LEPTIN ASSAY

Abstract: A trans-activation assay for leptin is described. Leptin response elements are located proximal to a promoter, and the promoter region is operatively linked to a reporter gene. When leptin binds with the receptor, the reporter gene is transcribed.

Description: TITLE OF THE INVENTION LEPTIN ASSAY FIELD OF THE INVENTION This invention relates to an assay for determining the presence of compounds which bind to a leptin receptor. This invention also relates to an assay for quantifying the amount of a leptin receptor binding agent present in a sample. BACKGROUND OF THE INVENTION Leptin is a product of the Ob gene (Zhang et al., 1995 Nature 372:425-432). In its absence, mice develop extreme obesity resembling morbid obesity of humans. They also develop diabetes resembling human type D diabetes (Zhang et al., 1995 Nature 372:425- 432), and become infertile (Chehab et ai, 1996 Nature Genetics 12:318- 320). Repletion of leptin leptin reverses these pathologies. Currently, the only manner in which preparations of recombinant leptin can be assayed is by injection of large quantities into obi ob mice (Campfield 1995 Science 269:546-549; Rentsch 1995 Biochem Biophys Res Comm 214:131 -136). Leptin activity is assessed based upon depression of feed intake and is highly variable among individuals. No mutually agreed upon standard of leptin activity exists. Therefore, a rapid, accurate assay for leptin bioactivity which utilizes small amounts of leptin would be desirable. DETAILED DESCRIPTION OF THE INVENTION This invention relates to a method for determining if a leptin receptor binding compound is present in a sample comprising: contacting the sample with a cell which comprises a) nucleic acids comprising a promoter region, said promoter region comprising a promoter and at least one leptin response element; said promoter region operatively linked to a reporter gene; and b) nucleic acids encoding a leptin receptor; and determining if transcription of the reporter gene occurs. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing functional assay of leptin using GTI -7 cells. Cells were either: mock transfected with respect to ob-r, transfected with lean rat Ob-r cDNA, or transfected human Ob-r cDNA and showed induction of luciferase activity in cell expressing Ob-r treated with leptin. Figure 2 is a graph showing that cells respond in a linear, dose-responsive manner to the addition of leptin as determined by increase in luciferase activity. GTI -7 cells were transiently transfected with human Ob-r and treated with various amounts of human leptin. Figure 3 is the DNA sequence for lean rat Ob-r. Figure 4 is the DNA sequence for human Ob-r which has been placed in the pCMV4 vector. The Gen Bank accession number for human ob-r is U43168. Nucleotides 141 -37-7-0 are used in pCMV4- Ob-4. Figure 5 is the DNA sequence for fatty rat Ob-r. As used throughout the specification and claims, the following definition shall apply: "Leptin receptor binding compound" means a compound which binds to a leptin receptor. "Leptin mimetic" means a compound other than leptin which binds to a leptin receptor and triggers a cascade of intracellular reactions which ultimately results in transcription of DNA under the control of leptin response elements. "Leptin response elements" means DNA sequences located within a promoter region which are responsive to a leptin receptor binding event, and in the presence of such an event, allow for transcription of the DNA under the control of the promoter. "Promoter region" means DNA located upstream of a protein or peptide encoding sequence, and includes at least one response element. "Promoter" includes full length promoters, minimal promoters and promoters which are less than full length, but include more nucleic acids than minimal promoters. "Ob-r" means Ob-receptor. One aspect of this invention is a cellular "trans" activation assay which can determine if a particular compound can bind to a leptin receptor. In general, the binding of a ligand to its membrane -bound receptor initiates an intercellular cascade of signals. Ultimately, a specific transcriptional control element is activated, leading to