OAT, OATP, and MRP Drug Transporters and the Remote Sensing and Signaling Theory.

The coordinated movement of organic anions (e.g., drugs, metabolites, signaling molecules, nutrients, antioxidants, gut microbiome products) between tissues and body fluids depends, in large part, on organic anion transporters (OATs) [solute carrier 22 (SLC22)], organic anion transporting polypeptides (OATPs) [solute carrier organic (SLCO)], and multidrug resistance proteins (MRPs) [ATP-binding cassette, subfamily C (ABCC)]. Depending on the range of substrates, transporters in these families can be considered multispecific, oligospecific, or (relatively) monospecific. Systems biology analyses of these transporters in the context of expression patterns reveal they are hubs in networks involved in interorgan and interorganismal communication. The remote sensing and signaling theory explains how the coordinated functions of drug transporters, drug-metabolizing enzymes, and regulatory proteins play a role in optimizing systemic and local levels of important endogenous small molecules. We focus on the role of OATs, OATPs, and MRPs in endogenous metabolism and how their substrates (e.g., bile acids, short chain fatty acids, urate, uremic toxins) mediate interorgan and interorganismal communication and help maintain and restore homeostasis in healthy and disease states.

The multidrug resistance protein 4 is expressed and functionally active in isolated bladder from pig.

Multidrug resistance proteins type 4 (MRP4) and 5 (MRP5) play pivotal roles in the transport of cyclic nucleotides in various tissues. However, their specific functions within the lower urinary tract remain relatively unexplored. This study aimed to investigate the effect of pharmacological inhibition of MRPs on cyclic nucleotide signaling in isolated pig bladder. The relaxation responses of the bladder were assessed in the presence of the MRP inhibitor, MK571. The temporal changes in intra- and extracellular levels of cAMP and cGMP in stimulated tissues were determined by mass spectrometry. The gene (ABCC4) and protein (MRP4) expression were also determined. MK571 administration resulted in a modest relaxation effect of approximately 26% in carbachol-precontracted bladders. The relaxation induced by phosphodiesterase inhibitors such as cilostazol, tadalafil, and sildenafil was significantly potentiated in the presence of MK571. In contrast, no significant potentiation was observed in the relaxation induced by substances elevating cAMP levels or stimulators of soluble guanylate cyclase. Following forskolin stimulation, both intracellular and extracellular cAMP concentrations increased by approximately 15.8-fold and 12-fold, respectively. Similarly, stimulation with tadalafil + BAY 41-2272 resulted in roughly 8.2-fold and 3.4-fold increases in intracellular and extracellular cGMP concentrations, respectively. The presence of MK571 reduced only the extracellular levels of cGMP. This study reveals the presence and function of MRP4 transporters within the porcine bladder and paves the way for future research exploring the role of this transporter in both underactive and overactive bladder disorders.NEW & NOTEWORTHY This study investigates the impact of pharmacological inhibition of MRP4 and MRP5 transporters on cyclic nucleotide signaling in isolated pig bladders. MK571 administration led to modest relaxation, with enhanced effects observed in the presence of phosphodiesterase inhibitors. However, substances elevating cAMP levels remained unaffected. MK571 selectively reduced extracellular cGMP levels. These findings shed light on the role of MRP4 transporters in the porcine bladder, opening avenues for further research into bladder disorders.

A conserved WXXE motif is an apical delivery determinant of ABC transporter C subfamily isoforms.

