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Drug Transport

By: Robert Gates, Biofiles Volume 6 Article 1

Protein-based drug transporters are found in most tissues including liver, kidney, intestine, and brain. Because of their complexity and genetic heterogeneity, these proteins are often produced as recombinant membrane preparations expressed in Sf9 cells. These transporters are particularly important in cancer treatment and multi-drug resistance research. Understanding the specific mechanisms of tumor cell transporters is becoming an essential aspect of chemotherapuetic drug design.

ABC-type transporters are located in the plasma membrane Figure 1 and control the translocation of many classes of molecules. Some allow the specific passage of inorganic ions, while others facilitate ATP-dependent translocation of organic compounds including short peptides, lipids, bile acids, glutathione, and glucuronide conjugates.

v6a1-figure-1

Figure 1. The Bile salt export pump (BSEP), encoded by ABCB11, is a member of the Multi Drug Resistance (MDR)/ TAP subfamily of ATP-binding cassette (ABC) transporters. Members of the ABC transporter superfamily are defined by the sequence and organization of their ATP-binding cassette (ABC) domains. The ABC domains contain several conserved sequences, including a Walker A motif, Walker B motif, and the ABC signature motif.

BSEP is a canalicular-specific exporter and is the major human bile acid transport protein. Mutations in this gene are associated with a severe human disease, type 2 Progressive Familial Intrahepatic Cholestasis (PFIC2). Structurally, BSEP contains two transmembrane domains, each consisting of six membrane-spanning domains, and two ABC domains.

Typically, ABC transporters are composed of multiple transmembrane domains (TMD) and one or more ATP-binding domains (ABC).

Membrane preparations containing ABC transporters show a baseline ATPase activity that varies for different transporters. Transported substrates increase this baseline ATPase activity, while inhibitors or slowly transported compounds inhibit the baseline ATPase activity and/or the ATPase activity measured in the presence of a stimulating agent. Both activation and inhibition studies can be performed.

Transport Proteins

BSEP human

Bile salt export pump; ABCB11 The vesicular transport assay determines the interaction of compounds with the BSEP transporter. The interaction is detected by changes in the initial rate of 3H-taurocholic acid transport by BSEP into membrane vesicles purified from Sf9 cells expressing the transporters. Membrane preparations from infected cells always contain some closed membrane vesicles that have an inside-out orientation (5–10% of total lipid). In the case of these inside-out vesicles, transport of substrates across the membrane takes molecules from the surrounding buffer and transports them into the vesicles.

The bile salt export pump (BSEP/ABCB11) belongs to the family of ATP-binding-cassette (ABC) transporters and has also been called the sister of P-glycoprotein (sister Pgp). Most ABC transporters transport substrates across the cell membrane using ATP as an energy source. BSEP is the major bile salt transporter in the liver canalicular membrane and is inhibited by a number of drugs or drug metabolites. This is potentially a significant mechanism for druginduced cholestasis. Dysfunction of individual bile salt transporters such as BSEP, due to genetic mutation, suppression of gene expression, disturbed signaling, or steric inhibition, is an important cause of cholestatic liver disease.

The quantity of transported molecules can be determined by methods such as HPLC, LC/MS/ MS separation and detection, and also by labeling with fluorescent or radioactive (3H-taurocholic acid) tags. BSEP mediates the transport of taurocholic acid (TC) very efficiently. Compounds that interact with the transporter modulate the initial rate of TC transport measured without any other compounds added. If a substance is a transported substrate of the transporter, it might compete with TC, thus reducing the rate of TC transport. If a compound is an inhibitor of the transporter, it will block the transport of TC into the membrane vesicles. Some compounds can be co-transported with TC, increasing the rate of TC transport compared to the control level.


MDR1 human

Pgp; ABCB1 The MDR1 protein is involved in cancer drug resistance and in the transport of hydrophobic drugs and xenobiotics in the bowel, kidney, liver, and the blood-brain barrier. Drugs interacting with this protein may be useful for the reversal of cancer drug resistance or increasing the absorption or brain entry of various pharmacological agents.

Detection of ATPase activity of the MDR1 protein is a measure of transporter activity. The assay is performed using purified membrane vesicles from Sf9 (Spodoptera frugiperda) cells, expressing high levels of MDR1 protein. The ABC transporters pump substrates out of the cell by using hydrolysis of ATP as an energy source. ATP hydrolysis yields inorganic phosphate (Pi), which can be detected by a simple colorimetric reaction. The amount of Pi liberated is proportional to the activity of the transporter.


