DE102006031913A1 - GAT1 assay - Google Patents

Abstract

A method for identifying an agent that modifies an enzymatic property of the GAT1 protein is proposed. Primary carriers are provided which contain the GAT1 protein in the region of their membranes. The primary carriers are deposited in the surface area of a biosensor electrode serving as a secondary carrier. A potential drug is provided. The potential drug is interacted with the GAT1 protein. The qualitative and / or quantitative influence of the potential active substance is determined by detecting an electrical action of the GAT1 protein or its change via the biosensor electrode as secondary carrier, e.g. B. by means of an electrical current measurement or voltage measurement.

Description

The The invention relates to a GAT1 assay, and more particularly to a method to identify substances that affect the transport activity of GAT-1 be influenced or transported by GATT.

The The invention further relates to an active ingredient complex, which according to the inventive method is or has been identified, a method of making 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 the drug.

The To bring about advances in pharmaceutical drug development a rapid increase in the number of potentially available therapeutic Means. It is however common a big Problem of formulating the appropriate means so that they are bioavailable, i.e. sufficient from the body be included for optimal efficacy or tolerability to secure.

Natural transport proteins play an important role in the uptake of different molecules.

GABA (Γ-aminobutyrate) is the predominant inhibitory neurotransmitter and in the nervous system of mammals widespread. GABA becomes specific at the synaptic cleft high-affine transporter displaced, of which at least four (GAT1-GAT4) were cloned. In situ hybridization has shown that GAT1 and GAT4 expression is brain specific.

Of the GAT1 GABA transporter (human 559 sequence) is approximately 2298 Nucleotides long and includes a predicted methionine initiating coding Sequence of about 1800 nucleotides, including stop codon. The coding Sequence located approximately between nucleic acids 235 and 2034 is coded for a GAT1 GABA transporter protein.

GABA activity At the GABA A receptor results in hyperpolarization of the synapses by inflowing Chloride ions. The GAT1 GABA Transporter pumps GABA from outside into the Cell. The hyperpolarization caused by the blockage of the GABA Transporters arises, leads to an inhibitory effect on cell contraction of the smooth Muscle.

For the function GAT1 therefore requires a chloride gradient.

in the Regarding the functional role and the expression pattern would be modulators the activity of the GAT1 GABA transporter of great Interest in the treatment of epilepsy and various cognitive Disorders.

Corresponding In the prior art, different methods and measuring devices known, with those properties of certain drugs quantitatively and can be qualitatively analyzed and described.

Also it is desirable apply prescribed and known active substances to the site complex, e.g. on the GAT1 protein, on a cell, a tissue or the like to the operation of this To influence and / or investigate the site of action.

So are incorporation or uptake studies of radioactively labeled substrates at GAT1-overexpressed Cells already described in the prior art.

Usual experiments on the whole organism are due to the complexity of the organisms and further Due to ethical and moral circumstances often questionable and the the results obtained have only a limited validity.

consequently procedures and facilities have been developed to isolated consideration of the mode of action of drugs to be tested on isolated centers of action or an investigation of the centers of action even possible is. This will include certain tissues, isolated cells and / or others biological entities, for example proteins or the like, in Isolated way of a viewing made accessible.

It For example, techniques using dyes are known or radioactive substances work, and changing transport and / or binding specificities exploit and represent. The study of the effect of substances in radioactive Eindungs- and recording assays but usually no Distinguish whether an active substance only binds or transports will (would have to all active substances are radioactively labeled).

If this procedure at all possible is, but it is disadvantageous in that it often only a small Resolving power and a high susceptibility to errors get. Often, such methods are also relatively insensitive.

Radioactive measurements require specially trained personnel and necessary safety equipment. Both methods can only be performed "offline" and require "mediators" in the form of staining reagents or radioactively mar 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.

It electrophysiological methods are also known, for example the so-called patch-clamp technique or two-electrode voltage-clamp technique, in which, for example, cells are isolated from an electrophysiological Investigation accessible are. In this approach, native cells become different Exposed to certain conditions and there are certain electrical activities that over the Cell membrane by proteins, protein complexes or the like are measured.

In spite of The achievable high accuracy are these known electrophysiological methods disadvantageous in that they due to their high metrological Effort, their susceptibility and their very low throughput for a fast, automatable and / or broad use are unsuitable and due to in the Usually native environment of the objects to be examined certain problems in the discrimination of unwanted Have signal components.

Furthermore These methods are very costly because, for example, experienced Staff for the implementation needed becomes. Furthermore, for the Patch-clamp method and the two-electrode voltage clamp method a cell culture unit or the attitude of Xenopus laevis frogs required.

A particularly favorable method for measuring electrochemical processes on, for example, membrane fragments is described in US Pat WO 02/074983 described for the purposes of the present application. A use of the method disclosed therein for testing on GAT1 proteins is not disclosed.

Of the The invention is therefore based on the object, a GAT1 protein assay in which in a particularly simple and yet reliable way and rapid drug testing, especially in mass production, possible at reasonable cost is.

Be solved These and other tasks, which are discussed in the beginning State of the art, by a method for identifying an active substance complex with all features of claim 1.