ATP-binding cassette transporter isoform C7 (ABCC7), also designated as cystic fibrosis transmembrane conductance regulator (CFTR), is exclusively targeted to the apical plasma membrane of polarized epithelial cells. Although the apical localization of ABCC7 in epithelia is crucial for the Cl- excretion into lumens, the mechanism regulating its apical localization is poorly understood. In the present study, an apical localization determinant was identified in the N-terminal 80-amino acid long cytoplasmic region of ABCC7 (NT80). In HepG2 cells, overexpression of NT80 significantly disturbed the apical expression of ABCC7 in a competitive manner, suggesting the presence of a sorting determinant in this region. Deletion analysis identified a potential sorting information within a 20-amino acid long peptide (aa 41-60) of NT80. Alanine scanning mutagenesis of this region in full-length ABCC7 further narrowed down the apical localization determinant to four amino acids, W57DRE60. This WDRE sequence was conserved among vertebrate ABCC7 orthologs. Site-directed mutagenesis showed that W57 and E60 were critical for the apical expression of ABCC7, confirming a novel apical sorting determinant of ABCC7. Furthermore, a WXXE motif (tryptophan and glutamic acid residues with two-amino acid spacing) was found to be conserved among the N-terminal regions of apically localized ABCC members with 12-TM configuration. The significance of the WXXE motif was demonstrated for proper trafficking of ABCC4 to the apical plasma membrane.Key words: apical plasma membrane, sorting, ATP-binding cassette transporter, CFTR, MRP4.

Multidrug resistance-associated protein 4 (Mrp4) is a novel genetic factor in the pathogenesis of obesity and diabetes.

Multidrug resistance protein 4 (Mrp4) is an efflux transporter known to transport several xenobiotics and endogenous molecules. We recently identified that the lack of Mrp4 increases adipose tissue and body weights in mice. However, the role of Mrp4 in adipose tissue physiology are unknown. The current study aimed at characterizing these specific roles of Mrp4 using wild-type (WT) and knockout (Mrp4-/- ) mice. Our studies determined that Mrp4 is expressed in mouse adipose tissue and that the lack of Mrp4 expression is associated with adipocyte hypertrophy. Furthermore, the lack of Mrp4 increased blood glucose and leptin levels, and impaired glucose tolerance. Additionally, in 3T3-L1 cells and human pre-adipocytes, pharmacological inhibition of Mrp4 increased adipogenesis and altered expression of adipogenic genes. Lack of Mrp4 activity in both of our in vivo and in vitro models leads to increased activation of adipose tissue cAMP response element-binding protein (Creb) and decreased plasma prostaglandin E (PGE) metabolite levels. These changes in Creb activation, coupled with decreased PGE levels, together promoted the observed metabolic phenotype in Mrp4-/- mice. In conclusion, our results indicate that Mrp4 as a novel genetic factor involved in the pathogenesis of metabolic diseases, such as obesity and diabetes.

Specific MRP4 Inhibitor Ceefourin-1 Enhances Apoptosis Induced by 6-Mercaptopurine in Jurkat Leukemic Cells, but Not in Normal Lymphoblast Cell Line CRL-1991.

Background and objectives: The multidrug resistance protein 4 (MRP4) is a member of the ABC transporter, which has been extensively related to many types of cancer including leukemia. MRP4 overexpression and activity over the efflux of some chemotherapeutic drugs are the main causes of chemoresistance. 6-mercaptopurine (6-MP) is a chemotherapeutic drug widely used in the consolidation and maintenance phases of leukemia treatment. However, 6-MP is a substrate of MRP4, which decreases its chemotherapeutic efficacy. Current research is focused on the development of MRP4 inhibitors to combat chemoresistance by allowing the accumulation of the drug substrates inside the cells. To date, the only specific MRP4 inhibitor that has been developed is ceefourin-1, which has been reported to inhibit MRP4 in many cancer cells and which makes it an excellent candidate to enhance the activity of 6-MP in a combined treatment in vitro of leukemic cells. Materials and methods: in the present work, we determined the enhancing activity of ceefourin-1 on the antiproliferative and apoptotic effect of 6-MP in leukemic Jurkat cells by trypan blue assay and flow cytometry. Besides, we determined the 6-MP and ceefourin-1 binding sites into MRP4 by molecular docking and molecular dynamics. Results: ceefourin-1 enhanced the apoptotic activity of 6-MP in Jurkat cells, while in CRL-1991 cells both antiproliferative and apoptotic effect were significantly lower. Ceefourin-1 additively cooperates with 6-MP to induce apoptosis in leukemic cells, but normal lymphoblast CRl-1991 showed resistance to both drugs. Conclusion: ceefourin-1 and 6-MP cooperates to trigger apoptosis in leukemic Jurkat cells, but the full mechanism needs to be elucidated in further works. In addition, our perspective is to test the cooperation between ceefourin-1 and 6-MP in samples from patients and healthy donnors.

