EP1797434A1 - Analyse relative a la proteine de type pept1 - Google Patents

Analyse relative a la proteine de type pept1

Info

Publication number
EP1797434A1
EP1797434A1 EP05790083A EP05790083A EP1797434A1 EP 1797434 A1 EP1797434 A1 EP 1797434A1 EP 05790083 A EP05790083 A EP 05790083A EP 05790083 A EP05790083 A EP 05790083A EP 1797434 A1 EP1797434 A1 EP 1797434A1
Authority
EP
European Patent Office
Prior art keywords
active substance
complex
protein
solution
peptl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05790083A
Other languages
German (de)
English (en)
Inventor
Natalie Watzke
Maarten Ruitenberg
Wolfgang Dörner
Renate Gauss
Béla KELETY
Joanna Tobien
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 EP1797434A1 publication Critical patent/EP1797434A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes

Definitions

  • the invention relates to a type-PepT protein assay, and more particularly to a method of identifying a substrate and / or modulators of a type PepTI protein.
  • the invention further relates to an active substance complex which has been identified according to the method according to the invention, to a method for producing a medicament which is a substrate of the type-PepTl protein or by conjugation with a substrate for a type-PepTl protein has been made to such a substrate, 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.
  • Natural transport proteins play in the uptake of various molecules, e.g. from the intestine an important role.
  • Polar or hydrophilic compounds are usually absorbed poorly in the intestine, since their transport via the cell membrane is energetically unfavorable, and requires energy.
  • Amino acids, di- and tripeptides, monosaccharides, vitamins, nucleosides, etc. are polar compounds whose incorporation is, however, essential for the organism. For such substances, there are usually specific transport systems.
  • PepTl In mammals, two peptide transporters PepTl and PepT2 have been detected so far.
  • PepTl is expressed in the small intestine, kidney, bile duct and pancreata
  • PepT2 is expressed in the kidney, the central nervous system (CNS), the peripheral nervous system (PNS), the lung, the mammary gland, the spleen, the colon and the pancreas is expressed (overview in Rubio-Aliaga & Daniel 2002).
  • CNS central nervous system
  • PNS peripheral nervous system
  • the lung the mammary gland
  • the spleen the colon and the pancreas is expressed (overview in Rubio-Aliaga & Daniel 2002).
  • the detection of active uptake of GIyGy into intestinal epithelial cells was successful (Newey & Smyth 1962).
  • the carrier is dependent on an inward H + gradient (Ganapathy & Leibach 1985).
  • PepTl but not PepT2
  • PepT1 is expressed in the gut
  • pharmacological agents that are substrates for PepT1, or that can be modified by PepTl can be detected and transported.
  • electrophysiological methods for example the so-called patch-clamp technique or voltage-clamp technique, in which, for example, cells are accessible in isolation to an electrophysiological examination.
  • patch-clamp technique or 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 invention is therefore based on the object to provide a type-PepTl-protein assay, in which in a particularly simple and yet reliable manner a rapid drug testing, especially in Massen ⁇ operation, is possible at a reasonable cost.
  • the present invention furthermore relates to an active substance or active substance complex according to claim 20, a method for producing a medicament according to claim 21, a test kit for carrying out the method according to the invention according to claim 22, a screening method according to claim 23, as well as uses of the method, the test kit and the medicament according to claims 24, 25 and 26, respectively.
  • Advantageous further developments are the subject of the respective dependent subclaims.
  • proton (s) together with 1 substrate molecule, naturally in the intestine normally 1 small peptide, in particular 1 di- or tripeptide, are shifted across the membrane.
  • the invention is based inter alia on the idea of the resulting charge shift - ie the ion transport of protons and / or of charged substrates, such as, for example, Di- or tripeptides or even electrically charged substrates - directly by attachment of enzyme preparations on a suitable surface, which is integrated into a flow-through system and / or in which a substrate jump can be carried out, as electrical current or as electrical potential change to measure and / or investigate the influence of different substances on the electrical quantities of current or potential.
  • charged substrates such as, for example, Di- or tripeptides or even electrically charged substrates - directly by attachment of enzyme preparations on a suitable surface, which is integrated into a flow-through system and / or in which a substrate jump can be carried out, as electrical current or as electrical potential change to measure and / or investigate the influence of different substances on the electrical quantities of current or potential.
  • the invention thus relates to a method for identifying potential substrates and / or inhibitors / activators of a type-PepTl protein which contains an enzymatic property and / or the transport behavior of the type-PepTl protein or a part thereof.
  • the method according to the invention comprises the following steps:
  • steps (c), (d) and / or (e) one or more of the following substeps are carried out:
  • a solution with 140 mmol / 1 KCl, 2 mmol / 1 MgCl 2 , 30 mmol / 1 Mes pH 6.0 or Hepes pH 7.0 and 25 mmol / 1 GIy- one can be used.
  • a solution with 140 mmol / 1 KCl, 2 mmol / l MgCl 2 , 30 mmol / l Mes pH 6.0 or Hepes pH 7.0 and 25 mmol / 1 Gly-Gly can be used as the activating solution.
