Virtual high-throughput screening and simulation studies of compounds from selected traditionally important medicinal plants for the identification of potential inhibitors of AcrB.

One of the significant human health concerns today is the emergence of drug-resistant bacteria and their propagation worldwide, rendering all available treatment options useless. One of the molecular mechanisms behind the evolution and dissemination of multidrug-resistant species is the overexpression of efflux pumps. AcrB is a major component of the AcrAB-TolC efflux pump belonging to the RND division and responsible for the extrusion of antibiotics from the bacterial cell leading to resistance. In this study, we applied the reverse chemogenomics technique to find inhibitors of AcrB to combat the issue of drug resistance. A total of 102 compounds from five different plants having medicinal properties were passed through Lipinski filter and ADMET screening was done to check their drug likeliness before docking against the AcrB protein and based on the -cDocker energy scores and toxicity analysis report, the compounds with best values were analyzed. A comparison was made between them and known inhibitors as well as antibiotics. Heat maps, frequency histograms, 2 D diagrams were generated, and the molecules were simulated for 60 ns using GROMACS. From the study, we have found Dihydrocapsaicin and Garcinexanthone-A to be a potential efflux pump inhibitors having all the characters of a promising drug candidate.Communicated by Ramaswamy H. Sarma.

Repurposing Approved Drugs as Fluoroquinolone Potentiators to Overcome Efflux Pump Resistance in Staphylococcus aureus.

Staphylococcus aureus is a versatile human commensal bacteria and pathogen that causes various community and hospital-acquired infections. The S. aureus efflux pump NorA which belongs to the major facilitator superfamily, confers resistance to a range of substrates. Many efflux pump inhibitors (EPIs) have been discovered, but none is clinically approved due to their undesirable toxicities. In this study, we have screened clinically approved drugs for possible NorA EPI-like activity. We identified six drugs that showed the best efflux pump inhibition in vitro, with a fractional inhibitory concentration index of ≤0.5, indicating synergism with hydrophilic fluoroquinolones. The mechanistic validation of efflux inhibitory potential was demonstrated in ethidium bromide-based accumulation and efflux inhibition assays. We further confirmed the functionality of EPIs by norfloxacin accumulation assay depicting more realistic proof of the conjecture. None of the EPIs disturbed membrane function or depleted the ATP synthesis levels in bacteria. Both raloxifene and pyrvinium displayed an increase in bactericidal activity of ciprofloxacin in time-kill kinetics, prolonged its post-antibiotic effect, and reduced the frequency of spontaneous resistant mutant development. The combination of EPIs with ciprofloxacin caused significant eradication of preformed biofilms. Moreover, in the murine thigh infection model, a single dose of pyrvinium combined with ciprofloxacin reduced the bacterial burden significantly compared to untreated control and ciprofloxacin alone, indicating the efficacy of the combination. Conclusively, this study represents approved drugs that can be repurposed and combined with antibiotics as NorA EPIs, having anti-biofilm properties to treat severe S. aureus infections at clinically relevant concentrations. IMPORTANCE Staphylococcus aureus is a frequent pathogen bacterium and the predominant cause of worsened nosocomial infections. Efflux pumps contribute to drug efflux and are reportedly associated with biofilm formation, thereby promoting difficult-to-treat biofilm-associated S. aureus infections. One strategy to combat these bacteria is to reduce active efflux and increase pathogen sensitivity to existing antibiotics. Repurposing approved drugs may solve the classical toxicity issues with previous efflux pump inhibitors and help reach sufficient plasma concentrations. We describe the in silico-based screening of FDA-approved drugs that identified six different molecules able to inhibit NorA pump (Major Facilitator Superfamily). Our study highlights that these compounds bind to and block the activity of the NorA pump and increase the sensitivity of S. aureus and methicillin-resistant S. aureus to fluoroquinolones. These drugs combined with fluoroquinolones significantly reduced the preformed biofilms and displayed significant efficacy in the murine thigh infection model when compared to untreated control and ciprofloxacin alone.

Overcoming vincristine resistance in cancer: Computational design and discovery of piperine-inspired P-glycoprotein inhibitors.