MRP4-mediated cAMP efflux is essential for mouse spermatozoa capacitation.

Mammalian spermatozoa must undergo biochemical and structural changes to acquire the capacity for fertilization, in a process known as capacitation. Activation of PKA enzymes is essential for capacitation, and thus cAMP levels are tightly regulated during this process. Previously, we demonstrated that during capacitation, bovine spermatozoa extrude cAMP through multidrug resistance-associated protein 4 (MRP4, also known as ABCC4), which regulates intracellular levels of the nucleotide and provides cAMP to the extracellular space. Here, we report the presence of functional MRP4 in murine spermatozoa, since its pharmacological inhibition with MK571 decreased levels of extracellular cAMP. This also produced a sudden increase in PKA activity, with decreased tyrosine phosphorylation at the end of capacitation. Blockade of MRP4 inhibited induction of acrosome reaction, hyperactivation and in vitro fertilization. Moreover, MRP4 inhibition generated an increase in Ca2+ levels mediated by PKA, and depletion of Ca2+ salts from the medium prevented the loss of motility and phosphotyrosine inhibition produced by MK571. These results were supported using spermatozoa from CatSper Ca2+ channel knockout mice. Taken together, these results suggest that cAMP efflux via MRP4 plays an essential role in mouse sperm capacitation.This article has an associated First Person interview with the first author of the paper.

Probenecid Increases the Concentration of 7-Chlorokynurenic Acid Derived from the Prodrug 4-Chlorokynurenine within the Prefrontal Cortex.

Recent advances in the understanding of depression have led to increasing interest in ketamine and the role that N-methyl-d-aspartate (NMDA) receptor inhibition plays in depression. l-4-Chlorokynurenine (4-Cl-KYN, AV-101), a prodrug, has shown promise as an antidepressant in preclinical studies, but this promise has not been realized in recent clinical trials. We sought to determine if transporters in the CNS could be playing a role in this clinical response. We used radiolabeled uptake assays and microdialysis studies to determine how 4-Cl-KYN and its active metabolite, 7-chlorokynurenic acid (7-Cl-KYNA), cross the blood-brain barrier (BBB) to access the brain and its extracellular fluid compartment. Our data indicates that 4-Cl-KYN crosses the blood-brain barrier via the amino acid transporter LAT1 (SLC7A5) after which the 7-Cl-KYNA metabolite leaves the brain extracellular fluid via probenecid-sensitive organic anion transporters OAT1/3 (SLC22A6 and SLC22A8) and MRP4 (ABCC4). Microdialysis studies further validated our in vitro data, indicating that probenecid may be used to boost the bioavailability of 7-Cl-KYNA. Indeed, we found that coadministration of 4-Cl-KYN with probenecid caused a dose-dependent increase by as much as an 885-fold increase in 7-Cl-KYNA concentration in the prefrontal cortex. In summary, our data show that 4-Cl-KYN crosses the BBB using LAT1, while its active metabolite, 7-Cl-KYNA, is rapidly transported out of the brain via OAT1/3 and MRP4. We also identify a hitherto unreported mechanism by which the brain extracellular concentration of 7-Cl-KYNA may be increased to produce significant boosting of the drug concentration at its site of action that could potentially lead to an increased therapeutic effect.

MRP4 is responsible for the efflux transport of mycophenolic acid ß-d glucuronide (MPAG) from hepatocytes to blood.