the transcription of DNA. It has been found, in accordance with this invention, that the binding of native leptin with its native leptin receptor initiates a cascade which activates leptin response elements, leading to the transcription of DNA. One such leptin response element which has been identified in accordance with this invention is an IRF-1 derived garnma-interferon activation sequence. IRF-1 derived activation sequences have been described in the art (Pine et al., 1994 EMBO J. 13:158-167) but their responsiveness to leptin-leptin receptor binding events has been heretofore unknown. This discovery of a leptin response element has allowed for the design of various assays which form the basis of this invention. A promoter region is constructed which contains a promoter of choice and one or more leptin response elements. The promoter region may contain be a hybrid promoter region, i.e. contain a promoter and response elements which do not naturally occur together, or they may be a naturally occurring promoter region. In a preferred embodiment the promoter is a well-characterized promoter, such as the heφes simplex virus thymidine kinase promoter, but any promoter which is known to function in the host cell chosen may be used. It is also preferred that the promoter be a minimal promoter, so that there are no transcriptional control sequences which may influence the activity of the leptin response element(s). The leptin-response element is preferably placed proximal to the promoter region. Intervening sequences may be present between the leptin response element and the promoter, provided they do not interfere with the functioning of the leptin response element. In accordance with this invention, a suitable leptin response element is an LRF-1 derived gamma interferon activation sequence 5'-CTGATTTCCC CGAAATGACG-3 * . In a preferred embodiment, the promoter region comprises a plurality of IRF- 1 derived gamma interferon activation sequences; and more preferably it comprises at least three such sequences. When a plurality of leptin response elements are present, they are preferably joined in tandem. However, there may be interviewing sequences present provided they do not interfere with the functioning of the leptin response elements. In order to detect transcription which occurs as a result of the leptin-leptin receptor binding, it is preferred to operatively link a reporter gene to the promoter region which contains at least one leptin response element. A reporter gene may be any gene which encodes a peptide which is easily detected, or otherwise allows for easy detection of transcription or translation. It generally encodes a protein which does naturally occur in the host cell or only is produced in small amounts by the host cell. Examples of well known reporter genes include: chloramphenicol acetyl transferase (CAT), green fluorescent protein (GFP), luciferase (either bacterial or firefly), and other enzyme-based detection systems such as β-galactosidase, alkaline phosphatase, and the like. In a particularly preferred embodiment, luciferase is a reporter gene. In alternative embodiments, the mRNA transcribed from the reporter gene DNA may be measured rather than the translation product. The reporter gene construct, which comprises a) the promoter region which contains a promoter and at least one leptin response element and b) the reporter gene operatively linked to the promoter region forms yet another aspect of this invention. It is preferably placed in an appropriate vector and in used to transfect a host cell. This vector comprises yet another aspect of this invention. The vector may be any known vector, including plasmids, cosmids and viral vectors which can function in a chosen host cell. The host cell may be any cell or cell line which is conveniently cultured. In general, a preferred host cell will be eukaryotic, preferably mammalian, and in particularly preferred embodiments it is a mouse hypothalamic cell (such as GTI-7), a mammalian neuroblastoma cell line, mouse fibroblast cell lines such as NIH/3T3 (ATCC CRL 1658) or L (ATCC CCL 1), Chinese hamster ovary CHO-Kl cells (ATCC CCL 61) or human embryonic kidney- derived 