Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/57509—Corticotropin releasing factor [CRF] (Urotensin)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
  • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
• • 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
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/575—Hormones
  • G01N2333/5751—Corticotropin releasing factor [CRF] (Urotensin)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
  • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

• the present invention refers to CRF or analogs thereof bearing a photoactivatable moiety and a label and their use in detecting CRF receptors and binding proteins and identifying and characterizing their binding site.
• Corticotropin-releasing factor appears to integrate the endocrine, autono ic, immunologic and behavioral responses to stress in the CNS.
• the 41 residue polypeptide (1) was originally characterized on the basis of its hypophysiotropic activity stimulating the release of adrenocorticotropic hormone (ACTH) (2) , which is known to stimulate the secretion of glucocorticoids from the adrenal cortex.
• ACTH adrenocorticotropic hormone
• CRF is the main regulator of the hypothalamus-pituitary-adrenal (HPA) axis leading to the release of glucocorticoids after exposure to stress.
• CRF exhibits its activity through G protein-coupled receptors.
• CRF receptor, type 1 (CRFR1) mainly found in pituitary and brain was independently cloned from human, mouse and rat brain, and a human Cushing's corticotropic cell tumor (3-6) .
• cDNAs coding for two splice variants of CRF receptor, type 2 (CRFR2a and CRFR2b) were cloned from brain, heart, and skeletal muscle (7-10) .
• urocortin (Ucn) a naturally occurring CRF analog, is the endogenous ligand to CRFR2 (11) .
• CRF binding protein Besides the CRF receptor, a 37 kDa CRF binding protein has been characterized. This protein which is not homologous to any known splice variant of CRFRl or CRFR2 was demonstrated to bind human/rat CRF (h/rCRF) but not ovine CRF (oCRF) with high affinity (12) .
• the very potent CRF antagonist astressin, cyclo(30-33) [D-Phe 12 , Nle 21 ' 38 , Glu 30 , Lys 33 ]h/rCRF-(12-41) with its amino acid sequence based on h/rCRF exhibited similar binding affinity to CRFRl as found for h/rCRF but did not bind to the CRF binding protein (13).
• Labeling through monofunctional photoaffinity probes is expected to provide higher yields than labeling with chemical cross-linking methods using bifunctional reagents. Additionally, photoactivation is assumed to be superior over thermal activation, because highly reactive species such as carbenes and nitrenes can be selectively formed after irradiation uder mild conditions. The carbenes or nitrenes formed can insert into X-H bonds and thereby attack groups that are normally inert to chemical affinity labeling (21) .
• PAL photoaffinity labeling
• the technical problem underlying the present invention is to provide CRF or analogs thereof which bind efficiently and with high affinity to the receptor resulting in an irreversible labeling of the receptor.
• the present invention provides CRF or analogs thereof bearing a photoactivatable moiety and a label.
• analog encompasses any variant or fragment of CRF which retains CRF ligand binding activity.
• the photoactivatable moiety and the label are adjacent to each other.
• the photoactivatable moiety should preferably be of such quality that the photoaffinity labeling can be performed under mild conditions at a suitable wavelength.