Mycophenolic acid (MPA) has become a cornerstone of immunosuppressive therapy, in particular for transplant patients. In the gastrointestinal tract, the liver and the kidney, MPA is mainly metabolized into phenyl-ß-d glucuronide (MPAG). Knowledge about the interactions between MPA/MPAG and membrane transporters is still fragmented. The aim of the present study was to explore these interactions with the basolateral hepatic MRP4 transporter. The inhibition of the MRP4-driven transport by various drugs which can be concomitantly prescribed was also evaluated. In vitro experiments using vesicles overexpressing MRP4 showed an ATP-dependent transport of MPAG driven by MRP4 (Michaelis-Menten constant of 233.9 ± 32.8 µM). MPA was not effluxed by MRP4. MRP4-mediated transport of MPAG was inhibited (from -43% to -84%) by ibuprofen, cefazolin, cefotaxime and micafungin. An in silico approach based on molecular docking and molecular dynamics simulations rationalized the mode of binding of MPAG to MRP4. The presence of the glucuronide moiety in MPAG was highlighted as key, being prone to make electrostatic and H-bond interactions with specific residues of the MRP4 protein chamber. This explains why MPAG is a substrate of MRP4 whereas MPA is not.

Effect of bile acids on the expression of MRP3 and MRP4: An In vitro study in HepG2 cell line.

INTRODUCTION AND OBJECTIVES: Free and conjugated bile acids (BA's) cannot cross cell membranes; therefore, a particular transport system is required by the cell. Members of the family of ABC (ATP-binding proteins) transporters transfer bile acids in and out of the cell, preventing their accumulation. High intracellular concentrations of bile acids, such as those observed in cholestasis, have been related to oxidative stress and apoptosis, which in many cases are the leading causes of hepatocyte damage. MRP3 and MRP4 (multidrug resistance-associated protein 3 and 4) proteins belong to the ABC subfamily C, and are transporters of the hepatocyte's basolateral membrane with a compensatory role. Both transporters' increased expression constitutes an essential role in the protective and adaptive responses of bile acid overload, such as cholestasis. This work aimed to analyze both transporters' mRNA and protein expression in an in vitro model of cholestasis using HepG2 cell line treated with main bile acids. METHODS: The expression of transporters was investigated through confocal microscopy immunofluorescence, Western Blot, and RT-qPCR after the main bile acids in HepG2 line cells. RESULTS: The results showed the relation between confluence and expression of both transporters in the plasma membrane. MRP3 showed atypical and heterogeneous distribution in this cell line. CDCA (chenodeoxycholic acid) at low concentrations induced the expression of mRNA of both transporters. In contrast, protein expression was induced by CA (cholic acid) at high concentrations. CONCLUSION: Primary bile acids (CDCA and CA) induce overexpression of the MRP4 and MRP3 transporters in the HepG2 cell line.

Predialysis and Dialysis Therapies Differently Affect Nitric Oxide Synthetic Pathway in Red Blood Cells from Uremic Patients: Focus on Peritoneal Dialysis.

Red blood cells (RBCs) have been found to synthesize and release both nitric oxide (NO) and cyclic guanosine monophosphate (cGMP), contributing to systemic NO bioavailability. These RBC functions resulted impaired in chronic kidney disease (CKD). This study aimed to evaluate whether predialysis (conservative therapy, CT) and dialysis (peritoneal dialysis, PD; hemodialysis, HD) therapies used during CKD progression may differently affect NO-synthetic pathway in RBCs. Our data demonstrated that compared to PD, although endothelial-NO-synthase activation was similarly increased, HD and CT were associated to cGMP RBCs accumulation, caused by reduced activity of cGMP membrane transporter (MRP4). In parallel, plasma cGMP levels were increased by both CT and HD and they significantly decreased after hemodialysis, suggesting that this might be caused by reduced cGMP renal clearance. As conceivable, compared to healthy subjects, plasma nitrite levels were significantly reduced by HD and CT but not in patients on PD. Additionally, the increased carotid intima-media thickness (IMT) values did not reach the significance exclusively in patients on PD. Therefore, our results show that PD might better preserve the synthetic NO-pathway in CKD-erythrocytes. Whether this translates into a reduced development of uremic vascular complications requires further investigation.

