EP1704406A4 - Systeme de detection d'adp a base de fluorescence - Google Patents

Systeme de detection d'adp a base de fluorescence

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Publication number
EP1704406A4
EP1704406A4 EP05702434A EP05702434A EP1704406A4 EP 1704406 A4 EP1704406 A4 EP 1704406A4 EP 05702434 A EP05702434 A EP 05702434A EP 05702434 A EP05702434 A EP 05702434A EP 1704406 A4 EP1704406 A4 EP 1704406A4
Authority
EP
European Patent Office
Prior art keywords
fluorescence
chelator
edta
adp
signal
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.)
Withdrawn
Application number
EP05702434A
Other languages
German (de)
English (en)
Other versions
EP1704406A2 (fr
Inventor
Gonzalo Castillo
Richard Leroy Mitchell
Jenny Ann Mulligan
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.)
MDS Pharma Services
Original Assignee
MDS Pharma Services
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 MDS Pharma Services filed Critical MDS Pharma Services
Publication of EP1704406A2 publication Critical patent/EP1704406A2/fr
Publication of EP1704406A4 publication Critical patent/EP1704406A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices

Definitions

  • the present invention is in the field of nucleotide detection.
  • it relates to the differential spectroscopic detection of nucleotides.
  • kinases have become a target for drug development for pharmaceutical companies. It has been estimated that up to 20% of all the targets in the pharmaceutical industry are currently kinases. The reason for this interest has to do with the fact that kinases play a crucial role in fundamental cellular processes. Disturbances in kinase activity may cause or be an indication of disease in humans, and targeting kinase molecules or receptors with drugs may alter the course of the disease.
  • Kinases are enzymes (biochemical catalysts) that transfer a phosphate group from ATP (adenosine triphosphate) to a substrate. As a result of the phosphate transfer, the ATP becomes dephosphorylated to form ADP (adenosine diphosphate). Once the substrate is phosphorylated in vivo, a biochemical pathway may be activated. A single kinase may have multiple substrates and, depending on which substrate is being phosphorylated, different pathways may be activated. Substrate selectivity is thus an important characteristic of kinases.
  • the biochemical reaction that kinases carry out is the following:
  • the substrate have an available hydroxyl group to accept the phosphate group being transferred by the kinase enzyme from the molecule of ATP.
  • Polypeptide or protein substrates thus generally contain tyrosine (tyrosine kinases) or serine/threonine (serine/threonine kinases) as the acceptor amino acid.
  • CK Creatine kinase
  • Creatine kinase is a "leakage" enzyme present in high concentration in the cytoplasm of myocytes and is the most widely used enzyme for evaluation of neuromuscular disease.
  • Current methods for CK detection involve multiple steps including the use of various other enzymes.
  • Detection of kinase activity for the purposes of drug discovery and drug profiling presents an interesting challenge for the pharmaceutical industry. Coupled detection systems with multiple enzymes, which are currently used in industry, are not practical due to the need for counter screens to rule out the effects of drugs on enzymes other than the one of interest. For example, in looking for CK inhibitors, hexokinase (HK), a coupled enzyme, can be affected by drugs that interact with the ATP binding site of CK because HK contains its own ATP binding site. [0008]
  • HK hexokinase
  • One approach that investigators searching for inhibitors of kinases in their drug discovery programs have traditionally used is the detection of substrate phosphorylation as a way to monitor kinase activity.
  • a currently used method to detect substrate phosphorylation is an ELISA based assay in which the detection of the phosphorylated substrate is done using a specific antibody. ELISA based systems show great sensitivity but many steps are required, a typical assay may run for hours, and many manipulations are needed, increasing the chance for error.
  • Another method currently used to monitor kinase activity is the detection and quantitation of the decrease of ATP as the assay progresses. This method is limited by the production of the product - in this case ADP - which, when accumulated, may inhibit the activity of the kinase. Application of such a methodology requires extensive assay development.
