Patent Application: US-92679804-A

Abstract:
the present invention relates to an antagonist or inhibitor of leptin signaling via the leptin receptor . the leptin antagonist binds to the leptin receptor , but is unable to induce jak - stat signal transduction via the leptin receptor . by binding to the leptin receptor , the leptin antagonist impairs binding of leptin to the leptin receptor and blocks leptin signaling .

Description:
the invention is further explained with the aid of the following illustrative examples . detection of binding site ii and iii in human and mouse leptin by structural superposition fssp structural similarity searches reveal that leptin shows the highest structural similarity with the cytokines of the il - 6 family and g - csf , and to a minor extent with other long chain cytokines , such as the growth hormone and placental lactogen . the crystal structures of human leptin ( 1ax8 ), human cntf ( 1cnt ), human il - 6 ( 1a1u ), bovine g - csf ( 1bgc ), vil6 ( 1i1r ), ovine placental lactogen ( 1f6f ), murine lif ( 11ki ) and human osm ( 1evs ) were superposed , using the fssp and prosup algorithms . human leptin residues overlapping with binding site ii or iii residues in the other cytokines were considered as possible binding site ii or iii residues in human leptin . a homology model was built for murine leptin , by replacing non - identical residues in human leptin structure by the optimal rotamer of the corresponding residue in mouse leptin , followed by energy minimization , using moe and the charmm22 force field . residues aligning with the possible binding site ii or iii residues in human leptin were considered as possible binding site ii or iii residues in human leptin . solvent - exposed residues in the predicted binding site ii and iii were mutated in the pmet7 - sigk - ha - mlep expression vector , and the mutant leptin was expressed in cos - 1 cells . the pcdm8 mleptin - ap vector is containing the mouse leptin sequence , followed by the human alkaline phosphatase sequence . the wild - type ( wt ) mouse leptin sequence was isolated from this vector by pcr , using the 5 ′ forward oligomeric primer 5 ′- gcgtccggaatccagaaagtccaggatg - 3 ′ ( seq id no : 6 ), containing a bspei restriction site , and the 3 ′ reverse primer 5 ′- cgctctagattagcattcagggctaacatcc - 3 ′ ( seq id no : 7 ), containing an xbai restriction site . the pmet7 - sigk - ha - lrlo plasmid contains the sigk signal peptide , followed by the ha tag sequence and the mouse leptin receptor sequence . the leptin receptor sequence was excised from this vector , using the bspei and xbai restriction enzymes , and the mouse leptin sequence was ligated into this opened vector . the resulting vector , pmet7 - sigk - ha - mlep , allows the expression of a fusion protein , consisting of the sigk signal peptide , followed by the ha - tag sequence , followed by a 4 amino acid ggsg linker , followed by amino acids 3 to 146 of mouse leptin . upon expression in eukaryotic cells , the sigk signal peptide is cleaved off and the ha - tagged protein is secreted in the medium . amino acid sequence ( seq id no : 8 ) of sigk - ha - mouse leptin : the arrow indicates the predicted cleavage site of the sigk signal peptide , the numbers above the sequence indicate the residue numbers in mouse leptin : the mouse leptin s120at121a mutation was introduced in the pmet7 - sigk - ha - mlep by pcr using primers o - 1821 and o - 1822 . the mouse leptin r20n mutation was introduced in the pmet7 - sigk - ha - mlep by pcr using primers o - 1701 and o - 1702 . the mouse leptin l13n mutation was introduced in the pmet7 - sigk - ha - mlep by pcr using primers o - 1769 and o - 1770 . all mutations were introduced using the quickchange method ( stratagene ) according to the manufacturer &# 39 ; s specifications . amino acid sequence ( seq id no : 15 ) of the mouse leptin mutants : vertical arrows indicate the position of the mutations , the numbers above the sequence indicate the residue numbers in mouse leptin : expression of the ha - tagged mouse leptin and ha - tagged mouse leptin mutants in cos - 1 cells cos - 1 cells were seeded at 4 × 10 5 cells / well in 6 - well plates , and grown overnight . the cells were then transfected using the polyethyleneimine ( pei ) transfection method with the pmet7 - sigk - ha - mlep or the r20n , l13n or s120at121a mutants in this vector . the medium was replaced after 4 hours transfection , and cells were incubated overnight , after which the medium was replaced by 2 ml opti - mem medium ( gibco - brl ). after another 72 hours , the opti - mem medium containing the secreted ha - tagged leptin or ha - tagged leptin mutant was collected , and cells were removed by centrifugation . cos - 1 cells were seeded at 8 × 10 6 cells per 175 cm 2 flask in dmem medium . ten 175 cm 2 flasks were transfected with the s120at121a pmet7 - sigk - ha - mlep mutant by transfection using polyethyleneimine . after 4 hours , the medium was replaced by fresh dmem medium , and cells were grown overnight . the medium was then replaced by 50 ml opti - mem , and cells were incubated in this medium for another 72 hours . the medium with the secreted s120at121a ha - tagged mouse leptin was collected and filtered through a 0 . 22 μm filter , and complete ( roche diagnostics ) protease inhibitor was added . the s120at121a ha - tagged mouse leptin was purified on a 1 ml anti - ha affinity column ( roche diagnostics ). the medium was loaded at a flow rate of 0 . 3 ml / min . the column was washed with 25 ml equilibration buffer ( 20 mm tris / hcl , ph 7 . 5 , 100 mm nacl , 0 . 1 mm edta )+ 0 . 05 % tween - 20 , followed by 10 ml equilibration buffer without tween - 20 . the s120at121a ha - mouse leptin was eluted with ha peptide ( 1 mg / ml ) in equilibration buffer . the s120at121a mouse leptin mutant does not induce jak - stat signaling of the mouse leptin receptor hek293t cells were seeded in 6 well plates at 4 × 10 5 cells / well and grown for 20 hours . the cells were then transfected with the pmet7 - mlrlo and pxp2d2 - rpap1 plasmids , using the standard calcium phosphate precipitation technique . one day after transfection , the cells were washed with phosphate buffered saline , and cultured in dmem medium supplemented with 10 % fetal calf serum and 50 μg / ml gentamycin . two days after transfection , the cells were dissociated with cell dissociation buffer ( invitrogen ) and resuspended in 2 ml of dmem medium supplemented with 10 % fetal calf serum and 50 μg / ml gentamycin . 50 μl of the cell suspension was seeded in each well of a black 96 - well plate ( costar ). in each well , 50 μl of the appropriate dilution of medium containing either ha - tagged mouse leptin , r20n mouse leptin , l13n mouse leptin or s120at121a mouse leptin was added . the pmet7 - mlrlo plasmid encodes the long form of the mouse leptin receptor , with a c - terminal myc tag . the pxp2d2 - rpap1 plasmid encodes the luciferase gene , under control of the stat3 - dependent rat pancreatitis - associated protein - 1 promoter . incubation of the transfected hek293t cells with wt ha - tagged mouse leptin leads to concentration - dependent luciferase activity . incubation of the transfected hek293t cells with the r20n mutant ha - tagged mouse leptin , the l13n mutant ha - tagged mouse leptin or the s120at121a mouse leptin does not lead to an appreciable increase of luciferase activity in the transfected cells . the s120at121a mouse leptin inhibits binding of wt leptin to the leptin receptor 8 × 10 6 cos - 1 cells were seeded in a 175 cm 2 flask , and grown overnight in dmem medium supplemented with 10 % fetal calf serum and 50 μg / ml gentamycin . the cells were transfected with the pmet7 - mlrcrm2 - his6 using the pei transfection method . pmet7 - mlrcrm2 - his6 encodes amino acids 407 - 604 , corresponding to the crm2 module of the murine leptin receptor , followed by a c - terminal his6 tag . after 4 hours , the medium was replaced by fresh dmem medium supplemented with 10 % fetal calf serum and 50 μg / ml gentamycin , and cells were grown overnight . the medium was then replaced by 50 ml opti - mem , and cells were incubated in this medium for another 72 hours . the medium with the secreted his - tagged crm2 leptin was collected and cells were removed by centrifugation . this medium is referred to as crm2 - his medium . 