WO2009103838A1 - Détection et quantification d'imines cycliques sur la base de la liaison compétitive aux récepteurs nicotiniques de l'acétylcholine - Google Patents

Détection et quantification d'imines cycliques sur la base de la liaison compétitive aux récepteurs nicotiniques de l'acétylcholine Download PDF

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Publication number
WO2009103838A1
WO2009103838A1 PCT/ES2009/070030 ES2009070030W WO2009103838A1 WO 2009103838 A1 WO2009103838 A1 WO 2009103838A1 ES 2009070030 W ES2009070030 W ES 2009070030W WO 2009103838 A1 WO2009103838 A1 WO 2009103838A1
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WO
WIPO (PCT)
Prior art keywords
alexafluor
cyclic imines
quantification
cyclic
nicotinic acetylcholine
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PCT/ES2009/070030
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English (en)
Spanish (es)
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WO2009103838A8 (fr
Inventor
Luis M. BOTANA LÓPEZ
Natalia VALARIÑO DEL RIO
Eva Fonfria Subiro
Jordi Molgo
Romulo Araoz
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Universidade De Santiago De Compostela
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Publication of WO2009103838A1 publication Critical patent/WO2009103838A1/fr
Publication of WO2009103838A8 publication Critical patent/WO2009103838A8/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
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9406—Neurotransmitters
    • G01N33/944—Acetylcholine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C251/00—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton
    • C07C251/02—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton containing imino groups
    • 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
    • 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
    • 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
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/4609—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates from reptiles
    • G01N2333/4613—Snake venom

