Role of the ATP-binding cassette drug transporters in the intestinal absorption of tanshinone IIB, one of the major active diterpenoids from the root of Salvia miltiorrhiza

There is an increasing use of herbal medicines worldwide, and the extracts from the root of Salvia miltiorrhiza are widely used in the treatment of angina and stroke. In this study, we investigated the mechanism for the intestinal absorption of tanshinone IIB (TSB), a major constituent of S. miltiorrhiza. The oral bioavailability of TSB was about 3% in rats with less proportional increase in its maximum plasma concentration (C_max) and area under the plasma concentration-time curve (AUC) with increasing dosage. The time to C_max (T_max) was prolonged at higher oral dosage. In a single pass rat intestinal perfusion model, the permeability coefficients (P_app) based on TSB disappearance from the lumen (P_lumen) were 6.2- to 7.2-fold higher (p < 0.01) than those based on drug appearance in mesenteric venous blood (P_blood). The uptake and efflux of TSB in Caco-2 cells were also significantly altered in the presence of an inhibitor for P-glycoprotein (PgP) or for multi-drug resistance associated protein (MRP1/2). TSB transport from the apical (AP) to basolateral (BL) side in Caco-2 monolayers was 3.3- to 5.7-fold lower than that from BL to AP side, but this polarized transport was attenuated by co-incubation of PgP or MRP1/2 inhibitors. The P_app values of TSB in the BL-AP direction were significantly higher in MDCKII cells over-expressing MDR1 or MRP1, but not in cells over-expressing MRP2-5, as compared with the wild-type cells. The plasma AUC_0-24hr in mdr1a and mrp1 gene-deficient mice was 10.2- to 1.7-fold higher than that in the wild-type mice. Furthermore, TSB significantly inhibited the uptake of digoxin and vinblastine in membrane vesicles containing PgP or MRP1. TSB also moderately stimulated PgP ATPase activity. Taken collectively, our findings indicate that TSB is a substrate for PgP and MRP1 and that drug resistance to TSB therapy and drug interactions may occur through PgP and MRP1 modulation.

Role of nanomedicine in reversing drug resistance mediated by ATP binding cassette transporters and P-glycoprotein in melanoma

Multidrug resistance (MDR) is one of the most common complex phenomenons exhibited by cancer cells. It is a very common property of melanoma postchemotherapy. MDR transporters, ATP binding cassette (ABC) transporters, play a critical role in conferring this property to melanoma cells. miRNA are post-transcriptional regulators that regulate the expression of these ABC transporters. Targeting these miRNA, in turn targeting ABC transporters with the help of nanodelivery systems to overcome drug resistance, is the primary focus for attaining successful treatment methods for drug-resistant melanoma. These delivery systems are endocytosed by the cancer cells and do not require ABC transporters for their delivery, being a promising therapeutic measure for melanoma.

Human multidrug resistance associated protein 4 resitance to camtothecins

Purpose The multidrug resistance associated protein (MRP) 4 is a member of the adenosine triphosphate (ATP)-binding cassette transporter family. Camptothecins (CPTs) have shown substantial anticancer activity against a broad spectrum of tumors by inhibiting DNA topoisomerase I, but tumor resistance is one of the major reasons for therapeutic failure. P-glycoprotein, breast cancer resistance protein, MRP1, and MRP2 have been implicated in resistance to various CPTs including CPT-11 (irinotecan), SN-38 (the active metabolite of CPT-11), and topotecan. In this study, we explored the resistance profiles and intracellular accumulation of a panel of CPTs including CPT, CPT-11, SN-38, rubitecan, and 10-hydroxy-CPT (10-OH-CPT) in HepG2 cells with stably overexpressed human MRP4. Other anticancer agents such as paclitaxel, cyclophosphamide, and carboplatin were also included.
Methods HepG2 cells were transfected with an empty vehicle plasmid (V/HepG2) or human MRP4 (MRP4/HepG2). The resistance profiles of test drugs in exponentially growing V/HepG2 and MRP4/HepG2 cells were examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) assay with 4 or 48 h exposure time of the test drug in the absence or presence of various MRP4 inhibitors. The accumulation of CPT-11, SN-38, and paclitaxel by V/HepG2 and MRP4/HepG2 cells was determined by validated high-performance liquid chromatography methods.
Results Based on the resistance folds from the MTT assay with 48 h exposure time of the test drug, MRP4 conferred resistance to CPTs tested in the order 10-OH-CPT (14.21) > SN-38 carboxylate (9.70) > rubitecan (9.06) > SN-38 lactone (8.91) > CPT lactone (7.33) > CPT-11 lactone (5.64) > CPT carboxylate (4.30) > CPT-11 carboxylate (2.68). Overall, overexpression of MRP4 increased the IC50 values 1.78- to 14.21-fold for various CPTs in lactone or carboxylate form. The resistance of MRP4 to various CPTs tested was significantly reversed in the presence of dl-buthionine-(S,R)-sulfoximine (BSO, a γ-glutamylcysteine synthetase inhibitor), MK571, celecoxib, or diclofenac (all MRP4 inhibitors). In addition, the accumulation of CPT-11 and SN-38 over 120 min in MRP4/HepG2 cells was significantly reduced compared to V/HepG2 cells, whereas the addition of celecoxib, MK571, or BSO significantly increased their accumulation in MRP4/HepG2 cells. There was no significant difference in the intracellular accumulation of paclitaxel in V/HepG2 and MRP4/HepG2 cells, indicating that P-glycoprotein was not involved in the observed resistance to CPTs in this study. MRP4 also conferred resistance to cyclophosphamide and this was partially reversed by BSO. However, MRP4 did not increase resistance to paclitaxel, carboplatin, etoposide (VP-16), 5-fluorouracil, and cyclosporine.
Conclusions Human MRP4 rendered significant resistance to cyclophosphamide, CPT, CPT-11, SN-38, rubitecan, and 10-OH-CPT. CPT-11 and SN-38 are substrates for MRP4. Further studies are needed to explore the role of MRP4 in resistance, toxicity, and pharmacokinetics of CPTs and cyclophosphamide.