  • Detection systems traditionally used to monitor the progress of kinase assays utilize spectrometric techniques such as fluorescence spectrometry, fluorescence polarization (Panvera, Molecular Devices, Chromagen), time-resolved fluorescence (Cis-Bio), absorbance spectrometry (MDS Pharma Services, Upstate), luminescence spectrometry (Promega), and non-spectrometric techniques such as scintillation (Perkin Elmer, Amersham) and chromatography (Caliper).
  • fluorescence spectrometry fluorescence polarization
  • Cis-Bio time-resolved fluorescence
  • MDS Pharma Services Upstate
  • luminescence spectrometry Promega
  • non-spectrometric techniques such as scintillation (Perkin Elmer, Amersham) and chromatography (Caliper).
  • Figure 1 shows superimposed fluorescence spectra of ADP and ATP with HEPES used as a control.
  • Figure 2 shows superimposed fluorescence spectra of ADP in the presence of EDTA (ADP-EDTA) and ATP in the presence of EDTA (ATP-EDTA) with HEPES-EDTA used as a control.
  • Figure 3 shows superimposed fluorescence spectra of ADP in the presence of EGTA (ADP-EGTA) and ATP in the presence of EGTA (ATP-EGTA) with HEPES- EGTA used as a control.
  • Figure 4 shows the results of fluorescence data read at 500 nm showing a 50 mM EDTA solution upon titration with ADP.
  • Figure 5 shows the results of fluorescence data showing ADP in the presence of EDTA upon titration with Ca2+ and Mg2+.
  • Figure 6 shows the results of fluorescence data showing ADP in the presence of EDTA upon titration with ATP.
  • Figure 7 shows the results of fluorescence data showing a series of ADP and ATP solutions of different concentrations upon addition of 50 mM of EDTA.
  • Figure 8 shows superimposed fluorescence spectra of selected nucleotides in the presence and absence of EDTA, with HEPES or HEPES-EDTA used as a control. DETAILED DESCRIPTION OF THE INVENTION
  • the inventors have developed technology for the detection of nucleotides in assays. It may be used as a means of monitoring biochemical activity in assays, such as, for example, kinase activity in a kinase assay. It is a spectroscopic detection system which provides a mechanism to study any activity in which the concentration of a diphosphorylated nucleoside changes with time. It can be used, for example, to monitor the conversion of a di- or triphosphorylated nucleoside to a mono- or diphosphorylated nucleoside respectively, or vice versa, independent of the substrate being used. A particular diphosphorylated nucleoside is ADP, and a particular triphosphorylated nucleoside is ATP.
  • this technology may be used to screen kinase targets against substrates for which there currently are no available detection methods.
  • this methodology could be used to detect and/or monitor the activity of enzymes that utilize ATP and generate ADP.
  • ATP and ADP are invariably present in any kinase reaction. Systems based on monitoring the consumption of ATP and/or production of ADP can thus permit the monitoring of any kinase reaction, independent of the substrate. Since ADP is not present in the reaction before it starts, the monitoring of the production of ADP can be an effective method for monitoring the activity of a kinase.
  • the inventors have determined that there is a difference in the spectroscopic spectra of ATP and ADP which can be enhanced by the addition of a chelator. More specifically, under certain circumstances, there is a fairly consistent difference in the fluorescence emission at around 450 to 550 nm in the fluorescence spectra of ADP and ATP molecules, as demonstrated in Figure 1.
  • the inventors have additionally determined that, in the presence of a chelator such as ethylenediaminetetraacetic acid (EDTA) there is a substantial increase in ADP fluorescence in the 450 to 550 nm range without a corresponding increase in ATP fluorescence, as shown in Figure 2.
  • a chelator such as ethylenediaminetetraacetic acid (EDTA)
  • FIG. 3 shows that there is a substantial increase in ADP fluorescence in the presence of the chelator ethylene glycol-bis (2-aminoethylether)-N,N,N',N,-tetraacetic acid (EGTA) without an increase in ATP fluorescence.
  • Figure 4 shows that the intensity of fluorescence at 500 nm is proportional to the amount of ADP added to a solution of EDTA.
  • the term "chelator” refers to any molecule which possesses at least one functional group which can coordinate to a metal, either covalently or non- covalently.
