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WO2004031235A1 - Regulation du recepteur humain de l'alpha-latrotoxine, independant du calcium - Google Patents

Regulation du recepteur humain de l'alpha-latrotoxine, independant du calcium Download PDF

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Publication number
WO2004031235A1
WO2004031235A1 PCT/EP2003/011059 EP0311059W WO2004031235A1 WO 2004031235 A1 WO2004031235 A1 WO 2004031235A1 EP 0311059 W EP0311059 W EP 0311059W WO 2004031235 A1 WO2004031235 A1 WO 2004031235A1
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WO
WIPO (PCT)
Prior art keywords
calcium
independent alpha
homolog
latrotoxin receptor
polypeptide
Prior art date
Application number
PCT/EP2003/011059
Other languages
English (en)
Inventor
Alex Smolyar
Original Assignee
Bayer Healthcare Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bayer Healthcare Ag filed Critical Bayer Healthcare Ag
Priority to AU2003267436A priority Critical patent/AU2003267436A1/en
Publication of WO2004031235A1 publication Critical patent/WO2004031235A1/fr

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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • GPCRs include receptors for such diverse agents as calcitonin, adrenergic hormones, endothelin, cAMP, adenosine, acetylcholine, serotonin, dopamine, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-1, rhodopsins, odorants, cytomegalovirus, G proteins themselves, effector proteins such as phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins such as protein kinase A and protein kinase C.
  • Still another embodiment of the present invention is a substantially purified Calcium-independent alpha-latrotoxin receptor (homolog 1) polypeptide encoded by any polynucleotide of the present invention.
  • homolog 1 Calcium-independent alpha-latrotoxin receptor
  • an expression vector of the present invention or a reagent of the present invention and a pharmaceutically acceptable carrier is provided.
  • Percent identity between a putative human calcium-independent alpha-latrotoxin receptor polypeptide variant and an amino acid sequence of SEQ ID NO:2 is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), and Henikoff &
  • the novel human CERL-2 homolog is highly expressed in the following cardiovascular related tissues: fetal heart, heart, pericardium, heart atrium (right), heart atrium (left), heart ventricle (left), heart apex, Purkinje fibers, interventricular septum, aortic smooth muscle cells, pulmonary artery smooth muscle cells, coronary artery endothelial cells. Expression in the above mentioned tissues demonstrates that the novel human CIRL-2 homolog or mRNA can be utilized to diagnose cardiovascular diseases. In addition, the activity of the novel human CIRL-2 homolog can be modulated to treat cardiovascular diseases.
  • effector cells accumulate at the site of allergic reaction in the airways and release toxic products that contribute to the acute pathology and eventually to the tissue destruction related to the disorder.
  • Other resident cells such as smooth muscle cells, lung epithelial cells, mucus-producing cells, and nerve cells may also be abnormal in individuals with asthma and may contribute to the pathology. While the airway obstruction of asthma, presenting clinically as an intermittent wheeze and shortness of breath, is generally the most pressing symptom of the disease requiring immediate treatment, the inflammation and tissue destruction associated with the disease can lead to irreversible changes that eventually make asthma a chronic disabling disorder requiring long-term management.
  • Athlete's foot (foot ringworm, caused by either Trichophyton or Epidermophyto ⁇ ), jock itch (groin ringworm, can be caused by a variety of fungi and yeasts), scalp ringworm, caused by Trichophyton or Microsporum), nail ringworm and body ringworm (caused by Trichophyton).
  • Skin growths which are abnormal accumulations of different types of cells, may be present at birth or develop later.
