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WO1998013518A1 - Procede pour detecter des modulateurs de la relaxation myocardique - Google Patents

Procede pour detecter des modulateurs de la relaxation myocardique

Info

Publication number
WO1998013518A1
WO1998013518A1 PCT/EP1997/005265 EP9705265W WO9813518A1 WO 1998013518 A1 WO1998013518 A1 WO 1998013518A1 EP 9705265 W EP9705265 W EP 9705265W WO 9813518 A1 WO9813518 A1 WO 9813518A1
Authority
WO
WIPO (PCT)
Prior art keywords
tsub
ppl
plb
vesicles
protein
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/EP1997/005265
Other languages
English (en)
Inventor
Isabelle Berrebi-Bertrand
Antoine Michel Alain Bril
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SmithKline Beecham Laboratoires Pharmaceutiques
Original Assignee
SmithKline Beecham Laboratoires Pharmaceutiques
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 SmithKline Beecham Laboratoires Pharmaceutiques filed Critical SmithKline Beecham Laboratoires Pharmaceutiques
Publication of WO1998013518A1 publication Critical patent/WO1998013518A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • 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
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue

Definitions

  • the invention relates to a novel process, in particular to a screening process for identifying compounds having potentially useful medical utility.
  • myocardial relaxation primarily depends upon the function of a specific enzyme located in the sarcoplasmic reticulum (SR) - SR-Ca 2+ ATPase. This enzyme mediates the uptake of Ca 2+ ions into the SR via a cardiac Ca ⁇ " pump.
  • SR sarcoplasmic reticulum
  • PLB phosphoprotein, phospholamban
  • PLB is a known regulatory subunit present in two forms - phosphorylated (PPLB) and the aforementioned dephosphorylated form.
  • the dephosphorylated form acts to suppress the activity of the Ca 2+ ATPase by decreasing the affinity of the pump for Ca 2+ . This inhibitory effect is relieved - thereby promoting cardiac relaxation - when PLB is phosphorylated to PPLB.
  • the phosphorylation of PLB is catalysed by a protein kinase (PK).
  • PK protein kinase
  • the dephosphorylation of PPLB is catalysed by a particular protein phosphatase (PP) PLB-PP.
  • the PLB-PP associated with cardiac SR is a type 1 enzyme or PP1.
  • the catalytic activity of PP1 is known to be controlled by a 37 kiloDalton (kDa) protein subunit, PPlCsub.
  • PPl-Tsub targeting subunits
  • the present invention provides a process for detecting modulators of myocardial relaxation, which process comprises assessing a modulating effect of a putative modulator upon the phosphorylation of phospholamban.
  • Suitable modulators include proteins and small molecules, especially small molecules. Suitable modulators are inhibitors or antagonists of the dephosphorylation of phospholamban .
  • Inhibitors or antagonists of the dephosphorylation of phospholamban are potentially useful for the treatment of cardiac disorders associated with an inhibition of myocardial relaxation, such as congestive heart failure, left ventricular hypertrophy and cardiac arrhythmias.
  • the invention is considered to extend to modulators, in particular the inhibitors or antagonists, identified by use of the process of the invention.
  • Suitable assessments of the modulating effects comprise detecting and/or characterising the said modulating effects.
  • the dephosphorylation of phospholamban is assessed via an interaction, suitably a binding interaction, between phospholamban and a protein phosphatase (PP), suitably PPlCsub and especially PPlCsub-PPl-Tsub complex.
  • PP protein phosphatase
  • the phospholamban is suitably located in sarcoplasmic reticulum vesicles.
  • a suitable source of PPl-Tsub has been obtained from an homosapiens PPP1R3 mRNA for protein phosphatasel.
  • Genbank accession number is X78578.
  • the PPl- Tsub clone was obtained already inserted in BlueScript plasmid.
  • the sequence of the human glycogen-associated regulatory subunit of type 1 protein phosphatase has been published in Diabetes 43, (10) 1234-1241 (1994).