Farther the subject of the present invention is a drug or drug complex, a method for producing a drug, a test kit for execution of the method according to the invention, a screening method, as well as uses of the method, the test kit and the drug. Advantageous developments are the subject the respective dependent Dependent claims.

in the Transport cycle of GAT1 become sodium and chloride ions together with GABA (γ-aminobutyrate) over the Membrane shifted. The invention is based on the idea, this GABA-dependent charge shift directly by attachment of enzyme preparations on a suitable Surface, which is integrated in a flow-through system or in which a substrate jump carried out can be measured as current flow and the influence of different Active substances on the measured variable current flow to investigate.

• (a) Bereitstellen einer Mehrzahl Primärträger, welche den Wirkortkomplex enthaltend das GAT1-Protein umfassen, insbesondere in einer Mehrzahl im Bereich einer Membran des jeweiligen Primärträgers enthalten,
• (b) Anlagern oder In-Kontakt-Bringen der Primärträger am oder im Oberflächenbereich eines Isolationsbereichs einer als Sekundärträger dienenden Biosensorelektrode in einem Messmedium, wobei der Sekundärträger bevorzugt gegenüber dem Messmedium und gegenüber den Primärträgern mittels des Isolationsbereichs mechanisch und elektrisch isoliert ist oder wird,
• (c) Bereitstellen mindestens eines potentiellen Wirkstoffkomplexes,
• (d) In-Kontakt-Bringen und In-Wechselwirkung-Bringen des potentiellen Wirkstoffkomplexes mit dem Wirkortkomplex der Primärträger oder Teilen davon, und
• (e) Bestimmen des qualitativen und/oder quantitativen Einflusses des potentiellen Wirkstoffkomplexes oder eines Teils davon auf enzymatische Eigenschaften des Wirkortkomplexes oder eines Teils davon durch Detektieren einer elektrischen Aktion oder des Wirkortkomplexes oder eines Teils davon oder eine Änderung dieser elektrischen Aktion über die Biosensorelektrode als Sekundärträger, dadurch gekennzeichnet, dass in den Verfahrensschritten (c), (d) und/oder (e) einer oder mehrere der folgenden Unterschritte durchgeführt werden:
• (f) Einbringen des Sekundärträgers mit den Primärträgern in einen Anlagerungspuffer und Detektieren einer elektrischen Aktion gemäß Verfahrensschritt (e)
• (g) Einbringen des Sekundärträgers mit den Primärträgern in ein zweites nicht-aktivierendes Medium und Detektieren einer elektrischen Aktion gemäß Verfahrensschritt (e), wobei im zweiten oder nicht-aktivierenden Medium ein Nicht-Substrat von GAT-1 vorgesehen ist,
• (h) Einbringen des Sekundärträgers mit den Primärträgern in ein drittes oder aktivierendes Messmedium und Detektieren einer elektrischen Aktion gemäß Verfahrensschritt (e), wobei das dritte oder aktivierende Messmedium dem zweiten oder nicht-aktivierenden Medium entspricht, jedoch zusätzlich ein Substrat des GAT1-Proteins enthält, insbesondere anstelle eines im zweiten oder nicht-aktivierenden Medium vorhandenen Nicht-Substrats.

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 inventive 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,
• (b) attaching or bringing into contact the primary carriers at or in the surface region of an isolation region of a biosensor electrode serving as a secondary carrier in a measurement medium, wherein the secondary carrier is or will be mechanically and electrically isolated from the measurement medium and from the primary carriers by means of the isolation region;
• (c) providing at least one potential active substance complex,
• (d) contacting and interacting the potential drug complex with the site complex of the primary carriers or portions thereof, and
• (E) determining the qualitative and / or quantitative influence of the potential active substance complex or a part thereof on enzymatic properties of the Wirkortkomplexes or a part thereof by detecting an electrical action or the Wirkortkomplexes or a part thereof or a change of this electrical action via the biosensor electrode as a secondary carrier , characterized in that in the process steps (c), (d) and / or (e) one or more of the following sub-steps are carried out:
• (f) introducing the secondary carrier with the primary carriers into an attachment buffer and detecting an electrical action according to the method step (s)
• (g) introducing the secondary carrier with the primary carriers into a second non-activating medium and detecting an electrical action according to process step (e), wherein a non-substrate of GAT-1 is provided in the second or non-activating medium,
• (h) introducing the secondary carrier with the primary carriers into a third or activating measuring medium and detecting an electrical action according to method step (e), wherein the third or activating measuring 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 inventive steps (f), (g), (h) - if appropriate with repetitions - preferred performed in the given order.

in the For purposes of the present invention, the term "enzymatic property" means the GATT protein at the same time always the transport behavior, e.g. through this Proteins mediated substrate and / or internal transport and thus decreases always referring to the influence of the protein discussed at the beginning on bioavailability potential agents. Wherever from the modification of the enzymatic Properties of the protein is, according to the invention also studies which includes whether a particular substance is transported by the GAT1 protein becomes.

Should checked whether a potential drug activates the GAT1 protein or inhibited, the potential drug may be present in all media.

Should checked whether a potential drug is being transported should the potential drug is present in the activating medium, but not in the accumulation buffer or in the non-activating medium.

• – K⁺
• – Cl^- nicht oder in niedrigen Konzentrationen, insbesondere in niedrigeren Konzentrationen als im zweiten oder nicht-aktivierenden Medium und als im dritten oder aktivierenden Medium
• – gegebenenfalls Mg⁺⁺ und
• – pH 6–8, besonders bevorzugt etwa 7.