P-glycoprotein (P-gp)/MDR-1 plays a major role in the development of multidrug resistance (MDR) by pumping the chemotherapeutic drugs out of the cancer cells and reducing their efficacy. A number of P-gp inhibitors were reported to reverse the MDR when co-administered with chemotherapeutic drugs. Unfortunately, none has approved for clinical use due to toxicity issues. Some of the P-gp inhibitors tested in the clinics are reported to have cross-reactivity with CYP450 drug-metabolizing enzymes, resulting in unpredictable pharmacokinetics and toxicity of co-administered chemotherapeutic drugs. In this study, two piperine analogs (3 and 4) having lower cross-reactivity with CYP3A4 drug-metabolizing enzyme are identified as P-glycoprotein (P-gp) inhibitors through computational design, followed by synthesis and testing in MDR cancer cell lines over-expressing P-gp (KB ChR 8-5, SW480-VCR, and HCT-15). Both the analogs significantly increased the vincristine efficacy in MDR cancer cell lines at low micromole concentrations. Specifically, 3 caused complete reversal of vincristine resistance in KB ChR 8-5 cells and found to act as competitive inhibitor of P-gp as well as potentiated the vincristine-induced NF-KB-mediated apoptosis. Therefore, 3 ((2E,4E)-1-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-5-(4-hydroxy-3-methoxyphenyl)penta-2,4-dien-1-one) can serve as a potential P-gp inhibitor for in vivo investigations, to reverse multidrug resistance in cancer.

Novel DiOC3 96-well real-time efflux assay for discovery of NorA efflux pump inhibitors in Staphylococcus aureus.

The NorA efflux pump is one of the most studied efflux systems in Staphylococcus aureus and confers multidrug resistance to a variety of dyes and antimicrobial compounds. Hence, inhibition of the NorA efflux pump might be a viable option for restoring susceptibility to antibiotics like fluoroquinolones. Fluorescent real-time efflux assays are important tools to identify putative efflux pump inhibitors. Nevertheless, the number of available compounds for usage in Staphylococcus aureus is limited. Previously, a 3-dipropyloxacarbocyanine iodide (DiOC3) efflux assay was published that circumvented problems associated with the usage of ethidium bromide, namely slow efflux and suggested mutagenicity. However, the DiOC3 assay protocol was cuvette - based and therefore needs to be adapted to the 96-well plate format. Hence, we optimized this assay for usage with 96-well plates. The new assay allows for rapid high-throughput efflux pump inhibitor screening.

Pharmacokinetic and Pharmacodynamic Factors Contribute to Synergism between Let-7c-5p and 5-Fluorouracil in Inhibiting Hepatocellular Carcinoma Cell Viability.

Pharmacological interventions for hepatocellular carcinoma (HCC) are hindered by complex factors, and rational combination therapy may be developed to improve therapeutic outcomes. Very recently, we have identified a bioengineered microRNA let-7c-5p (or let-7c) agent as an effective inhibitor against HCC in vitro and in vivo. In this study, we sought to identify small-molecule drugs that may synergistically act with let-7c against HCC. Interestingly, we found that let-7c exhibited a strong synergism with 5-fluorouracil (5-FU) in the inhibition of HCC cell viability as manifested by average combination indices of 0.3 and 0.5 in Hep3B and Huh7 cells, respectively. By contrast, coadministration of let-7c with doxorubicin or sorafenib inhibited HCC cell viability with, rather surprisingly, no or minimal synergy. Further studies showed that protein levels of multidrug resistance-associated protein (MRP) ATP-binding cassette subfamily C member 5 (MRP5/ABCC5), a 5-FU efflux transporter, were reduced around 50% by let-7c in HCC cells. This led to a greater degree of intracellular accumulation of 5-FU in Huh7 cells as well as the second messenger cyclic adenosine monophosphate, an endogenous substrate of MRP5. Since 5-FU is an irreversible inhibitor of thymidylate synthetase (TS), we investigated the interactions of let-7c with 5-FU at pharmacodynamic level. Interestingly, our data revealed that let-7c significantly reduced TS protein levels in Huh7 cells, which was associated with the suppression of upstream transcriptional factors as well as other regulatory factors. Collectively, these results indicate that let-7c interacts with 5-FU at both pharmacokinetic and pharmacodynamic levels, and these findings shall offer insight into molecular mechanisms of synergistic drug combinations. SIGNIFICANCE STATEMENT: Combination therapy is a common strategy that generally involves pharmacodynamic interactions. After identifying a strong synergism between let-7c-5p and 5-fluorouracil (5-FU) against hepatocellular carcinoma cell viability, we reveal the involvement of both pharmacokinetic and pharmacodynamic mechanisms. In particular, let-7c enhances 5-FU exposure (via suppressing ABCC5/MRP5 expression) and cotargets thymidylate synthase with 5-FU (let-7c reduces protein expression, whereas 5-FU irreversibly inactivates enzyme). These findings provide insight into developing rational combination therapies based on pharmacological mechanisms.