293 cells (ATCC CRL 1573). The host cell should either have an abundance of leptin receptors on its surface or it can be transformed to express an abundance of leptin receptors on its surface, the latter situation being the preferred one. Any leptin receptor may be used; human and rodent ones are preferred. Nucleic acids encoding human leptin receptors have been described (Tartaglia et al., 1995 Cell 83: 1263- 1271 which is hereby incoφorated by reference) and are shown in Figure 4. Mouse genes, can also be used, especially the position 26 to 2775, based on the sequence given in Gen Bank. Nucleic acids encoding leptin receptors from wild-type YdXs, fatty rats (fa/fa genotype) (shown in Figure 3) and rats heterozygous for the ob receptor gene (shown in Figure 5) are described in co-pending U.S. application Serial Nos. and 60/013,969, Attorney Docket Nos. 19462PV filed February 22, 1996 and 19462PV2, filed March 22, 1996, both of which are hereby incoφorated by reference. By transfecting cells with various leptin receptors, the biological response of receptors from different sources can be studied and compared. Also, leptin receptors containing particular desired mutations can be constructed according to known molecular biological techniques and the structure and function of the mutants can be studies using the assay of this invention. Another aspect of this invention is a set of vectors suitable for transfecting a host cell so that it can be used in the assays of this invention. The set of vectors comprises a first vector which contains a leptin receptor construct. The leptin receptor construct includes nucleic acids encoding a desired leptin receptor or mutated form of the receptor. It may be under the control of its native promoter or any other desired heterologous promoter. Optionally it may also contain other expression-control elements, such as enhancers and sequences which assist in expressing the receptor on the membrane. The set of vectors also comprises a second vector comprising the receptor gene construct described previously. In a preferred assay of this invention, compounds which are suspected of being leptin receptor ligands can be assayed. In this embodiment, cells which express a leptin receptor (either natural or recombinant) are transfected with the reporter gene construct described previously and is transfected with a leptin receptor construct, if the cell does not naturally express an abundance of the leptin receptor. The putative leptin receptor ligand is placed in contact with the transfected cells, and the presence of the reporter gene transcription or translation product is detected. This may be compared to the amount of transcription or translation measured in a control assay, where an identically transfected cell is placed in contact with leptin. A further advantage of the assay of this invention is that it is dose-responsive; i.e. as more leptin ligand-leptin receptor binding occurs, transcription and/or translation of reporter gene increases, and therefore allows for a quantitative determination of leptin receptor binding activity. A counterscreen may also be employed as a part of this assay. In the counterscreen, a second cell of the same cell type is transfected with the reporter gene construct described previously, but is not transfected with the leptin receptor gene construct. The putative leptin receptor ligand is placed in contact with the transfected cells and the presence of of the reporter gene transcription or translation is detected. Those putative leptin receptor ligands which activate the reporter gene construct only in the presence of leptin receptor are determined to be specific leptin agonists. Using this embodiment of the assay, leptin agonists and antagonists may be identified. A leptin agonist is a compound which binds to the leptin receptor, such as a leptin mimetic, and produces a cellular response which is at least about equivalent to that of leptin, and which may be greater than that of leptin. Such compounds would be useful in situations where leptin insufficiency causes obesity, diabetes or infertility. Also using this embodiment of the assay, leptin antagonists may be