Preferably, the first medium or the attachment buffer

• - K ⁺
• - Cl ^- 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
• Optionally Mg ⁺⁺ and
• PH 6-8, more preferably about 7.

• – Na⁺,
• – Cl^-,
• – gegebenenfalls Mg⁺⁺,
• – pH 6–8, besonders bevorzugt etwa 7,

The second medium or non-activating medium preferably contains:

• - Na ⁺ ,
• - Cl ^- ,
• Optionally Mg ⁺⁺ ,
• PH 6-8, more preferably about 7,

• – Na⁺,
• – Cl^-,
• – gegebenenfalls Mg⁺⁺,
• – pH 6–8, besonders bevorzugt etwa 7,
• – enthält, sowie
• – ein Substrat des GAT1-Proteins, weiter bevorzugt GABA in einer Konzentration zwischen 1 μmol/l und 10 mmol/l.

The third medium or activating medium preferably contains:

• - Na ⁺ ,
• - Cl ^- ,
• Optionally Mg ⁺⁺ ,
• PH 6-8, more preferably about 7,
• - contains, as well
• A substrate of the GAT1 protein, more preferably GABA in a concentration of between 1 μmol / l and 10 mmol / l.

Prefers is used as a measuring medium and in particular as a second or non-activating Medium and / or particularly preferred as the third or activating Medium media used with GABA, especially with a concentration from about 1 μmol / l to 10 mmol / l.

For the function GATT therefore requires a chloride gradient. Therefore, the Resting / annealing solution be chloride-free and contain gluconate. The measuring solutions included Chloride. Instead of gluconate, every other anion should also be used GAT1 is not transported, contained in the deposition medium can.

Prefers is the active site complex or part thereof a monomer or an oligomer used a GATT protein.

Further preferred is one identified by the method according to the invention Active substance or active substance complex, which has an enzymatic property an active site complex containing a GAT1 protein, or a part of it modified.

Further is used a Wirkortkomplex or a part thereof, which auf a GAT1 protein derived from a tissue of a mammal, e.g. from the small intestine, kidney or liver, or derived therefrom.

Especially the GAT1 protein is derived from mammalian cells and is cloned before.

Preferably the site complex containing the GAT1 protein is derived from the organisms rat, Pig, guinea pig, mouse, rabbit or human or is from these genetically derived.

According to the invention, aqueous measuring solutions and, in particular, aqueous electrolyte solutions are used as the measuring solution or measuring medium which serve as addition buffer, non-activating agent as well as activating solutions or media.

All used measuring solutions contain a buffer known to those skilled in the art, preferably selected from: MOPS, HEPES, MES, Tris, PIPES and others. a., as they are e.g. published in "Buffers, Calbiochem", but also publications and textbooks of Chemistry, biology or biotechnology can be taken. According to the invention these agents at pH 6-8, preferably buffer at about pH 7. The choice of the appropriate PH range or the buffer can depend on the substrate (active substance complex) and be determined by the expert by routine experimentation.

In a particularly preferred embodiment, an attachment buffer composed of K-gluconate, MgCl ₂ , Hepes, pH 7 / NMG, and DTT is also used. However, any other cation species of the 1st or 2nd main group of PSE or choline ^{+ can also} be used, the cation concentration being approximately as high as in the activating or non-activating solution.

• – Zumischen oder Injizieren des Wirkstoffkomplexes, eines Teils und/oder einer Vorstufe davon,
• – Austauschen oder Zumischen des Messmediums oder eines Teils davon und/oder
• – chemisches oder physikalisches Umsetzen oder Reagieren des Messmediums oder eines Teils davon oder des Wirkstoffs, eines Teils und/oder einer Vorstufe davon

durchgeführt werden.According to the invention, the method steps (c) and / or (d), optionally also (f) to (h) by means of

• Admixing or injecting the active substance complex, a part and / or a precursor thereof,
• - replace or mix the measuring medium or a part thereof and / or
• - chemical or physical reaction or reaction of the measuring medium or a part thereof or the active ingredient, a part and / or a precursor thereof

be performed.

The means that on the one hand the active substance complex or a part thereof directly by injecting or adding into the medium can be transported to the site complex. Simply designed such a process when the respective measuring medium exchanged is, for example, in a continuous manner. Next exists the possibility, that the active substance only by a chemical or physical Reacting or reacting in the medium to be released. This can for example, by irradiation.

Of the qualitative and / or quantitative influence of the potential active substance or active substance complex is inventively detected by detecting an electrical Action determines which by the Wirkortkomplex or a part and especially by the GAT1 protein.

When electrical action is used by the site complex or a part thereof generated electric power or an electric generated therefrom Potential, which is generated in each case by charge or mass transport or displacement, conformational changes, ligand binding, annealing or release, or a combination thereof.

These electrical action is determined with and without potential drug, and by comparing both sets of measurements it is examined whether the potential drug influences the enzymatic properties of the GAT1 protein.

Like in the WO02 / 074983 the primary carriers are described with the Wirkortkomplexen on a secondary carrier, namely brought the biosensor electrode and especially with their isolation area in contact and attached there.

at consist of the configuration of the biosensor electrode as a secondary carrier diverse Options.