• Examples of the photoactivatable moiety are the 4-(l-azi- 2,2,2-trifluoroethyl)-benzoyl residue or the phenylalanine analog thereof.
• the label can be a radioactive marker, e.g. 125 I, or a fluorescent marker, e.g. fluorescein, or via biotin which interacts with avidin carrying a fluorescent group.
• a radioactive marker e.g. 125 I
• a fluorescent marker e.g. fluorescein
• Preferred embodiments of the invention are a CRF agonist, 4-(l-azi-2,2,2-trifluoroethyl)benzoyl-[ 125 I]- tyrosine°oCRF (compound 3) , and CRF antagonists based on the amino acid sequence of astressin carrying the 4-(l-azi- 2,2,2-trifluoroethyl)-benzoyl (ATB) residue and a histidine or tyrosine by choice for specific radiolabeling, e.g.
• ATB- cyclo(30-33) [ 125 I-His 13 , Nle 21 ' 38 , Glu 30 , Ala 32 , Lys 33 ]h/rCRF-(13-41) (compound 6) and ATB-cyclo(30- 33) [Nle 21 ' 38 , Glu 30 , 125 I-Tyr 32 , Lys 33 ]h/rCRF- (13-41) (compound 7) .
• the synthesis of the compounds of the invention can be performed by linking the photoactivatable moiety, e.g. ATB, to the CRF or CRF analog and subsequent labeling, e.g. iodination.
• the synthesis of compounds 4 and 5 is performed by linking 4-(l-Azi-2,2,2-trifluoroethyl)benzoic acid to cyclo(30-33) [Nle 21 ' 38 , Glu 30 , Ala 32 , Lys 33 ]h/rCRF- (13-41) and cyclo(30-33) [Nle 21 ' 38 , Glu 30 , Tyr 32 , Lys 33 ]h/rCRF-(13-41) .
• Cyclization of the peptides on the resin prior to coupling of the phenyldiazirine to the N- ter inus of the peptides is chosen because of the probable sensitivity of the diazirine group towards tetrakistriphenylphosphine palladium (0) (23) .
• Subsequent iodination with 125 ⁇ a t histidine 13 or tyrosine 32 furnishes compounds 6 and 7 with a specific activity of 82 TBq mmol, respectively.
• the [ 125 I]Tyr°oCRF analog bears the 4-(l-azi-2,2,2- trifluoroethyl)benzoyl residue at its N-terminus, where the disturbance of ligand binding is supposed to be minimal (2, 24, 25).
• the immediate proximity of the photoactivatable part to the radioactive tracer in the molecule facilitates the identification and purification of peptide fragments after photoaffinity labeling experiments.
• CRF-R1 with a molecular weight of approximately 75kDa was detected with the new CRF analog in HEK 293 cells, permanently transfected with the CRFRl gene.
• the compounds of the invention can be used for the detection of CRF receptors and binding proteins and for the identification of the binding site of these proteins.
• the photoaffinity labeling technique of the present invention is advantageous towards chemical cross-linking methods when identifying the ligand binding site within a receptor molecule as on irradiation of the photoactivatable ligand, a highly reactive short living species is formed, which then irreversibly binds with high yield to its receptor.
• the affinity tagged receptor polypeptide identified by the label is stable so that it can be further purified, e.g. by HPLC. It can then be cleaved into fragments, and the binding site can be identified by amino acid sequence analysis.
• FIG. 1 Synthetic route for the photoactivatable diazirine 1 according to document (26) and its linkage to Tyr°oCRF 1-41 to generate 2 and its 125 iodinated analog 3.
• FIG. 2. (A.) Displacement of [ 125 I-Tyr°]oCRF bound to membranes from transfected HEK 293 cells by oCRF ( • ) or ovine photoCRF 2 ( D ) • Data are the mean of triplicates of a representative experiment. (Inset) Scatchard plots of the binding of oCRF ( • ) and ovine photoCRF 2 ( ⁇ ) ⁇
• Fig. 3 Stimulation of intracellular cAMP accumu ⁇ lation in Y79 cells by oCRF ( » ) , ovine photoCRF 2 ( O) , and ovine photoCRF 2 (x) in the presence of 100 nM recombinant human [D-Phe 12 , Nle 21 ' 38 ]CRF- (12-41) .