  • the chelator may be multidentate or coordinate in a unidentate manner.
  • the chelator may be a macrocycle such as a porphyrin.
  • the chelator may chelate a metal by donating or sharing pi electrons. Chelators further derivatized to increase spectroscopic detection are also included.
  • the functional group of the chelator may be negatively or positively charged, or neutral.
  • Suitable functional groups include carboxylato, thiolato, hydrido, cyano, carbonato, thiocarcamato, thiocarboxylato, thiophosphinato amino, phophoro, hydrazino, nitrilo, hydrazido, oxime, and thioether.
  • chelators include ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), ethylene, and ethylene glycol-bis(2- aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA).
  • EDTA ethylenediaminetetraacetic acid
  • DTPA diethylenetriaminepentaacetic acid
  • EGTA ethylene glycol-bis(2- aminoethylether)-N,N,N',N'-tetraacetic acid
  • Suitable spectrometric techniques include fluorescence spectrometry, ultraviolet and infrared absorption and transmission spectrometry, luminescence spectrometry, Raman spectrometry, and phosphorescence spectrometry. Most preferred is fluorescence spectrometry.
  • Suitable fluorescence spectrometry techniques include the excitation of a sample with, for example, a xenon lamp, laser-induced fluorescence using lifetime fluorescence in order to increase detection sensitivity, fluorescent polarization which may boost the desired signal and lower background noise to improve sensitivity, and time-resolved fluorescence.
  • metals such as lanthanides, may be used to change the spectrometric characteristics, particularly the fluorescence spectrometric characteristics, of nucleotide-chelator interactions. It is known that tertiary complexes may be formed between certain organic molecules, chelators, and lanthanides (Dia andis EP, Christopoulos TK. Anal. Chem., 1990, 14:1149, Anal. Christopoulos
  • the peak generated by a fluorescence signal is typically broad, and the ability to precisely select of a wavelength of excitation and/or emission largely depends on the calibration of the instrument and the fluorescence detection system, or reader, used.
  • a Spex FluoroMax Spectrofluorometer (serial number 2093, Spex Industries, New Jersey) with a single well reader was used with a passband of 0.1 nm.
  • a Molecular Devices FLEXstation plate reader (serial number FX 01090, California) was used, which has a passband of 10 nm and therefore cannot detect the fluorescence signal as precisely as the SPEX Fluoromax scanner.
  • ATP A-7699 Sigma Ultra, LOT#053k7042, Adenosine 5'-triphosphate disodium salt
  • EGTA E0396 Sigma Ultra, Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'- tetraacetic acid
  • Experiment 1 Difference in Fluorescence Spectra of ATP and ADP in the range of 450 to 500 nm
  • a solution containing either 5 mM ADP or ATP in 10 mM HEPES pH 8.0 buffer was analyzed for fluorescence emission within the ranges of 400 to 600 nm using a SPEX Fluoromax fluorescence scanner.
  • the excitation wavelength used for this experiment was 405 nm. This experiment shows that there is a weak fluorescence emission difference between ADP and ATP measured at around 500 nm.
  • Experiment 2 Selective Interaction of EDTA and EGTA with ADP over ATP
  • a fluorescence scan was performed using a SPEX Fluoromax on a solution containing 10 mM ADP or 10 mM ATP in 10 mM HEPES pH 8.0 buffer in the presence or absence of 50 mM EDTA.
  • the fluorescence spectra of the solution containing ADP is greatly enhanced in the presence of EDTA.
  • ATP does not show an increase in fluorescence in the presence of EDTA.
  • Experiment 5 Titration of Solution Containing ADP and EDTA with ATP
  • ATP Since ATP does not seem to fluoresce in the presence of EDTA, and ADP does, the effect of the presence of ATP on the EDTA-ADP induced fluorescence was examined.
  • 10 mM ATP completely disrupts the interaction between ADP and EDTA. This observation suggests that ATP can directly interact with EDTA; however, it does not cause an increase in fluorescence under these conditions.
  • Experiment 6 Solution of ADP Titrated with EDTA
  • EDTA will be incubated in the presence and absence of terbium and in the presence or absence of increasing concentrations of nucleotides (ATP, ADP). Fluorescence emission will be monitored with the use of a SPEX Fluoromax fluorescence scanner.
  • EDTA will be incubated with europium and in the presence or absence of increasing concentrations of nucleotides (ATP, ADP).