Especially Preferably, a biosensor electrode is used as secondary carrier, in which an electrically conductive and more solid Electrode area is provided with at least one electrode, which opposite the measuring medium and opposite the primary carriers electrically and mechanically isolated by providing an isolation region in the form of a solid supported membrane, which layered structure is composed of a lower layer of an organic Thio compound as the lowest and the electrode facing each layer and an upper layer of an amphiphilic organic compound.

In a preferred embodiment of the method according to the invention a biosensor electrode is used as a secondary carrier, in which a Electrode in the electrode area made of gold or a gold alloy is, with a monolayer of a long-chain alkanethiol as an undercoat and a monolayer of a lipid as a topcoat thereon.

In Another preferred embodiment is a biosensor electrode used as a secondary carrier, in which one of the electrodes of the biosensor electrode covers as a secondary carrier Area of the isolation area as a membrane structure in the manner of a solid-supported double-layer membrane or bilayer membrane is or is formed.

in the Regard to the primary carriers, which the site complex and the GAT1 proteins in the area of their membrane contain different possibilities, with the objective of the procedure.

For the inventive method can as a primary carrier in each case a eukaryotic cell, a prokaryotic 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 a modified form, in particular in purified and / or modified Shape, to be used.

When Primary vehicles can too each a vesicle, a liposome or a micellar structure be used. The membrane fragments can also attach planar, i.e. as non-spherical Structure.

Especially Advantageously, the inventive method designed when the Sensor arrangement of biosensor electrode as a secondary carrier and attached to it primary carriers in A measuring chamber, measuring range or measuring vessel flows around the measuring medium or flows against becomes.

So let yourself for example, a change in concentration due to a change of the measuring medium with regard to the active substance complex or a part thereof easily realize. Also can be by such a measurement in the frame a flow system reliable with high time resolution the experimental conditions according to the invention to adjust. Also with an automated pipetting device can the inventive method be carried out advantageously.

Especially economical and for a statistical evaluation accessible the method according to the invention is designed in particular, when a plurality of tests are performed consecutively, in particular by successively replacing the medium to be measured, if necessary with Intermediate washing or rinsing the measuring space.

Important is in the present inventive method comparing the action of the active site complex in the case of the active ingredient complex with a situation where the potential drug complex is not present. This can be done for example by the Change between non-activating to be activated medium in the presence or absence of the potential Complex of active substances and comparison of the measured values obtained.

potential active substances to be tested are particularly preferably monoclonal antibodies, antibody fragments, polyclonal antibodies and peptides. It can however, other substances that are suspected to be can develop a corresponding effect, are added. These substances are preferably dissolved in Administered form.

Especially preferred substances used as test substances in the test system according to the invention can be examined are low molecular weight compounds. Such compounds often have none or only minor side effects when used as an active principle in a pharmaceutical composition can be used. Another advantage such substances is the possibility oral administration.

Examples include cyclic pentapeptides, as described by Haubner et. al., J. Am. Chem. Soc. 1996, 118, 7641-7472, have been described. According to the invention, small peptides, amino acids and amino acid analogs, 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 the active substance complex can be found both in the attachment buffer, in the non-activating Medium or added in the activating medium or released there become.

Also is an intermediate step an incubation of the Wirkortkomplexe or the primary carrier carrying them the active ingredient complex conceivable.

Of It is also conceivable that between steps (f) and (h) a washing step performed is by introducing the secondary carrier with the Primary carriers in a fourth or wash medium, which is preferred with the non-activating Medium is identical.

Further It is conceivable that right before the step (h) at a particular preferred embodiment the method according to the invention the step (f) is repeatedly performed.

It is preferred if the step (f) before the actual Measurement series, i. before steps (g) and (h) are performed for the first time, for at least 5 minutes (min), preferred for at least 10 minutes, more preferably for at least 15 minutes, even more preferred for at least 20 minutes, and more preferably for at least 30 minutes. The steps (g) and (h) according to the invention for at least 0.5 seconds (sec), at most but 5 sec and preferably for about 1-2 sec carried out. After the first implementation of steps (g) and (h) becomes step (f) for at least 1 second to at most 120 sec performed before given to for at least 30 sec to 90 sec, and more preferably for 50 sec to 70 sec.

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 addition medium has Chloridi in concentrations of about 0 mmol / l 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 one Medium have sodium 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. These media, ie the non-activating medium and the activating medium also has 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.

For the function GAT1 therefore requires a chloride gradient. Therefore, the Resting / annealing solution be chloride-free and contain gluconate. The measuring solutions included Chloride. Instead of gluconate, every other anion should also be used GAT1 is not transported, contained in the deposition medium can.

Of Further contains the activating medium is a substrate of the GAT1 protein GABA. Other possible Substrates are other neurotransmitters.

The Concentration may vary depending on the application. In competition studies GABA can range from about one or a few hundred μmol / l to to tens of mmol / l, e.g. in the range of about 50 μmol / l to about 5 mmol / L, preferably in the range of about 100 μmol / L to about 1.5 mmol / l. In drug tests for activation and / or inhibition of the GAT1 protein may also be useful for a concentration in the range above e.g. in the range of about 1 mmol / L to about 100 mmol / L.

According to the invention, 10 mmol / l valine, 140 mmol / l NaCl, 2 mmol / l MgCl ₂ , 30 mmol / l Hepes pH 7 / NMG and 200 μM DTT are alternatively used as non-activating medium.

