WO2006013109A1 - Procede pour la verification de l'interaction d'une substance avec une molecule cible - Google Patents

Procede pour la verification de l'interaction d'une substance avec une molecule cible Download PDF

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Publication number
WO2006013109A1
WO2006013109A1 PCT/EP2005/008510 EP2005008510W WO2006013109A1 WO 2006013109 A1 WO2006013109 A1 WO 2006013109A1 EP 2005008510 W EP2005008510 W EP 2005008510W WO 2006013109 A1 WO2006013109 A1 WO 2006013109A1
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WO
WIPO (PCT)
Prior art keywords
target molecule
pattern
diffusion coefficients
cell
diffusion
Prior art date
Application number
PCT/EP2005/008510
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English (en)
Inventor
Hanna Jankevics
Michael Prummer
Horst Vogel
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Epfl Swiss Federal Institute Of Technology-Lausanne
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Publication date
Application filed by Epfl Swiss Federal Institute Of Technology-Lausanne filed Critical Epfl Swiss Federal Institute Of Technology-Lausanne
Publication of WO2006013109A1 publication Critical patent/WO2006013109A1/fr

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Classifications

    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • 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

Definitions

  • the present invention relates to a method for testing a substance interacting with a target molecule in a cell by fluorescence correlation spectroscopy (FCS).
  • the target molecule is a nuclear hormone receptor, e.g. the estrogen receptor.
  • US 2002/0072076 describes a determination of the interaction of a substance with a hormone receptor in a cell by FCS wherein alterations of the diffusion coefficient of a fluorescently labelled hormone receptor in the absence or in the presence of a test compound is determined.
  • the method allows determination whether or not the test substance is a substance interacting with the hormone receptor. The method, however, does not allow a clarification of the effect of the test substance on the hormone receptor.
  • the present invention provides a method for testing a substance interacting with a target molecule in a cell, comprising: a) providing a cell containing a target molecule carrying a fluorescent label, b) adding a test substance to the cell and determining a pattern or distribution of diffusion coefficients for the fluorescently labelled target molecule by FCS, c) optionally comparing the pattern of diffusion coefficients obtained in step
  • step (b) with a reference pattern/distribution and d) optionally classifying the effect of the test substance on the target molecule based on the pattern of diffusion coefficients obtained in step
  • the target molecule may be any molecule, which can carry a fluorescent label, e.g. a peptide, polypeptide or nucleic acid, e.g. DNA or RNA.
  • a fluorescent label e.g. a peptide, polypeptide or nucleic acid, e.g. DNA or RNA.
  • the target molecule is a polypeptide or protein, e.g. a hormone receptor such as the estrogen receptor (ER).
  • the target molecule may be located in the cytoplasm, in the nucleus, in an organelle or on and/or adjacent to a cell membrane.
  • the cell is preferably a living cell, e.g. a prokaryotic, or eukaryotic, e.g. an animal or plant cell. More preferably, the cell is a mammalian, e.g. human cell.
  • the target molecule carries a fluorescent label.
  • Fluorescent labels can be added externally and subsequently bound to the target molecule, e.g. chemical compounds like ligands which specifically interact with biological macromolecules or macromolecular complexes or macromolecular structures on the cell surface, inside the cell membrane, or inside of the cell.
  • cellular components might be labeled either chemically, biochemically, by expressing suitable mutant proteins, e.g. fusion proteins between the protein of interest and different fluorescent proteins such as GFP and YFP or, fusion proteins between the protein of interest and human alkylguanin-DNA-alkyltransferase which subsequently are selectively modified with a fluorescent label (Keppler et al.
  • labelled target molecules are fusion proteins of a fluorescent protein, e.g. GFP or YFP with nuclear hormone receptor.
  • fluorescent protein e.g. GFP or YFP with nuclear hormone receptor.
  • suitable labelled target molecules are:
  • YFP-labeled full length steroid receptor coactivator-3 SRC-3
  • [YFP-FL] YFP-labeled receptor interaction domain (RID) of SRC-3
  • [YFP-RID] YFP-labeled point mutated YFP-ER (not interacting with SRCs)
  • further test systems may comprise the testing of specific nuclear hormone receptor interaction partners such as DNA, co- activator proteins, co-repressor proteins, chaperone proteins, proteasomes and proteins involved in the general transcription machinery.
  • specific nuclear hormone receptor interaction partners such as DNA, co- activator proteins, co-repressor proteins, chaperone proteins, proteasomes and proteins involved in the general transcription machinery.
  • the present invention is based on the finding that the determination of diffusion coefficients for the fluorescently labelled target molecule by FCS in the presence of a test substance under defined test conditions in a heterogeneous environment gives a pattern or distribution of diffusion coefficients which is characteristic for the effect of the test substance on the target molecule.
  • the diffusion coefficient is preferably determined by comparing a measured intensity autocorrelation function with a model function assuming one or more, e.g. two diffusive components.
  • the determination of a pattern of diffusion coefficients requires a plurality of measurements. The measurements may be carried out under identical and/or different test conditions. In addition, a number of distributions may be determined under different test conditions, e.g.
  • test substance by varying the concentration of the test substance, physical parameters such as temperature, pressure, irradiation (e.g. UV, VIS 1 IR), stress (e.g. chemical or mechanical stress) or environmental conditions (e.g. medium composition, presence of chemical compounds or surfaces with particular composition and/or structure) or any combination thereof, and a part of the whole set of distributions of the target molecule may be used to assess the effect of a test substance.
  • physical parameters e.g. UV, VIS 1 IR
  • stress e.g. chemical or mechanical stress