Substrates and inhibitors of human multidrug resistance associated proteins and the implications in drug development

Human contains 49 ATP-binding cassette (ABC) transporter genes and the multidrug resistance associated proteins (MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP4/ABCC4, MRP5/ABCC5, MRP6/ABCC6, MRP7/ABCC10, MRP8/ABCC11 and MRP9/ABCC12) belong to the ABCC family which contains 13 members. ABCC7 is cystic fibrosis transmembrane conductance regulator; ABCC8 and ABCC9 are the sulfonylurea receptors which constitute the ATP-sensing subunits of a complex potassium channel. MRP10/ABCC13 is clearly a pseudo-gene which encodes a truncated protein that is highly expressed in fetal human liver with the highest similarity to MRP2/ABCC2 but without transporting activity. These transporters are localized to the apical and/or basolateral membrane of the hepatocytes, enterocytes, renal proximal tubule cells and endothelial cells of the blood-brain barrier. MRP/ABCC members transport a structurally diverse array of important endogenous substances and xenobiotics and their metabolites (in particular conjugates) with different substrate specificity and transport kinetics. The human MRP/ABCC transporters except MRP9/ABCC12 are all able to transport organic anions, such as drugs conjugated to glutathione, sulphate or glucuronate. In addition, selected MRP/ABCC members may transport a variety of endogenous compounds, such as leukotriene C(4) (LTC(4) by MRP1/ABCC1), bilirubin glucuronides (MRP2/ABCC2, and MRP3/ABCC3), prostaglandins E1 and E2 (MRP4/ABCC4), cGMP (MRP4/ABCC4, MRP5/ABCC5, and MRP8/ABCC11), and several glucuronosyl-, or sulfatidyl steroids. In vitro, the MRP/ABCC transporters can collectively confer resistance to natural product anticancer drugs and their conjugated metabolites, platinum compounds, folate antimetabolites, nucleoside and nucleotide analogs, arsenical and antimonial oxyanions, peptide-based agents, and in concert with alterations in phase II conjugating or biosynthetic enzymes, classical alkylating agents, alkylating agents. Several MRP/ABCC members (MRPs 1-3) are associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. Drug targeting of these transporters to overcome MRP/ABCC-mediated multidrug resistance may play a role in cancer chemotherapy. Most MRP/ABCC transporters are subject to inhibition by a variety of compounds. Based on currently available preclinical and limited clinical data, it can be expected that modulation of MRP members may represent a useful approach in the management of anticancer and antimicrobial drug resistance and possibly of inflammatory diseases and other diseases. A better understanding of their substrates and inhibitors has important implications in development of drugs for treatment of cancer and inflammation.

Efflux pumps of Mycobacterium tuberculosis play a significant role in antituberculosis activity of potential drug candidates