  • PPlCsub and anti PPlCsub are both commercially available: for example both can be obtained from Euromedex [PPlCsub, ref 14-110 and anti human PPlCsub, ref 06-22].
  • PLB has been produced in rabbit reticulocyte lysate using an "in vitro" transcription/translation system (Promega). This methodology offers several advantages including rapid protein synthesis and 35s Methionine labelling. The products can be rapidly analysed by SDS PAGE using autoradiography and immunoblot experiments.
  • PLB protein appears to be produced in its multimeric form in rabbit reticulocyte lysate and migrated slowly. When lysate denatured, the PLB protein co-migrated with the control form purified from dog RS (about 25 Kd).
  • Any conventional methodology can be employed to assess the interaction between PLB and PPl-Tsub, for example biosensor technology, radiobinding method, scintillation proximity assays, co-immunoprecipitation assays, native page electrophoresis or cross linking experiments methods.
  • radiolabelled 35S-PPl -Tsub is used, the effect of a putative modulator was then assessed by determining the effect of the putative modulator upon the transfer of the label to PLB using conventional radioisotopic methods.
  • immobilised SR vesicles which may be solubilized or non-solubilised.
  • the PLB of the immobilised vesicles retained binding capacity as demonstrated by interaction with PLB monclonal antibodies.
  • polyclonal antibodies raised against PPl-Tsub are able to bind to SR vesicles, suggesting the presence of viable PPl-Tsub in the immobilised preparations.
  • immobilised SR vesicles for example dog vesicles, form a further part of this invention, preferably using aminosilane coated micro-cuvettes.
  • Micro-cuvettes are available with a choice of derivatised sensor surfaces, including a carboxymethyl-dextran hydrogel, or an aminosilane surface linker specifically developed for large molecules and cells.
  • a binding agent such as bis (sulfosuccinimidyl) suberate (BS3,
  • ImM is used to cross link the vesicles to the immobilisation agent surface.
  • the vesicles are immobilised using known technology, for example that described below.
  • the monoclonal PLB antibodies are obtained from Upstate Biotechnology (ref 05-205 according to Suzuki and Wang, J Biol.chem 261 :7018 1986).
  • the pharmacological viability of the PPl-Tsub may be validated by demonstrating an interaction between the PP 1 -Tsub and polyclonal antibodies raised against active PPl-Tsub
  • Activation A baseline is established with sodium phosphate buffer which is then replaced with 200 ul of BS3 (Pierce, 21579) solution for 10 minutes, followed by aspiration and washing thoroughly with buffer.
  • the sodium phosphate buffer was then changed to a suitable buffer for the binding stage, eg PBS.
  • Dog SR Vesicles were solubilised with 1% Zwittergent in a cuvette and successfully immobilized onto aminosilane surface.
  • Binding and dissociation are increasingly important measures of biological activity and function.
  • Affinity sensors is an optical biosensor system for studying biomolecular interactions in real-time. It allows reactions to be watched as they happen, so revealing the dynamics as well as the strength of binding. Analysis is carried out very rapidly without the need for labels. Binding and dissociation are seen as shifts in resonance angle arising as one partner or more in free solution binds to, or dissociate from, the other partner immobilized at the surface of the sensor.
  • Skeletal muscle SR vesicles-anti-PPI-Tsub binding as PPl-Tsub is also expressed in skeletal muscle.
  • Dog SR vesicles were prepared according to Jones et al., J. Biol. Chem 1979,254,530-539.
  • Monoclonal antibodies raised in mouse against PLB are obtained from Upstate Biotechnology (ref 05-205 according to Suzuki and Wang, J Biol Chem 261 :7018 1986). Polyclonal antibodies raised in sheep against PPl-Tsub were obtained from Philip Cohen (University of Dundee).

