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  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

• Subject of the present invention is a method to determine whether a certain molecule is capable of binding the leptin receptor and, in relation to the biological effects thereof, of mimicking, inhibiting or potentiating the biological action of leptin itself on the cells used as targets.
• Leptin is a protein belonging to the cytokine family, produced by adipocytes, and its involvement in body weight homeostasis has been clearly demonstrated.
• Obesity is the result of a positive energy balance determined in the organism by an increase in the caloric intake/energy consumption ratio. It is known that over 30% of the adult population in the industrial world suffers from obesity. This disease represents one of the most important public health problems, given that it is associated with type II diabetes, hypertension, hyperlipidemia and an increase in mortality rate. Leptin is a protein of 16 Kilodalton whose levels in blood are correlated with body weight index in rodents and in humans (Maffei et al . , 1995).
• Leptin therefore carries out the function of an adipose tissue sensor by acting directly upon the brain, and in particular as mentioned above, upon the centres governing the feeling of satiety and the energy consumption (Spiegelman et al , 1996; Caro et al . , 1996).
• leptin receptor OB-Rb
• OB-Rb leptin receptor
• OB-Rb is a protein related to the gpl30 -family of cytokine receptors
• W096EP2291 supplies some information concerning the possibility of selecting compounds which modulate leptin 's effects using a transduction signal and a STAT-responsive DNA element for the transcription of a reporter gene.
• hypothalamic neuropeptides have been implicated as leptin-sensitive regulators of energy balance (Schwartz et al., 1996; Stephens et al . , 1995; Qu et al . , 1996) .
• Leptin inhibits the expression of NPY mRNA in the arcuate nucleus (Schwartz et al . , 1996; Schindler et al . , 1995) and the release of NPY immunoreactive material from isolated hypothalamus (Stephens et al . , 1995). Finally, the obesity syndrome of ob/ob mice is attenuated by NPY deficiency (Erickson et al. , 1996) .
• leptin' s anti- obesity activity is mediated at least in part by the inhibition of NPY expression and release.
• the object of the present invention is the reconstitution of the said effect characterizing the action of leptin in vivo in a cellular system in vitro.
• Such reconstitution is carried out firstly by using cells which express the leptin receptor naturally, or through genetical engineering. Subsequently, by means of a series of operations which are known per se but whose combination had never before been considered, it is possible to detect variations in NPY production or in choline acetyltransferase activity, two of the nodal points in the signal cascade through which leptin exercises its biological action within the cell.
• the parameters considered in fact refer to neuropeptide Y and to choline acetyltransferase, namely, two molecules of proteinic nature.
• the induction of reporter genes whose expression is controlled by means of leptin-responsive regulative elements has already been described (Baumann et al . , 1996; Rosenblum et al . , 1996), direct effects of leptin on the production of endogenous neuronal peptides or proteins, in particular NPY or choline acetyl transferase has not been described before.
• the method subject of the invention thus proves capable of assaying biological activity of all compounds which show they are capable of binding the leptin receptor.
• this assay is based on the detection of leptin 1 s capacity of inducing the production in neuronal cell lines of a neuronal protein (NPY) whose involvement in the brain's neuro-transmissive mechanism has been demonstrated, it can also be used to determine in a detailed the neuronal signalling mechanism involved in the action of leptin.
• NPY neuronal protein
• Figure 1 is a graph showing that in SN-56 cells stably transfected with an expression vector for human leptin receptor leptin induces the activation of cellular STAT transcription factors. Cells were not treated (-) or treated for 15 min. with leptin at the indicated concentrations. The arrows denote DNA binding of STAT3 homodimers, STAT1.STAT3 heterodimers, and STAT1 homodimers (Zhong et al . , 1994).
• Figure 2 is a graph showing that SN-56 cells stably transfected with an expression vector for human leptin receptor SN-56.L64 (IRBM-1) deposited on June 5th 1997 at the Advanced Biotechnology Center (ABC) , with access number PD97001, respond to the addition of leptin by decreasing the production of neuropeptide Y.
• the graph shows that native SN-56 cells do not respond to leptin, and that ciliary neurotrophic factor (CNTF) , which shares anti-obesity effects with leptin, also down- regulates the production of neuropeptide Y.
• D experiments performed in the presence of 10 ⁇ M dexamethasone .
• C 10 ng/ml CNTF
• L 100 ng/ml leptin.
• Figure 3 is a graph showing that SN-56 cells stably transfected with an expression vector for human leptin receptor (SN-56/OB-Rb) respond to the addition of leptin by increasing the cellular enzymatic activity of choline acetyltransferase.
