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WO2008006536A1 - Analyse de la gat1 - Google Patents

Analyse de la gat1 Download PDF

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Publication number
WO2008006536A1
WO2008006536A1 PCT/EP2007/006073 EP2007006073W WO2008006536A1 WO 2008006536 A1 WO2008006536 A1 WO 2008006536A1 EP 2007006073 W EP2007006073 W EP 2007006073W WO 2008006536 A1 WO2008006536 A1 WO 2008006536A1
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WO
WIPO (PCT)
Prior art keywords
active substance
complex
medium
protein
gat1
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/EP2007/006073
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German (de)
English (en)
Inventor
Natalie Watzke
Kerstin Diekert
Renate Gauss
Petr Obrdlik
Maarten Ruitenberg
Wolfgang Dörner
Bela Kelety
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.)
Iongate Biosciences GmbH
Original Assignee
Iongate Biosciences GmbH
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 Iongate Biosciences GmbH filed Critical Iongate Biosciences GmbH
Publication of WO2008006536A1 publication Critical patent/WO2008006536A1/fr
Anticipated expiration legal-status Critical
Ceased 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
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • 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
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the invention relates to a GATl assay, and more particularly to a method of identifying substances that affect the transport activity of GAT-I or GATl.
  • the invention further relates to an active substance complex which has been or has been identified according to the method according to the invention, a method for producing a drug, a test kit for carrying out the method according to the invention, a screening method and the uses of the method according to the invention, of the test kit according to the invention and of the medicament.
  • GABA ⁇ -aminobutyrate
  • GABA is the predominant inhibitory neurotransmitter and is widely distributed in the mammalian nervous system. GABA is displaced at the synaptic cleft by specific high-affinity transporters, of which at least four (GAT1-GAT4) have been cloned. In situ hybridization has shown that GAT 1 and GAT4 expression are brain specific.
  • the GATl GABA transporter (human 559 sequence) is approximately 2298 nucleotides long and contains a predictive a methionine-initiating coding sequence of about 1800 nucleotides, including stop codon.
  • the coding sequence located approximately between nucleic acids 235 and 2034, encodes a GAT 1 GABA transporter protein.
  • GABA activity at the GABA A receptor results in hyperpolarization of the synapses due to inflowing chloride ions.
  • the GAT 1 GABA transporter pumps GABA into the cell from the outside.
  • the hyperpolarization resulting from the blockage of the GABA transporter leads to an inhibitory effect on smooth muscle cell contraction.
  • modulators of the activity of the GAT 1 GABA transporter would be of great interest for the treatment of epilepsy and various cognitive disorders.
  • Radioactive measurements require specially trained personnel and necessary safety equipment. Both methods can only be performed “offline” and require “mediators” in the form of color reagents or radioactively labeled substrates. What is determined is not the immediate enzyme activity, but the change in a substrate concentration after completion of the transport. These Substrates capture transport activity very slowly over the course of minutes.
  • electrophysiological methods for example the so-called patch-clamp technique or two-electrode voltage-clamp technique, in which, for example, cells are accessible in isolation from an electrophysiological examination.
  • patch-clamp technique or two-electrode voltage-clamp technique
  • native cells are exposed to different conditions and certain electrical activities mediated across the cell membrane by proteins, protein complexes or the like contained therein are measured.
  • the present invention relates to an active ingredient or active ingredient complex, a method for producing a drug, a test kit for carrying out the erfmdungsgespecializeden method, a screening method, as well as uses of the method, the test kit and the drug.
  • Advantageous developments are the subject of the respective dependent claims.
  • GATl sodium and chloride ions are shifted across the membrane together with GABA ( ⁇ -ammobutyrate).
  • GABA ⁇ -ammobutyrate
  • the invention is based on the idea of measuring this GABA-dependent charge shift directly as binding of enzyme preparations on a suitable surface, which is integrated in a flow-through system or in which a substrate jump can be carried out, as the current flow and the influence of different active substances on the measured current flow to investigate.