Active efflux of drugs mediated by efflux pumps that confer drug resistance is one of the mechanisms developed by bacteria to counter the adverse effects of antibiotics and chemicals. To understand these efflux mechanisms in Mycobacterium tuberculosis, we generated knockout (KO) mutants of four efflux pumps of the pathogen belonging to different classes. We measured the MICs and kill values of two different compound classes on the wild type (WT) and the efflux pump (EP) KO mutants in the presence and absence of the efflux inhibitors verapamil and L-phenylalanyl-L-arginyl-β-naphthylamide (PAβN). Among the pumps studied, the efflux pumps belonging to the ABC (ATP-binding cassette) class, encoded by Rv1218c, and the SMR (small multidrug resistance) class, encoded by Rv3065, appear to play important roles in mediating the efflux of different chemical classes and antibiotics. Efflux pumps encoded by Rv0849 and Rv1258c also mediate the efflux of these compounds, but to a lesser extent. Increased killing is observed in WT M. tuberculosis cells by these compounds in the presence of either verapamil or PAβN. The efflux pump KO mutants were more susceptible to these compounds in the presence of efflux inhibitors. We have shown that these four efflux pumps of M. tuberculosis play a vital role in mediating efflux of different chemical scaffolds. Inhibitors of one or several of these efflux pumps could have a significant impact in the treatment of tuberculosis. The identification and characterization of Rv0849, a new efflux pump belonging to the MFS (major facilitator superfamily) class, are reported.

Role of multidrug resistance associated proteins in drug development

The Multidrug Resistance Associated Proteins (MRPI, MRP2, MRP3, MRp4, MRp5, MRP6, MRP7, MRPS and MRP9) belong to the ATP-binding cassette superfamily (ABCC family) of transporters expressed differentially in the liver, kidney, intestine and blood-brain barrier. MRps transport a structurally diverse array of endo- and xenobiotics and their metabolites (in particular conjugates) and are subject to induction and inhibition by a variety of compounds. An increased efflux of natural product anticancer drugs and other anticancer agents by MRPs in cancer cells is associated with tumor resistance. These transporting proteins play a role in the absorption, distribution and elimination of various compounds in the body. There are increased reports on the clinical impact of genetic mutations of genes encoding MRP1-9. Therefore, MRPs have an important role in drug development, since a better understanding of their function and regulating mechanism can help minimize and avoid drug toxicity, unfavorable drug-drug interactions, and to overcome drug resistance.

ABCB1 polymorphism predicts escitalopram dose needed for remission in major depression

The ATP-binding cassette family of transporter proteins, subfamily B (MDR/TAP), member 1 (ABCB1) (P-glycoprotein) transporter is a key component of the blood–brain barrier. Many antidepressants are subject to ABCB1 efflux. Functional polymorphisms of ABCB1 may influence central nervous system bioavailability of antidepressants subject to efflux. Single-nucleotide polymorphisms (SNPs) at rs1045642 (C3435T) of ABCB1 have been associated with efflux pump efficiency. This may explain part of the interindividual variation in antidepressant dose needed to remit. Individuals (N=113) with DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition) major depressive disorder (MDD) were treated with escitalopram (ESC) or venlafaxine (VEN) over 8 weeks. The17-item Hamilton Depression Rating Scale was assessed serially, blind to genotype. SNP rs1045642 of ABCB1 along with two SNPs previously reported to be in linkage disequilibrium with it (rs2032582 and rs1128503) were genotyped. Demographic features, clinical features, P450 metabolizer status and 5-HTTLPR (serotonin-transporter-linked promoter region) genotype were controlled for. Carriers of rs1045642 TT needed on average 11 mg of ESC to remit, whereas TC and CC carriers required 24 and 19 mg, respectively (P=0.0001). This equates to a 2.0- (95% confidence interval=1.5–3.4; P<0.001) fold greater ESC dose needed to remit for C carriers compared with TT carriers at rs1045642. Of VEN-treated subjects carrying TT genotype at rs1045642, 73.3% remitted compared with 12.5% for CC genotype (odds ratio=6.69; 95% confidence interval=1.72–25.9, P=0.006). These data suggest that antidepressant dose needed to remit can be predicted by an ABCB1 SNP. This has the potential clinical translation implications for dose selection and remission from MDD.

The last 10 amino acid residues beyond the hydrophobic motif are critical for the catalytic competence and function of protein kinase Cá*

The segment C-terminal to the hydrophobic motif at the V5 domain of protein kinase C (PKC) is the least conserved both in length and in amino acid identity among all PKC isozymes. By generating serial truncation mutants followed by biochemical and functional analyses, we show here that the very C terminus of PKCα is critical in conferring the full catalytic competence to the kinase and for transducing signals in cells. Deletion of one C-terminal amino acid residue caused the loss of ~60% of the catalytic activity of the mutant PKCα, whereas deletion of 10 C-terminal amino acid residues abrogated the catalytic activity of PKCα in immune complex kinase assays. The PKCα C-terminal truncation mutants were found to lose their ability to activate mitogen-activated protein kinase, to rescue apoptosis induced by the inhibition of endogenous PKC in COS cells, and to augment melatonin-stimulated neurite outgrowth. Furthermore, molecular dynamics simulations revealed that the deletion of 1 or 10 C-terminal residues results in the deformation of the V5 domain and the ATP-binding pocket, respectively. Finally, PKCα immunoprecipitated using an antibody against its C terminus had only marginal catalytic activity compared with that of the PKCα immunoprecipitated by an antibody against its N terminus. Therefore, the very C-terminal tail of PKCα is a novel determinant of the catalytic activity of PKC and a promising target for selective modulation of PKCα function. Molecules that bind preferentially to the very C terminus of distinct PKC isozymes and suppress their catalytic activity may constitute a new class of selective inhibitors of PKC.