Definitions

  • Gymnodimines and spirolides were discovered in the first half of the 1990s (Hu, T., et al. 2001. J Nat Prod, 64, 308-12; Molgó, J., et al. 2007. Phycotoxins. Chemistry and Biochemistry. , Botana, LM, 319-335; Seki, T., et al. 1995. Tetrahedron Lett, 36, 7093-7096). They are toxins produced by planktonic species that accumulate in the body of filtering shellfish that feed on them. As a consequence, these toxins reach man by the consumption of contaminated mollusks. The presence of gymnodimine and spirolide cyclic imines in shellfish can now be detected by two techniques.
  • the first one consists of intraperitoneal injection to mice of a certain weight of a food extract (Munday, R., et al. 2004. Toxicon, 44, 173-8; Richard, D., et al. 2000. Harmful Algal Blooms, 383; Yasumoto, T., et al. 1978. Bulletin of Japanese Society of Science and Fisheries, 44, 1249-1255).
  • This technique in addition to ethical problems for requiring the sacrifice of large numbers of animals, presents technical problems such as the large number of false positives and the lack of selectivity for different groups of toxins.
  • the second detection technique is performed by liquid chromatography coupled to mass spectrometry (LC-MS).
  • LC-MS liquid chromatography coupled to mass spectrometry
  • this method perfectly differentiates the different toxins, it requires very expensive and sophisticated equipment and technically very qualified personnel.
  • the LC-MS technique would not detect new toxin analogues not yet characterized or for which there are no standards (very common in the field of marine toxins) and which, however, may have a high toxic activity (Fux, E. , et al. 2007. J Chromatogr A, 1157, 273-80).
  • Fluorescence polarization and surface plasmon resonance are techniques widely used for the detection of interactions at the molecular level (Alfonso, C, et al. 2005. Anal Biochem, 344, 266-74; Kakehi, K., et al. 2001 Anal Biochem, 297, 111-6; Karlsson, R. 2004. J Mol Recognit, 17, 151-61). Fluorescence polarization is based on the ability of fluorescent molecules in solution to induce changes in the plane of polarization of light depending on their size. The technique requires fluorescent marking of one of the molecules that will interact, preferably the smallest.
  • Competing compound molecule that binds to nicotinic acetylcholine receptors at the same binding sites as cyclic imines.
  • Marker molecule that binds through a covalent bond to another and whose physical or chemical properties allow the detection by laboratory techniques of the presence of the compound formed by the union of both in a mixture.
  • Problem solution any solution in which it is desired to detect the presence or absence of cyclic imines, obtained from the extraction of mollusks, plankton, seawater, or any other origin that may be of interest.
  • the object of the invention is to provide a process for quantifying cyclic imines.
  • the method consists in a) contacting the nicotinic acetylcholine receptor with a test solution and with a competing compound; b) detect the binding or interaction between the nicotinic acetylcholine receptor and the competing compound; c) calculate the concentration of cyclic imines contained in the problem solution.
  • This method is based on the fact that the signal detected when the receptor binds to the competitor compound will be smaller the greater the amount of cyclic imines in the problem solution, that bind to the receptor competing with the competitor compound and inhibiting the latter's binding to the receiver.
  • the problem solution is contacted with the nicotinic receptor prior to the addiction of the competing compound.
  • the time that the problem solution must be in contact with the nicotinic receptor before the addition of the competing compound can be from 0 minutes to hours, depending on the affinities of the competing compound and the compound that is desired to be detected in the problem solution (cyclic imines ) for nicotinic receptors.
  • cyclic imine patterns such as gymnodimine or spirolide marine toxins
  • a calibration curve is obtained that will allow the concentration of cyclic imines in a problem solution to be calculated from the degree of inhibition of the nicotinic competitor-receptor compound interaction.
  • the competing compound is preferably selected from ⁇ -bungarotoxin, ⁇ -conotoxins, cobratoxin, cyclic imines or epibatidine.
  • the competing compound is preferably labeled with the following AlexaFluor® 488, tetramethylrhodamine, AlexaFluor® 555, AlexaFluor® 680, AlexaFluor® 647 AlexaFluor® 594, Oregon Green® 514, fluorescein, Texas Red®-R, biotin markers , HRP (horseradish peroxidase), radioactive iodine or tritium.
  • the detection method is preferably selected from i. the fluorescence polarization technique when the marking is preferably performed with the fluorescent markers AlexaFluor® 488, tetramethylrhodamine, AlexaFluor® 555, AlexaFluor® 680, AlexaFluor® 647 AlexaFluor® 594, Oregon Green 514, Texas Red®-R or fluorescein.
  • the technique of surface plasmon or optical mirror resonance biosensors when immobilization is preferably carried out by chemical reaction or biotin dizzying, iii.
  • AlexaFluor® 594 Oregon Green® 514, fluorescein, Texas Red®-R, biotin,
  • HRP horseradish peroxidase
  • radioactive iodine radioactive iodine or tritium
  • the invention is also directed towards a kit for the quantification of cyclic imines comprising at least the nicotinic acetylcholine receptor and a competing compound in the union with the nicotinic acetylcholine receptor.
  • the competing compound of this kit is preferably selected from ⁇ -bungarotoxin, ⁇ -conotoxins, cobratoxin, cyclic imines or epibatidine.
  • the competing compound of this kit is preferably labeled with AlexaFluor 488, AlexaFluor® 488, tetramethylrhodamine, AlexaFluor® 555, AlexaFluor® 680, AlexaFluor® 647 AlexaFluor® 594, Oregon Green® 514, fluorescein, Texas Red® -R, biotin, HRP (horseradish peroxidase), radioactive iodine or tritium.
  • the technique described in the present invention allows the use of nicotinic receptors of different origin, either of muscular or nervous origin, of any species that expresses this type of receptors or recombinants, isolated from tissues or cell cultures.
  • the process described in the present invention would be useful for quantifying any other substance that binds competitively with a compound labeled to nicotinic acetylcholine receptors at the same binding site as cyclic imines.
  • FIG. 1 Gymnodimine calibration curve (standard purchased from the Institute for Marine Biosciences, National Research Council, Suite, NS, Canada) in a test competition with ⁇ -bungarotoxin for binding to nicotinic receptors using fluorescence polarization.
  • Several dilutions of gymnodimine 2000, 1000, 500, 100 and 50 nM were prepared and mixed with nicotinic acetylcholine receptor. After a 2 hour incubation at room temperature, a 40 nM dilution of fluorescently labeled ⁇ -bungaro toxin was added with the Alexa Fluor 488 fluorophore. After a 30 minute incubation the fluorescence polarization values were measured.
  • Fluorescence polarization is detected for each well using a Hidex P ⁇ ate Chamaleon spectrofluorimeter (Turku, Finland). The length of Excitation light wave is selected with a 485 nm filter, while the fluorescence emitted by the sample is detected with polarization filters at 535 nm (vertical and horizontal).
  • the constant G is determined for each combination of filters and fluorescent molecule experimentally, in this case it has the value of 0.98.
  • the percentage response for each calibration concentration or sample is calculated considering as the maximum response or 100% the ⁇ -bungarotoxin with receptor and as a minimum response or 0% the ⁇ -bungarotoxin alone without receptor.
  • the response values of the calibration solutions are plotted against the corresponding toxin concentration and a calibration curve is obtained by a mathematical adjustment.
  • the response values obtained for the problem solution are substituted in the mathematical equation and the concentration of toxin containing said problem solution is thus calculated.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de quantification d'imines cycliques basé sur la liaison compétitive aux récepteurs nicotiniques de l'acétylcholine. Les imines cycliques gymnodimines et spirolides peuvent être considérées comme des contaminants dans les mollusques marins destinés à la consommation humaine. On a utilisé la capacité de ces imines cycliques à se lier aux récepteurs nicotiniques de l'acétylcholine de manière compétitive avec l'α-bungarotoxine pour la mise au point d'une méthode de détection de ces toxines en solution. L'invention se base sur la quantification de la liaison des récepteurs nicotiniques à l'α-bungarotoxine au moyen de techniques permettant de détecter les interactions moléculaires, telles que la mesure des changements de polarisation de fluorescence ou la surveillance des changements de l'indice de réfraction au voisinage d'une surface. Le signal de liaison de l'α-bungarotoxine au récepteur est inhibé par la présence d'imines cycliques en solution, ce qui permet la quantification de ces toxines marines au moyen d'un pré-étalonnage.
PCT/ES2009/070030 2008-02-21 2009-02-17 Détection et quantification d'imines cycliques sur la base de la liaison compétitive aux récepteurs nicotiniques de l'acétylcholine WO2009103838A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ESP200800608 2008-02-21
ES200800608A ES2325049B2 (es) 2008-02-21 2008-02-21 Deteccion y cuantificacion de iminas ciclicas basada en la union competitiva a receptores nicotinicos de acetilcolina.