  • Noncancerous (benign) growth and cancerous (malignant) growth types are distinguished.
  • Moles (nevi) are small, usually dark, skin growths that develop from pigment-producing cells in the skin (melanocytes). Most moles are harmless. However, noncancerous moles can develop into malignant melanoma.
  • Skin tags are soft, " small, flesh-colored or slightly darker skin flaps that appear mostly on the neck, in the armpits, or in the groin. Lipomas are soft deposits of fatty material that grow under the skin, causing round or oval lumps.
  • aplastic anemia Several congenital and familiar forms of aplastic anemia have been described, including Fanconi's anemia, Shwachman-Diamond syndrome, familiar aplastic anemia, and aplasia associated with dyskeratosis congenita or amegakaryocytic thrompocytopenia.
  • Type I and Type II diabetes can be treated with agents that mimic insulin action or that treat diabetic complications by reducing blood glucose levels.
  • agents that reduces new blood vessel growth can be used to treat the eye complications that develop in both diseases.
  • any. of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans. Diagnostic methods
  • Purified calcium-independent alpha-latrotoxin receptor polypeptides comprising a glutathione-S- transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution.
  • Human calcium-independent alpha-latrotoxin receptor polypeptides comprise the amino acid sequence shown in SEQ ID NO:2.
  • the test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.
  • TaqMan quantitative analysis Specific primers and probe are designed according to the recommendations of PE Applied Biosystems; the probe can be labeled at the 5' end FAM (6-carboxy-fluorescein) and at the 3' end with TAMRA (6-carboxy-tetramethyl-rhodamine). Quantification experiments are performed on 10 ng of reverse transcribed RNA from each sample. Each determination is done in triplicate.
  • FAM 6-carboxy-fluorescein
  • TAMRA 6-carboxy-tetramethyl-rhodamine
  • Human embryonic kidney 293 cells transfected with a polynucleotide which expresses human calcium-independent alpha-latrotoxin receptor are scraped from a culture flask into 5 ml of Tris HCl, 5 mM EDTA, pH 7.5, and lysed by sonication. Cell lysates are centrifuged at 1000 rpm for 5 minutes at 4 °C. The supernatant is centrifuged at 30,000 x g for 20 minutes at 4 °C.
  • Female conscious SHR (Moellegaard/Denmark, 220 - 290 g) are equipped with implantable radiotelemetry, and a data aquisition system (Data Sciences, St. Paul, MN, USA), comprising a chronically implantable transducer/transmitter unit equipped with a fluid-filled catheter is used.
  • the transmitter is implanted into the peritoneal cavity, and the sensing catheter is inserted into the descending aorta.
  • Compounds are tested against a vehicle treated control group. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal) prior to formalin or capsaicin administration.
  • application routes i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal
  • TH free-floating tyrosine hydroxylase
  • Rotarod Test We use a modification of the procedure described by Rozas and Labandeira-Garcia (1997), with a CR-1 Rotamex system (Columbus Instruments, Columbus, OH) comprising an IBM-compatible personal computer, a CIO-24 data acquisition card, a control unit, and a four-lane ⁇ rotarod unit.
  • the rotarod unit consists of a rotating spindle (diameter 7.3 cm) and individual compartments for each mouse.
  • the system software allows preprogramming of session protocols with varying rotational speeds (0-80 rpm). Infrared beams are used to detect when a mouse has fallen onto the base grid beneath the rotarod.
  • Tumor cells or fragments, of mammary adenocarcinoma origin are implanted directly into a surgically exposed and reflected mammary fat pad in rodents! The fat pad is placed back in its original position and the surgical site is closed. Hormones may also be administered to the rodents to support the growth of the tumors. Compounds are administered p.o., i.p., i.v., i.m., or s.c according to a predetermined schedule. Tumor and body weights are measured and recorded 2-3 times weekly. Mean tumor weights of all groups over days post inoculation are graphed for comparison. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p 0.05 as compared to the control group.