MDR1B from rat

The MDR1 protein is involved in cancer drug resistance and in the transport of hydrophobic drugs and xenobiotics in the bowel, kidney, liver, and the blood-brain barrier. In rodents, there are two MDR1 genes, MDR1A and MDR1B, while in human, there is a single MDR1 gene. Based on function and tissue distribution in rodents, the equivalent of the human MDR1 gene product (PgP) is the product of the rodent MDR1B gene. There have been no reported significant differences in function, substrate specificity, or substrate affinity between these two proteins

Detection of the ATPase activity of the Mdr 1b protein is a measure of transporter activity. The assay is performed using purified membrane vesicles from Sf9 (Spodoptera frugiperda) cells, expressing high levels of Mdr 1b protein. The ABC transporters pump substrates out of the cell by using hydrolysis of ATP as an energy source. ATP hydrolysis yields inorganic phosphate (Pi), which can be detected by a simple colorimetric reaction. The amount of Pi liberated is proportional to the activity of the transporter.


MRP2 human

The vesicular transport assay determines the interaction of compounds with the MRP2 transporter. The interaction is detected by changes in the initial rate of 3H-β-estradiol 17-(β-Dglucuronide) transport by MRP2 into membrane vesicles purified from Sf9 cells expressing the transporters. Membrane preparations from infected cells always contain some closed membrane vesicles that have an inside-out orientation (5-10% of total lipid). In the case of these inside-out vesicles, transport of substrates across the membrane takes molecules from the surrounding buffer and transports them into the vesicles.

MRP2 (ABCC2) is an organic anion transporter found in the liver, kidney, and gut epithelium apical membranes. The transport of glucuronate conjugates plays a role in the detoxification of endogenous and xenobiotic substances, and may cause multidrug resistance (MDR) in tumor cells.

The quantity of transported molecules can be determined by methods such as HPLC, LC/MS/ MS separation and detection, and also by labeling with fluorescent or radioactive (3H-β-estradiol 17-(β-D-glucuronide) tags. MRP2 mediates the transport of β-estradiol 17-(β-D-glucuronide) (E217βG) very efficiently. Compounds that interact with the transporter modulate the initial rate of E217βG transport measured without any other compounds added. If a substance is a transported substrate of the transporter, it might compete with E217βG, thus reducing the rate of E217βG transport. If a compound is an inhibitor of the transporter, it will block the transport of E217βG into the membrane vesicles. Some compounds can be co-transported with E217βG increasing the rate of E217βG transport compared to the control level.


MRP2 from rat

Detection of ATPase activity of the Mrp2 protein is a measure of transporter activity. The assay is performed using purified membrane vesicles from Sf9 (Spodoptera frugiperda) cells, expressing high levels of Mrp2 protein. ABC transporters pump substrates out of the cell by using hydrolysis of ATP as an energy source. ATP hydrolysis yields inorganic phosphate (Pi), which can be detected by a simple colorimetric reaction. The amount of Pi liberated is proportional to the activity of the transporter.

MRP2 (ABCC2) is an organic anion transporter found in liver, kidney, and gut epithelium apical membranes. The transport of glucuronate conjugates plays a role in the detoxification of endogenous and xenobiotic substances, and may cause multidrug resistance (MDR) in tumor cells. The rat Mrp2 transporter shows 72.3% sequence identity and 85.6% sequence similarity with human MRP2. Both transporters are expressed on the canalicular membrane of the liver and are known to be responsible for the transport of some organic molecules and their conjugates to the bile.


MXR human

MXR membrane vesicles are purified from recombinant baculovirus transduced Sf9 cells or selected, MXR over-expressing mammalian cells. Membrane preparations from transporter expressing cells always contain some closed membrane vesicles that are inside-out orientation (5–10% of total lipid). In the case of these insideout vesicles, the transport of substrates across the membrane takes molecules from the buffer in which the membrane is suspended and transports them into the vesicles. The rate of this transport is temperature and ATP dependent.

The quantity of transported molecules can be determined by methods such as HPLC, LC/MS/ MS separation and detection, and also by labeling with fluorescent or radioactive (3H labeled MTX) tags.