identified. A leptin antagonist is a compound which can bind to the leptin receptor, but produces a lesser response than that of native leptin. Such compounds would be useful in the treatment of anorexia and cachexia. In another aspect of this invention, novel leptin response elements can be identified by inserting various putative leptin response elements proximal to a promoter which controls the transcription of a reporter gene. A cell transfected with this construct and expressing natural or recombinant leptin receptors is contacted with leptin. The resulting transcription of the reporter gene is compared with that which occurs when the leptin response element is an LRF-1 derived gamma interferon activation sequence. Novel leptin response elements which are identified using this assay form yet another aspect of this invention. In another embodiment of this invention, the potency of a leptin preparation can be determined, and quantified. In this embodiment, the leptin-containing preparation is contacted with the cell containing the reporter gene construct and which is expressing leptin receptors (either recombinant or native). The amount of transcription and/or translation of the reporter gene is measured and is compared to that obtained using preparations of known potency. In yet another aspect of this invention, the biological activity of various leptin receptors can be studied and compared. In this embodiment, a first cell is transfected with a reporter gene construct and a first leptin receptor. The first leptin receptor can be any leptin receptor or variant or mutant whose activity is to be determined. The first cell is put into contact with leptin and the reporter gene transcription or translation is measured. This amount is compared to that obtained under the same assay conditions using a second leptin receptor whose activity is known or is otherwise to be used as a reference. The following non-limiting Examples are presented to better illustrate the invention. EXAMPLE 1 Vector Construction AH32, an expression vector transactivated by leptin, was constructed as follows. Two complimentary oligonucleotides containing the Stat Binding Element (SBE) from the IRF- 1 gene (Sims et ai, 1993 Mol Cell Biol. 13:690-702; and Pine et al., 1994 EMBO J. 13: 158-167; both of which are hereby incoφorated by reference) were synthesized, kinased and annealed using standard molecular biology techniques (Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 2nd Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The sequences of the oligonucleotides used is 5'-CTGATTTCCC CGAATGACG-3' and 5'-CGTCATTTCG GGGAAATCAT-3'. The double stranded oligonucleotides were cloned into a Sma I site of pTKLuc. Plasmid pTKLuc contains a promoter sequence from -35 to + 10 of the heφes simplex virus thymidine kinase gene (McKnight et al., 1982 Science, 217:316-324) upstream of the firefly luciferase gene found in pZLuc (Schadlow, et al., 1992 Mol. Biol Cell 3:941 -951). The ligated DNA was used to transform E. coli using DH5α competent cells (Gibco/BRL, Gaithersburg, MD). DNA obtained from the transformed colonies was analyzed by restriction digest analysis and the orientation and number of copies of SBE oligonucleotides found in each of the transformants was confirmed by sequencing through the insert using a sequencing kit (US Biochemical, Cleveland, OH). Clone AH32 was found to contain 3 copies of the SBE inserted in the same orientation and was used for further studies with a human fibroblast cell line (WI- 38 VA13 subline 2RA; ATCC No. CCL 75.1). Cells transfected with the AH32 showed a 6- 10 fold increase in the luciferase activity after 6h of treatment with LFN-α or LFN-γ. Primers ROBR 35 (5'-TTGGAGGACT ATGGGTGTC-3') and ROBR 37 (5'- CTACTGGAAT GGAACCTGG-3') were used to obtain the full length coding sequence for the rat OB-R, (approximately 3.7 kb in size) by the PCR using rat hypothalamic cDNA as the template. Long PCR produced the full length fragments by a modified method of Barnes (Barnes, 1994, Proc. Natl. Acad. Sci. 91 :2216-2220, which is hereby incoφorated by reference). For the long PCR reactions, Taq Extender (Stratagene, La Jolla, CA) and