Study of Endogen Substrates, Drug Substrates and Inhibitors Binding Conformations on MRP4 and Its Variants by Molecular Docking and Molecular Dynamics.

Multidrug resistance protein-4 (MRP4) belongs to the ABC transporter superfamily and promotes the transport of xenobiotics including drugs. A non-synonymous single nucleotide polymorphisms (nsSNPs) in the ABCC4 gene can promote changes in the structure and function of MRP4. In this work, the interaction of certain endogen substrates, drug substrates, and inhibitors with wild type-MRP4 (WT-MRP4) and its variants G187W and Y556C were studied to determine differences in the intermolecular interactions and affinity related to SNPs using protein threading modeling, molecular docking, all-atom, coarse grained, and umbrella sampling molecular dynamics simulations (AA-MDS and CG-MDS, respectively). The results showed that the three MRP4 structures had significantly different conformations at given sites, leading to differences in the docking scores (DS) and binding sites of three different groups of molecules. Folic acid (FA) had the highest variation in DS on G187W concerning WT-MRP4. WT-MRP4, G187W, Y556C, and FA had different conformations through 25 ns AA-MD. Umbrella sampling simulations indicated that the Y556C-FA complex was the most stable one with or without ATP. In Y556C, the cyclic adenosine monophosphate (cAMP) and ceefourin-1 binding sites are located out of the entrance of the inner cavity, which suggests that both cAMP and ceefourin-1 may not be transported. The binding site for cAMP and ceefourin-1 is quite similar and the affinity (binding energy) of ceefourin-1 to WT-MRP4, G187W, and Y556C is greater than the affinity of cAMP, which may suggest that ceefourin-1 works as a competitive inhibitor. In conclusion, the nsSNPs G187W and Y556C lead to changes in protein conformation, which modifies the ligand binding site, DS, and binding energy.

ABCC4/MRP4 contributes to the aggressiveness of Myc-associated epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is a complex disease comprising discrete histological and molecular subtypes, for which survival rates remain unacceptably low. Tailored approaches for this deadly heterogeneous disease are urgently needed. Efflux pumps belonging to the ATP-binding cassette (ABC) family of transporters are known for roles in both drug resistance and cancer biology and are also highly targetable. Here we have investigated the association of ABCC4/MRP4 expression to clinical outcome and its biological function in endometrioid and serous tumors, common histological subtypes of EOC. We found high expression of ABCC4/MRP4, previously shown to be directly regulated by c-Myc/N-Myc, was associated with poor prognosis in endometrioid EOC (P = .001) as well as in a subset of serous EOC with a "high-MYCN" profile (C5/proliferative; P = .019). Transient siRNA-mediated suppression of MRP4 in EOC cells led to reduced growth, migration and invasion, with the effects being most pronounced in endometrioid and C5-like serous cells compared to non-C5 serous EOC cells. Sustained knockdown of MRP4 also sensitized endometrioid cells to MRP4 substrate drugs. Furthermore, suppression of MRP4 decreased the growth of patient-derived EOC cells in vivo. Together, our findings provide the first evidence that MRP4 plays an important role in the biology of Myc-associated ovarian tumors and highlight this transporter as a potential therapeutic target for EOC.

Human multidrug resistance protein 4 (MRP4) is a cellular efflux transporter for paracetamol glutathione and cysteine conjugates.