A Two-Tiered In Vitro Approach to De-Risk Drug Candidates for Potential Bile Salt Export Pump Inhibition Liabilities in Drug Discovery.

Hepatocellular accumulation of bile salts by inhibition of bile salt export pump (BSEP/ABCB11) may result in cholestasis and is one proposed mechanism of drug-induced liver injury (DILI). To understand the relationship between BSEP inhibition and DILI, we evaluated 64 DILI-positive and 57 DILI-negative compounds in BSEP, multidrug resistance protein (MRP) 2, MRP3, and MRP4 vesicular inhibition assays. An empirical cutoff (5 µM) for BSEP inhibition was established based on a relationship between BSEP IC50 values and the calculated maximal unbound concentration at the inlet of the human liver (fu*Iin,max, assay specificity = 98%). Including inhibition of MRP2-4 did not increase DILI predictivity. To further understand the potential to inhibit bile salt transport, a selected subset of 30 compounds were tested for inhibition of taurocholate (TCA) transport in a long-term human hepatocyte micropatterned co-culture (MPCC) system. The resulting IC50 for TCA in vitro biliary clearance and biliary excretion index (BEI) in MPCCs were compared with the compound's fu*Iin,max to assess potential risk for bile salt transport perturbation. The data show high specificity (89%). Nine out of 15 compounds showed an IC50 value in the BSEP vesicular assay of <5µM, but the BEI IC50 was more than 10-fold the fu*Iin,max, suggesting that inhibition of BSEP in vivo is unlikely. The data indicate that although BSEP inhibition measured in membrane vesicles correlates with DILI risk, that measurement of this assay activity is insufficient. A two-tiered strategy incorporating MPCCs is presented to reduce BSEP inhibition potential and improve DILI risk. SIGNIFICANCE STATEMENT: This work describes a two-tiered in vitro approach to de-risk compounds for potential bile salt export pump inhibition liabilities in drug discovery utilizing membrane vesicles and a long-term human hepatocyte micropatterned co-culture system. Cutoffs to maximize specificity were established based on in vitro data from a set of 121 DILI-positive and -negative compounds and associated calculated maximal unbound concentration at the inlet of the human liver based on the highest clinical dose.

Effects of Chrysin and Its Major Conjugated Metabolites Chrysin-7-Sulfate and Chrysin-7-Glucuronide on Cytochrome P450 Enzymes and on OATP, P-gp, BCRP, and MRP2 Transporters.

Chrysin is an abundant flavonoid in nature, and it is also contained by several dietary supplements. Chrysin is highly biotransformed in the body, during which conjugated metabolites chrysin-7-sulfate and chrysin-7-glucuronide are formed. These conjugates appear at considerably higher concentrations in the circulation than the parent compound. Based on previous studies, chrysin can interact with biotransformation enzymes and transporters; however, the interactions of its metabolites have been barely examined. In this in vitro study, the effects of chrysin, chrysin-7-sulfate, and chrysin-7-glucuronide on cytochrome P450 enzymes (2C9, 2C19, 3A4, and 2D6) as well as on organic anion-transporting polypeptides (OATPs; 1A2, 1B1, 1B3, and 2B1) and ATP binding cassette [P-glycoprotein, multidrug resistance-associated protein 2, and breast cancer resistance protein (BCRP)] transporters were investigated. Our observations revealed that chrysin conjugates are strong inhibitors of certain biotransformation enzymes (e.g., CYP2C9) and transporters (e.g., OATP1B1, OATP1B3, OATP2B1, and BCRP) examined. Therefore, the simultaneous administration of chrysin-containing dietary supplements with medications needs to be carefully considered due to the possible development of pharmacokinetic interactions. SIGNIFICANCE STATEMENT: Chrysin-7-sulfate and chrysin-7-glucuronide are the major metabolites of flavonoid chrysin. In this study, we examined the effects of chrysin and its conjugates on cytochrome P450 enzymes and on organic anion-transporting polypeptides and ATP binding cassette transporters (P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2). Our results demonstrate that chrysin and/or its conjugates can significantly inhibit some of these proteins. Since chrysin is also contained by dietary supplements, high intake of chrysin may interrupt the transport and/or the biotransformation of drugs.