  • Tumor cells or fragments, of prostatic adenocarcinoma origin are implanted directly into a surgically exposed dorsal lobe of the prostate in rodents.
  • the prostate is externalized through an ' abdominal incision so that the tumor can be implanted specifically in the dorsal lobe while verifying that the implant does not enter the seminal vesicles.
  • the successfully inoculated prostate is replaced in the abdomen and the incisions through the abdomen and skin are closed.
  • Hormones may also be administered to the rodents to support the growth of the tumors.
  • Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule.
  • l-2xl0 4 CD34 + cells were plated in triplicate in 24-well plates with 1ml Iscoves modified Dulbecco medium (IMDM) (Invitrogen) containing 10% fetal bovine serum (FCS, Invitrogen), 1% Glutamine (Invitrogen) supplemented with SCF (25ng/ml) (PeproTech), different concentration of Erythropoietin (O.OlU/ml - lU/ml) (Erypo® FS 4000, Cilag) with or without compounds. Control cells were incubated with 0.1-0.2%) DMSO instead of compounds. The cultures were incubated at 37°C in a fully humidified atmosphere with 5% C0 2 . After 9 to 14 days cells were harvested, counted and stained with phycoeiythrin (PE)-conjugated mAb against Glycophorin A (Pharmingen) to analyze differentiation.
  • IMDM Iscoves modified Dulbec
  • a J mice are exposed to the smoke from 2 unfiltered cigarettes per day for 6 days per week for 14 weeks. Non-smoking, age-matched animals are used as controls. Animals are orally dosed with test compound or vehicle 1 hour before and 7 hours after smoke exposure. This twice-daily dosing regime is continued throughout the smoke exposure period. On day 7 of the weekly exposure, animals are given only 1 dose of test compound and are not exposed to cigarette smoke.
  • the potency of a test compound is evaluated by comparison of the tobacco smoke induced increase in LMI in animals dosed with either the test compound or just the vehicle used for administration of the compound.
  • Rats are anesthetized by intramuscular administration of ketamine (75 mg/kg) and xylazine (15 mg/kg).
  • the abdomen is opened through a midline incision, and a polyethylene catheter (BECTON DICKINSON, PE50) is implanted into the bladder through the dome.
  • the catheter is tunneled through subcutis of the animal by needle (14G) to neck.
  • the inguinal region is incised, and a polyethylene catheter (BECTON DICKINSON, PE50) filled with saline (Otsuka) is inserted into a femoral vein.
  • the catheter is tunneled through subcutis of the animal by needle to neck.
  • the forward primer sequence was: Primerl cgcaaataccgtcaaacaga.
  • the reverse primer sequence was Primer2 ggctccggtaaatgatgaac Probe 1 agcaggaatgggcttgcaaagttg, labeled with FAM (carboxyfluorescein succinimidyl ester) as the , reporter dye and TAMRA (carboxytetramethylrhodamine) as the quencher, was used as a probe.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention porte sur un récepteur humain de l'alpha-latrotoxine, indépendant du calcium, sur des réactifs régulant ledit récepteur, et sur des réactifs se fixant aux produits géniques dudit récepteur, qui peuvent jouer un rôle dans la prévention, l'amélioration, ou la correction de dysfonctionnements ou maladies incluant de manière non limitative: les troubles cardio-vasculaires, respiratoires, gastro-intestinaux et hépatiques, du métabolisme, inflammatoires, hématologiques, neurologiques, et de la reproduction, et le cancer, le diabète et l'obésité.
PCT/EP2003/011059 2002-10-07 2003-10-07 Regulation du recepteur humain de l'alpha-latrotoxine, independant du calcium WO2004031235A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003267436A AU2003267436A1 (en) 2002-10-07 2003-10-07 Regulation of human calcium-independent alpha-latrotoxin receptor