  • the invention thus relates to a method for identifying an active substance complex which modifies an enzymatic property and / or the transport behavior of an active site complex which contains a GAT1 protein or a part thereof.
  • the erfmdungsgelaute method comprises the following steps: (a) providing a plurality of primary carriers which comprise the active site complex containing the GAT1 protein, in particular in a plurality in the region of a membrane of the respective primary carrier,
  • step (h) introducing the secondary carrier with the primary carriers into a third or activating measurement medium and detecting an electrical action according to process step (e), wherein the third or activating measurement medium corresponds to the second or non-activating medium but additionally contains a substrate of the GAT1 protein , in particular instead of a non-substrate present in the second or non-activating medium.
  • the erfmdungsgesorgeen steps (f), (g), (h) are - optionally carried out with repetitions - preferably in the predetermined order.
  • the term "enzymatic property" of the GAT1 proteme also always means the transport behavior, e.g. The mediated by these proteins substrate and / or ion transport and thus always refers to the initially discussed influence of the protein on the bioavailability of potential drugs. Wherever there is talk of the modification of the enzymatic properties of the protein, according to the invention investigations are also included which show whether a certain substance is transported by the GAT1 proteme.
  • the potential active substance can be present in all media.
  • the potential active substance In order to check whether a potential active substance is being transported, the potential active substance should be be present medium, but not in the accumulation buffer or in the non-activating medium.
  • the first medium or the attachment buffer Preferably, the first medium or the attachment buffer
  • - Cl is not or in low concentrations, in particular in lower concentrations than in the second or non-activating medium and as in the third or activating medium
  • pH 6-8 more preferably about 7.
  • the second medium or non-activating medium preferably contains:
  • pH 6-8 more preferably about 7
  • the third medium or activating medium preferably contains:
  • a substrate of the GATl protein more preferably GABA in a concentration of between 1 ⁇ mol / l and 10 mmol / l.
  • the quiescence / addition solution may be chloride free and contain gluconate.
  • the doses contain chloride.
  • any other anion that is not transported by GATl should also be able to be included in the deposition medium.
  • the active site complex or part thereof used is a monomer or an oligomer of a GAT1 protein.
  • an active ingredient or active substance complex identified by the process according to the invention, which modifies an enzymatic property of an active site complex which contains a GAT1 protein or a part thereof.
  • an active site complex or a part thereof based on a GAT1 protein derived from a tissue of a mammal e.g. from the small intestine, kidney or liver, or derived therefrom.
  • the GAT1 protein originates from mammalian cells and is present in cloned form.
  • the active site complex containing the GATl protein is derived from or derived from the organisms rat, pig, monkey, mouse, rabbit or human.
  • aqueous solutions and, in particular, aqueous electrolyte solutions are used as the measurement solution or measuring medium which, as an addition buffer, does not activate and activating solutions or media.
  • All measuring solutions used contain a buffer known to those skilled in the art, preferably selected from: MOPS, HEPES, MES, Tris, PIPES and the like. a., as they are e.g. in "Buffers, Calbiochem", but also publications and textbooks of chemistry, biology or biotechnology can be found. According to the invention, these agents should buffer at pH 6 to 8, preferably at about pH 7. The choice of the appropriate pH range or of the buffer can depend on the substrate (active substance complex) and can be determined by a person skilled in the art by routine experiments.
  • an attachment buffer is also used, composed of K-gluconate, MgCl 2 , Hepes, pH 7 / NMG, and DTT.
  • an attachment buffer composed of K-gluconate, MgCl 2 , Hepes, pH 7 / NMG, and DTT.
  • any other cation species of the 1st or 2nd main group of the PSE or choline "1" can also be used, the cation concentration being approximately as high as in the activating or non-activating solution.
  • the active substance complex or a part thereof can be transported directly to the site complex by injection or admixing into the measuring medium.