  • environmental conditions e.g. medium composition, presence of chemical compounds or surfaces with particular composition and/or structure
  • the distribution of the diffusion coefficients for a test compound may be compared with one or several reference patterns or distributions of diffusion coefficients. These reference patterns may be obtained by determining a pattern of diffusion coefficients with a substance having an already known effect on the target molecule.
  • the present invention it may be determined, if a test substance binds or interacts with the target molecule at all. Additionally, the present invention allows a classification of the effect of the test substance on the target molecule, e.g. by determining the class of ligands the test compounds belong to.
  • the test compound may be. a partial or complete agonist or a partial or complete antagonist of the target molecule.
  • the method of the present invention is particularly useful for clinical diagnostics and high-throughput drug screening.
  • HEK293 Endothelial breast cancer cells
  • HEK293 Human Embryo Kidney cells 293
  • CHO Chinese Hamster Ovary cells
  • HEK293, HeLa, CHO and MCF-7 cells were regularly maintained in DMEM/F-12 with Glutamax-I (Invitrogen Corporation, UK) with 2.2 % Fetal Calf Serum in 5 % CO 2 incubator at 37C.
  • Glutamax-I Invitrogen Corporation, UK
  • 2.2 % Fetal Calf Serum in 5 % CO 2 incubator at 37C.
  • FCS measurements were performed using the Confocor2-LSM510 microscope (Zeiss, Jena, Germany) equipped with an C-Apochromat 40x 1.2 water immersion objective.
  • the YFP fluorescence was excited with a 488-nm line from an argon ion laser and detected through a pinhole with a diameter of 70 ⁇ m, and a 530-600 nm band pass filter. A laser power of below 3 kW/cm 2 for the FCS experiments.
  • the LSM510 was used to localize cells with a low expression level, and to position the laser beam for the FCS measurement in different areas of the cell.
  • the coordinates of the laser beam in the LSM510 images for different zooms and scan speeds were determined by recording the coordinates of the bleached spot seen on a cover glass slide containing fluorescent glue, after a short pulse of the FCS laser at maximum intensity settings.
  • the cover slide was a kind gift from Zeiss (Zeiss, Jena, Germany).
  • Rhodamine 6G Rho ⁇ G
  • PBS solution pH 7.4.
  • Rho ⁇ G diffusion was obtained as an average of 20 consecutive measurements, at the experimental conditions used for the subsequent study.
  • the confocal volume changed slightly for every measurement day, and in turn also the characteristic diffusion times. Therefore it was necessary to normalize the diffusion times retrieved in experiments made at different days to one standard rhodamine diffusion time. All diffusion times given in this work are scaled to a Rho ⁇ G diffusion time of 21 ⁇ s.
  • FCS data analysis In the data analysis the raw data are described by a suitable correlation function. To fit the correlation function to the raw data, an iterative procedure was performed with the Levenberg-Marquardt algorithm to minimize chi 2 .
  • the mean BF and the standard deviation of the mean were calculated for the different sample conditions and used in further evaluations. Normally, at least ten cells were investigated of the same experimental conditions.
  • the mean BF for the different ligands was plotted versus the ligand concentration to get dose-response curves. These dose-response curves were used to evaluate the potency of the different ligands to induce immobile ERs.
  • a half-maximal value of the effective concentration (EC50) for the bleaching was calculated from these curves for each ligand in order to separate their individual potency to immobilize the ER inside the nuclei.
  • "immobile” corresponds to the timescale of FCS, i.e. to those particles which move so slowly that their fluorophores are bleached during passage across the detection volume.
  • the autocorrelation functions of YFP chimeras were fitted to one- , two-, or three-component models of free 3D diffusion, plus two dark-states for all in vivo measurements, to derive the translational diffusion coefficients and the number of molecules diffusing through the confocal volume.
  • the second model will be preferred over the first.
  • the F-distribution can be found tabulated in a number of books, and it is also available from different software packages (e.g. Mathematica 4.2, Wolfram Research Inc., Champaign, IL, USA).
  • Weighted histograms are mean histograms that are constructed in the following manner: the amplitude of the bin in these weighted histograms is made by adding the fraction of each diffusion coefficient that falls into that bin. The fraction of the diffusion coefficient is retrieved from the 2P* fit.
  • Diffusion coefficient histograms at different ligand concentrations In order to compare the distributions of diffusion coefficients caused by the different ER ligands in vivo and to compare data from different ligand concentrations, the 2P* model with two dark-states was used. The reason for keeping the same model for all ligand concentrations was that the change of model will change the diffusion coefficients retrieved, and hence to be able to directly compare the data they all have to be fitted with the same model.
  • the first diffusive particle was kept fixed to increase the number of successful fits. This is due to the sensitivity of the data fitting procedure.
  • the fixed value was chosen based on a first run through the data leaving the two diffusion parameters free. This value was then kept the same for all ligand concentrations, because the choice of this value will affect the second diffusion coefficient as well.
  • Figure 1 shows a discrimination of different concentrations of an added ligand (E2) by comparison of the measured distributions of the diffusion coefficient.
  • Figure 2 shows a discrimination of different added ligands by comparing the measured distributions of the diffusion coefficient of the receptor.
  • Antagonists (ICI), partial agonists (4OHT), and agonists (E2, BPA) exhibit different diffusion patterns. Even though the partial agonist 4OHT acts as an antagonist in MCF-7 cells its diffusive behavior differs significantly from that of the full antagonist ICI. This detailed discrimination goes far beyond a pure detection of binding events (Fig. 2A ⁇ D). A determination of the ligand binding curves of the different ligands to the receptor from the bleaching (Fig. 2E) and the mobility distributions (Fig. 2F).
  • Figure 3 shows a discrimination of the effect of different ligands on the estrogen receptor and 2 different coactivator segments, RID and FL.