• the graph shows that native SN-56 do not respond to leptin, and that CNTF, which shares anti-obesity effects with leptin, also increases the activitity of choline acetyltransferase.
• Subject of the present invention is a method to determine whether a certain molecule is capable of binding leptin ' s receptor and, in relation to the subsequent biological effects thereby, of mimicking, inhibiting or potentiating the biological action of leptin itself on cells used as targets.
• the method in question makes it possible to detect NPY production and enzymatic activity of choline acetyltransferase (parameters associated with leptin' s biological action) , once the molecule of interest binds with the receptor of leptin itself.
• this cell assay can supply a ⁇ wide range of data referred to leptin.
• leptin a cell assay can supply a ⁇ wide range of data referred to leptin.
• NPY production, choline acetyltransferase activity it is possible to determine also the type of biological effect resulting from the link of said molecule with the receptor of leptin itself.
• ciliary neurotrophic factor CNTF
• CNTF ciliary neurotrophic factor
• leptin antagonists in all compounds considered which can bind the leptin receptor but cannot produce any biological response in target cells. A compound of this kind may prove useful in preparing drugs suitable in the treatment of anorexia and cachexia .
• NPY cellular immunoreactivity is preferably performed by radioimmunassay (RIA) .
• RIA radioimmunassay
• Other methods described in the art and likewise employed to assess NPY cellular levels may be for instance immunologic and immunoenzymatic methods, or else, if the quantity of messenger RNA must be detected, Northern Blot, RNA-ase protection, or reverse transcription by polymerase chain reaction (RT-PCR) .
• RT-PCR polymerase chain reaction
• cell line SN-56.L64 obtained by engineering of a cell line (SN-56) derived from septal neurons, although it contains no neurons from the hypothalamus (i.e. a region in which leptin acts physiologically and in which the relevant receptor OB-Rb is physiologically expressed) the cell line also constitutes surprisingly enough a reference system for the method's actuation.
• IRBM-1 cell line SN-56.L64
• leptin receptor Any natural or modified leptin receptor can be used for the invention's purpose, although the human rather than the rodent type is preferable.
• the structural sequences coding for human leptin receptors have been described in the art (Tartaglia et al . , 1995 Cell 83:1263-1271). Genes for mouse receptors can also be used, particularly the one in GenBank data bank, containing the sequence from nucleotide 27 to nucleotide 2775.
• GenBank data bank containing the sequence from nucleotide 27 to nucleotide 2775.
• the sequence of the rat receptor also described in the art (Phillips et al . , 1996, Nature Gen. 13:18-19) may also be used.
• an expression vector containing a cDNA for human leptin receptor nucleotides 141-3770, containing the sequence coding for the whole receptor form OB-Rb.
• pCMV4 vector (Andersson, 1989) is preferably used but any other expression vector compatible with the cells on which the assay must be carried out can be used and with the necessity of obtaining a stable expression of leptin receptor.
• the compounds considered ligands capable of binding the leptin receptor are placed in contact with the presumed ligand, then levels of neuropeptide Y and choline acetyltransferase are measured.
• a counterscreen may be constituted by a second cell of the same type which has not been transfected by the leptin receptor.
• cells capable of expressing the (either natural or recombinant) leptin receptor are placed simultaneously in contact with the compounds of interest and leptin, before related NPY production or choline acetyltransferase variation are measured. The result is compared with that obtained placing an identical cell in contact with leptin alone. It is thus possible to determine whether a certain compound is capable of inhibiting or potentiating leptin ' s natural action, which may be useful to produce drugs for pathologies in which there is not a total lack of leptin, but rather a deficit of, or a resistance to, leptin at cellular level.
• the subject of the present invention is a method for the selection of molecules capable of mimicking, potentiating or inhibiting the effects of interaction between leptin and cells displaying on the membrane the receptor thereof, comprising the following operations a) contacting said molecules with said cells; and b) detecting the variation in said cells of the neuropeptide Y production, or of the enzymatic activity of choline acetyltransferase, caused by the binding of said molecule to said receptor.
• the cells can be also engineered. Engineering can be carried out by a series of operations in combination on any cell line, extensively described in the example 1 below, including the following essential operations: - transferaction of the cell line with an expression vector;
• the cell line concerned is SN-56 the result is the line SN-56.L64 (IRBM-1), also subject of the present invention.
• the qualitative and/or quantitative NPY assay can be carried out with immunoenzymatic methods.
• Cell line SN-56.L64 obtained from cell line SN-56 by a cell engineering process extensively described in example 1, in which the stable expression of leptin receptor has been guaranteed, was deposited on June 5th 1997 at the Advanced Biotechnology Center, ABC in Genoa, with access number PD97001.