the Expand Long Template PCR System (Boehringer Mannheim, Indianapolis, IN) were used in combination. The standard PCR reaction mix, in a final volume of 20 μl, consisted of 40 ng of template (rat cDNA), 200 ng of primers, 500 mM dNTPs, 1 X Buffer 3 from the Expand kit, and 0.2 ml of the Expand kit enzyme mix. Reactants were assembled in thin walled reaction tubes. The amplification protocol was 1 cycle of 92°C for 30 sec, followed by 35 cycles at 92°C for 30 sec, 54°C for 1 min. and 68°C for 5 min. using a Perkin Elmer 480 Thermal Cycler. The DNA sequence of the full length products was determined using ABI PRISM Dye terminator cycle sequencing with AmpliTaq DNA polymerase, FS (Perkin Elmer, Foster City), and a number of PCR induced errors were detected. To eliminate these PCR induced mutations, restriction fragments from clones containing the correct DNA sequence were assembled. Three different restriction fragments were used to construct a full length clone derived from fa/fa hypothalamic cDNA, with a C at nucleotide 880. The expression construct was obtained by ligating a fragment from a hind III polylinker site to a Nde I site at nucleotide 502, a fragment from the Nde I site at 502 to a Sac I site at 1522, and a fragment from the Sac I site at 1522 to a Xba I polylinker site into the Hind lU and Xba I sites of pcDNA 3 (Invitrogen, San Diego, CA). The wild type (lean) clone, with an A at nucleotide 880, was constructed by replacing the Nde I by Sac I fragment from 502 to 1522 with a fragment derived from lean hypothalamic cDNA. pCHI 10, a vector expressing β-galactosidase under control of the SV40 early promoter (Hall et al., 1983, J Mol Appl Gen 2: 101- 109), was obtained from Pharmacia Biotech, Inc. (Piscataway, NJ). A fragment of the human Ob receptor cDNA, nucleotides 141 -3770, containing the entire coding region was obtained using PCR and subcloned into pCMV4 (Andersson, S. 1989 J Biol. Chem. 264:8222-8229) a gift of D.W. Russell. EXAMPLE 2 Cell Culture GTI -7 cells, a mouse hypothalamic cell line, a gift of P. L. Mellon (Liposits et al., 1991 Endocrinology 129: 1575-1583) were maintained in Dulbecco-modified Eagle Medium (DMEM) (Gibco/BRL, Gaithersburg, MD), 10 % bovine calf serum, 50 μg/ml streptomycin, and 50 U/ml penicillin (growth medium) at 37°C in an atmosphere of 95% air, 5% Cθ2- Cells were seeded at approximately 4x lO^/cm^ and passaged before confluence. EXAMPLE 3 Transfection of GTI-7 Cells 18 hours before transfection, cells were plated at 3.5 x 10-5 cells per 35 mm tissue culture dish. The plasmids AH32, appropriate Ob-r plasmid and pCHl 10 were introduced into cells using liposome- mediated transfection. Specifically, 300 ng of each plasmid was added to 200 μl of Optimem™ (Gibco/BRL, Gaithersburg, MD). 10 μg of Lipofectamine™ (Gibco/BRL, Gaithersburg, MD) was added to a second 200 μl volume of Optimem™. The two solutions were mixed, incubated at room temperature for 30 minutes and adjusted to 600 μl with Optimem™. Cells were washed twice with phosphate buffered saline. The DNA-liposome complex was applied to cells. Cells were incubated for 6 hours at which time 2 ml of growth medium was added to cells. 16 hours later, the transfection mixture was removed and 2 ml fresh growth medium was added. EXAMPLE 4 Luciferase Assay Approximately 24 hours after transfection, various amounts of recombinant human leptin were added to cells. Cells were then incubated for 24 hours. Cell culture medium was removed and cells were washed promptly with 2 ml of ice-cold calcium and magnesium- free phosphate buffered saline. Cells were then prepared for assay with 150 μl Promega reporter lysis reagent (Promega, Madison, WI) according to the manufacturer's protocol. 20 μl of cell lysates was then assayed for luciferase activity using a Dynatech ML 3000 luminometer (Dynatech, Chantilly, VA) using cycle mode after initiating the reaction with 100 μl of Promega luciferase assay reagent. The β-galactosidase activity in 50 μl of each cell lysate was determined using a β-galactosidase assay (Promega, Madison, WI). Results were normalized by dividing the relative light units obtained for each sample by the β-galactosidase activity.