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  • Health & Medical Sciences (AREA)
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Abstract

La présente invention a trait à un procédé pour la vérification de l'interaction d'une substance avec une molécule cible dans une cellule par la spectroscopie à corrélation de fluorescence par l'addition d'une substance test à la cellule et la détermination d'une distribution de coefficients de diffusion pour la molécule cible marquée par fluorescence. De préférence, la molécule cible est un récepteur hormonal nucléaire, par exemple, un récepteur d'oestrogène.
PCT/EP2005/008510 2004-08-06 2005-08-05 Procede pour la verification de l'interaction d'une substance avec une molecule cible WO2006013109A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP04018731.2 2004-08-06
EP04018731 2004-08-06

Publications (1)

Publication Number Publication Date
WO2006013109A1 true WO2006013109A1 (fr) 2006-02-09

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2186827A1 (fr) 2008-11-14 2010-05-19 HS LifeSciences Ltd. Clonage ADNc dirigé par un marqueur de substitution d'ARN induits sélectivement
US7892756B2 (en) 2006-10-19 2011-02-22 Monell Chemical Senses Center Human salty taste receptor and methods of modulating salty taste perception

Citations (3)

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US20030022224A1 (en) * 2001-07-19 2003-01-30 Olympus Optical Co., Ltd. Method of detecting binding reaction between protein and test substance
US6582907B1 (en) * 1999-12-09 2003-06-24 Pharmacia & Upjohn Company Use of fluorescence correlation spectroscopy to identify compounds that bind to target species under isothermal denaturing conditions
US20040142386A1 (en) * 1993-01-18 2004-07-22 Evotec Biosystems Gmbh Method and a device for the evaluation of biopolymer fitness

Patent Citations (3)

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US20040142386A1 (en) * 1993-01-18 2004-07-22 Evotec Biosystems Gmbh Method and a device for the evaluation of biopolymer fitness
US6582907B1 (en) * 1999-12-09 2003-06-24 Pharmacia & Upjohn Company Use of fluorescence correlation spectroscopy to identify compounds that bind to target species under isothermal denaturing conditions
US20030022224A1 (en) * 2001-07-19 2003-01-30 Olympus Optical Co., Ltd. Method of detecting binding reaction between protein and test substance

Non-Patent Citations (4)

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Title
MODOS K ET AL: "Maximum-entropy decomposition of fluorescence correlation spectroscopy data: application to liposome-human serum albumin association", EUROPEAN BIOPHYSICS JOURNAL SPRINGER-VERLAG GERMANY, vol. 33, no. 1, February 2004 (2004-02-01), pages 59 - 67, XP002350974, ISSN: 0175-7571 *
N. KAHYA ET AL: "Lipid domain formation and dynamics in giant unilamellar vesicles explored by fluorescence correlation spectroscopy", JOURNAL OF STRUCTURAL BIOLOGY, vol. 147, July 2004 (2004-07-01), pages 77 - 89, XP002350975 *
WACHSMUTH MALTE ET AL: "Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching.", BIOPHYSICAL JOURNAL, vol. 84, no. 5, May 2003 (2003-05-01), pages 3353 - 3363, XP002352320, ISSN: 0006-3495 *
WEIDEMANN T ET AL: "Counting Nucleosomes in Living Cells with a Combination of Fluorescence Correlation Spectroscopy and Confocal Imaging", JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB, vol. 334, no. 2, 21 November 2003 (2003-11-21), pages 229 - 240, XP004470971, ISSN: 0022-2836 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7892756B2 (en) 2006-10-19 2011-02-22 Monell Chemical Senses Center Human salty taste receptor and methods of modulating salty taste perception
EP2186827A1 (fr) 2008-11-14 2010-05-19 HS LifeSciences Ltd. Clonage ADNc dirigé par un marqueur de substitution d'ARN induits sélectivement

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