Methotrexate (MTX) is a transported substrate of the MXR transporter with low affinity and high capacity. The vesicular transport assay provides information on any interaction between the MXR transporter and the test compound that would affect the transport of the reporter substrate (3H-Methotrexate ) into the membrane vesicles. If a test compound is an activator or inhibitor of the MXR transporter, it competes with MTX, thus reducing the rate of MXR mediated MTX transport. Distributed for SOLVO Biotechnology, Inc.

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Selected Antibodies to Transport Proteins

Product Name Host Clone No. Form Gene Symbol Species Reactivity Application Prestige Antibody Cat. No.
Anti-ABCB10 (N-term) rabbit - IgG fraction of antiserum ABCB10, human human ELISA (i) - SAB1300312-100UG
Anti-ABCB6 (C-term) rabbit - IgG fraction of antiserum ABCB6, human human ELISA (i) IHC WB - SAB1300078-100UG
Anti-ABCB7 (C-term) rabbit - IgG fraction of antiserum ABCB7, human human ELISA (i) IHC WB - SAB1300313-100UG
Anti-ABCC4 rabbit - affinity isolated antibody ABCC4, human human IHC (p) PA Yes HPA002476-100UL
Anti-ABCC9 rabbit - affinity isolated antibody ABCC9, human human IHC (p) PA Yes HPA007279-100UL
Anti-AF130358.1 rabbit - affinity isolated antibody AF130358.1, human human IHC (p) PA Yes HPA027071
Anti-CDH17 rabbit - affinity isolated antibody CDH17, human human IHC (p) PA Yes HPA023616-100UL
Anti-CDH17 rabbit - affinity isolated antibody CDH17, human human IHC (p) PA Yes HPA023614-100UL
Anti-CFTR rabbit - affinity isolated antibody CFTR, human human IHC (p) PA Yes HPA021939-100UL
Anti-Dopamine Transporter (N-terminal) rabbit - affinity isolated antibody SLC6A3, human Slc6a3, rat Slc6a3, mouse human mouse (predicted) rat WB - D6944-25UL
D6944-200UL
Anti-GABA Transporter GAT-3 rabbit - affinity isolated antibody Slc6a11, rat rat IHC - G8407-.1ML
Anti-LST-3TM12 (ab1) rabbit - affinity isolated antibody LST-3TM12, human human WB - SAB2101401-50UG
Anti-LST-3TM12 (ab2) rabbit - affinity isolated antibody LST-3TM12, human human WB - SAB2101402-50UG
Anti-MRP8/ABCC11 goat - affinity isolated antibody ABCC11, human bovine canine human ELISA (i) WB - SAB2500647-100UG
Anti-OSTB rabbit - affinity isolated antibody OSTbeta, human human IHC (p) PA Yes HPA008533-100UL
Anti-PMAT(Slc29a4) (N-term) rabbit - IgG fraction of antiserum PMAT, human human ELISA (i) IHC WB - SAB1300519-100UG
Anti-SLC10A2 goat - affinity isolated antibody Slc10a2*, mouse mouse rat ELISA (i) WB - SAB2500947-100UG
Anti-SLC10A5 rabbit - affinity isolated antibody SLC10A5, human human IHC (p) PA WB Yes HPA025966-100UL
Anti-SLC10A5 rabbit - IgG fraction of antiserum SLC10A5, human human IHC WB - AV43773-100UG
Anti-SLC10A7 rabbit - affinity isolated antibody SLC10A7, human human WB - SAB2102163-50UG
Anti-SLC15A1 rabbit - affinity isolated antibody SLC15A1, human human IHC (p) PA Yes HPA002827-100UL
Anti-SLC16A1 rabbit - affinity isolated antibody SLC16A1, human human IF (i) IHC (p) PA WB Yes HPA003324-100UL
Anti-SLC16A3 rabbit - affinity isolated antibody SLC16A3, human human IHC (p) PA WB Yes HPA021451-100UL
