Multidrug resistance associated proteins and uses thereof

Details Multidrug resistance associated proteins and uses thereof Multidrug resistance associated proteins and uses thereof

1999-03-31

2004-07-06

Park, Hankyel T. (Department: 1648)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S006120, C435S069100, C530S389100, C536S023100

Reexamination Certificate

active

06759238

ABSTRACT:

Throughout this application, certain publications are referenced by number. Full citations for these publications may be found listed at the end of the specification and preceding the Claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art. A Sequence Listing is provided.
FIELD OF THE INVENTION
The present invention relates generally to to multi-drug resistance, and more particularly to materials such as multi-drug resistant proteins (MRP), and to possible diagnostic and therapeutic uses thereof. The invention further relates to methods for identifying treatments refractive to drug resistance.
BACKGROUND OF THE INVENTION
Microbial and cellular resistance to drug therapy is a major and long-standing problem to the treatment of disease and infection, including cancer. Cross-resistance between different anti-microbial and anti-cancer agents, which are structurally and functionally distinct, is a relatively common phenomenon called multi-drug resistance (MDR).
With respect to cancers, some malignant tumors respond poorly to chemotherapy, indicating that the target cells are intrinsically resistant. Other tumors initially respond well to chemotherapy, but appear to develop resistance, indicating a selection process or cellular response to the chemotherapeutic agent(s). The broad-spectrum resistance characteristic of MDR, therefore, is of great clinical significance.
MDR was initially described in cultured tumor cells which following selection for resistance to a single anti-tumor agent became resistant to a range of chemically diverse anti-cancer agents (52). These MDR cells exhibited a decrease in intracellular drug accumulation due to active efflux by transporter proteins. The so-called “multi-drug transporters” are membrane proteins capable of expelling a broad range of toxic molecules from the cell (53). These multi-drug transporters belong to the ATP-binding cassette (ABC) superfamily of transport proteins that utilize the energy of ATP hydrolysis for activity (53, 57). In microorganisms, multi-drug transporters play an important role in conferring antibiotic resistance on pathogens.
Several mechanisms have been described as responsible for MDR. The most well characterized gene conferring drug resistance by an ATP-dependent efflux mechanism is the MDR1 gene product, P-glycoprotein (Pgp). a member of the ABC cassette family of transporters. Pgp removes hydrophobic drugs of diverse chemical structures from cells as an efflux pump (55).
Another transporter protein, capable of conferring drug resistance, the multi-drug resistance protein (MRP), has been identified in a number of MDR human tumor cell lines that do not appear to express Pgp (52). The presence of MRP at the cell surface of such cells has been associated with alterations in drug accumulation and distribution (52). Expression of MRP causes a form of multi-drug resistance similar to that conferred by Pgp (52). The two proteins, however, are only distantly related. MRP has also been shown to be a primary active transporter of a structurally diverse range of organic anionic conjugates. Like Pgp, MRP has a broad substrate specificity. In addition to hydrophobic compounds, MRP is able to transport metallic oxyanions and glutathione and other conjugates, including peptidyl leukotrienes (52). This is in contrast to Pgp. (Stride, B D, et al., 1997, Mol. Pharmacol., 52:344-53). The mechanism by which MRP transports these compounds and mediates multi-drug resistance is not understood. In addition, topoisomerase II has been associated with MDR. Like Pgp, MRP is expressed in normal human tissues in addition to tumor cells (52). In normal cells, MRP appears to be located within the cytoplasm, indicating that it may function differently in normal cells as compared with tumor cells (52). Homologs of human Pgp and MRP have been found in microorganisms such as Plasmodium falciparum, candida albicans, Saccharomyces cerevisiae and Lactococcus lactis (53).
Although MDR1 was cloned some time ago, proteins in animal cells that were functionally similar were not readily identified. MRP has been described which in some cell types confer a drug resistance phenotype similar to the MDR1 gene (58). The prototype MRP1 gene was first described in 1992. Subsequently, MRP2 (cMOAT) was cloned. Both MRP1 and MRP2 act to efflux anionic compounds, including drugs or endogenous compounds. Several yeast MRP homologues have been identified (49) and recently, additional human homologues have been identified in the EST databases. Particularly, Borst and colleagues searched the EST database and identified four additional family members (MRP2, MRP3, MRP4, and MRP5). Nonetheless, the human MRP homologues have until now remained functionally undefined. MRP3 has been described as exhibiting high expression in some cell lines but not in others, with overexpression of MRP3 in resistant lines being identified in several doxorubicin-resistant and cisplatin-resistant cell lines (49). MRP5 was identified as being very widely expressed. MRP4 in contrast to MRP3 and MRP5, was not reportedly overexpressed in any cell line analyzed (49). Importantly, the EST-based primary MRP4 sequence determined lacks several crucial pieces of information including: (1) a classic Walker A motif which is a signature of the ATP-binding domain found in ABC cassette transporter members; (2) more than 90% of the protein sequence; and (3) any functional marker. MRP3, MRP4 and MRP5 have been localized to a different chromosome than MRP1 and MRP2, indicating that they are not merely alternative splicing products (49).
ABC transporters are integral membrane proteins involved in ATP-dependent transport across biological membranes. Members of this superfamily play roles in a number of phenomena of biomedical interest, including cystic fibrosis (CFTR) and multi-drug resistance. Many ABC transporters are predicted to consist of two functional domains, a membrane-spanning domain and a cytoplasmic domain. The latter contain conserved nucleotide-binding motifs with the former containing substrate binding or recognition sites. Attempts to determine the structure of ABC transporters and of their separate domains have not yet been successful (57).
The ABC transporters of glutathione S-conjugates and related amphiphilic anions have been identified as MRP1 and MRP2. These 190-kDa membrane glycoproteins have been cloned. MRP1 and MRP2 have been shown to be unidirectional, ATP-driven, export pumps with an amino acid identity of 49% in humans. MRP1 is detected in the plasma membrane of many cell types, including erythrocytes. MRP2, also known as canalicular MRP (cMRP) or canalicular multispecific organic anion transporter (cMOAT), has been localized to the apical domain of polarized epithelia, such as the hepatocyte canalicular membrane and kidney proximal tubule luminal membrane. Physiologically important substrates of both transporters include glutathione S-conjugates, such as leukotriene C4, as well as bilirubin glucuronides, 17&bgr;-glucuronosyl estradiol and glutathione disulfide. Both transporters have been associated with multiple drug resistance of malignant tumors because of their capacity to pump drug conjugates and drug complexes across the plasma membrane into the extracellular space. The substrate specificity of MRP1 and MRP2 studied in inside-out oriented membrane vesicles is very different from MDR1 (Pgp). MRP1 and MRP2 have been called conjugate transporting ATPases, functioning in detoxification and, because of their role in glutathione disulfide export, in the defense against oxidative stress (54).
A cDNA encoding another ATP-binding cassette transporter, MOAT-B, has been reportedly cloned and mapped (56). Comparison of the MOAT-B predicted protein with other transporters revealed that it is most closely related to MRP. cMOAT, and the yeast organic anion transporter YCF1. Although MOAT-B is closely related to these transporters, it is distinguished by the absence of a approx

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