Effect of hydroxyamines derived from lapachol and norlachol against Staphylococcus aureus strains carrying the NorA efflux pump.

Isolated substances and those organically synthesized have stood out over the years for their therapeutic properties, including their antibacterial activity. These compounds may be an alternative to the production of new antibiotics or may have the ability to potentiate the action of preexisting ones. In this context, the objective of this study was to evaluate the in vitro antibacterial and efflux pump inhibitory activity of hydroxyamines derived from lapachol and norlachol, more specifically the compounds 2-(2-Hydroxyethylamino)-3-(3-methyl-2-butenyl)-1,4 dihydro-1,4-naphthalenedione, 2-(2-Hydroxyethylamino)-3-(2-methyl-propenyl)[1,4]naphthoquinone and 2-(3-Hydroxypropylamino)-3-(3-methyl-2-butenyl)-[1,4]naphthoquinone, against Staphylococcus aureus strains carrying the NorA efflux pump mechanism. The substances were synthesized from 2-hydroxy-quinones, lapachol and nor-lapachol, obtaining the corresponding 2-methoxylated derivatives via dimethyl sulfate alkylation in a basic medium, which then reacted chemoselectively with 2-ethanolamine and 3-propanolamine to form the corresponding amino alcohols. All three molecules underwent a virtual structure-based analysis (docking). The antibacterial activity of the substances was measured by determining their Minimum Inhibitory Concentration (MIC) and a microdilution assay was performed to verify efflux pump inhibition using the substances at a sub-inhibitory concentration. The results were subjected to statistical analysis using an analysis of variance (ANOVA) followed by Bonferroni's post hoc test. The substances obtained MIC values ≥1024 µg/mL, however, a significant reduction of their MICs was observed when the substances were associated with norfloxacin and ethidium bromide, with this effect being attributed to efflux pump inhibition. Following a virtual analysis based on its structure (docking), information regarding the affinity of new ligands for the ABC efflux pump were observed, thus contributing to the understanding of their mechanism of molecular interactions and the discovery of functional ligands associated with a reduction in bacterial resistance.

Aloe-emodin induces hepatotoxicity by the inhibition of multidrug resistance protein 2.

BACKGROUND: Aloe-emodin (AE) is among the primary bioactive anthraquinones present in traditional Chinese medicinal plants such as Rheum palmatum L. Multidrug resistance protein 2 (ABCC2/ MRP2) is an important efflux transporter of substances associated with cellular oxidative stress. However, the effects of traditional Chinese medicine on this protein remain unclear. PURPOSE: The aim of this research is to study the role of ABCC2 in AE-induced hepatotoxicity. METHODS: The expression of ABCC2 protein and mRNA levels were analyzed by Western-Blotting and qRT-PCR, respectively. The intracellular oxidative stress caused by AE was evaluated by quantifying the levels of intracellular reactive oxygen species, malondialdehyde, glutathione reduced and oxidized glutathione. The levels of adenosine triphosphate, mitochondrial membrane potential and mitochondrial DNA were explored to evaluate the effects of AE on mitochondrial function. The effects of AE on cell apoptosis and cell cycle were detected by flow cytometry. To further clarify the key role of ABCC2 in AE induced cytotoxicity, we used pCI-neo-ABCC2 plasmid to over express ABCC2 protein, and small interfering RNA was used to knockdown ABCC2 in HepG2 cells. Additionally, we investigated the impact of AE on ABCC2 degradation pathway and the hepatotoxic effects of AE in mice. RESULTS: AE was found to inhibit ABCC2 transport activity, downregulate ABCC2 expression and altered intracellular redox balance. Induction of oxidative stress resulted in depletion of intracellular glutathione reduced, mitochondria dysfunction and activation of apoptosis. ABCC2 overexpression significantly reduced AE-induced intracellular oxidative stress and cell death, which was enhanced by ABCC2 knockdown. Furthermore, AE was observed to promote ABCC2 degradation through induction of autophagy and hepatotoxicity was induced in mice by promoting ABCC2 degradation. CONCLUSIONS: The inhibition of ABCC2 is a novel effect of AE that triggers oxidative stress and apoptosis. These findings are helpful in understanding the toxicological effects of AE-containing medicinal plants.