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US41627002P 2002-10-07 2002-10-07
US60/416,270 2002-10-07
US46096703P 2003-04-08 2003-04-08
US60/460,967 2003-04-08

Publications (1)

Publication Number Publication Date
WO2004031235A1 true WO2004031235A1 (fr) 2004-04-15

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2003/011059 WO2004031235A1 (fr) 2002-10-07 2003-10-07 Regulation du recepteur humain de l'alpha-latrotoxine, independant du calcium

Country Status (2)

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AU (1) AU2003267436A1 (fr)
WO (1) WO2004031235A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7468350B2 (en) 2004-06-16 2008-12-23 Pneumrx, Inc. Glue composition for lung volume reduction
US7553810B2 (en) 2004-06-16 2009-06-30 Pneumrx, Inc. Lung volume reduction using glue composition
US7608579B2 (en) 2004-06-16 2009-10-27 Pneumrx, Inc. Lung volume reduction using glue compositions
US7678767B2 (en) 2004-06-16 2010-03-16 Pneumrx, Inc. Glue compositions for lung volume reduction
US7766938B2 (en) 2004-07-08 2010-08-03 Pneumrx, Inc. Pleural effusion treatment device, method and material

Citations (3)

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WO2000052039A2 (fr) * 1999-03-04 2000-09-08 Icos Corporation Materiaux de lectomedine et procedes
EP1103563A1 (fr) * 1998-07-23 2001-05-30 Takeda Chemical Industries, Ltd. Nouvelle proteine receptrice conjuguee a la proteine g et adn de cette derniere
WO2001075440A2 (fr) * 2000-03-31 2001-10-11 Mitsubishi Pharma Corporation Genes associes a la schizophrenie

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1103563A1 (fr) * 1998-07-23 2001-05-30 Takeda Chemical Industries, Ltd. Nouvelle proteine receptrice conjuguee a la proteine g et adn de cette derniere
WO2000052039A2 (fr) * 1999-03-04 2000-09-08 Icos Corporation Materiaux de lectomedine et procedes
WO2001075440A2 (fr) * 2000-03-31 2001-10-11 Mitsubishi Pharma Corporation Genes associes a la schizophrenie

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [online] 7 August 2003 (2003-08-07), "Toxicity related gene, Seq ID 3966", XP002269003, retrieved from EBI Database accession no. ADB58940 *
DATABASE UNIPROT [online] 1 May 1999 (1999-05-01), "Latrophilin 2 splice variant babbe", XP002269002, retrieved from EBI Database accession no. O97808 *
DATABASE UNIPROT [online] 1 May 1999 (1999-05-01), "Latrophilin 2 splice variant bbbaf", XP002269000, retrieved from EBI Database accession no. O97815 *
DATABASE UNIPROT [online] 1 May 1999 (1999-05-01), "Latrophilin 2 splice variant bbbbe", XP002269001, retrieved from EBI Database accession no. O97816 *
DATABASE UNIPROT [online] 1 May 1999 (1999-05-01), "Latrophilin 2 splice variant bbbbf", XP002268999, retrieved from EBI Database accession no. O97817 *
ICHTCHENKO K ET AL: "A NOVEL UBIQUITOUSLY EXPRESSED ALPHA-LATROTOXIN RECEPTOR IS A MEMBER OF THE CIRL FAMILY OF G-PROTEIN-COUPLED RECEPTORS", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 274, no. 9, 26 February 1999 (1999-02-26), pages 5491 - 5498, XP000828192, ISSN: 0021-9258 *
MATSUSHITA H ET AL: "The latrophilin family: multiply spliced G protein-coupled receptors with differential tissue distribution", FEBS LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 443, no. 3, 29 January 1999 (1999-01-29), pages 348 - 352, XP004259173, ISSN: 0014-5793 *
NAGASE T ET AL: "PREDICTION OF THE CODING SEQUENCES OF UNIDENTIFIED HUMAN GENES. XI. THE COMPLETE SEQUENCES OF 100 NEW CDNA CLONES FROM BRAIN WHICH CODE FOR LARGE PROTEINS IN VITRO", DNA RESEARCH, UNIVERSAL ACADEMY PRESS, JP, vol. 5, 1998, pages 277 - 286, XP000828191, ISSN: 1340-2838 *
WHITE ET AL: "Isolation and characterisation of a human homologue of the latrophilin gene from a region of 1p31.1 implicated in breast cancer", ONCOGENE, BASINGSTOKE, HANTS, GB, vol. 17, no. 26, 31 December 1998 (1998-12-31), pages 3513 - 3519,ERRATUM-3519, XP002107075, ISSN: 0950-9232 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7468350B2 (en) 2004-06-16 2008-12-23 Pneumrx, Inc. Glue composition for lung volume reduction
US7553810B2 (en) 2004-06-16 2009-06-30 Pneumrx, Inc. Lung volume reduction using glue composition
US7608579B2 (en) 2004-06-16 2009-10-27 Pneumrx, Inc. Lung volume reduction using glue compositions
US7678767B2 (en) 2004-06-16 2010-03-16 Pneumrx, Inc. Glue compositions for lung volume reduction
USRE46209E1 (en) 2004-06-16 2016-11-22 Pneumrx, Inc. Glue composition for lung volume reduction
USRE47231E1 (en) 2004-06-16 2019-02-12 Pneumrx, Inc. Glue composition for lung volume reduction
US7766938B2 (en) 2004-07-08 2010-08-03 Pneumrx, Inc. Pleural effusion treatment device, method and material

Also Published As

Publication number Publication date
AU2003267436A1 (en) 2004-04-23

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