Claims: WHAT IS CLAIMED IS: 1. A method for determining if a leptin receptor binding compound is present in a sample comprising: contacting the sample with a cell which comprises a) nucleic acids comprising a promoter region, said promoter region comprising a promoter and at least one leptin response element; said promoter region operatively linked to a reporter gene; and b) nucleic acids encoding a leptin receptor; and determining if transcription of the reporter gene occurs. 2. A method according to Claim 1 wherein the leptin response element is an IRF- 1 derived gamma-interferon activation sequence. 3. A method according to Claim 2 wherein the IRF- 1 derived gamma interferon activation sequence is CTG ATT TCC CCG AAA TGA CG. 4. A method according to Claim 3 wherein the promoter region comprises a plurality of leptin response elements. 5. A method according to Claim 4 wherein the promoter region comprises at least three IRF-1 derived gamma- interferon activated sequences. 6. A method according to Claim 3 wherein the leptin receptor is a recombinant leptin receptor. 7. A method according to Claim 6 wherein the leptin receptor is human or rodent. 8. A method according to Claim 7 wherein the nucleic acid comprising the leptin receptor comprises a sequence selected from the group of sequences consisting of those in Figures 3, 4 and 5. 9. A method according to Claim 3 wherein the transcription of the reporter gene occurs in proportion to the amount of leptin receptor binding compound present. 10. A nucleic acid comprising a promoter region, said promoter region comprising a promoter and at least one leptin response element; said promoter region operatively linked to a reporter gene. 1 1. A nucleic acid according to Claim 10 wherein the leptin response element is CTG ATT TCC CCG AAA TGA CG. 12. A nucleic acid according to Claim 1 1 wherein the promoter is a minimal promoter. 13. A nucleic acid according to Claim 12 wherein the reporter gene is luciferase. 14. A vector comprising nucleic acids comprising a promoter region, said promoter region comprising a promoter and at least one leptin response element, and said promoter region operatively linked to a reporter gene. 15. A set of vectors comprising a) a first vector comprising a promoter region, said promoter region comprising a promoter and at least one leptin response element, said promoter region operatively linked to a reporter gene; and b) a second vector comprising nucleic acids encoding a leptin receptor. 16. A set of vectors according to Claim 15, wherein the second vector comprises a sequence selected from the group of sequences consisting of those in Figures 3, 4 and 5. 17. A cell which has been transfected with a) a vector comprising nucleic acids comprising a promoter region, said promoter region comprising a promoter and at least one leptin-response element, and said promoter region operatively linked to a reporter gene; and b) nucleic acids encoding a leptin receptor. 18. A method for quantifying the amount of a leptin- receptor binding compound present in a sample comprising: contacting the sample with a cell which has been transfected with a) nucleic acids comprising a promoter region, said promoter region comprising a promoter and at least one leptin response element; said promoter region operatively linked to a reporter gene; and b) nucleic acids encoding a leptin receptor; and determining the amount of transcription of the reporter gene that occurs. 19. A method of determining if a putative leptin response element is a leptin response element comprising: providing a cell transfected with a construct comprising a promoter region, said promoter region comprising a promoter and the putative leptin response element, said promoter region operatively linked to a reporter gene, and said cell expressing leptin receptor; and contacting said cell with leptin. 20. A method for determining the leptin binding ability of a putative leptin receptor comprising: providing a cell which expresses the putative leptin receptor, said cell also comprising nucleic acids comprising a promoter region, said promoter region comprising a promoter and at least one leptin response element, and wherein said promoter region is operatively linked to a reporter gene; and contacting the cell with leptin.

Inventors: CHEN FANG, CULLY DORIS F, HESS JOHN W, QURESHI SAJJAD A, ROSENBLUM CHARLES I, TOTA MICHAEL R, VAN DER PLOEG LEONARDUS, CHEN, FANG, CULLY, DORIS, F., HESS, JOHN, W., QURESHI, SAJJAD, A., ROSENBLUM, CHARLES, I., TOTA, MICHAEL, R., VAN DER PLOEG, LEONARDUS
Applicants: CHEN FANG, CULLY DORIS F, HESS JOHN W, MERCK & CO INC, PLOEG LEONARDUS V D, QURESHI SAJJAD A, ROSENBLUM CHARLES I, TOTA MICHAEL R, CHEN, FANG, CULLY, DORIS, F., HESS, JOHN, W., MERCK & CO., INC., QURESHI, SAJJAD, A., ROSENBLUM, CHARLES, I., TOTA, MICHAEL, R., VAN DER PLOEG, LEONARDUS
Application year: 1997
Main classification: G01N33
IPC codes: C07K14, C12N1, C12N5, C12N15, C12Q1, G01N33