  • steps (f) and (g) according to the invention are preferably carried out in the predetermined sequence, if necessary with repetitions.
  • step (f) it can be particularly favorable if, before step (f), a step (f) is carried out once, incubating with a conditioning buffer instead of a non-activating solution, the addition buffer corresponding to the non-activating solution, but the compensator not included in the solution.
  • a conditioning buffer instead of a non-activating solution, the addition buffer corresponding to the non-activating solution, but the compensator not included in the solution.
  • the potential drug should only be present in the activating solution, but not in the non-activating solution.
  • the potential active substance can be present in all media.
  • the active site complex or part thereof used is preferably a monomer or an OHomer of a PepTI protein.
  • type-PepT1 protein means that the protein has the typical characteristics of the PepT1 protein, in particular including proteins which, in their transport and substrate recognition properties as well as their sensitivity to inhibitors and activators, belong to the PepT1 Or are similar to the renal isoform PepT 2. Both isoforms can easily be used in the present test system.
  • a site complex or portion thereof will be used which is based on a type-PepTl protein derived from a tissue of a mammal, e.g. from the small intestine, kidney, bile duct or Pankre ⁇ as, or is derived therefrom.
  • the type-PepTl protein is derived from mammalian cell lines and is cloned.
  • the site complex comes from the organism pig, mouse, sheep or human or is derived from these genetically.
  • the expression "enzymatic property" of the type-PepTl protein always means the transport behavior, eg the peptide or proton transport mediated by these proteins, and thus also refers to the influence of the protein discussed at the outset.
  • investigations are included according to the invention which are intended to show whether a the substance of the type-PepTl protein is transported and / or whether a certain substance modifies the transport properties of the type-PepTl protein, ie is an inventive modulator of the type-PepTl protein.
  • Wirkstoff ⁇ complex means either a potential substrate whose transport via the type-PepTl protein is to be investigated, or a so-called modulator which inhibits, for example, the transport of previously determined substrates or activates.
  • the active substance complex or a part thereof can be transported directly into the site of the active site complex by injection or admixing into the measurement solution.
  • a process is particularly simple if the respective measuring solution is simply exchanged, for example in a continuous manner.
  • the active substance is released only by a chemical or physical reaction or reaction in the measurement solution. This can also be done for example by supplying radiation.
  • the qualitative influence and / or the quantitative influence of the potential drug or drug complex is determined according to the invention by detecting an electrical action which is mediated by the site complex or a part thereof and in particular by the type-PepTl protein.
  • this electrical action is determined with and without potential drug, and by comparing both series of measurements it is examined whether the potential drug affects the enzymatic properties of the type-PepTl protein.
  • the primary carriers are brought into contact with the active site complexes on a secondary carrier, namely the biosensor electrode and in particular with its isolation region, and are deposited there in particular.
  • a biosensor electrode is used as a secondary carrier, in which an electrically conductive and festConsequentlyar ⁇ tiger electrode region is provided with at least one electrode which is electrically insulated from the measuring solution and with respect to the primary carriers by providing an isolation region in the form of a solid-supported membrane which is built up in layers from a lower layer of an organic thio compound as the lowest layer and the electrode respectively facing the layer and from an upper layer of an amphiphilic organic compound.
  • a biosensor electrode is used as the secondary carrier, in which a gold electrode is provided in the electrode region with a monolayer of a long-chain alkanethiol as a lower layer thereon and a monolayer of a lipid as a topcoat thereon.
  • the primary carriers which are to contain the site complex and in particular the type-PepTl proteins in the region of their membrane, different possibilities result, wherein the respective objective of the method can be taken into account.
  • the primary carrier is 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 dressings thereof in native form and / or in a modified form, in particular in purified and / or modified form.
  • a vesicle, a liposome or a micellar structure is used as the primary carrier.
  • the membrane fragments can also deposit planar, i. as a non-spherical structure.
  • the method according to the invention is particularly advantageous if the sensor arrangement of biosensor electrode as secondary carrier and primary carriers mounted thereon in a measuring space, measuring area or measuring vessel flows around or flows against the measuring solution.
  • a change in concentration with respect to the active substance complex or a part thereof can be easily achieved by changing the measurement solution.
  • the test conditions according to the invention can advantageously be adjusted in an easy manner and reliably with high time resolution. Also with the aid of an automated pipetting device, the method according to the invention can be advantageously carried out.