  • Such a process is simple if the respective measuring medium is exchanged, for example in a continuous manner.
  • the active substance is released only by a chemical or physical reaction or reaction in the measuring medium. This can be done for example by irradiation.
  • the qualitative and / or quantitative influence of the potential drug or drug complex is determined according to the invention by detecting an electrical action mediated by the active site complex or a part thereof and in particular by the GAT1 protein.
  • the electric action used is an electric current generated by the site complex or a part thereof or an electric potential generated therefrom, which is generated by charge or mass transport or displacement, conformational changes, ligand binding, annealing or release, or a Combination of it.
  • This electrical action is determined with and without potential drug, and by comparing both series of measurements, it is examined whether the potential drug has an influence on the enzymatic properties of the GAT1 protein.
  • the primary carriers are brought into contact with the active site complexes on a secondary carrier, namely the biosensor electrode and, above all, with their isolation region, and are deposited there.
  • a secondary carrier namely the biosensor electrode and, above all, with their isolation region, and are deposited there.
  • a biosensor electrode as a secondary substrate, in which an electrically conductive and fixed-body electrode region is provided with at least one electrode which is electrically and mechanically isolated from the measuring medium and from the primary carriers by providing an insulating region in the form of a solid-body-supported membrane , which is constructed in a layered manner from a lower layer of an organic thio compound as the lower and the electrode respectively facing layer and an upper layer of an amphiphilic organic compound.
  • a biosensor electrode is used as a secondary carrier, wherein an electrode in the electrode region of gold or a gold alloy, with a monolayer of a long-chain alkanethiol as a lower layer and a monolayer of a lipid as a top layer on it.
  • a biosensor electrode is used as a secondary carrier, in which the region of the isolation region covering one electrode of the biosensor electrode as a secondary carrier is or is formed as a membrane structure in the manner of a solid-body-supported double-layer membrane or bilayer membrane.
  • each eukariontician cell a prokariontician cell, in particular an oocyte, a bacterium, a virus, an organelle or constituents thereof, in particular membrane fragments, or associations thereof in native form and / or in modified form, in particular purified and / or modified form.
  • a vesicle, a liposome or a micellar structure can also be used as the primary carrier.
  • the membrane fragments can also attach planar, i. as a non-spherical structure.
  • the erfmdungsgedorfe method is particularly advantageous if the sensor assembly of biosensor electrode as a secondary carrier and P ⁇ mar- trolls attached thereto in a measuring chamber, measuring range or measuring vessel flows around the medium or is flown.
  • a change in concentration with respect to the active substance complex or a part thereof can be easily achieved by changing the measuring medium.
  • such measuring in the context of a flow system can reliably set the experimental conditions with high time resolution. Even with an automated pipettor, the erfmdungsgedorfe method can be advantageously carried out.
  • the erfmdungsgerate method designed when successively a plurality of tests is performed, in particular by successively replacing the measuring medium, possibly with intervening washing or coils of the measuring space. It is important in the present inventive method to compare the action of the active site complex in the present active substance complex with a situation in which the potential active substance complex is not present. This can be done, for example, by changing between activating medium to be activated in the presence or absence of the potential active substance complex and comparing the measured values obtained.
  • Potential active substances to be tested are particularly preferably monoclonal antibodies, antibody fragments, polyclonal antibodies and peptides. However, other substances that are suspected of having an effect can also be added. These substances are preferably administered in dissolved form.
  • Particularly preferred substances that can be tested as test substances in the test system according to the invention are low molecular weight compounds. Such compounds often have little or no side effects when used as an active principle in a pharmaceutical composition. Another advantage of such substances is the possibility of oral administration.
  • cyclic pentapeptides as described by Haubner et. al., J. Am. Chem. Soc. 1996, 118, 7641-7472.
