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.

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 (P_i), which can be detected by a simple colorimetric reaction. The amount of P_i 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 (P_i), which can be detected by a simple colorimetric reaction. The amount of P_i 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.