Protein-based Drug Transport
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 chemotherapeutic 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.
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 etween 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.
Selected Antibodies to Transport Proteins | ||||||||
|---|---|---|---|---|---|---|---|---|
| Product Name | Host | Clone No. | Form | Gene Symbol | Species Reactivity | Application | Prestige Antibody | Product 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 |
Selected Transporter Modulators | ||
|---|---|---|
| Name | Description | Product 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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