Modulation of drug resistance in Staphylococcus aureus by 132-hydroxy-(132-R/S)-pheophytin isolated from Sargassum polyceratium.

Efflux pumps are integral parts of the bacterial plasma membrane that are responsible for many cases of antibiotic resistance. Modulators of drug resistance are regarded as the most suitable new antibacterial therapies. We evaluated the extracts of Sargassum polyceratium and the isolated compound pheophytin (Sp-1) for antibiotic modifying activity in strains of Staphylococcus aureus with efflux pump. The minimum inhibitory concentrations (MICs) for norfloxacin, tetracycline and erythromycin were determined by the microdilution broth method, in the absence and presence of the extract at a sub-inhibitory concentration (MIC/4). The extracts and isolated compounds showed no significant antimicrobial activity, but they changed the antibiotic activity, decreasing bacterial resistance by 2 to 4x. Using a checkerboard method, it was also possible to observe the synergistic effect (ΣFIC ≤ 0.5) between Sp-1 and the antibiotics erythromycin and norfloxacin. The results indicate that the seaweed Sargassum polyceratium and pheophytin are potential sources of an antibiotic adjuvant that modulates bacterial resistance, acting as a putative efflux pump inhibitor.

Vielanin P enhances the cytotoxicity of doxorubicin via the inhibition of PI3K/Nrf2-stimulated MRP1 expression in MCF-7 and K562 DOX-resistant cell lines.

BACKGROUND: Cancer cells that are resistant to structurally and mechanically unrelated anticancer drugs are said to have multidrug resistance (MDR). The overexpression of the ATP-binding cassette (ABC) transporter is one of the most important mechanisms of MDR. Vielanin P (VP), a dimeric guaiane from the leaves of Xylopia vielana, has the potential to reverse multidrug resistance. PURPOSE: To evaluate the meroterpenoid compound VP as a low cytotoxicity MDR regulator and the related mechanisms. METHODS: Cell viability was determined by CCK-8 and MTT assays. Apoptosis and the accumulation of doxorubicin (DOX) and 5(6)-carboxyfluorescein diacetate (CFDA) were determined by flow cytometry. We determined mRNA levels by quantitative real-time polymerase chain reaction (qRT-PCR). Protein levels were analyzed by Western blotting and immunofluorescence. RESULTS: In the MCF-7 and K562 DOX-resistant cell lines, VP treatment (10 µM or 20 µM) enhanced the activity of chemotherapeutic agents. We found that VP selectively inhibited MRP1 mRNA but not MDR1 mRNA. VP enhanced DOX-induced apoptosis and reduced colony formation in the presence of DOX in drug-resistant cells. Moreover, VP increased the accumulation of DOX and the MRP1-specific substrate CFDA. In addition, VP reversed MRP1 protein levels and the accumulation of DOX and CFDA in MRP1-overexpressing MCF-7 and K562 cells. Thus, the mechanism of MDR reversal by VP is MRP1-dependent. Furthermore, we found that the inhibitory effect of VP on MRP1 is PI3K/Nrf2-dependent. CONCLUSION: These results support the potential therapeutic value of VP as an MDR-reversal agent by inhibiting MRP1 via PI3K/Nrf2 signaling.

Antimicrobial activity of Phyllanthus amarus Schumach. & Thonn and inhibition of the NorA efflux pump of Staphylococcus aureus by Phyllanthin.

The aim of this study was to evaluate the antimicrobial activity of ethanoic extract of P. amarus (PAEE) and its compound Phyllanthin, as well as, investigate if these natural products could modulate the fluoroquinolone-resistance in S. aureus SA1199-B by way of overexpression of the NorA efflux pump. Microdilution tests were carried out to determine the minimal inhibitory concentration (MIC) of the PAEE or Phyllanthin against several bacterial and yeast strains. To evaluate if PAEE or Phyllanthin were able to act as modulators of the fluoroquinolone-resistance, MICs for Norfloxacin and ethidium bromide were determined in the presence or absence of PAEE or Phyllanthin against S. aureus SA1199-B. PAEE showed antimicrobial activity against Gram-negative strains, meanwhile Phyllanthin was inactive against all strains tested. Addition of PAEE or Phyllanthin, to the growth media at sub-inhibitory concentrations enhanced the activity of the Norfloxacin as well as, Ethidium Bromide, against S. aureus SA1199-B. These results indicate that Phyllanthin is able to modulate the fluoroquinolone-resistance possibly by inhibition of NorA. This hypothesis was supported by in silico docking analysis which confirmed that Phyllantin is a NorA ligand. Thus, this compound could be used as a potentiating agent of the Norfloxacin activity in the treatment of infections caused by fluoroquinolone-resistant S. aureus.