According to the invention, 10 mmol / l GABA, 140 mmol / l NaCl, 2 mmol / l MgCl ₂ , 30 mmol / l Hepes pH 7 / NMG and 200 μmol / l DTT are alternatively used as activating medium in a particularly preferred embodiment of the inventive method ,

According to one In another aspect, the present invention provides an active ingredient or an active substance complex which has an enzymatic property a Wirkortkomplexes containing a GAT1 protein, or modified part or a plurality of properties.

• – die Bindung, Anlagerung oder Freigabe des Wirkstoffkomplexes oder eines Teils davon oder des Messmediums oder eines Teils davon,
• – der Transport oder die Verschiebung des Wirkstoffkomplexes oder eines Teils davon oder des Messmediums oder eines Teils davon,
• – die chemische Umsetzung oder Reaktion des Wirkstoffkomplexes oder eines Teils davon oder des Messmediums oder eines Teils davon,
• – eine Konformationsänderung oder Bewegung des Wirkortkomplexes oder eines Teils davon oder eine beliebige Kombination dieser Prozesse.

The enzymatic property used is:

• The binding, attachment or release of the active substance complex or a part thereof or the measuring medium or a part thereof,
• The transport or the displacement of the active substance complex or a part thereof or the measuring medium or a part thereof,
• The chemical reaction or reaction of the active substance complex or a part thereof or the measuring medium or a part thereof,
• A conformational change or movement of the active site complex or a part thereof or any combination of these processes.

• – Identifizieren eines Wirkstoffs und/oder eines Wirkstoffkomplexes, welcher eine enzymatische Eigenschaft eines Wirkstoffkomplexes, welcher ein GAT1-Protein enthält, oder eines Teils davon oder eine Mehrzahl von Eigenschaften geeignet modifiziert,
• – Herstellen und/oder Isolieren des Wirkstoffs, des Wirkstoffkomplexes, eines Teils und/oder eines Derivats davon,
• – Aufreinigen des Wirkstoffs, Wirkstoffkomplexes, Teils und/oder Derivats,
• – Mischen und/oder Portionieren des Wirkstoffs, Wirkstoffkomplexes, Teils und/oder Derivats mit einer pharmazeutisch verträglichen Trägersubstanz.

Furthermore, 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 or a part thereof or a plurality of properties,
• Preparing and / or isolating the active substance, the active substance complex, a part and / or a derivative thereof,
• Purifying the active substance, active substance complex, part and / or derivative,
• - Mixing and / or portioning of the active ingredient, active ingredient complex, part and / or derivative with a pharmaceutically acceptable carrier.

• – mindestens einen Primärträger beispielsweise mit dem GAT1-Protein,
• – einen Messbereich,
• – gegebenenfalls der erfindungsgemäße Anlagerungspuffer, das nicht-aktivierende Medium oder das aktivierende Medium und
• – weiterhin gegebenenfalls mindestens einen potentiellen Wirkstoffkomplex.

Furthermore, the present invention provides a test kit for carrying out the method according to the invention for identifying an active substance complex. This test kit points to:

• At least one primary carrier, for example with the GAT1 protein,
• A measuring range,
• If appropriate, the addition buffer according to the invention, the non-activating medium or the activating medium and
• - Further, if appropriate, at least one potential active substance complex.

• – eines unbekannten Wirkstoffs, Wirkstoffkomplexes, eines Teils und/oder Derivats davon,
• – des Vorhandenseins eines unbekannten Wirkstoffs, Wirkstoffkomplexes, Teils und/oder Derivats davon,
• – des Vorhandenseins eines bekannten Wirkstoffs, Wirkstoffkomplexes, Teils und/oder Derivats davon,
• – der Konzentration eines unbekannten Wirkstoffs, Wirkstoffkomplexes, Teils und/oder Derivats davon,
• – der Konzentration eines bekannten Wirkstoffs, Wirkstoffkomplexes, Teils und/oder Derivats davon.

According to the invention, a screening method for identifying is also provided for identifying:

• An unknown active substance, active substance complex, a part and / or derivative thereof,
• The presence of an unknown active substance, active substance complex, part and / or derivative thereof,
• The presence of a known active substance, active substance complex, part and / or derivative thereof,
• - The concentration of an unknown drug, drug complex, part and / or Deri vats of it,
• The concentration of a known active substance, active substance complex, part and / or derivative thereof.

These in terms of sizes or properties of the used method according to the invention can be combined as desired.

there is the active substance, the active substance complex, a part or derivative thereof an enzymatic property of a site complex that is a GAT1 protein contains or a part of it modified.

According to one Another aspect of the present invention is the inventive method used to identify a drug complex, to find of inhibitors, activators, partial or temporary inhibitors or modulators of an enzymatic property of a site of action complex, which contains a GAT1 protein, for example a human GAT1 protein.

Of Further, the test kit according to the invention used according to the invention for finding inhibitors, activators, partial or temporary inhibitors or modulators of a GAT1 protein-containing active site complex, for example, a human GAT1 protein.

Of Further, the drug is used according to the invention for inhibition, partial or temporary Inhibition, activation or other modulation of a GAT1 protein containing active complex or a part thereof, for example a human GAT1 protein.

Furthermore is a z. B. aqueous Measuring medium provided, in which arranged the primary carrier and the secondary carrier become. The electrode area is compared to the measuring medium, the primary carriers and across from the biological units preferably largely electrically isolated.