  • small peptides, amino acids and amino acid analogues, steroids, nucleotides and other organic chemical substances having a molecular weight of ⁇ 5000, preferably ⁇ 3000 and particularly preferably ⁇ 2000, are attributed to the low molecular weight substances. It can be added to the active substance complex both in the addition buffer, in the non-activating medium and in the activating medium or released there.
  • a washing step is carried out between steps (f) and (h) by introducing the secondary carrier with the primary carriers into a fourth or washing medium, which is preferably identical to the non-activating medium.
  • step (f) is carried out repeatedly before step (h) in a particularly preferred embodiment of the method according to the invention.
  • step (f) is initiated before the beginning of the actual measurement series, i. before the steps (g) and (h) are carried out for the first time for at least 5 minutes (min), preferably for at least 10 min, more preferably for at least 15 min, even more preferably for at least 20 min and especially preferably for at least 30 min is performed.
  • steps (g) and (h) are carried out for at least 0.5 seconds (sec), but not more than 5 seconds and preferably for about 1-2 seconds.
  • step (f) is carried out for at least 1 second to at most 120 seconds, preferably for at least 30 seconds to 90 seconds, and particularly preferably for 50 seconds to 70 seconds.
  • the addition medium has potassium ions in concentrations in the range of about 10 mmol / l to about 200 mmol / l, preferably in the range of about 140 mmol / l.
  • the attachment medium has chloride ions in concentrations of about 0 mmol / 1 to 100 mmol / l, preferably in the range of about 4 mmol / l, in any case significantly less than the non-activating medium and the activating medium.
  • the other media ie the non-activating medium and the activating medium, have sodium ions in concentrations ranging from about 10 mmol / l to about 200 mmol / l, preferably in the range of about 140 mmol / l.
  • These media, ie the non-activating medium and the activating medium additionally have chloride ions in concentrations in the range of about 10 mmol / l to about 200 mmol / l, preferably in the range of about 140 mmol / l.
  • the quiescent / addition solution may be chloride free and contain gluconate.
  • the measuring solutions contain chloride. Instead of gluconate, any other anion that is not transported by GATl should also be able to be included in the deposition medium.
  • the activating medium contains a substrate of the GATl protein, preferably GABA.
  • Other possible substrates are other neurotransmitters.
  • the concentration may vary depending on the application.
  • GABA may be used in the range of about one or a few hundred ⁇ mol / l to a few tens of mmol / l, eg in the range of about 50 ⁇ mol / 1 to about 5 mmol / l, preferably in the range of about 100 ⁇ mol / l to about 1.5 mmol / l.
  • a concentration in the range above may also be expedient, for example in the range from about 1 mmol / l to about 100 mmol / l.
  • 10 mmol / l VaIm, 140 mmol / l NaCl, 2 mmol / l MgCl 2 , 30 mmol / l Hepes pH 7 / NMG and 200 ⁇ M DTT are alternatively used as non-activating medium.
  • 10 mmol / 1 GABA, 140 mmol / l NaCl, 2 mmol / l MgCl 2 , 30 mmol / l Hepes pH 7 / NMG and 200 ⁇ mol / 1 DTT are alternatively used as activating medium in a particularly preferred embodiment of the inventive method ,
  • the present invention provides a drug or drug complex which modifies an enzymatic property of an active site complex containing a GAT1 protein, or a portion thereof, or a plurality of properties.
  • the enzymatic property used is:
  • the present invention provides a method for producing a medicament comprising the steps of: Identifying an active substance and / or an active substance complex which suitably modifies an enzymatic property of an active substance complex which contains a GAT1 protein, of a part thereof or a plurality of properties,
  • test kit for carrying out the erfmdungsgeschreiben method for identifying a drug complex. This test kit points to:
  • At least one primary carrier for example with the GAT1 protein
  • a screening method for identifying namely for identifying: an unknown active substance, active substance complex, a part and / or derivative thereof,
  • the active substance, the active ingredient complex, a part or derivative thereof is an enzymatic property of a Wirkortkomplexes which contains a GATl proteme, or a part thereof modified.
