Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 

 

 

Title:  Gene encoding organic anion transporter

United States Patent:  6,432,631

Issued:  August 13, 2002

Inventors:  Cihlar; Tomas (San Mateo, CA)

Assignee:  Gilead Sciences, Inc. (Foster City, CA)

Appl. No.:  330245

Filed:  June 10, 1999

Abstract

This invention is concerned with human organic anion transporter ("hOAT"). Isolated nucleic acid encoding hOAT is provided, along with isolated hOAT polypeptide. hOAT nucleic acid and/or hOAT polypeptide are employed in transgenic animals, recombinant cells, replicable vectors and analytical procedures for identifying hOAT agonists or antagonists, assays for identifying hOAT alleles and/or isotypes, screening tests for nephrotoxic or neurologically active compounds, and determination of drug-drug interactions within the kidney or brain.

DETAILED DESCRIPTION OF THE INVENTION

hOAT polypeptide is defined as a polypeptide sequence that is at least about 85% homologous by amino acid sequence (ordinarily at least about 90%, and preferably at least about 95%).

An expressed sequence tag (EST) of approximately 200 bp having high homology to a segment of hOAT is found in the GenBank EST entries under accession no. R25797. Invention hOAT nucleic acids per se as defined herein exclude any expressed sequence tag (EST) or other nucleic acid sequences found in public databases on the filing date (such databases being expressly incorporated by reference), including the sequence of accession no. R25797 as well as the rat ROAT1 and OAT1, and mouse NKT, sequences of the prior art. However, other inventive subject matter such as isolated protein, methods for screening and the like as set forth above do not (unless expressly stated to the contrary) exclude the use of the R25797 sequence or its expression product.

"Homology" is defined as the percentage of residues in a candidate amino acid sequence that is identical with the residues in the reference sequence hOAT after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for the alignment are well know in the art. One computer program which may be used or adapted for purposes of determining whether a candidate sequence falls within this definition is "Align 2", authored by Genentech, Inc., which was filed with user documentation in the United States Copyright Office, Washington, D.C. 20559, on Dec. 10, 1991.

"Isolated" hOAT nucleic acid is one that has been separated from its environment as it is found in nature, i.e., from the genome in the case of DNA or from cellular environment in the instance of RNA.

"Isolated" hOAT polypeptide is one that has been separated from its normal cellular environment, and includes hOAT protein that is homogeneous by SDS-PAGE using silver stain.

In calculating amino acid sequence homology the candidate and reference sequences are aligned in the fashion that produces the maximum number of aligned residues, with insertions and deletions of residues represented by gaps in the aligned sequences. For example, a 120 residue polypeptide containing a 100 residue reference sequence fragment fused at its N-terminus to a 6 residue polyhistidine affinity tag, but with a single substitution in the hOAT domain, is calculated to be 99% homologous to the hOAT reference sequence since the sequence of the fragment corresponds exactly to the maximally aligned hOAT reference sequence except for a single residue substitution and the 6 residue N-terminal fusion. Thus, if the alignment-maximizing comparison of the candidate and reference sequences reveals an insertion (or deletion) of one or more amino acid residues, then these residues are ignored for the purposes of making the homology calculation. Applicant recognizes that this convention gives rise to theoretical 100% homology between 2 differing sequences, but has chosen to establish his own definition for the purposes of this filing.

Analysis of homology is based on any one or more of the sequences imputed from the nucleic acid used to express the hOAT, the sequence of the product as first produced in vitro, or the sequence after any post-translational modification. Thus, if the reference and candidate sequences are identical when expressed, but a glutamine residue is later deaminated to glutamic acid, the first candidate is 100% homologous, but the deaminated sequence is not.

For the purposes herein "hOAT activity" means any one or more of the functions performed by hOAT in the human, including in particular the transport of organic anions.

It is not necessary for a polypeptide to have anion transport activity in order to fall under the definition of hOAT herein. For example, in some embodiments hOAT polypeptides possess at least one immune epitope that is capable of substantial cross-reaction with an antibody raised against reference sequence hOAT, and thus are useful in immunoassays for hOAT, but may possess mutations that render the polypeptide incapable of anion transport.

The hOAT polypeptides of this invention comprise substitutions for, deletions of, or insertions of any amino acid residue adjacent to any of the reference sequence amino acid residue sites.  Substitutional hOATs are those in which at least one amino acid residue in the reference sequence has been removed and a different amino acid inserted in its place at the same position. One or more residues are substituted.

Alanine is a common substitution for any residue, and is commonly used in alanine scanning to identify functional residues, but it is within the scope of this invention to substitute other residues into the hOAT reference sequence. The introduced residues generally are naturally occurring amino acids, commonly G, A, Y, V, L, I, S, T, D, E, Q, C, M, N, F, P, W, K, R or H (using conventional single letter code; EP 323,149). Suitable residues also include hydroxyproline, beta-hydroxyaspartic acid, gamma-carboxyglutamic acid, hydroxylysine or norleucine, to be employed as alternatives to their namesakes.