Of the Electrode area has e.g. at least one electrode. These For one thing, it can be used as a mechanically stable material area be formed, in particular as a plate, as a wire and / or the like.

A particularly simple arrangement of the sensor electrode device results when the electrode is essentially on the surface of the carrier deposited material layer is formed. It can work to act a vapor-deposited or sputtered material layer. The Material layer for forming the electrode preferably has a layer thickness from about 10 to 200 nm.

The GAT1 protein may each be in substantially native form and / or in modified, in particular purified, microbiological and / or molecular biological changed Form are used. For one thing, this can be specific native Properties tested and pharmacologically examined. on the other hand are also molecular biological or genetically engineered changes on certain aspects, such as transport or the pharmacological mode of action of an active substance to analyze.

Especially it is advantageous that primary carrier of a each substantially uniform type of primary carriers provided become. This is in terms of a clear statement and analysis of a drug test of importance and relates on the geometric, physical, chemical, biological and molecular biological properties of the primary carriers.

The previous one also applies to those provided in the primary carrier Site complexes and the GAT1 protein. Here are Wirkortkomplexe and GAT1 proteins each of a substantially uniform type provided, in particular with regard to their geometric, physical, chemical, biological and molecular biological properties. additionally should the biological units in an advantageous manner with regard to their orientation and / or their ability to be activated roughly uniform in relation to the respective primary carrier be.

As already executed, The invention is based inter alia on the object, the effect of Substances for the function of GAT1 proteins of an investigation accessible close. Substances that enhance the effect of this transport protein are useful as potential agents, e.g. for treatment of epilepsy and various cognitive disorders of commercial interest. Also would be substrates of the protein transported by this interesting new molecules.

The procedure according to the invention 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 becomes.

Furthermore is in contrast to the patch-clamp technique or voltage-clamp technique higher Throughput possible.

EXAMPLE 1: Preparation of the sensor chip

The gold surface was mercaptanized by placing in a 1 mmol / l / l octadecylmercaptan solution, rinsed with 2-propanol (absolute) or distilled water and dried. It was then covered with diphytanoyl-phosphatidylcholine (in decane) and overlayed with an addition buffer (140 mmol / l K-gluconate, 2 mmol / l MgCl ₂ , 30 mmol / l Hepes, pH 7 / NMG, 2 mmol / l DTT) ,

EXAMPLE 2: Preparation of the proteinaceous Membrane fragment suspension

The Membrane fragments were prepared according to the following protocol: 1 gram of cell pellet 1 ml of sucrose buffer (0.25 mol / l sucrose, 5 mmol / l Tris, pH 7.5, ca. 2 mmol / l DTT, PMSF *, Complete **); * 50 μl PMSF as saturated ethanolic solution to 100 ml of buffer; ** Complete / Fa. Roche, one tablet per 50ml Buffer. Cells pottern until homogeneous mass is formed (alternatively a Parr bomb was also used), 10 min / 680g / 4 ° C; 10 minutes / 6100 g / 4 ° C; Got over 1 h / 100,000 g / 4 ° C centrifuge; Take up pellet in 5 mmol / l Tris pH 7.5 so that a very cloudy Mixture arises; with addition of 70% sucrose (in 5 mmol / l Tris) to 51.3% sucrose, centrifuge tubes with 44.4%, 30.7% and 8.6% sucrose in 5 mmol / l Tris pH 7.5 overcoat; 1 h 40 min / 100,000 g / 4 ° C in Centrifuge the swing out rotor; Remove bands with Pasteur pipette and pellet in attachment buffer (see above); 30 minutes / 150,000 g / 4 ° C; pellets in accumulation buffer.

Example 3: Production of the GAT1 sensor chip

10 .mu.l of the membrane suspension (protein concentration about 0.1-1.0 mg ml ^-1 ) were applied to the sensor chip and incubated overnight in the refrigerator (at <8 ° C).

The capacity of the protein-loaded membranes was about 300-1000 nF cm ^-2 , the conductivity G _1s at about 10-100 nS cm ^-2 .

Example 4: Activity measurement on GAT1-expressing membrane fragments

The flow cell with integrated protein-loaded sensor chip was first flushed with addition buffer (see above, 200 μmol / l DTT instead of 2 mmol / l). This was followed by the non-activating solution (10 mmol / l valine, 140 mmol / l NaCl, 2 mmol / l MgCl ₂ , 30 mmol / l Hepes pH 7 / NMG, 200 μmol / l DTT) and by electromechanical 3/2 Directional valve was then switched to activating solution (10 mmol / l GABA, 140 mmol / l NaCl, 2 mmol / l MgCl ₂ , 30 mmol / l Hepes pH 7 / NMG, 200 .mu.mol / l 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

The Measurements were made as above, however, in the activating and nonactivating solution 140 mmol / l NaCl through 140 mmol / l KCl or 140 mmol / l Choline-Cl exchanged. That's it Cosubstrate sodium is no longer available for the GABA transport available. Under these conditions there can be no sodium chloride GABA cotransport occur.

Example 6: Activity Measurement on Control Membranes

The Measurements were made as in the GAT1-expressing membranes. there There were no signals when switching from non-activating (without GABA) on activating solution (with GABA).

1 shows the current response of the GAT1-loaded biosensor to a GABA concentration jump in the absence and presence of sodium.

2 shows the current response of the control membrane-loaded biosensor to a GABA concentration jump.