Paracetamol (acetaminophen, APAP) overdose is a leading cause of acute drug-induced liver failure. APAP hepatotoxicity is mediated by the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). NAPQI is inactivated by conjugation with glutathione (GSH) to APAP-GSH, which is further converted into its cysteine derivative APAP-CYS. Before necrosis of hepatocytes occurs, APAP-CYS is measurable in plasma of the affected patient and it has been proposed as an early biomarker of acetaminophen toxicity. APAP-GSH and APAP-CYS can be extruded by hepatocytes, but the transporters involved are unknown. In this study we examined whether ATP-binding cassette (ABC) transporters play a role in the cellular efflux of APAP, APAP-GSH, and APAP-CYS. The ABC transport proteins P-gp/ABCB1, BSEP/ABCB11, BCRP/ABCG2, and MRP/ABCC1-5 were overexpressed in HEK293 cells and membrane vesicles were produced. Whereas P-gp, BSEP, MRP3, MRP5, and BCRP did not transport any of the compounds, uptake of APAP-GSH was found for MRP1, MRP2 and MRP4. APAP-CYS appeared to be a substrate of MRP4 and none of the ABC proteins transported APAP. The results suggest that the NAPQI metabolite APAP-CYS can be excreted into plasma by MRP4, where it could be a useful biomarker for APAP exposure and toxicity. Characterization of the cellular efflux of APAP-CYS is important for its development as a biomarker, because plasma concentrations might be influenced by drug-transporter interactions and upregulation of MRP4.

Roles of ABCC1 and ABCC4 in Proliferation and Migration of Breast Cancer Cell Lines.

ABCC1 and ABCC4 utilize energy from ATP hydrolysis to transport many different molecules, including drugs, out of the cell and, as such, have been implicated in causing drug resistance. However recently, because of their ability to transport signaling molecules and inflammatory mediators, it has been proposed that ABCC1 and ABCC4 may play a role in the hallmarks of cancer development and progression, independent of their drug efflux capabilities. Breast cancer is the most common cancer affecting women. In this study, the aim was to investigate whether ABCC1 or ABCC4 play a role in the proliferation or migration of breast cancer cell lines MCF-7 (luminal-type, receptor-positive) and MDA-MB-231 (basal-type, triple-negative). The effects of small molecule inhibitors or siRNA-mediated knockdown of ABCC1 or ABCCC4 were measured. Colony formation assays were used to assess the clonogenic capacity, MTT assays to measure the proliferation, and scratch assays and Transwell assays to monitor the cellular migration. The results showed a role for ABCC1 in cellular proliferation, whilst ABCC4 appeared to be more important for cellular migration. ELISA studies implicated cAMP and/or sphingosine-1-phosphate efflux in the mechanism by which these transporters mediate their effects. However, this needs to be investigated further, as it is key to understand the mechanisms before they can be considered as targets for treatment.

Aryl hydrocarbon receptor ligands increase ABC transporter activity and protein expression in killifish (Fundulus heteroclitus) renal proximal tubules.

Many widespread and persistent organic pollutants, for example, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and some polychlorinated biphenyls, activate the aryl hydrocarbon receptor (AhR) causing it to translocate to the cell nucleus where it transactivates target genes, increasing expression of a number of xenobiotic metabolizing enzymes as well as some transporters. AhR's ability to target transporters within the kidney is essentially unexplored. We show here that exposing isolated killifish (Fundulus heteroclitus) renal proximal tubules to micromolar ß-naphthoflavone (BNF) or nanomolar TCDD roughly doubled the transport activity of Multidrug resistance-associated proteins Mrp2 and Mrp4, P-glycoprotein (P-gp) and Breast cancer resistance protein (Bcrp), all ATP-driven xenobiotic efflux pumps and critical determinants of renal xenobiotic excretion. These effects were abolished by actinomycin D and cycloheximide and by the AhR antagonist, α-naphthoflavone, indicating that increased transport activity was dependent on transcription and translation as well as ligand binding to AhR. Quantitative immunostaining of renal tubules exposed to BNF and TCDD showed increased luminal membrane expression of Mrp2, Mrp4, P-gp and Bcrp. Thus, in these renal tubules, the four ABC transporters are targets of AhR action.

Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors.

BACKGROUND/AIMS: Signaling of Gs protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear. METHODS: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA. RESULTS: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors. CONCLUSION: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.

Structural patterns of the human ABCC4/MRP4 exporter in lipid bilayers rationalize clinically observed polymorphisms.

The ABCC4/MRP4 exporter has a clinical impact on membrane transport of a broad range of xenobiotics. It is expressed at key locations for drug disposition or effects such as in the liver, the kidney and blood cells. Several polymorphisms and mutations (e.g., p.Gly187Trp) leading to MRP4 dysfunction are associated with an increased risk of toxicity of some drugs. So far, no human MRP4 structure has been elucidated, precluding rationalization of these dysfunctions at a molecular level. We constructed an atomistic model of the wild type (WT) MRP4 and the p.Gly187Trp mutant embedded in different lipid bilayers and relaxed them for hundreds of nanoseconds by molecular dynamics simulations. The WT MRP4 molecular structure confirmed and ameliorated the general knowledge about the transmembrane helices and the two nucleotide binding domains. Moreover, our model elucidated positions of three generally unresolved domains: L1 (linker between the two halves of the exporter); L0 (N-terminal domain); and the zipper helices (between the two NBDs). Each domain was thoroughly described in view of its function. The p.Gly187Trp mutation induced a huge structural impact on MRP4, mainly affecting NBD 1 structure and flexibility. The structure of transporter enabled rationalization of known dysfunctions associated with polymorphism of MRP4. This model is available to the pharmacology community to decipher the impact of any other clinically observed polymorphism and mutation on drug transport, giving rise to in silico predictive pharmacogenetics.

Octadecyloxyethyl Adefovir Exhibits Potent in vitro and in vivo Cytotoxic Activity and Has Synergistic Effects with Ara-C in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) continues to be a deadly disease, with only 50-70% of patients achieving complete remission and less than 30% of adults having sustained long-term remissions. In order to address these unmet medical needs, we carried out a high-throughput screen of an in-house library of on- and off-patent drugs with the OCI/AML-2 cell line. Through this screen, we discovered adefovir dipi-voxil (adefovir-DP) as being active against human AML. In addition to adefovir-DP, there are second-generation formulations of adefovir, including octadecyloxyethyl adefovir (ODE-adefovir) and hexadecyloxypropyl adefovir (HDP-adefovir), which were designed to overcome the pharmacokinetic problems of the parent compound adefovir. Given the known clinical benefit of nucleoside analogs for the treatment of AML, we undertook studies to evaluate the potential benefit of adefovir-based molecules. In AML cell lines and patient samples, adefovir-DP and ODE-adefovir were highly potent, whereas HDP-adefovir was significantly less active. Interestingly, ODE-adefovir was remarkably less toxic than adefovir-DP towards normal hematopoietic cells. In addition, ODE-adefovir at a dose of 15 mg/kg/day showed potent activity against human AML in a NOD/SCID mouse model, with a reduction of human leukemia in mouse bone marrow of > 40% in all mice tested within 20 days of treatment. Based on its chemical structure, we hypothesized that the cytotoxicity of ODE-adefovir toward AML was through cell cycle arrest and DNA damage. Indeed, ODE-adefovir treatment induced cell cycle arrest in the S phase and increased levels of pH2Ax, indicating the induction of DNA damage. Furthermore, there was an increase in phospho-p53, transactivation of proapoptotic genes and activation of the intrinsic apoptotic pathway. Subsequent investigation unveiled strong synergism between ODE-adefovir and ara-C, making their coadministration of potential clinical benefit. Expression of MRP4, a nucleoside transporter, appeared to influence the response of AML cells to ODE-adefovir, as its inhibition potentiated ODE-adefovir killing. Taken together, our findings indicate that clinical development of ODE-adefovir or related compounds for the treatment of AML is warranted.