  • the method according to the invention is used for identifying an active substance complex for finding inhibitors, activators, partial or temporary inhibitors or modulators of an enzymatic property of an active site complex which contains a GAT1 protein, for example a human GAT1 proteme.
  • test kit according to the invention is used according to the invention for finding inhibitors, activators, partial or temporary inhibitors or modulators a GATl protein-containing active site complex, such as a human GATl proteme.
  • the medicament according to the invention is used for the inhibition, partial or temporary inhibition, activation or other modulation of a GATl protease-containing active substance complex or a part thereof, for example a human GATl proteme.
  • a z As wassriges measuring medium provided in which the primary carriers and the secondary carriers are arranged.
  • the electrode region is preferably largely electrically insulated from the measuring medium, the primary carriers and from the biological units.
  • the electrode area has e.g. at least one electrode.
  • this can itself be designed as a mechanically stable material region, in particular as a plate, as a wire and / or the like.
  • the electrode is essentially designed as a material layer deposited on the surface of the carrier. It may be a vapor-deposited or sputtered material layer.
  • the material layer for forming the electrode preferably has a layer thickness of about 10 to 200 nm.
  • the GAT1 proteme can in each case be used in substantially native form and / or in a modified, in particular purified, microbiologically and / or molecularly modified form. On the one hand, this allows specific native properties to be tested and pharmacologically examined. On the other hand, molecular-biological or genetically engineered changes are also suitable certain aspects, for example, the transport or the pharmacological mode of action of an active substance.
  • magnetic carriers of a respectively substantially uniform type of primary carrier are provided. This is important with regard to the most unambiguous statement and analysis of a drug test and refers to the geometric, physical, chemical, biological and molecular-biological properties of the primary carriers.
  • one of the objects of the invention is to make the effect of substances on the function of GAT1 proteins amenable to investigation.
  • Substances that modulate the action of this transport protein are considered to be potential drugs, e.g. for the treatment of epilepsy and various cognitive disorders of commercial interest.
  • substrates of the protein transported by this were interesting new molecules.
  • the inventive method offers the following advantages:
  • the measurement of the enzyme activity according to the method proposed according to the invention requires no mediators or labeled substrates. A single measurement takes only a few seconds. The measurement is sensitive to all substrates. If a substance does not irreversibly inhibit the reaction, several measurements can be taken with an enzyme-loaded sensor. Substrates need not be chemically modified because the current response or potential response of the protein is induced by a rapid solution change.
  • the gold surface was made by inserting in a 1 mmol / l / l
  • the membrane fragments were prepared according to the following protocol: per liter cell pellet ImI sucrose buffer (0.25 mol / l sucrose, 5 mmol / l Tris, pH 7.5, about 2 mmol / l DTT, PMSF *, Complete **) ; * 50 ⁇ l PMSF as a saturated ethanolic solution to 100 ml buffer; ** Complete / Fa. Roche, one tablet per 50ml buffer.
  • the capacity of the protein-loaded membranes was about 300-1000 nF cm “2 , the conductivity G ls at about 10-100 nS cm "
  • the flow cell with integrated protembeladenem sensor chip was first with accumulation buffer (see above, 200 .mu.mol / 1 DTT instead of 2 mmol / 1) impressspult. This was followed by the non-activating solution (10 mmol / 1 valine, 140 mmol / l NaCl, 2 mmol / l MgCl 2 , 30 mmol / l Hepes pH 7 / NMG, 200 ⁇ mol / 1 DTT) and by electromechanical 3/2 Directional valve was then switched to activating solution (10 mmol / 1 GABA, 140 mmol / 1 NaCl, 2 mmol / 1 MgCl 2 , 30 mmol / 1 Hepes pH 7 / NMG, 200 .mu.mol / 1 DTT). The cotransport of sodium, chloride and GABA leads to a positive signal, as positive charge carriers are shifted to the membrane.