  • the method according to the invention is particularly economical and accessible for a statistical evaluation when a plurality of tests are carried out successively, in particular by successive exchanging of the measuring solution, optionally with interposed washing or rinsing of the measuring chamber.
  • the present inventive method it is important to compare the action of the active site complex in the case of the active substance complex present with a situation in which the potential active substance complex is not present. This can be done, for example, by changing between non-activating solution to be activated in the presence or absence of the potential active substance complex and comparing the measured values obtained.
  • Potential modulators to be tested are particularly preferably monoclonal antibodies, antibody fragments, polyclonal antibodies and peptides. However, it is also possible to add other substances which are suspected of having a corresponding effect. These substances are preferably administered in dissolved form. Particularly preferred substances that can be investigated as possible potential substrates and / or modulators 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, 1 18, 7641-7472.
  • small peptides, amino acids and amino acid analogs, steroids, nucleotides and other organochemical substances having a molecular weight of ⁇ 5000, preferably ⁇ 3000 and more preferably ⁇ 2000, are attributed to the low molecular weight substances.
  • an intermediate step is an incubation of the active site complexes or of the primary carriers carrying them with the active substance complex.
  • a washing step is carried out between steps (f) and (g) by introducing the secondary carrier with the primary carriers into a [washing] solution, which is preferably identical to the holding buffer.
  • aqueous measuring solutions and in particular aqueous electrolyte solutions are used as the measuring solution, which are referred to as addition buffer, non-activating and activating solution.
  • All measurement solutions used contain a pH-stabilizing agent known to those skilled in the art, preferably selected from the list: MOPS, HEPES, MES, Tris, PIPES, etc., as published eg in "Buffers, Calbiochem", but also with ease According to the invention, these known agents should have a stabilizing effect in the range from pH 6 to 8, preferably about 7.
  • the choice of the suitable pH range and agent may depend on the substrate (drug complex) and easily determined by the skilled worker by routine experimentation.
  • the attachment buffer comprises at least one cation species, preferably selected from the list consisting of K +, Ca ++, Na +, Li +, Mg ++, choline +, Rb +, Sr ++ in a concentration which is preferably about as high as the Ka ⁇ tion concentration in the non activating and in the activating solution.
  • this concentration can be between 1 ⁇ mol / 1 and 1 mol / 1, preferably between 10 and 200 mmol / l, particularly preferably between 120-160 mmol / l.
  • the non-activating solution likewise contains at least one cation species in a concentration which is preferably approximately as high as the cation concentration in the addition buffer.
  • attachment buffer It is preferably a cation of the list described for the attachment buffer, preferably it corresponds to the cation or the cations of the attachment buffer.
  • non-activating solution may optionally comprise a compensator.
  • This compensator serves to ensure that when switching from non-activating solution to solution to be activated, e.g. By changing the ionic strength, no site-independent, false-positive signal is detected.
  • the compensator is preferably an amino acid, and particularly preferably the amino acid glycine, if the dipeptide Gly-Gly is used as the substrate for inhibition measurements in the activating solution.
  • the type of compensator used depends on the type of substrate used.
  • the compensator used is preferably an amino acid with a pK value which is approximately equal to the p j value of the di- or tripeptide.
  • an acid or a base which has a pK value which corresponds approximately to that of the investigated target molecule is used as the compensator.
  • the concentration of the compensator generally depends on the concentration of the substrate, and is generally between 0 to 100 mmol / l. In principle, the concentration of the compensator is varied until an action site-independent electrical action due to the change from non-activating to activating solution no longer occurs.
  • the activating solution may contain the compensator regardless of whether it is contained in the non-activating solution.
  • the activating solution likewise contains at least one cation species in a concentration which is preferably approximately as high as the cation concentration in the addition buffer.
  • the attachment buffer It is preferably a cation of the list described for the attachment buffer, it preferably corresponds to the cation or the cations of the attachment buffer. Furthermore, it contains a substrate of the type-PepTl protein, preferably a di- or tripeptide, particularly preferably Gly-Gly for inhibition or activation measurements.
  • substrates are, for example, monosaccharides, vitamin nucleosides and also beta-lactam antibiotics and similarly structured compounds.
  • these are peptide-like compounds.
  • the concentration can vary between> 0 and 1 M.
  • the compensator may also be present in the activating solution if such a better signal-to-background ratio, i. that absence of site-independent, false-positive signals when changing from non-activating solution to akti ⁇ fourender solution can be achieved.
  • Cations which are suitable according to the invention are all cations which (a) do not inhibit PepTl and (b) do not trigger any measuring artifacts.
  • Zn and Cu are known to inhibit PepTl.