Selectivity in the Efflux of Glucuronides by Human Transporters: MRP4 Is Highly Active toward 4-Methylumbelliferone and 1-Naphthol Glucuronides, while MRP3 Exhibits Stereoselective Propranolol Glucuronide Transport.

Xenobiotic and endobiotic glucuronides, which are generated in hepatic and intestinal epithelial cells, are excreted via efflux transporters. Multidrug resistance proteins 2-4 (MRP2-MRP4) and the breast cancer resistance protein (BCRP) are efflux transporters that are expressed in these polarized cells, on either the basolateral or apical membranes. Their localization, along with expression levels, affects the glucuronide excretion pathways. We have studied the transport of three planar cyclic glucuronides and glucuronides of the two propranolol enantiomers, by the vesicular transport assay, using vesicles from baculovirus-infected insect cells expressing human MRP2, MRP3, MRP4, or BCRP. The transport of estradiol-17ß-glucuronide by recombinant MRP2-4 and BCRP, as demonstrated by kinetic values, were within the ranges previously reported. Our results revealed high transport rates and apparent affinity of MRP4 toward the glucuronides of 4-methylumbelliferone, 1-naphthol, and 1-hydroxypyrene (Km values of 168, 13, and 3 µM, respectively) in comparison to MRP3 (Km values of 278, 98, and 8 µM, respectively). MRP3 exhibited lower rates, but stereoselective transport of propranolol glucuronides, with higher affinity toward the R-enantiomer than the S-enantiomer (Km values 154 vs 434 µM). The glucuronide of propranolol R-enantiomer was not significantly transported by either MRP2, MRP4, or BCRP. Of the tested small glucuronides in this study, BCRP transported only 1-hydroxypyrene glucuronide, at very high rates and high apparent affinity (Vmax and Km values of 4400 pmol/mg/min and 11 µM). The transport activity of MRP2 with all of the studied small glucuronides was relatively very low, even though it transported the reference compound, estradiol-17ß-glucuronide, at a high rate (Vmax = 3500 pmol/mg/min). Our results provide new information, at the molecular level, of efflux transport of the tested glucuronides, which could explain their disposition in vivo, as well as provide new tools for in vitro studies of MRP3, MRP4, and BCRP.

Quantitative Evaluation of Drug Resistance Profile of Cells Expressing Wild-Type or Genetic Polymorphic Variants of the Human ABC Transporter ABCC4.

Broad-spectrum resistance in cancer cells is often caused by the overexpression of ABC transporters; which varies across individuals because of genetic single-nucleotide polymorphisms (SNPs). In the present study; we focused on human ABCC4 and established cells expressing the wild-type (WT) or SNP variants of human ABCC4 using the Flp-In™ system (Invitrogen, Life Technologies Corp, Carlsbad, CA, USA) based on Flp recombinase-mediated transfection to quantitatively evaluate the effects of nonsynonymous SNPs on the drug resistance profiles of cells. The mRNA levels of the cells expressing each ABCC4 variant were comparable. 3-(4,5-Dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay clearly indicated that the EC50 values of azathioprine against cells expressing ABCC4 (WT) were 1.4-1.7-fold higher than those against cells expressing SNP variants of ABCC4 (M184K; N297S; K304N or E757K). EC50 values of 6-mercaptopurine or 7-Ethyl-10-hydroxy-camptothecin (SN-38) against cells expressing ABCC4 (WT) were also 1.4-2.0- or 1.9-fold higher than those against cells expressing the SNP variants of ABCC4 (K304N or E757K) or (K304N; P403L or E757K); respectively. These results indicate that the effects of nonsynonymous SNPs on the drug resistance profiles of cells expressing ABCC4 can be quantitatively evaluated using the Flp-In™ system.