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Urology & Nephrology (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un procédé pour détecter des modulateurs de la relaxation myocardique. Ce procédé consiste à évaluer un effet modulateur d'un modulateur putatif sur la phosphorylation du phospholamban et des vésicules du réticulum sarcoplasmique utilisées dans ce procédé.
PCT/EP1997/005265 1996-09-26 1997-09-24 Procede pour detecter des modulateurs de la relaxation myocardique Ceased WO1998013518A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR9611725A FR2753722A1 (fr) 1996-09-26 1996-09-26 Procede de detection de modulateurs de relaxation cardiaque et modulateurs ainsi obtenus
FR96/11725 1996-09-26

Publications (1)

Publication Number Publication Date
WO1998013518A1 true WO1998013518A1 (fr) 1998-04-02

Family

ID=9496079

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1997/005265 Ceased WO1998013518A1 (fr) 1996-09-26 1997-09-24 Procede pour detecter des modulateurs de la relaxation myocardique

Country Status (2)

Country Link
FR (1) FR2753722A1 (fr)
WO (1) WO1998013518A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000063426A3 (fr) * 1999-04-15 2001-02-08 Devgen Nv Procedes de criblage de composes
US8498704B2 (en) 2010-02-03 2013-07-30 Cardiac Pacemakers, Inc. Sympathetic stimulation for improved myocardial relaxation

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1354671A (zh) * 1998-11-02 2002-06-19 加利福尼亚大学董事会 抑制受磷蛋白活性以治疗心脏病和心力衰竭的方法
EP1728516A1 (fr) * 1998-11-02 2006-12-06 The Regents of the University of California Methode pour l'inhibition de l'activité de la phospholamban pour le traitement de maladies cardiaques et de l'insuffisance cardiaque
EP1317289B1 (fr) 2000-09-11 2009-02-25 The Regents of the University of California Mutante de PLB dominante négative pour le traitement des maladies cardiaques

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993019754A1 (fr) * 1992-03-27 1993-10-14 Smithkline Beecham Plc Derives de phenol et de pyridinol utilises comme agents lusitropes
WO1996032139A1 (fr) * 1995-04-11 1996-10-17 The Regents Of The University Of California Procede de modulation in vivo de la contractilite du muscle cardiaque

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993019754A1 (fr) * 1992-03-27 1993-10-14 Smithkline Beecham Plc Derives de phenol et de pyridinol utilises comme agents lusitropes
WO1996032139A1 (fr) * 1995-04-11 1996-10-17 The Regents Of The University Of California Procede de modulation in vivo de la contractilite du muscle cardiaque

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
E. G. KRANIAS: "Regulation of calcium transport by protein phosphatase activity associated with cardia sarcoplasmic reticulum.", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 260, no. 20, 15 September 1985 (1985-09-15), pages 11006 - 11010, XP002053755 *
I. EDES & E. G. KRANIAS: "Review: Regulation of cardiac sarcoplasmic reticulum function by phospholamban", MEMBRANE BIOCHEMISTRY, vol. 7, no. 3, 1987 - 1988, pages 175 - 192, XP002053752 *
K. L. KOSS & E. G. KRANIAS: "Phospholamban: a prominent regulator of myocardial contractibility", CIRCULATION RESEARCH, vol. 79, no. 6, December 1996 (1996-12-01), pages 1059 - 1063, XP002053754 *
K. TAKISHIMA ET AL.: "A spin-label study of the effects of drugs on calcium release from isolated sarcoplasmic reticulum vesicles.", JOURNAL OF BIOCHEMISTRY, vol. 87, no. 1, 1980, TOKYO, JP, pages 305 - 312, XP002053753 *
Z. AHMAD ET AL.: "Autonomic regulation of type 1 protein phosphatase in cardiac muscle.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 264, no. 7, 5 March 1989 (1989-03-05), MD US, pages 3859 - 3863, XP002053756 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000063426A3 (fr) * 1999-04-15 2001-02-08 Devgen Nv Procedes de criblage de composes
US8498704B2 (en) 2010-02-03 2013-07-30 Cardiac Pacemakers, Inc. Sympathetic stimulation for improved myocardial relaxation
US9278217B2 (en) 2010-02-03 2016-03-08 Cardiac Pacemakers, Inc. Sympathetic stimulation for improved myocardial relaxation

Also Published As

Publication number Publication date
FR2753722A1 (fr) 1998-03-27

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