  • the present invention provides an active substance or an active substance complex which modifies an enzymatic property of a type of a PepTl protein-containing active site complex or a part thereof and which is identified according to the method according to the invention for identifying an active substance complex has been.
  • the present invention provides a method for producing a medicament comprising the steps of:
  • Identification of an active substance and / or an active substance complex which suitably modifies an enzymatic property of an active substance complex, which contains one type of a PepT1 protein, or a part thereof or a plurality of properties, if appropriate with the aid of the method according to the invention .
  • test kit for carrying out the method according to the invention for identifying an active substance complex. This test kit points to:
  • a screening method for identification is also created, namely for identifying: an unknown active substance, active substance complex, a part and / or derivative thereof,
  • the process according to the invention for identifying an active substance complex is used for finding inhibitors, activators, partial or temporary inhibitors or modulators of an enzymatic property of an active site complex which contains a type-PepT1 protein ,
  • test kit according to the invention is used according to the invention for finding modulators, i. Inhibitors or activators, e.g. partial or temporary inhibitors of an active site complex containing one type of PepTI protein.
  • modulators i. Inhibitors or activators, e.g. partial or temporary inhibitors of an active site complex containing one type of PepTI protein.
  • the site complex contains the PepTl protein.
  • the medicament according to the invention is used for the inhibition, partial or temporary inhibition, activation or other modulation of a type-peptide complex containing active ingredient.
  • a z. B. aqueous measuring solution in which the primary carrier and the secondary carrier are arranged.
  • the electrode area is preferably largely electrically isolated from the measuring solution (activating or non-activating solution), the primary carriers and, compared with the biological units.
  • the electrode region has, for example, at least one electrode. On the one hand, 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 designed essentially 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 type-PepTl protein may in each case be provided in substantially native form and / or in modified, in particular purified, microbiologically and / or molecularly-modified form.
  • certain native properties can be tested and examined pharmacologically.
  • molecular biological or genetically engineered changes are also suitable for analyzing certain aspects, for example the transport or the pharmacological mode of action of an active substance.
  • primary carriers of a respectively substantially uniform type of primary carriers are provided. This is important in terms of the most unambiguous statement and analysis of a drug test and relates to the geometric, physical, chemical, biological and molecular-biological properties of the primary carriers.
  • Wirkort ⁇ provided in the primary carrier complex and the type-PepTl protein.
  • Wirkortkomplexe and type PepTl protein are provided each of a substantially uniform type, in particular with regard to their geometric, physical, chemi ⁇ 's, biological and molecular biological properties.
  • the biological units should advantageously be approximately uniform with regard to their orientation and / or with regard to their activatability with respect to the respective primary carrier.
  • the invention is inter alia based on the object, the effect of substances on the function of type-PepTl - Proteins of a study to make accessible. Substances which modulate the action of this transport protein are of potential commercial interest as potential neuroprotective agents. Also, substrates of the protein transported by it would possibly be important new molecules.
  • the measurement of the transport activity or the transport properties according to the method proposed according to the invention requires no mediator 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 carried out with a sensor loaded with enzyme. Substrates do not have to be chemically modified since the current response or potential response of the protein is induced by a rapid solution change.
  • the capacity of the protein-loaded membranes was about 1000 nF cm '2 , the conductivity G 13 at about 10 nS cm ' 2 .
  • Glibenclamide is an inhibitor of PepTl (IC 50 in oocytes about 100 ⁇ M). The inhibition of the current amplitude by glibenclamide proves that the detected signal is actually a specific PepT1 signal. Complete inhibition with glibenclamide is not possible due to the poor solubility of glibenclamide.
  • FIG. 1 shows, in the form of a graph, schematically the transport current I (t) produced by the PepTI protein as electrical action measurable by the biosensor electrode.
  • Fundamental advantages of the present invention are the high mechanical stability of the intended sensor arrangement and, associated therewith, the high 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 to conventional methods, whereby corresponding test methods can be worked out and carried out inexpensively.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne une analyse relative à la protéine de type PepT1 et en particulier un procédé d'identification d'un substrat et/ou d'un modulateur de la protéine PepT1.
EP05790083A 2004-10-01 2005-09-30 Analyse relative a la proteine de type pept1 Withdrawn EP1797434A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102004048391A DE102004048391B4 (de) 2004-10-01 2004-10-01 Typ-PepT1-Protein-Assay
PCT/EP2005/010601 WO2006037573A1 (fr) 2004-10-01 2005-09-30 Analyse relative a la proteine de type pept1