3 Current response of the GATT-loaded biosensor to a GABA concentration jump in different concentrations.

The The abscissa of the figures denotes the time t, respectively, where t = 0 indicates the time at which GABA is flushed into the system. The ordinate of the figures thus denotes the measured electrical Current I (t), as a function of time t, passing through the biosensor electrode is measured.

Shown in 1 three measuring tracks A, B and C, which correspond to different measuring conditions with respect to the substrate concentration at GABA.

Lane A of the 1 Figure 4 shows a measurement in which the GAT1 protein moieties are normally activated by the addition of 10 mmol / L GABA at time t = 0, in the presence of 140 mmol / l NaCl. GABA transport uses the GAT1 protein to transfer sodium and chloride ions via the primary carriers, eg via membrane fragments or Ve to move on the biosensor membrane and the electrode. In the transport cycle of GABA net positive electronic charge is shifted across the membrane, which leads to a positive current signal. This can be recognized by the measured positive current signal.

Lane B in 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 in the exchange of NaCl with KCl, sodium chloride GABA transport can no longer take place.

Lane C in 1 shows a measurement in which, in the presence of 140 mmol / l NaCl again by means of the GAT1 protein sodium and chloride ions on the primary carrier to the biosensor membrane and the electrode are moved to. This is analogous to track A in 1 recognizable by the measured positive current signal.

Like from the 1 As can be seen, the cotransport of sodium ions, chloride ions and GABA leads to a positive signal, since only such positive charge carriers can be shifted to the membrane.

2 shows the measured according to another embodiment of the method according to the invention with a biosensor electrode electric current I as a function of time t, using control membranes in which no GAT1 is present. When switching from non-activating (without GABA) to activating solution (with GABA), no signal beyond the noise is detectable. This is due to the lack of an ion transport function, which in the measurements too 1 generated or mediated by GAT1.

In 3 Four measurement tracks are shown that correspond to different measurement conditions with respect to the substrate concentration at GABA. At 10 mmol / l GABA the GAT1 protein units are activated normally, 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) is detectable. At the lowest concentration of 0.5 mmol / l, the signal is only slightly above the background noise. In summary shows 3 thus, the relevance of the presence of GABA for the transport activity of the GAT1 units.

basic Advantages of the present invention are the high mechanical stability of the intended Sensor arrangement and, consequently, the high operational readiness, the easy handling and low susceptibility. In use The sensor arrangement arise in the context of pharmacological Drug testing a long life, high reliability, a low susceptibility to interference and especially one opposite usual Method relevant increased test throughput, resulting in appropriate test procedures economical prepared and carried out can be.

Furthermore is in contrast to other techniques the signal quality better, in the sense of a better signal-to-noise ratio.

Claims (26)