• SN-56.L64 (IRBM-1) : engineering of cells SN-56 for the stable expression of leptin receptor
• a leptin receptor expression vector was constructed by subcloning a fragment (nucleotides 141-3770 of the sequence deposited in Gen Bank; accession number U43168) of the human leptin receptor (OB-Rb) cDNA into the vector pCMV4 (Andersson, 1989) .
• the mouse septal neuron- neuroblastoma hybrid cell line SN-56 (Lee et al . , 1990) (a kind gift of Dr. J.
• Total cell extracts were obtained BY resuspending the cell pellet in 5 volumes of 10 mM Hepes pH 7,9; 0.4 M NaCl ; 0.1 mM EGTA, 5% glycerol, 50 mM NaF, lOmM Na4P2 ⁇ 7, 0.5 MM dithiotreitol, 0.5 mM phenilmethilsulphonylfluoride, 10 ⁇ g/ml trasylol, 2 ⁇ g/ml leupeptin.
• Cell lysate was centrifugated at 100,000 x revolutions for 10 min, and an aliquot of the supernatant (10 ⁇ g of protein) was used for the determination of the DNA binding activity of STAT factors by assaying electrophoretic mobility shift, in accordance with the procedure described by Sadowsky and Gilman (Sadowsky et al., 1993), using the high affinity SIE m67 oligonucleotide (Wagner et al . , 1990).
• the probe was labelled by filling in 5' protruding end with Klenow enzyme in presence of (a 32 P)dATP and (a 32 PdCTP) (3000 Ci/mmol) .
• the cells (in the example, which are provided only for illustrative purposes and do not limit the scope of the present invention we refer as described to wild type SN-56 cells and to the same cells transfected with an expression vector for the leptin receptor) have been plated on 100 mm dishes (1.5xl0 6 cells per dish) containing the culture medium as described in the general information section.
• the cells were treated for three days with dexametasone (lO ⁇ M) , human leptin (100 ng/ml) or CNTF (10 ng/ml) or with no factor. Cells were washed with PDS and centrifugated.
• Cells were extracted with 200 ⁇ l of 0.1 N HCl and cell extracts were centrifugated for 10 min at 14000 x rpm. The pellet was resuspended in 1 N NaOH and 0.1 volumes of a 0.5 M sodium phosphate buffer, pH 8.2. Aliquots in duplicate of cell extracts were subjected to radioimmunoassay to assess the presence of
• stabilized SN-56 cells for the expression of recombinant human leptin receptor respond to leptin addition by decreasing neuropeptide Y production.
• Dexametasone described as an inducer of NPY production in neuronal cell lines (Higuchi et al . , 1988), doubles NPY immunoreactivity in cells SN-56.
• leptin inhibits NPY production in presence of dexametasone.
• the diagram in figure 2 shows that wild type cells SN-56 do not respond to leptin, and that the neurotrophic ciliary factor which has anti-obesity effects similar to those of leptin, negatively regulates neuropeptide Y production.
• EXAMPLE 3 Assay on cells SN-56 ,L64 (IRBM-1) for the assessment of biological activity of leptin based on the activity of choline acetyltransferase.
• Cells (SN-56 and SN-56.L64 (IRBM-1) were seeded on a 24-well culture dish (3 x 10 4 cells per well) containing
• Opti-MEM GibcoBRL
• Opti-MEM Penicillin and streptomycin
• the culture medium was removed by aspiration and the cells lyzed in 250 ⁇ l of 20 mM Tris-Hcl, 0.1% Triton X-100, pH 7.5. Lysates were clarified by centrifugation (10 min at 10000 x g) and aliquots in duplicates were tested for choline acetyltransferase activity with the Fonnum modified method (Fonnum, 1975) .
• reaction mix containing 15 ⁇ l of cell extract and 50 ⁇ l of the reaction buffer (50 mM Tris-Hcl pH 7.5; 400 mM NaCl ; 10 mM choline chloride; 0.1 mM eserine hemisulphate; lOmM EDTA; 1 mg/ml of bovine serum albumin; 5 ⁇ M acetyl- coenzyme A; 25 nCi [ 14 C] acetyl-coenzyme A) were incubated for lh at 37 °C, and subsequently the labelled acetylcholine was identified by separation according to procedures known in the art (31) . Finally results were normalized to the content of total cellular proteins.
• cells SN-56.L64 stably transfected with an expression vector for the human leptin receptor respond to leptin addition with an increase of cell enzymatic activity in choline acetyltransferase.
• the diagram shows wild type SN-56 cells which are insensitive to leptin and that CNTF, which has an anti-obesity effect similar to that of leptin, causes the increase of the intracellular activity of choline acetyltransferase.

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• 1997
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