• Data is the mean ⁇ SEM values (bars) of duplicates of a representative experiment.
• FIG. 4 Photoaffinity cross-linking of ovine 1 5 ⁇ - photoCRF 3 to HEK 293 cell membrane ho ogenates. Lanes: 1-5, extracts of cells stably transfected with cDNA coding for rCRFRl; 6 and 7, extracts of nontransfected HEK 293 cells. Radioactive ovine photoCRF was bound in the absence of oCRF (lanes 1, 5, and 6) or in the presence of 100 nM (lane 2), 1 ⁇ M (lane 3), 10 ⁇ M (lanes 4 and 7) oCRF or 1 ⁇ M vasoactive intestinal peptide (lane 5) .
• FIG 5. (A) Plot of radioactivity of membrane components covalently labeled with ovine 1 5 I-photo CRF 3 and purified with RPHPLC. (B) Pooled fractions were subjected to SDS/PAGE in 7.5% gels.
• a permanent cell line was established from HEK 293 cells stably transfected with cDNA coding for rCRFRl.
• a pool of HEK cell clones was employed in the following experiments. Binding results obtained with individual HEK cell clones did not differ significantly from the results of binding experiments with the cell clone pool. Scatchard analysis indicated that oCRF was bound with a K ⁇ value of 7.8 ⁇ 6.3 nM at a high-affinity site and a K ⁇ value of 137 ⁇ 90 nM at a low-affintiy site.
• EC50 values of 0.5 ⁇ 0.2 nM and 0.4 ⁇ 0.1 nM were determined for oCRF and photoCRF, respectively (Fig. 2B) .
• Non-transfected cells did not show significant binding or cAMP accumulation. This observation was also confirmed by photoaffinity-labeling experiments.
• Ovine 125 I-photoCRF 3 did not bind to membranes of non-transfected HEK 293 cells (see Fig. 4) .
• Compound 7 did not bind to membranes of non-transfected HEK 293 cells.
• Statistical analysis of the described binding and cAMP data with the program ANOVA revealed no significant differences between the K d and K ⁇ values for astressin and compound .
• Both peptides exhibited high potency to reduce the stimulatory potency of oCRF to produce cAMP in transfected HEK 293 cells and Y79 cells.
• the freshly prepared tracer 7 was stored free of any carrier protein, and the photoaffinity labeling experiments were performed in buffer solutions in the absence of BSA.
• a 66 kDa cross-link was identified with SDS PAGE after irradiation at 360 nm of a mixture of compound 7 and membranes of HEK 293 cells permanently transfected with rCRFRl. No cross-link could be identified without light activation at 360 nm.
• Binding of compound 7 to the receptor could be efficiently inhibited by addition of 1 ⁇ M ATB-cyclo(30-33) [Nle 21 ' 38 , Glu 30 , Tyr 32 , Lys 33 ]h/rCRF-(31-41) (compound 5) but not 1 ⁇ M vasoactive intestinal peptide (VIP) in agreement with the assumed specificity of this photoprobe.
• VIP vasoactive intestinal peptide
• the compounds of the invention can be used for the specific irreversible labeling and tracking of receptors in various tissue membranes, of CRF binding proteins, as well as in cytological investigations using a fluorescent analog of 2, 4 or 5, e.g. on cell sorting, receptor internalization, trafficking.
• the resin was filtered off, washed three times with 0.5 ml of NMP, added to 750 ⁇ l of cleavage mixture (75 ⁇ g of crystalline phenol, 25 ⁇ l of EDT, 50 ⁇ l of thioanisole and 50 ⁇ l of dH 2 0, 1 ml of TFA) and stirred for 1.4 hr.
• the resin was filtered off and the peptide precipitated in 20 ml of ice cold ether. After filtration, the crude peptide was dissolved in 2 ml of TFA and 50 ml of 20% MeCN in 0.1 % TFA/water and lyophilized.
• the mixture was pipetted onto a Bond Elut C ⁇ g cartridge (Varian Associates) , prewetted with 5 ml of MeOH, then 5 ml of 0.1 % TFA/water.
