Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-07-12
2004-01-20
Eyler, Yvonne (Department: 1647)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S024300, C435S069100, C435S320100, C435S325000, C530S350000
Reexamination Certificate
active
06680379
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to specific proteins as well as recombinant versions of these proteins which are organic anion transporters. These proteins include human membrane proteins. preferentially found in kidney and liver cells. The present invention also relates to nucleotide sequences encoding these novel organic anion transporters. In another aspect, the present invention relates to methods for using these proteins as in vitro screening agents to identify substrates for the proteins and inhibitors which block transport activity. In yet another aspect, these proteins may be used in in vitro assays to predict drug pharmacokinetics, drug distribution and drug toxicity. The invention also discloses antibodies that specifically bind to these proteins and which may be used in diagnostic assays.
BACKGROUND OF THE INVENTION
The following describes certain relevant art, none of which is admitted to be prior art to the inventions described herein.
The liver and kidney are two organs that can extract a variety of organic anions from circulation, including endogenous compounds such as bile acids and bilirubin and xenobiotics such as sulfobromophthalein (BSP) and p-aminohippurate (PAH). The liver and kidney thus have the critical functions of bile secretion, detoxification, and drug elimination. Since the cell membrane represents a hydrophobic barrier that prevents influx of charged or hydrophilic molecules, hepatocytes and kidney epithelial cells express proteins on their basolateral membranes to facilitate transport of organic anions into the cell. The liver and kidney contain membrane proteins that transport very specific compounds such as taurocholic acid or prostaglandins. However, they also have proteins that exhibit a wider substrate specificity. Recently, transporters of the latter type have been cloned from rat liver and kidney. One such protein is called the organic anion transporting polypeptide, oatp (Jacquemin et al. (1994)
PNAS
91:133-37). This protein belongs to a family of related transporters, members of which consists of oatp1, oatp2, oatp3, and prostaglandin transporters. These proteins share homology at the amino acid level (Noe et al. (1997)
PNAS
94: 10346-50; Kanai et al. (1995)
Science
268: 866-69; Abe et al. (1998)
J. Biol. Chem.
273: 22395-401). When expressed in Xenopus oocytes or in mammalian cells, members of the oatp family were shown to transport many types of organic anions including taurocholic acid, BSP, and conjugated steroid hormones. The related prostaglandin transporters show high affinity transport of prostaglandins.
Shortly after the cloning of oatp1, members of another organic anion transporter family, OAT1 and OAT2, were isolated from rat kidney and liver, respectively (Sekine et al. (1997)
J. Biol. Chem
272: 18526-29; Sekine et al. (1998)
FEBS Letters
429: 179-82). Rat OAT1 and OAT2 are homologous to each other but show no significant homology to members of the oatp family. However, they show some homology to the family of organic cation transporters (OCTs), suggesting that these two families of proteins share a common origin. Rat OAT1 and OAT2 can transport many different organic anions. OAT1, when expressed in oocytes, can transport p-aminohippurate (PAH), methotrexate, and glutarate. OAT1 is most likely the molecular entity responsible for the classical p-aminohippurate/&agr;-ketoglutarate exchanger found in the kidney proximal tubule. OAT2, expressed predominantly in the liver, has been shown to transport organic anions such as salicylate, methotrexate, and &agr;-ketoglutarate. Therefore, OAT1 and OAT2 belong to yet another family of multi-specific organic anion transporters distinct from the oatp family.
SUMMARY OF THE INVENTION
The present invention is directed to a group of polypeptides, preferentially expressed in the liver and kidneys of humans and which have activity as organic anion transporters. We refer to these polypeptides as “human organic anion transporters” or “hOAT polypeptides”. These proteins and their properties are described in detail below.
The invention also concerns, nucleic acids encoding hOAT polypeptides, cells, tissues and animals containing such nucleic acids, antibodies to the polypeptides, assays utilizing the polypeptides, and methods relating to all of the foregoing.
A first aspect of the invention features an isolated, enriched, or purified nucleic acid molecule encoding an hOAT polypeptide or encoding a fragment of an hOAT polypeptide.
In preferred embodiments the isolated nucleic acid comprises a nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or a functional derivative thereof, a nucleic acid sequence that hybridizes to the nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or a functional derivative thereof.
The nucleic acid may be isolated from a natural source by cDNA cloning or subtractive hybridization; the natural source may be mammalian (human) blood, semen, or tissue and the nucleic acid may be synthesized by the triester or other method or by using an automated DNA synthesizer. Preferably, the nucleic acid is isolated from mammalian kidney or brain, most preferably from liver.
Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones can be obtained directly from total DNA or from total RNA. The cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 10
6
-fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
In another preferred embodiment, the nucleic acid molecules of the invention comprise nucleotide sequences that (a) have the nucleic acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or a functional derivative thereof, or encode polypeptides having the full-length amino acid sequences set forth in SEQ ID NO:7, SEQ ID NO:8; SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or a functional derivative thereof, (b) is the complement of the nucleotide sequence of (a); (c) hybridizes under highly stringent conditions to the nucleotide molecules of (a) and encodes a naturally occurring hOAT polypeptide; (d) encodes an hOAT polypeptide having the full-length amino acid sequence of the sequence set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12 except that it lacks one or more of the domains selected from the group consisting of an extracellular domain, a transmembrane domain, and an intracellular domain; or (e) is the complement of the nucleotide sequence of (d).
The nucleic acid molecules of the invention are isolated, enriched, or purified preferably from a mammal, more preferably from a human.
Hybridization techniques may be used to isolate a nucleic acid molecule of interest. Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. Stringency is controlled by varying salt or denaturant concentrations. Highly stringent conditions may mean conditions that are at least as stringent as the following: hybridization in 50% formamide, 5×SSC, 50 mM NaH
3
PO
4
, pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5×Denhart solution at 42° C. overnight; washing with 2×SSC, 0.1% SDS at 45° C.; and washing with 0.2×SSC, 0.1% SDS a
DeBerry Regina M.
Eyler Yvonne
Metabasis Therapeutics, Inc.
Paul Hastings Janofsky & Walker LLP
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