Anti-SLC16A7/MCT2 goat - affinity isolated antibody SLC16A7, human chimpanzee human ELISA (i) WB - SAB2500948-100UG
Anti-SLC18A2 rabbit - affinity isolated antibody SLC18A2, human human IHC (p) PA Yes HPA016856-100UL
Anti-SLC1A1 rabbit - affinity isolated antibody SLC1A1, human human IHC (p) PA Yes HPA020086-100UL
Anti-SLC1A2 rabbit - affinity isolated antibody SLC1A2, human human IHC (p) PA Yes HPA009172-100UL
Anti-Slc22A17 rabbit - affinity isolated antibody SLC22A17, human human mouse rat ELISA (i) IF (i) WB - SAB3500306-100UG
Anti-SLC22A17 rabbit - affinity isolated antibody SLC22A17, human human IHC (p) PA Yes HPA002728-100UL
Anti-SLC22A2 rabbit - IgG fraction of antiserum SLC22A2, human human WB - AV43847-100UG
Monoclonal Anti-SLC22A2 mouse 2D2 purified immunoglobulin SLC22A2, human human ELISA (i) WB - WH0006582M1-100UG
Anti-SLC22A3 rabbit - affinity isolated antibody SLC22A3, human human IHC (p) PA Yes HPA029750-100UL
Anti-SLC22A7 rabbit - affinity isolated antibody SLC22A7, human human WB - SAB2102178-50UG
Anti-SLC22A8 rabbit - affinity isolated antibody SLC22A8, human human WB - SAB2102179-50UG
Anti-SLC25A26 rabbit - affinity isolated antibody SLC25A26, human human IHC (p) PA Yes HPA026887-100UL
Monoclonal Anti-SLC27A4 mouse 1F4-1B10 purified immunoglobulin SLC27A4, human human ELISA (i) WB - WH0010999M1-100UG
Anti-SLC29A1 rabbit - affinity isolated antibody SLC29A1, human human IHC (p) PA WB Yes HPA012384-100UL
Anti-SLC29A2 rabbit - affinity isolated antibody SLC29A2, human human IHC (p) PA Yes HPA018168-100UL
Anti-SLC36A4 rabbit - affinity isolated antibody SLC36A4, human human IHC (p) PA Yes HPA017887-100UL
Anti-SLC44A2 rabbit - affinity isolated antibody SLC44A2, human human IHC (p) PA Yes HPA003228-100UL
Anti-SLC6A1 rabbit - affinity isolated antibody SLC6A1, human human WB - SAB2102222-50UG
Anti-SLC6A2 rabbit - affinity isolated antibody SLC6A2, human human WB - SAB2102224-50UG
Monoclonal Anti-SLC6A4 mouse 2A9 purified immunoglobulin SLC6A4, human human ELISA (i) - WH0006532M6-100UG
Monoclonal Anti-SLC6A5 mouse 3B3 purified immunoglobulin SLC6A5, human human ELISA (i) WB - WH0009152M1-100UG
Anti-SLC6A6 rabbit - affinity isolated antibody SLC6A6, human human IHC (p) PA Yes HPA015028-100UL
Anti-SLC6A7 rabbit - affinity isolated antibody SLC6A7, human human IHC (p) PA Yes HPA028907-100UL
Anti-SLC7A3 rabbit - affinity isolated antibody SLC7A3, human human IHC (p) PA Yes HPA003629-100UL
Anti-SLCO3A1 rabbit - affinity isolated antibody SLCO3A1, human human WB - SAB2102229-50UG
Anti-SLCO5A1 rabbit - affinity isolated antibody SLCO5A1, human human IHC (p) PA Yes HPA025062-100UL
Anti-TAP1 rabbit - affinity isolated antibody TAP1, human human WB - SAB2102370-50UG
Anti-TETRAN rabbit - affinity isolated antibody TETRAN, human human IHC WB - AV45054-50UG
Anti-Vesicular Acetylcholine Transporter (VAChT) rabbit - affinity isolated antibody Slc18a3, rat SLC18A3, human human rat IF (i) IHC (p) WB - V5387-.2ML
Anti-Vesicular Monoamine Transporter (VMAT-2) guinea pig - whole antiserum Slc18a2, rat rat IHC (f ) - V6637