Dissecting isoform selectivity of P13K inhibitors: the role of non-conserved residues in the catalytic pocket

The last few years have seen the identification of numerous small molecules that selectively inhibit specific class I isoforms of PI3K (phosphoinositide 3-kinase), yet little has been revealed about the molecular basis for the observed selectivities. Using site-directed mutagenesis, we have investigated one of the areas postulated as being critical to the observed selectivity. The residues Thr⁸⁸⁶ and Lys⁸⁹⁰ of the PI3Kγ isoform project towards the ATP-binding pocket at the entrance to the catalytic site, but are not conserved. We have made reciprocal mutations between those residues in the β isoform (Glu⁸⁵⁸ and Asp⁸⁶²) and those in the α isoform (His⁸⁵⁵ and Gln⁸⁵⁹) and evaluated the potency of a range of reported PI3K inhibitors. The results show that the potencies of β-selective inhibitors TGX221 and TGX286 are unaffected by this change. In contrast, close analogues of these compounds, particularly the α-isoform-selective compound (III), are markedly influenced by the point mutations. The collected data suggests two distinct binding poses for these inhibitor classes, one of which is associated with potent PI3Kβ activity and is not associated with the mutated residues, and a second that, in accord with earlier hypotheses, does involve this pair of non-conserved amino acids at the catalytic site entrance and contributes to the α-isoform-selectivity of the compounds studied.

Chemotoxicity of doxorubicin and surface expression of P-glycoprotein (MDR1) is regulated by the Pseudomonas aeruginosa toxin Cif

P-glycoprotein (Pgp), a member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily, is a major drug efflux pump expressed in normal tissues, and is overexpressed in many human cancers. Overexpression of Pgp results in reduced intracellular drug concentration and cytotoxicity of chemotherapeutic drugs and is thought to contribute to multidrug resistance of cancer cells. The involvement of Pgp in clinical drug resistance has led to a search for molecules that block Pgp transporter activity to improve the efficacy and pharmacokinetics of therapeutic agents. We have recently identified and characterized a secreted toxin from Pseudomonas aeruginosa, designated cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif). Cif reduces the apical membrane abundance of CFTR, also an ABC transporter, and inhibits the CFTR-mediated chloride ion secretion by human airway and kidney epithelial cells. We report presently that Cif also inhibits the apical membrane abundance of Pgp in kidney, airway, and intestinal epithelial cells but has no effect on plasma membrane abundance of multidrug resistance protein 1 or 2. Cif increased the drug sensitivity to doxorubicin in kidney cells expressing Pgp by 10-fold and increased the cellular accumulation of daunorubicin by 2-fold. Thus our studies show that Cif increases the sensitivity of Pgp-overexpressing cells to doxorubicin, consistent with the hypothesis that Cif affects Pgp functional expression. These results suggest that Cif may be useful to develop a new class of specific inhibitors of Pgp aimed at increasing the sensitivity of tumors to chemotherapeutic drugs, and at improving the bioavailability of Pgp transport substrates.

The P-Loop domain of yeast Clp1 mediates interactions between CF IA and CPF factors in Pre-mRNA 3′ end formation

 Cleavage factor IA (CF IA), cleavage and polyadenylation factor (CPF), constitute major protein complexes required for pre-mRNA 3' end formation in yeast. The Clp1 protein associates with Pcf11, Rna15 and Rna14 in CF IA but its functional role remained unclear. Clp1 carries an evolutionarily conserved P-loop motif that was previously shown to bind ATP. Interestingly, human and archaean Clp1 homologues, but not the yeast protein, carry 5' RNA kinase activity. We show that depletion of Clp1 in yeast promoted defective 3' end formation and RNA polymerase II termination; however, cells expressing Clp1 with mutant P-loops displayed only minor defects in gene expression. Similarly, purified and reconstituted mutant CF IA factors that interfered with ATP binding complemented CF IA depleted extracts in coupled in vitro transcription/3' end processing reactions. We found that Clp1 was required to assemble recombinant CF IA and that certain P-loop mutants failed to interact with the CF IA subunit Pcf11. In contrast, mutations in Clp1 enhanced binding to the 3' endonuclease Ysh1 that is a component of CPF. Our results support a structural role for the Clp1 P-loop motif. ATP binding by Clp1 likely contributes to CF IA formation and cross-factor interactions during the dynamic process of 3' end formation.