Piperine enhances the bioavailability of silybin via inhibition of efflux transporters BCRP and MRP2.

BACKGROUND: Although silybin serves as a well-known hepatoprotective agent with prominent anti-inflammatory, anti-oxidant and anti-fibrotic activities, its low bioavailability limits its application in the treatment of chronic liver diseases. However, novel formulation products with increased solubility were not sufficient to achieve pharmacologically meaningful concentrations of silybin in the clinical studies even used at high dosage. HYPOTHESIS/PURPOSE: We hypothesized that inhibiting efflux transporter(s) and/or glucuronidation by piperine might enhance the bioavailability and efficacy of silybin. METHODS: Pharmacokinetics of silybin given alone or in-combination with piperine was determined by a validated LC-MS method. A CCl4 induced rat model of liver injury was prepared and verified for comparing the effects of silybin and combination treatment. To investigate the underlying mechanism, the inhibition effects of piperine on transportation of silybin were performed in Caco-2 and transfected MDCKII cell lines as well as sandwich-cultured rat hepatocytes (SCH). Human liver microsomes incubation was used for exploring the modulation effects of piperine on the phase-2 metabolism of silybin. RESULTS: In the present study, we demonstrated for the first time that piperine as a bioenhancer increased the bioavailability of silybin (146%- 181%), contributing to a boosted therapeutic effect in CCl4-induced acute liver-injury rat model. The underlying mechanisms involved that piperine enhanced the absorption of silybin by inhibiting the efflux transporters including MRP2 and BCRP but not MDR1 in Caco-2 and transfected MDCKII cell lines. Moreover, piperine could inhibit the biliary excretion of silybin and conjugated metabolites in sandwich-cultured rat hepatocytes. Notably, we found that piperine did not affect the phase-2 metabolism of silybin. CONCLUSION: Efflux transporters play an important role in the pharmacokinetic behavior of flavolignans, and modulating these transporters by bioenhancer such as piperine could enhance the in vivo absorption of silybin, leading to more effective treatments.

Discovery of novel multidrug resistance protein 4 (MRP4) inhibitors as active agents reducing resistance to anticancer drug 6-Mercaptopurine (6-MP) by structure and ligand-based virtual screening.

Multidrug resistance protein 4 (MRP4/ABCC4) is an ATP-binding cassette (ABC) transporter. It is associated with multidrug resistance (MDR), which is becoming a growing challenge to the treatment of cancer and infections. In the context of several types of cancer in which MRP4 is overexpressed, MRP4 inhibition manifests striking effects against cancer progression and drug resistance. In this study, we combined ligand-based and structure-based drug design strategy, by searching the SPECS chemical library to find compounds that are most likely to bind to MRP4. Clustering analysis based on a two-dimensional fingerprint was performed to help with visual selection of potential compounds. Cell viability assays with potential inhibitors and the anticancer drug 6-MP were carried out to identify their bioactivity. As a result, 39 compounds were tested and seven of them reached inhibition above 55% with 6-MP. Then compound Cpd23 was discovered to improve HEK293/MRP4 cell sensibility to 6-MP dramatically, and low concentration Cpd23 (5 µM) achieved the equivalent effect of 50 µM MK571. The accumulation of 6-MP was determined by validated high-performance liquid chromatography methods, and pretreatment of the HEK293/MRP4 cells with 50 µM MK571 or Cpd23 resulted in significantly increased accumulation of 6-MP by approximately 1.5 times. This compound was first reported with a novel scaffold compared with previously known MRP4 inhibitors, which is a hopeful molecular tool that can be used for overcoming multidrug resistance research.

Activation of soluble guanylyl cyclase with inhibition of multidrug resistance protein inhibitor-4 (MRP4) as a new antiplatelet therapy.