Publications (1)

Publication Number Publication Date
EP1797434A1 true EP1797434A1 (fr) 2007-06-20

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EP05790083A Withdrawn EP1797434A1 (fr) 2004-10-01 2005-09-30 Analyse relative a la proteine de type pept1

Country Status (4)

Country Link
US (1) US20080248486A1 (fr)
EP (1) EP1797434A1 (fr)
DE (1) DE102004048391B4 (fr)
WO (1) WO2006037573A1 (fr)

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Publication number Priority date Publication date Assignee Title
DE102006031913A1 (de) * 2006-07-10 2008-01-17 Iongate Biosciences Gmbh GAT1-Assay

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Publication number Priority date Publication date Assignee Title
US5658782A (en) * 1993-10-20 1997-08-19 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education On Behalf Of The Oregon Health Sciences University A Non-Profit Organization Amino acid transporters and uses
DE10112505C1 (de) * 2001-03-15 2003-01-30 Iongate Biosciences Gmbh Sensoranordnung und Vorrichtung zur amperometrischen und/oder potentiometrischen, pharmakologischen Wirkort- und/oder Wirkstofftestung sowie Verfahren zur amperometrischen und/oder potentiometrischen, pharmakologischen Wirkort- und/oder Wirkstofftestung
US7666610B2 (en) * 2002-03-29 2010-02-23 Chugai Seiyaku Kabushiki Kaisha Expressing transporters on viral envelopes

Non-Patent Citations (1)

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Title
See references of WO2006037573A1 *

Also Published As

Publication number Publication date
WO2006037573A1 (fr) 2006-04-13
DE102004048391B4 (de) 2007-08-16
WO2006037573B1 (fr) 2006-06-08
DE102004048391A1 (de) 2006-04-13
US20080248486A1 (en) 2008-10-09

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