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Optics & Photonics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Méthode et trousse de détection d'un nucléotide, ou de détection différentielle d'un nucléotide dans un mélange d'au moins deux nucléotides dans une solution. On ajoute un agent chélateur à la solution et on détecte un signal généré ou modifié par l'adjonction du chélateur en utilisant un système de détection spectroscopique. On peut également ajouter un lanthanide à la solution avant de détecter le signal. De préférence, le système de détection spectroscopique est un système à base de fluorescence.
EP05702434A 2004-01-16 2005-01-17 Systeme de detection d'adp a base de fluorescence Withdrawn EP1704406A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US53673804P 2004-01-16 2004-01-16
PCT/IB2005/000290 WO2005069725A2 (fr) 2004-01-16 2005-01-17 Systeme de detection d'adp a base de fluorescence

Publications (2)

Publication Number Publication Date
EP1704406A2 EP1704406A2 (fr) 2006-09-27
EP1704406A4 true EP1704406A4 (fr) 2008-05-28

Family

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EP05702434A Withdrawn EP1704406A4 (fr) 2004-01-16 2005-01-17 Systeme de detection d'adp a base de fluorescence

Country Status (4)

Country Link
EP (1) EP1704406A4 (fr)
JP (1) JP2007518094A (fr)
CA (1) CA2553218A1 (fr)
WO (1) WO2005069725A2 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996038724A1 (fr) * 1995-05-31 1996-12-05 Board Of Regents, The University Of Texas System Cations metalliques de lanthanides pour la detection et la separation simultanees lors de l'electrophorese capillaire
US5980861A (en) * 1996-03-12 1999-11-09 University Of Massachusetts Chelator compositions and methods of synthesis thereof

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4962045A (en) * 1988-05-02 1990-10-09 The Perkin-Elmer Corporation Time-resolved fluorimetric detection of lanthanide labeled nucleotides
JP4140929B2 (ja) * 1997-01-17 2008-08-27 旭化成ファーマ株式会社 １，５ａｇまたはａｄｐの測定法
GB0030727D0 (en) * 2000-12-15 2001-01-31 Lumitech Uk Ltd Methods and kits for detecting kinase activity
WO2004068115A2 (fr) * 2003-01-30 2004-08-12 Bellbrook Labs, Llc Procede relatif a des essais de reactions a transfert de groupe

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996038724A1 (fr) * 1995-05-31 1996-12-05 Board Of Regents, The University Of Texas System Cations metalliques de lanthanides pour la detection et la separation simultanees lors de l'electrophorese capillaire
US5980861A (en) * 1996-03-12 1999-11-09 University Of Massachusetts Chelator compositions and methods of synthesis thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHRISTOPOULOS T K ET AL: "ENZYMATICALLY AMPLIFIED TIME-RESOLVED FLUORESCENCE IMMUNOASSAY WITH TERBIUM CHELATES", ANALYTICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY. COLUMBUS, US, vol. 64, no. 4, 15 February 1992 (1992-02-15), pages 342 - 345, XP000261107, ISSN: 0003-2700 *
DIAMANDIS E P ET AL: "EUROPIUM CHELATE LABELS IN TIME-RESOLVED FLUORESCENCE IMMUNOASSAYS AND DNA HYBRIDIZATION ASSAYS", ANALYTICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY. COLUMBUS, US, vol. 62, no. 22, 15 November 1990 (1990-11-15), pages 1149A - 1150A,1152, XP000165602, ISSN: 0003-2700 *
P. PREM ET AL: "The effect of various chelating agents on the mobilization of iron from reticulocytes in the presence and abscence of pyridolal isonicotinoyl hydrazone", BBA, vol. 802, 1984, Amsterdam, NL, pages 477 - 489 *
See also references of WO2005069725A2 *

Also Published As

Publication number Publication date
JP2007518094A (ja) 2007-07-05
EP1704406A2 (fr) 2006-09-27
WO2005069725A2 (fr) 2005-08-04
WO2005069725A3 (fr) 2006-04-06
CA2553218A1 (fr) 2005-08-04

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