8 × 10 6 cos - 1 cells were seeded in a 175 cm 2 flask , and grown overnight in dmem supplemented with 10 % fetal calf serum and 50 μg / ml gentamycin . the cells were transfected with the pcdm8 ap - mlep plasmid using the pei transfection method . the pcdm8 ap - mlep plasmid allows expression of a fusion protein , consisting of murine leptin linked to the c - terminus of human secreted alkaline phosphatase ( seap ). after 4 hours , the medium was replaced by fresh dmem medium , and cells were grown overnight . the medium was then replaced by 50 ml opti - mem , and cells were incubated in this medium for another 72 hours . the medium with the secreted seap - leptin was collected and cells were removed by centrifugation . this medium is referred to as seap - leptin medium . maxisorp plates ( nunc ) were coated overnight at 6 ° c . with 0 . 25 μg / ml anti - his5 antibody ( qiagen ). plates were washed four times with phosphate buffered saline ph 7 . 5 containing 0 . 1 % tween 20 . the free protein binding sites on the plates were blocked by incubation with 1 % casein in phosphate buffered saline ph 7 . 5 at 37 ° c . for two hours . plates were washed four times with phosphate buffered saline ph 7 . 5 containing 0 . 1 % tween 20 . the plates were incubated for 2 hours at 37 ° c . with the undiluted medium containing the his - tagged crm2 of the murine leptin receptor . plates were washed four times with phosphate buffered saline ph 7 . 5 containing 0 . 1 % tween 20 . the plates were then incubated with seap - leptin medium and different concentrations of either ha - tagged mouse leptin or s120at121a leptin antagonist . addition of s120at121a mouse leptin or wt leptin , but not of mutant r20n inhibits the binding of the seap - leptin to the his - tagged crm2 of the murine leptin receptor . the results are shown in fig3 . the s120at121a mouse leptin mutant inhibits the jak - stat signaling of the mouse leptin receptor hek 293t cells were seeded in 6 well plates at 4 × 10 5 cells / well and grown for 20 hours . the cells were then transfected with the pmet7 - mlrlo and pxp2d2 - rpap1 plasmids , using the standard calcium phosphate precipitation technique . one day after transfection , the cells were washed with phosphate buffered saline , and cultured in dmem medium supplemented with 10 % fetal calf serum and 50 μg / ml gentamycin . two days after transfection , the cells were dissociated with cell dissociation buffer ( invitrogen ) and resuspended in 2 ml of dmem medium , supplemented with 10 % fetal calf serum and 50 μg / ml gentamycin . 50 μl of the cell suspension was seeded in each well of a black 96 - well plate ( costar ). opti - mem medium , containing wt ha - tagged leptin ( see , example 4 ) was diluted 16 fold in opt - mem . 50 μl of this diluted medium was added to the cells , together with 50 μl of dilution of anti - ha purified ( see example 5 ) ha tagged s120at121a leptin . the cells were then incubated overnight . the medium was removed and the cells were incubated with 50 μ of lysis buffer ( 25 mm tris , ph 7 . 8 ; 2 mm edta ; 2 mm dtt ; 10 % glycerol ; 1 % triton x - 100 ) for 10 minutes . 35 μl of luciferase substrate buffer ( 20 mm tricine ; 1 . 07 mm ( mgco 3 ) 4 mg ( oh ) 2 . 5h 2 o ; 2 . 67 mm mgso 4 . 7h 2 o ; 0 . 1 mm edta ; 33 . 3 mm dtt ; 270 mm coenzyme a ; 470 mm luciferin ; 530 mm atp ; final ph 7 . 8 ) was then added to the lysate , and the light emission was measured in a topcount chemiluminescence counter ( packard ). the results are shown in fig4 . incubation of the transfected hek293t cells with the wt ha - tagged mouse leptin induces luciferase activity . co - 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