fact, they had other pharmacological properties, as well as a relatively low affinity for MDR transporters. An example of this first generation P-gp inhibitors is verapamil. The second generation included more specific with less side-effect inhibitors, such as dexverapamil or dexniguldipine. A third generation of P-gp inhibitors comprised compounds such as tariquidar, with high affinity to P-gp at nanomolar concentrations. These generations of inhibitors of P-gp have been examined in preclinical and clinical studies; however, these trials have largely failed to demonstrate an improvement in therapeutic efficacy. Therefore, new and innovative strategies, such as the fallback to natural products, the design of peptidomimetics and dual activity ligands emerged as a fourth generation of P-gp inhibitors. The chemistry of P-gp inhibitors, as well as their in vitro, in vivo and clinical trials are discussed, and the most recent advances concerning Pgp modulators are reviewed.

3′-O, 4′-O-aromatic acyl substituted 7,8-pyranocoumarins: a new class of P-glycoprotein modulators

P-glycoprotein (Pgp) overexpression in tumour cells leads to multidrug resistance (MDR) and causes failure in cancer chemotherapy. We have previously identified (±)-praeruptorin A (PA) as a potential lead compound for Pgp modulators. In this study we investigated the MDR-reversing activities of PA derivatives. Series 7,8-pyranocoumarins with various C-3' and C-4' side chains had been semi-synthesized and their MDR-reversing activity was investigated in Pgp-overexpressing MDR tumour cell line HepG2/Dox and in a KB V1 xenograft animal model. All 7,8-pyranocoumarins exhibited equal or higher activity in modulating Pgp. DCK (12), DMDCK (15), 16, 21, 23 and 24 at 4 µm achieved 91%∼99% decrease in IC50 value (concentration inhibiting cell growth by 50%) of anticancer agents vinblastine, doxorubicin, puromycin and paclitaxel, and were more active than others. DMDCK also remarkably enhanced the growth inhibitory effect of paclitaxel on KB V1 xenografts (P < 0.05), showing a potency required for clinical usage. Mechanistic studies suggested that these 7,8-pyranocoumarins might reverse Pgp-MDR through directly binding to substrate binding site(s) or allosteric site(s) on Pgp therefore impairing Pgp-mediated drug transport. Results from the study suggested that 3'-O, 4'-O-aromatic acyl substituted 7,8-pyranocoumarins could serve as a new class of Pgp modulator. Acyls play an important role in maintaining and enhancing the Pgp-modulating ability of pyranocoumarins. 3,4-Dimethoxyl substituted aromatic acyls, bearing a methoxy that might interact with Pgp as hydrogen bond accepter, were shown to be the most potent for reversing MDR.

Modulation of Mrp1 (ABCc1) and Pgp (ABCb1) by Bilirubin at the Blood-CSF and Blood-Brain Barriers in the Gunn Rat

Accumulation of unconjugated bilirubin (UCB) in the brain causes bilirubin encephalopathy. Pgp (ABCb1) and Mrp1 (ABCc1), highly expressed in the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) respectively, may modulate the accumulation of UCB in brain. We examined the effect of prolonged exposure to elevated concentrations of UCB on expression of the two transporters in homozygous, jaundiced (jj) Gunn rats compared to heterozygous, not jaundiced (Jj) littermates at different developmental stages (2, 9, 17 and 60 days after birth). BBB Pgp protein expression was low in both jj and Jj pups at 9 days (about 16–27% of adult values), despite the up-regulation in jj animals (2 and 1.3 fold higher than age matched Jj animals at P9 and P17–P60, respectively); Mrp1 protein expression was barely detectable. Conversely, at the BCSFB Mrp1 protein expression was rather high (60–70% of the adult values) in both jj and Jj at P2, but was markedly (50%) down-regulated in jj pups starting at P9, particularly in the 4th ventricle choroid plexuses: Pgp was almost undetectable. The Mrp1 protein down regulation was accompanied by a modest up-regulation of mRNA, suggesting a translational rather than a transcriptional inhibition. In vitro exposure of choroid plexus epithelial cells obtained from normal rats to UCB, also resulted in a down-regulation of Mrp1 protein. These data suggest that down-regulation of Mrp1 protein at the BSCFB, resulting from a direct effect of UCB on epithelial cells, may impact the Mrp1-mediated neuroprotective functions of the blood-cerebrospinal fluid barrier and actually potentiate UCB neurotoxicity.