The intracellular levels of cyclic GMP are controlled by its rate of formation through nitric oxide-mediated stimulation of soluble guanylate cyclase (sGC) and its degradation by phosphodiesterases. Multidrug resistance protein 4 (MRP4) expressed in human platelets pumps cyclic nucleotides out of cells. In search for new antiplatelet strategies, we tested the hypothesis that sGC activation concomitant with MRP4 inhibition confers higher antiplatelet efficacy compared with monotherapy alone. This study was undertaken to investigate the pharmacological association of the sGC activator BAY 60-2770 with the MRP4 inhibitor MK571 on human washed platelets. Collagen- and thrombin-induced platelet aggregation and ATP-release reaction assays were performed. BAY 60-2770 (0.001-10 µM) produced significant inhibitions of agonist-induced platelet aggregation accompanied by reduced ATP-release. Pre-incubation with 10 µM MK571 alone had no significant effect on platelet aggregation and ATP release, but it produced a left displacement by about of 10-100-fold in the concentration-response curves to BAY 60-2770. Pre-incubation with MK571increased and decreased, respectively, the intracellular and extracellular levels of cGMP to BAY 60-2770, whereas the cAMP levels remained unchanged. The increased VASP-serine 239 phosphorylation in BAY 60-2770-treated platelets was enhanced by MK571. In Fluo-4-loaded platelets, BAY 60-2770 reduced the intracellular Ca2+ levels, an effect significantly potentiated by MK571. Flow cytometry assays showed that BAY 60-2770 reduces the αIIbß3 integrin activation, which was further reduced by MK571 association. Blocking the MRP4-mediated efflux of cGMP may be a potential mechanism to enhance the antiplatelet efficacy of sGC activators.

High-content screening of clinically tested anticancer drugs identifies novel inhibitors of human MRP1 (ABCC1).

Multidrug resistance protein 1 (MRP1/ABCC1), an integral transmembrane efflux transporter, belongs to the ATP-binding cassette (ABC) protein superfamily. MRP1 governs the absorption and disposition of a wide variety of endogenous and xenobiotic substrates including various drugs across organs and physiological barriers. Additionally, its overexpression has been implicated in multidrug resistance in chemotherapy of multiple cancers. Here, we describe the development of a high content imaging-based screening assay for MRP1 activity. This live cell-based automated microscopy assay is very robust and allows simultaneous detection of cell permeable, non-toxic and potent inhibitors. The validity of the assay was demonstrated by profiling a library of 386 anti-cancer compounds, which are under clinical trials, for interactions with MRP1. The assay identified 12 potent inhibitors including two known MRP1 inhibitors, cyclosporine A and rapamycin. On the other hand, MRP1-inhibitory activity of tipifarnib, AZD1208, deforolimus, everolimus, temsirolimus, HS-173, YM201636, ESI-09, TAK-733, and CX-6258 has not been previously reported. Inhibition of MRP1 activity was further validated using flow cytometry and confocal microscopy for the respective detection of calcein and doxorubicin in MRP1-overexpressing cells. Among the identified compounds, tipifarnib, AZD1208, rapamycin, deforolimus, everolimus, TAK-733, and temsirolimus resensitized MRP1-overexpressing H69AR cells towards vincristine, a cytotoxic chemotherapeutic agent, by 2-6-fold. Using purified HEK293 membrane vesicles overexpressing MRP1, MRP2, MRP3, and MRP4, we also demonstrated that the identified compounds exert differential and selective response on the uptake of estradiol glucuronide, an endogenous MRP substrate. In summary, we demonstrated the effectiveness of the high content imaging-based high-throughput assay for profiling compound interaction with MRP1.

Inhibition of Breast Cancer Resistance Protein and Multidrug Resistance Associated Protein 2 by Natural Compounds and Their Derivatives.

The food and dietary supplements we consume contain a wide variety of plant secondary metabolites and other compounds, which, like drugs, can be absorbed, metabolized, distributed, and excreted from the body. In the intestine, these compounds can interact with transport proteins such as the multidrug resistance associated protein 2 (MRP2, ABCC2) and the breast cancer resistance protein (BCRP, ABCG2) that regulate the absorption of drugs and other compounds. Inhibition of these transporters by dietary components could lead to increased exposure and adverse effects of concomitantly administered drugs. Therefore, we screened a library of 124 natural compounds and their derivatives using the vesicular transport assay to evaluate their inhibitory potential on MRP2 and BCRP. Of the library compounds, 36% were identified as BCRP inhibitors, whereas the number was only 3.2% for MRP2. BCRP inhibitors are described by higher molecular weight, number of rings, aromaticity, and LogD7.4 than noninhibitors. IC50 values were measured for six dual inhibitors, among which three novel inhibitors, gossypin, nordihydroguaiaretic acid, and octyl gallate, were identified. Our results confirm that flavonoids are avid inhibitors of BCRP, and flavones and flavonols appear to be important subclasses of flavonoids for this inhibition. The strong inhibition of BCRP transport by some compounds suggests that their presence at high levels in the diet could cause food-drug interactions, but this seems to be a minor cause of concern for MRP2.