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WO2009103838A1 true WO2009103838A1 (fr) 2009-08-27
WO2009103838A8 WO2009103838A8 (fr) 2009-12-10

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2484362A2 (fr) * 2009-09-30 2012-08-08 Universidade De Santiago De Compostela Utilisation de la gymnodimine, de ses analogues et de ses dérivés pour le traitement et/ou la prévention de maladies neurodégénératives associées aux protéines tau et -amyloïde

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020045618A1 (en) * 1999-04-26 2002-04-18 Nielsen Simon Feldbaek Heteroaryl diazacycloalkanes, their preparation and use
US20050131003A1 (en) * 2002-04-18 2005-06-16 Hui-Fang Chang Heterocyclic compounds

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020045618A1 (en) * 1999-04-26 2002-04-18 Nielsen Simon Feldbaek Heteroaryl diazacycloalkanes, their preparation and use
US20050131003A1 (en) * 2002-04-18 2005-06-16 Hui-Fang Chang Heterocyclic compounds

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ARAOZ, R. ET AL.: "Neurotoxins in axenic oscillatorian cyanobacteria: coexistence of anatoxin-a and homoanatoxin-a determined by ligand-binding assay and GC/MS", MICROBIOLOGY, vol. 151, 2005, pages 1263 - 1273 *
KAWAY, H. ET AL.: "Epibatidine binds to four sites on the torpedo nicotinic acetylcholine receptor", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 366, 2008, pages 834 - 839 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2484362A2 (fr) * 2009-09-30 2012-08-08 Universidade De Santiago De Compostela Utilisation de la gymnodimine, de ses analogues et de ses dérivés pour le traitement et/ou la prévention de maladies neurodégénératives associées aux protéines tau et -amyloïde
EP2484362A4 (fr) * 2009-09-30 2013-04-10 Univ Santiago Compostela Utilisation de la gymnodimine, de ses analogues et de ses dérivés pour le traitement et/ou la prévention de maladies neurodégénératives associées aux protéines tau et -amyloïde

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ES2325049B2 (es) 2010-03-16
ES2325049A1 (es) 2009-08-24
WO2009103838A8 (fr) 2009-12-10

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