A method of identifying an active agent complex which modifies an enzymatic property of an active site complex containing a GAT1 protein, comprising the steps of: (a) providing a plurality of primary carriers containing the GATT protein-containing active site complex, in particular in a plurality in the region of a membrane (b) attaching or bringing into contact the primary carriers at or in the surface region of an isolation region of a biosensor electrode serving as a secondary carrier in a measurement medium, the secondary carrier preferably mechanically and electrically isolated from the measurement medium and from the primary carriers by means of the isolation region (c) providing at least one potential drug complex; (d) contacting and, in particular, interacting the potential drug complex with the site complex of the primary carriers or portions thereof, and (e) determining of the qualitative and / or quantitative influence of the potential active substance complex or a part thereof on enzymatic properties of the active site complex or a part thereof by detecting an electrical action of the active site complex or a part thereof or changing the electrical action via the biosensor electrode as secondary carrier, wherein in the process steps (c), (d) and / or (e) one or more of the following sub-steps are performed: (f) introducing the secondary carrier with the primary carriers into an attachment buffer and detecting an electrical action according to or in the sense of method step (e) , (g) introducing the secondary carrier with the primary carriers into a second non-activating medium and detecting an electrical action according to or in the sense of method step (e), (h) introducing the secondary carrier with the primary carriers into a third or activating measuring medium and detecting one electrical action according to or in the sense of method step (e), wherein the third or activating medium the second or non-activating medium, but additionally containing a substrate of the GAT1 protein. The method of claim 1, wherein a biosensor electrode is used as secondary carrier, in which an electrically conductive and more solid Electrode area is provided with at least one electrode, which opposite the measuring medium and opposite the primary carriers preferred electrically and mechanically isolated by providing an isolation region in the form of a solid supported membrane, which layer is built up from a lower layer of a organic thio compound as the bottom and the electrode respectively facing layer and from an upper layer of an amphiphilic organic compound. Method according to one of the preceding claims, wherein in which a biosensor electrode is used as a secondary carrier in which an electrode of gold is provided in the electrode area with a monolayer of a long-chain alkanethiol as a lower layer on top and a monolayer of a lipid as a topcoat on top. Method according to one of the preceding claims, wherein in which a biosensor electrode is used as a secondary carrier in which the one electrode of the biosensor electrode as a secondary carrier covering Area of the isolation area as a membrane structure in the manner of a solid-supported double-layer membrane or bilayer membrane is or is formed. Method according to one of the preceding claims, wherein which as the primary carrier respectively a eukaryotic cell, a prokaryotic cell, an oocyte, a bacterium, a virus, an organelle or constituents, in particular Membrane fragments, or bandages thereof in native or modified form, in particular in purified and / or modified form is used. Method according to one of the preceding claims, wherein which as the primary carrier respectively a vesicle, a liposome or a micellar structure is used. Method according to one of the preceding claims, wherein which uses a GAT1 protein, which consists of a tissue of a mammal, Preferably brain, intestine or kidney, or genetically derived from this is derived or is. Method according to one of the preceding claims, wherein which the GAT1 protein from the organism pig, mouse, sheep or human originates or is derived from this genetically. Method according to one of the preceding claims, wherein which the GAT1 protein at least partially in the primary carrier a Membrane spanning formed or used or is. Method according to one of the preceding claims, wherein which is used as an electrical action through the Wirkortkomplex or a part thereof, or one of them generated electric potential, which is generated by each Charge or mass transport or shift, conformational changes, ligand binding, annealing or release, or a combination thereof. Method according to one of the preceding claims, wherein which is used as an enzymatic property: - the connection, Addition or release of the active substance complex or a part thereof or the measuring medium or a part thereof, - the transport or the displacement of the active substance complex or a part thereof or the measuring medium or a part thereof, - the chemical Reaction or reaction of the active substance complex or a part thereof or the measuring medium or a part thereof, - a conformation change or movement of the active site complex or a part thereof or - any Combination of these processes. Method according to one of the preceding claims, wherein which as the measuring medium an aqueous Measuring medium is used and in particular an aqueous electrolyte solution. Method according to one of the preceding claims, wherein wherein the method steps (c) and / or (d) are performed by: - To mix or injecting the drug complex, a part or a preform from that, - Change the measuring medium or a part thereof and / or - chemical or physically reacting or reacting the measuring medium or a part thereof or the active substance complex, a part or a preform of it. Method according to one of the preceding claims, wherein which a sensor assembly of biosensor electrode as a secondary carrier and attached to it primary carriers in A measuring chamber, measuring range or measuring vessel flows around the measuring medium or flows against becomes. Method according to one of the preceding claims, wherein which consecutively performs a plurality of tests by successively exchanging the measuring medium, if necessary with intermediate washing or rinsing of the measuring space. Method according to one of the preceding claims, in in which a washing step is carried out between steps (g) and (h) by introducing the secondary carrier with the primary carriers into one fourth or washing medium. Method according to one of the preceding claims, in which immediately before step (h) step (f) is repeatedly performed. Method according to one of the preceding claims, - in which the first medium or the attachment buffer - K ⁺ - Cl ^- 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 - optionally Mg ⁺⁺ , PH 6-8, more preferably about 7, contains, - in which the second or non-activating medium - Na ⁺ , - Cl ^- , - optionally Mg ⁺⁺ , - pH 6-8, more preferably about 7, contains , and - in which the third or activating medium - Na ⁺ , - Cl ^- , - optionally Mg ⁺⁺ , - pH 6-8, more preferably about 7, and - a substrate of the GATT protein, preferably GABA between 1 μmol / contains 1 and 10 mmol / l. Method according to one of the preceding claims, wherein which as a measuring medium and in particular as a second or non-activating Medium and / or particularly preferred as the third or activating Medium media used with GABA, especially with a Concentration of about 1 μM to 10 mmol / l. Active ingredient or complex of active substances, - which one an enzymatic property of a site complex that is a GAT1 protein contains or part of it modified, - which according to a Method according to one of the claims 1 to 19 is identified. Method for producing a medicament with the steps: - Identify an active substance and / or an active substance complex, which is a certain enzymatic property of a Wirkortkomplexes, which ein Contains GAT1 protein, or a part thereof or a plurality of properties modified by a method according to any one of claims 1 to 19 - Produce and / or isolating the active ingredient, the active agent complex, a Part and / or derivative thereof, - purifying the active substance, Active substance complex, part or derivative, - Mixing and / or portioning the active ingredient, the complex of active ingredients, the part or derivative with a pharmaceutically acceptable Carrier. Test kit for performing the method after a the claims 1 to 19, With: - at least a primary carrier, - one Measuring range, - one first medium or annealing buffer, a second or non-activating one Medium, a third or activating medium for receiving a potential drug complex and - at least one more potential complex of active substances. Screening method for identification: - one unknown active substance, active substance complex, part or derivative from that, - of Presence of an unknown drug, drug complex, Part or derivative thereof, - the presence of a known active substance, active substance complex, part or derivative thereof, - the concentration an unknown active substance, active substance complex, part or derivative from that, - of the Concentration of a known active substance, active substance complex, part or derivatives thereof or - one any combination of the aforementioned sizes or properties by Using a method according to one of claims 1 to 19 and / or the test kit according to claim 22, wherein the active ingredient, the active substance complex, a part or the derivative thereof an enzymatic Property of an active site complex containing a GAT1 protein, or modified part of it. Use of the method according to one of claims 1 to 19, for finding inhibitors, partial or temporary inhibitors, Activators or modulators of an enzymatic property of a Wirkortkomplexes, which contains a GAT1 protein. Use of the test kit of claim 22 for retrieval of inhibitors, activators, partial or temporary inhibitors or modulators of an enzymatic property of a site complex, which contains a GAT1 protein. Use of a medicament prepared according to the method of claim 21, - For inhibition, partial or temporary inhibition or modulation of an enzymatic property of a Wirkortkomplexes containing a GAT1 protein.