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Selected Transporter Modulators

Name Description Catalog No.
1-Benzoyl-5-methoxy-2-methylindole-3-acetic acid Putative inhibitor of multidrug resistance-associated protein 1 (MRP1). B1183-5MG
Berberine chloride form Fluorescent stain for heparin in mast cells An alkaloid with weak antibiotic properties. Substrate for MDR efflux pumps. Antimicrobial activities of berberine is potentiated by the MDR inhibitor 5´-methoxyhydnocarpin (5´-MHC). Berberine upregulates the expression of Pgp in hepatoma cells. Treatment with berberine potentially results in the reduced accumulation of chemotherapeutic drugs. B3251-5G
B3251-10G
B3251-25G
Berberine hemisulfate salt Fluorescent stain for heparin in mast cells An alkaloid with weak antibiotic properties. Substrate for MDR efflux pumps. Antimicrobial activities of berberine is potentiated by the MDR inhibitor 5´-methoxyhydnocarpin (5´-MHC). Berberine upregulates the expression of Pgp in hepatoma cells. Treatment with berberine potentially results in the reduced accumulation of chemotherapeutic drugs. B3412-10G
1-(4-Chlorobenzyl)-5-methoxy-2- methylindole-3-acetic acid Putative inhibitor of multidrug resistance-associated protein 1 (MRP1). C1610-5MG
Chloroquine diphosphate salt DNA intercalator. Also used to increase transfection efficiency. Standard anti-malarial drug. Substrate for MRP in multidrug resistant cell line and inhibits photoaffinity labeling of MRP by quinolinebased photoactive drug IAAQ (N-[4-[1-hydroxy-2-(dibutylamino) ethyl]quinolin-8-yl]-4-azidosalicylamide). C6628-25G
C6628-50G
C6628-100G
C6628-250G
1-Deoxyforskolin from Coleus forskohlii P-GPefflux inhibitor D1290-1MG
9-Deoxyforskolin from Coleus forskohlii P-GPefflux inhibitor D4665-1MG
1-(4-Fluorobenzyl)-5-methoxy-2-methylindole-3-acetic acid Putative inhibitor of multidrug resistance-associated protein 1 (MRP1). F2927-5MG
Indomethacin Cyclooxygenase (COX) inhibitor that is relatively selective for COX-1. I7378-5G
I7378-10G
I7378-25G
I7378-100G
Ko143 Ko143 has been used as a positive control inhibitor on functions of BCRP in MCF7 and BCRP over-expressing MCF7/MX100 cell lines using a BCRP prototypical substrate mitoxantrone.
K2144-1MG
K2144-5MG
Mefenamic acid An NSAID. Circumvents MRP-mediated multidrug resistance. Specifically and significantly potentiates the cytotoxicity of anthracyclines as well as teniposide, VP-16 and vincristine. M4267-50G
M4267-500G
PGP-4008 Selectively inhibits P-glycoprotein. P6490-2MG
Probenecid Useful as an inhibitor of the organic anion transporter, MRP. P8761-25G
P8761-100G
Reversin 121 Peptide chemosensitizer, inhibitor of P-glycoprotein. R1276-3MG
Reversin 205 Peptide chemosensitizer, inhibitor of P-glycoprotein. R1401-10MG
Staurosporine from Streptomyces sp. Potent inhibitor of phospholipid/calcium-dependent protein kinase. Inhibits the upregulation of VEGF expression in tumor cells. Partially reverses MDR, sensitizing cells with MDR phenotype to cytotoxic agents. Inhibits Pgp phosphorylation. S4400-.1MG
S4400-.5MG
S4400-1MG
Staurosporine from Streptomyces sp. Potent inhibitor of phospholipid/calcium-dependent protein kinase. Inhibits the upregulation of VEGF expression in tumor cells. Partially reverses MDR, sensitizing cells with MDR phenotype to cytotoxic agents. Inhibits Pgp phosphorylation. S5921-.1MG
S5921-.5MG
S5921-1MG
Staurosporine solution from Streptomyces sp. Potent inhibitor of phospholipid/calcium-dependent protein kinase. Inhibits the upregulation of VEGF expression in tumor cells. Potent cell-permeable inhibitor of protein kinase C. Induces apoptosis in Jurkat cells. S6942-200UL
Sulindac Treatment of human colorectal cancer cell lines induces MRP1 and MRP3 but not other members of the MRP family. Reported to significantly increase the cytotoxicity of the anthracyclines as well as teniposide, VP-16 and vincristine. Nonsteroidal anti-inflammatory; preferential inhibitor of COX-1. S8139-5G
S8139-25G
Tolmetin sodium salt dihydrate An NSAID. Circumvents MRP-mediated multidrug resistance. Significantly increases the cytotoxicity of the anthracyclines as well as teniposide, VP-16 and vincristine. T6779-1G
T6779-5G
R(+)-Verapamil monohydrochloride hydrate Inhibitor of P-glycoprotein; less active enantiomer of (±)-verapamil. V106-5MG
V106-25MG
Zomepirac sodium salt An NSAID. Circumvents MRP-mediated multidrug resistance. Significantly increases the cytotoxicity of the anthracyclines as well as teniposide, VP-16 and vincristine. Z2625-250MG
Z2625-1G
Z2625-5G

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