• Five milliliters of dH 2 0 followed by 5 ml of 0.1 % TFA/water were passed through the column in order to separate the iodinated peptide from free iodine and BSA.
• the iodinated peptide was then eluted from the column by the addition of 5 ml of 80% MeCN in 0.1 % of TFA/H 2 0.
• the specific activity of the peptide 82 TBq/mmol.
• the peak tubes of radioactivity were pooled and 3-mercaptoethanol was added to a final concentration of 0.5 M.
• the iodinated tracer 3 (Fig. 1) was stored in aliquots at -20°C and typically used for binding assays and photoaffinity labeling experiments for 2 months.
• the CRF peptides were synthesized with Fmoc chemistry on TentaGel S RAM resin (0.1 mmole scale, Rapp, Tubingen, F.R.G.) with a model ABI 433A peptide synthesizer (Applied Biosystems) .
• the crude peptides were purified by preparative reverse-phase HPLC (RPHPLC) performed on a Waters Prep Nova- Pak HR iQ silica gel column (5 x 30 cm, 6- ⁇ m particle size, 6-nm pore size) with a mixture of aqueous 0.1% trifluoroacetic acid (TFA) and MeCN.
• the mass spectra of the purified peptides were measured with ESI (electrospray ion) MS on a Micromass AutoSpec-T tandem mass spectrometer.
• the purified CRF peptides were subjected to analytical RPHPLC on a Vydac C ⁇ Q silica gel column (0.46 x 25 cm, 5- ⁇ m particle size, 30-nm pore size) with solvents A (0.1% TFA in water) and B (80% MeCN in 0.1% TFA in water) at a flow rate of 1 ml/min.
• the samples were eluted with 5% B for 5 min. and then with a linear gradient of 5-95% B in 30 min.
• ATB-cyclo(30-33) [ 125 I-His 13 , Nle 21 ' 38 , Glu 30 , Ala 32 , Lys 33 ]h/rCRF- (13-41) (Compound 6 ) and ATB-cyclo(30-
• Human embryonic kidney cells 293 (Graham, Smiley, Russell & Nairn, 1977) (supplied by Dr. C. Stevens and G. Sharraa, The Salk Institute, La Jolla) were grown in Dulbecco's modified eagle medium (GIBCO BRL, Gaitherburg, MD, USA, cat. no.: 041-01885M) supplemented with 10% fetal calf serum (Sigma, St. Louise, MO, USA, cat. no.: F-7524) and brought to a final concentration of 4 mM L-glutamine (GIBCO BRL, cat. no.: 043-05030), 0.45% glucose. They were maintained as described (31) .
• the rat CRFRl gene fragment (1284 bp, BamHI, EcII26II fragment) was subcloned into the vector pcDNA3 (Invitrogen, San Diego, Ca, USA) .
• the recombinant plasmid (pCDNA3-rCRF ] was isolated, and purified with the Qiagen plasmid preparation system (Qiagen, Hilden, Germany) . The ligation sites were verified by DNA sequence analysis.
• HEK 293 cells were transfected with pCDNA3-rCRF-Rl utilizing the calcium/BBS transfection method (32) . Sixteen hours after transfection, the medium was removed and replaced by selection medium (600 ⁇ g/ml Geneticin in medium) . Cells were grown until confluent and split 1:2 with further selection. Following one to two weeks of growing under selection conditions, all cells were geneticin- resistant and grew normally.
• the cells obtained according to Example 5 were dislodged from the cell culture flasks with a cell scraper into ice cold PBS buffer. The cells were precipitated at 150 g for 10 min. at 4°C, resuspended in 1 x PBS buffer and recentrifuged. The supernatant was entirely removed and the wet weight of the cell pellet was determined.
• the cells were suspended in 3 ml/g cells of CRF membrane buffer (50 mM Tris/Cl, 5 mM MgCl 2 , 2 mM EGTA, 500 ⁇ l Trasylol (FBA, New York, USA), 1 mM DTT, pH 7.4) and treated for 10 strokes (each 2 s) with the medium sized polytron tool at power level 5.