Phytochemical Inhibition of Multidrug Resistance Protein-1 as a Therapeutic Strategy for Hemangioendothelioma.

AIMS: Hemangiomas are endothelial cell tumors and the most common soft tissue tumors in infants. They frequently cause deformity and can cause death. Current pharmacologic therapies have high-risk side-effect profiles, which limit the number of children who receive treatment. The objectives of this work were to identify the mechanisms through which standardized berry extracts can inhibit endothelial cell tumor growth and test these findings in vivo. RESULTS: EOMA cells are a validated model that generates endothelial cell tumors when injected subcutaneously into syngeneic (129P/3) mice. EOMA cells treated with a blend of powdered natural berry extracts (NBE) significantly inhibited activity of multidrug resistance protein-1 (MRP-1) compared to vehicle controls. This resulted in nuclear accumulation of oxidized glutathione (GSSG) and apoptotic EOMA cell death. When NBE-treated EOMA cells were injected into mice, they generated smaller tumors and had a higher incidence of apoptotic cell death compared to vehicle-treated EOMA cells as demonstrated by immunocytochemistry. Kaplan-Meier survival curves for tumor-bearing mice showed that NBE treatment significantly prolonged survival compared to vehicle-treated controls. INNOVATION: These are the first reported results to show that berry extracts can inhibit MRP-1 function that causes apoptotic tumor cell death by accumulation of GSSG in the nucleus of EOMA cells where NADPH oxidase is hyperactive and causes pathological angiogenesis. CONCLUSIONS: These findings indicate that berry extract inhibition of MRP-1 merits consideration and further investigation as a therapeutic intervention and may have application for other cancers with elevated MRP-1 activity. Antioxid. Redox Signal. 26, 1009-1019.

Dihydromyricetin reverses MRP2-mediated MDR and enhances anticancer activity induced by oxaliplatin in colorectal cancer cells.

Dihydromyricetin (DMY), extracted from the Chinese herbal medicine Ampelopsis grossedentata, possesses antitumor potential in different types of human cancer cells. Hence, its effects on drug resistance and molecular mechanisms in colorectal cancer (CRC) are still unknown. In our present study, we observed that DMY enhanced the chemosensitivity to oxaliplatin (OXA). DMY increased OXA-induced apoptosis and reduced 5(6)-carboxy-2',7'-dichlorofluorescein accumulation in OXA-resistant CRC HCT116/L-OHP cells. Our mechanistic study suggested that DMY treatment inhibited multidrug resistance protein 2 (MRP2) expression levels and promoter activity, indicating that DMY reduced not only MRP2 transcriptional and translational levels but also its function. Additional experiments indicated that the nuclear translocation of nuclear factor-erythroid 2 p45 related factor 2, a MRP2 regulator, was also inhibited by DMY. In summary, our study provided the first direct evidence that the inhibitory effects of DMY on MRP2 expression in OXA-resistant CRC cells were closely associated with the inhibition of nuclear factor-erythroid 2 p45 related factor 2 signaling. DMY could be a potential candidate for CRC chemotherapy.

Inhibition of the multidrug efflux pump LmrS from Staphylococcus aureus by cumin spice Cuminum cyminum.

Staphylococcus aureus is a serious causative agent of infectious disease. Multidrug-resistant strains like methicillin-resistant S. aureus compromise treatment efficacy, causing significant morbidity and mortality. Active efflux represents a major antimicrobial resistance mechanism. The proton-driven multidrug efflux pump, LmrS, actively exports structurally distinct antimicrobials. To circumvent resistance and restore clinical efficacy of antibiotics, efflux pump inhibitors are necessary, and natural edible spices like cumin are potential candidates. The mode of cumin antibacterial action and underlying mechanisms behind drug resistance inhibition, however, are unclear. We tested the hypothesis that cumin inhibits LmrS drug transport. We found that cumin inhibited bacterial growth and LmrS ethidium transport in a dosage-dependent manner. We demonstrate that cumin is antibacterial toward a multidrug-resistant host and that resistance modulation involves multidrug efflux inhibition.