These substitutions may be conservative, i.e., the substituting residue is structurally or functionally similar to the substituted residue. Other substitutions will be less conservative in that they constitute an exchange between different structural or functional classes of residues. For the purposes herein, these classes are as follows: 1. Electropositive: R, K, H; 2. Electronegative: D, E; 3. Aliphatic: V, L, I, M; 4. Aromatic: F, Y, W; 5. Small: A, S, T, G, P, C; 6. Charged: R, K, D, E, H; 7. Polar: S, T, Q, N, Y, H, W; and 8. Small Hydrophilic: C, S, T. Intergroup substitutions generally will have greater effects on protein function than conservative (intraclass) substitutions. Thus, it is particularly within the scope of this invention to introduce conservative substitutions into hOAT and, if the results are not satisfactory, to introduce non-conservative substitutions at the sites. Typically, however, proline, glycine, and cysteine substitutions or insertions into the sequence are not favored. An example of an expressed variant is a change at codon 498 from AGC to ATC, resulting in expression of isoleucine in place of serine. Other variants are introduced into DNA encoding hOAT without resulting in a change in protein sequence, e.g. from ATC to ATT at codon 453 or from GGG to GGT at codon 491.

hOAT variants are readily identified by methods apparent to the ordinary artisan. For example, sites shown by alanine scanning to influence selected biological activity are subjected to saturation mutagenesis to identify the optimal modification for the activity in question, e.g. selectivity for transport of a particular anion.

hOATs representing combinations of sequence variants are within the scope of this invention. 2, 3, 4, 5, or more substitutions, deletions or insertions are introduced into hOAT as defined herein. Typically, a deletion of a single residue will be accompanied by an insertion within 1 to about 3 residues of the deletion site. Generally, deletions of larger domains unnecessary for anion transport activity need not be accompanied by an insertion. The results of individual amino acid substitutions are generally additive except when the residues interact with each other directly or indirectly. They are readily screened using the same procedures described in Sweet et al. or Sekine et al. (supra) in order to identify those having the properties of reference sequence hOAT or the desired modified properties.

Included within the scope of this invention are hOATs having one or more amino acids inserted immediately adjacent to a hOAT amino acid at any position in the reference sequence. Insertional hOATs generally will have a polypeptide structure comprising the sequence NH2 -PP-A-(X)nl -B-PP-COOH, wherein X is the inserted residue(s) (which may be the same or different), n1 is an integer (generally 1-30, typically 1 or 2), either A or B are the designated residue sites for insertion and PP represents the remainder of the hOAT or a bond at the hOAT N or C terminus.

The invention includes fusions of hOAT and selected antibody recognition sequences (heterologous polypeptides) for immunoaffinity purification of hOAT from cell culture, fusions of hOAT sequences with affinity tags such as FLAG or polyhistidine, and chimeric sequences (particularly fusions of hOAT sequence fragments with fragments of other receptors of the 12-transmembrane spanning region class).

Also included within the scope of this invention are hOATs in which a glycosylation site is introduced or removed from the reference sequence, whether by substitution, insertion or deletion of one or more amino acid residues. Such changes will result in the installation or removal of the sequence NXS or NXT, where X can be any residue. Thus, asparagine can be substituted for any residue located 2 residues N-terminal to serine or threonine to introduce a glycosylation site. Alternatively, single glycosylation can be omitted by substituting glycosylated asp with any residue, deleting site-adjacent serine or threonine substituting any residue into the glycosylation site to perturb the NXS or NST sequence.

Also included within the scope of this invention are deletional hOATs, i.e., hOATs in which one or more amino acid residues of the reference sequence have been removed at a designated site, whereby flanking residues are now joined by a peptide bond in the ordinary fashion. It generally is not preferred to delete P, C or G residues.

Typically, deletions or insertions are relatively small, on the order of 1 to 10 residues and generally no more than 2, although deletions or insertions can be quite large if they are not in critical portions of the reference sequence, or the additional sequence is to be removed at some point during post-translational or post-recovery processing. The number of residues that are deleted or inserted in part will depend upon whether or not they are found in secondary structural components such as helices or sheets (whereupon only 1 or, preferably 2 residues are inserted or deleted), or are in less structurally confined domains such as loops, where larger numbers of residues may be deleted or inserted without unduly perturbing the structure of hOAT.

The hOATs of this invention may be subject to post-translational covalent modification, e.g. deamidation of asparagine or glutamine, or oxidation of cysteine residues. Glycosylation can be variant or absent depending upon the host cell used to express the variant or absent such modifications are included within the scope of this invention. If hOAT is glycosylated in recombinant cell culture, it preferably is glycosylated with carbohydrates characteristic of mammalian cells, although it also may bear fungal (such as yeast) glycosylation patterns. Glycosylation is acceptable which is characteristic of expression of hOAT from one or more of fibroblast, kidney, brain, lung, skin, neural liver or bone marrow cells or cell lines, or from any mammalian cell line such as CHO or embryonic kidney cells.

Naturally occurring human alleles are included within the scope of this invention. The readily are identified by obtaining nucleic acid samples from individuals in a population, sequencing hOAT from such individuals and determining residues at which variation is found. Once each variation is identified, it is straight-forward to determine the frequency of the putative allele in other individuals by PCR using primers specific for the domain in question, or such other methods as are conventional in the field for determining proportions of alleles in human populations.

Claim 1 of 5 Claims

What is claimed is:

1. A method for identifying an inhibitor of the hOAT mediated transport process comprising (a) providing a recombinant cell culture expressing biologically active hOAT having the amino acid sequence of SEQ ID No. 2, and providing a covalently modified form of a nucleotide phosphonate analogue a candidate inhibitor and (b) determining whether or not the analogue is transported by hOAT in the recombinant cell culture.
 


____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]