• the nuclei were precipitated for 5 min at 600 g in the cold. The supernatant was carefully removed with a Pasteur pipette and collected on ice. The pellet was reextracted with the same amount of membrane buffer using some strokes of the polytron. The nuclei were again precipitated from this suspension as described.
• the combined supernatant ⁇ were centrifuged at 10,000 g for 15 min to precipitate the membranes.
• the pellet was resuspended with 3 ml/g of cells in storage buffer (membrane buffer containing 20% glycerol) with 10 strokes of a glass Teflon homogenizer.
• a micro BCA assay (Pierce, Rockford, USA) was performed with 2 ⁇ l and 4 ⁇ l of the suspension to estimate the total protein concentration (about 2.5 ⁇ g/ ⁇ l) .
• the membranes were frozen in liquid nitrogen and stored at -70 ° C until use.
• Photoaffinity labeling experiments were in principle performed in the same manner as mentioned above except that the incubation buffer used was without BSA.
• a concentration series of either oCRF (0, 100 nM, 1 ⁇ M, 10 ⁇ M) or VIP (1 ⁇ M) and 180,000 cpm of 3 per tube HEK 293 membrane homogenates of either transfected or non- transfected cells (75 ⁇ g of protein/tube) were added and incubated for the indicated time. Before photolysis, the pellets were washed three times, resuspended in 300 ⁇ l of buffer and irradiated at 360 nm for 30 min (4°C, 8 cm distance from the lamps) .
• HEK 293 and human Y79 retinoblastoma cells were incubated with different CRF analogs in the presence of 1 or 5 mM 3-isobutyl-l- methylxanthine (37'C, 30 min), respectively.
• the incubation medium of the Y79 cells contained additionally 1 mg/ml BSA and 0.05 mg/ml ascorbic acid.
• compound 2 or the photoactivatable astressin analogs were used, all experiments were performed in the dark. After removal of the medium, cells were lyzed with aqueous 6% trichloroacetic acid (100*C, 5 min) .
• the cell lysates were stored at -70°C until assayed with a RIA kit (Amersham, Little Chalfont) . Data analysis was achieved with the sigmoidal dose-response curve fitting programs ALLFIT. Statistical significance was determined across groups by one-way ANOVA.
• Membrane protein (250 ⁇ g) was labeled with 1.1 x 10 7 cpm of 3 (2.82 pmol) .
• One-tenth of the sample was dissolved in 50% ethanolic formic acid (100 ⁇ l) and subjected to RPHPLC using a Vydac C4 silica gel column (0.46 x 25 cm, 5 ⁇ m particle size, 30 nm pore size.) Elution was accomplished with a mixture of aqueous 0.5% trifluoroacetic acid and EtOH.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Molecular Biology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Immunology (AREA)
• Genetics & Genomics (AREA)
• Zoology (AREA)
• Toxicology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Endocrinology (AREA)
• Gastroenterology & Hepatology (AREA)
• Engineering & Computer Science (AREA)
• Biophysics (AREA)
• Urology & Nephrology (AREA)
• Cell Biology (AREA)
• Biomedical Technology (AREA)
• Hematology (AREA)
• Biotechnology (AREA)
• Microbiology (AREA)
• Food Science & Technology (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Peptides Or Proteins (AREA)

Priority Applications (1)

Applications Claiming Priority (4)

Publications (1)

Family

ID=8219815

Family Applications (1)

Country Status (4)

Families Citing this family (1)

Family Cites Families (5)

• 1996
  • 1996-11-14 EP EP96939046A patent/EP0866856A1/de not_active Withdrawn
  • 1996-11-14 CA CA 2237548 patent/CA2237548A1/en not_active Abandoned
  • 1996-11-14 JP JP9518589A patent/JP2000500968A/ja active Pending
  • 1996-11-14 WO PCT/EP1996/005011 patent/WO1997018306A1/en not_active Application Discontinuation

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events