  • Example 5 Activity measurement without the cosubstrate sodium
  • Fig. 1 shows the current response of the loaded with GATl
  • FIG. 2 shows the current response of the control membrane-loaded biosensor to a GABA concentration jump.
  • FIG. 3 Current response of the GATl-loaded biosensor to a GABA concentration jump in different concentrations.
  • the ordinate of the figures thus denotes the measured electric current I (t), as a function of the time t, which is measured via the biosensor electrode.
  • Shown in FIG. 1 are three measuring tracks A, B and C, which correspond to different measuring conditions with respect to the substrate concentration at GABA.
  • GABA transport by means of the GATl protein, sodium and chloride ions are transferred via the primer carriers, e.g. via membrane fragments or vesicles, towards the biosensor membrane and the electrode.
  • the primer carriers e.g. via membrane fragments or vesicles
  • Lane B in Fig. 1 shows a measurement using 140 mmol / l KCl instead of NaCl. Without the cosubstrate of the sodium ions, which is no longer available for GABA transport when NaCl is replaced by KCl, sodium chloride GABA transport can no longer take place.
  • Lane C in FIG. 1 shows a measurement in which, in the presence of 140 mmol / l NaCl again by means of the GATl protein, sodium and chloride ions are moved toward the biosensor membrane and the electrode via the primary carriers. This can be seen analogously to track A in FIG. 1 on the measured positive current signal.
  • Fig. 2 shows the according to another embodiment of the inventive method with a biosensor electrode measured electric current I as a function of time t, using control membranes in which no GATl is present.
  • a biosensor electrode measured electric current I as a function of time t, using control membranes in which no GATl is present.
  • FIG. 3 shows four measuring tracks which correspond to different measuring conditions with respect to the substrate concentration at GABA.
  • the GAT1 protein units are normally activated, at 5 mmol / l and even more strongly at 2.5 mmol / l already a clearly visible decrease of the maximum of the measured positive electric current I (t) can be detected.
  • the signal is only slightly above the background noise.
  • Figure 3 thus shows the relevance of the presence of GABA for the transport activity of the GAT1 entities.
  • Fundamental advantages of the present invention are the high mechanical stability of the proposed sensor arrangement and, associated with it, the great operational readiness, the ease of handling and the low susceptibility to interference.
  • the use of the sensor arrangement results in a long service life, high reliability, low susceptibility to interference and, in particular, a significantly increased test throughput compared with conventional methods, whereby corresponding test methods can be elaborated and carried out inexpensively.
  • the signal quality is better, for a better signal-to-noise ratio.

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Abstract

La présente invention concerne un procédé d'identification d'une matière active, laquelle modifie une propriété enzymatique de la protéine GAT1. Des transporteurs primaires sont mis à disposition, lesquels contiennent la protéine GAT1 dans la zone de leurs membranes. Les transporteurs primaires sont déposés dans la zone superficielle d'une électrode de biocapteur servant de transporteur secondaire. Une matière active potentielle est mise à disposition. La matière active potentielle est amenée en interaction avec la protéine GAT1. On détermine l'influence qualitative et/ou quantitative de la matière active potentielle en détectant une action électrique de la protéine GAT1 ou sa modification par l'intermédiaire de l'électrode de biocapteur comme transporteur secondaire, par exemple au moyen d'une mesure électrique du courant ou de la tension.
PCT/EP2007/006073 2006-07-10 2007-07-09 Analyse de la gat1 Ceased WO2008006536A1 (fr)

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DE102006031913.3 2006-07-10
DE200610031913 DE102006031913A1 (de) 2006-07-10 2006-07-10 GAT1-Assay

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US20040259073A1 (en) * 2003-03-07 2004-12-23 Ariang Hassibi Charge perturbation signature methods and devices for membrane analysis

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WO2006037573A1 (fr) * 2004-10-01 2006-04-13 Iongate Biosciences Gmbh Analyse relative a la proteine de type pept1

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