WO2009071301A2 - Procédé permettant de détecter des effecteurs de l'activité protéase de cis/trans isomérases - Google Patents

Procédé permettant de détecter des effecteurs de l'activité protéase de cis/trans isomérases Download PDF

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WO2009071301A2
WO2009071301A2 PCT/EP2008/010303 EP2008010303W WO2009071301A2 WO 2009071301 A2 WO2009071301 A2 WO 2009071301A2 EP 2008010303 W EP2008010303 W EP 2008010303W WO 2009071301 A2 WO2009071301 A2 WO 2009071301A2
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cis
trans
trans isomerase
molecule
proenzyme
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PCT/EP2008/010303
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WO2009071301A3 (fr
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Gunter Fischer
Cordula Schiene-Fischer
Tobias AUMÜLLER
Günther JAHREIS
Gerhard KÜLLERTZ
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Publication of WO2009071301A2 publication Critical patent/WO2009071301A2/fr
Publication of WO2009071301A3 publication Critical patent/WO2009071301A3/fr

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    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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
    • C12Q1/533—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving isomerase
    • 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
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

  • the present invention relates to a method for finding cis / trans isomerase effectors and for quantifying the cis / trans isomerase inhibiting or activating effect of corresponding effectors.
  • Enzymes that can catalyze the cis / trans isomerization of peptide bonds are referred to as cis / trans isomerases.
  • the cis / trans isomerases include the peptidyl prolyl cis / trans isomerases (PPIases) and the secondary amide peptide bond cis / trans isomerases (APIases).
  • APIases differ from PPIases in that their cis / trans isomerase activity is directed to the cis / trans isomerization of secondary amide peptide bonds (Rail-Fischer C. et al .: Nature Structural Biology., 9 (6): 419-). 424, 2002; Rail-Fischer C. et al.: Biological Chemistry 383 (12): 1865-1873, 2002). APIases and the inhibition of their APIase activity are of great importance in human and veterinary medicine (e.g. US 2006100130).
  • PPIases are the families of cyclophilins (Galat A.: Eur. J. Biochem 216 (1993) 689-707,hacker J. & Fischer G: Mol. Microbiol 10 (1993) 445-456), FKBP's and counted the parvulins.
  • cyclophilins refers to enzymes which have a PPIase activity and which can be determined, for example, by conventional methods of sequence comparison with known cyclophilins (eg: A. Galat: Arch. Bioch. & Biophys., 371 (1999) 149-162; IT Chou Sc CS. Gasser: Plant Mol. Biol. 35 (1997) 873-892; D.
  • cyclophilins is also intended to include those enzymes which, in addition to homologies to known cyclophilins, also have homologies to other PPlases, such as the enzymes recently discovered and termed FCBs (B. Adams et al.: Journal of Biological Chemistry 280 (2005) 24308-24314).
  • a special feature of cis / trans isomerases of the FKBP family is that their cis / trans isomerase activity can be inhibited by FK506.
  • FK506 A special feature of cis / trans isomerases of the FKBP family is that their cis / trans isomerase activity can be inhibited by FK506.
  • the FKBPs and the inhibition of their cis / trans isomerase activity are of great importance in human and veterinary medicine as well as in biochemical and biotechnological questions, as has been described for example in numerous publications (eg KR 2002024377, US Pat. EP 1687443, JP 2006166845, AU 2002317841, EP 1666053, WO 2006042406, WO 2006012256, WO 2005063964).
  • the cis / trans isomerase activity of representatives of the parvulin family can be significantly inhibited neither with cyclosporin A nor with FK506 at an inhibitor concentration ⁇ 1 ⁇ M with a 10-fold molar excess of inhibitor.
  • a specific irreversible inhibition by the natural product juglone (5-hydroxy-l, 4-naphthoquinone) could be detected ( Hennig et al.: Biochemistry 37 (1998) 5953-5960).
  • Juglone is a natural product isolated from walnut having both bacteriostatic and fungicidal and cytotoxic properties against eukaryotic cells (eg: TJ Monks et al.: Toxicol, Appl. Pharmacol. 112 (1992) 2-16; N. Didry et al. : Pharmacy 49 (1994) 681-683).
  • the first group comprises all eukaryotic enzymes with a specificity for substrates with (PO 3 H 2 ) Ser- / (PO 3 H 2 ) Thr residues before the amino acid residue proline.
  • These include, among others, the human Pinl (hPinl) and the ESS1 / PTF1 from yeast (eg: KP Lu et al .: Nature 380 (1996) 544-547; SD Hanes et al .: Yeast 5 (1998) 55-72, J Hani et al.: FEBS Lett. 365 (1995) 198-202).
  • the reversible phosphorylation of Ser / Thr residues plays a central role in the regulation of basic cellular processes.
  • the regulation of the eukaryotic cell cycle is, for example, subject to the principle of a temporally very precise sequence of activations of various signal transduction cascades. This process is mainly controlled by proline-specific Ser / Thr phosphatases and kinases.
  • the reversible phosphorylation of proteins on Ser / Thr residues leads to structural changes of proteins and thus regulates their biological activity, for example with regard to their stability, enzymatic activity or theirs Binding affinity to other proteins (EA Nigg Bioessays 17 (1995) 471-480).
  • PPIase inhibitors such as sirolimus or everolimus show comparable effects in transplantation immunology as cyclosporin A, without these active ingredients inhibiting in vitro the protein phosphatase calcineurin (eg Lisk W. et al.: Transplantation Proceedings 38 (2006) 69-73) , In addition, these drugs do not have the observed cancer-causing effect such as cyclosporin A (eg: Kauffman et al.: Transplantation 80 (2005) 883-889).
  • PPIase inhibitors which are used therapeutically in transplantation immunology and do not inhibit calcineurin, have an inhibitory effect on tumor growth and tumor angiogenesis (eg: J. Andrassy et al .: Transplantation 80 (2005) 171-174; SH Kim et al.: J. Pathology 164 (2004) 1567-1574; H. Yang et al.: J. of Surgical Res. 123 (2005) 312-319).
  • Known assays which detect the acceleration of cis / trans isomerization of peptide bonds by cis / trans isomerases can be subdivided into direct and indirect methods.
  • Direct assays (a-c) exploit physical measurable parameters whose size is directly related to the isomerization of cis / trans bonds.
  • Indirect tests (d, e) either exploit the altered nature of a substance used for detection due to the isomerization or use isomer-specific reaction principles.
  • Isomer-Specific Mobility If the electrophoretic mobility of suitable cis / trans isomerase substrates for ice and trans isomer is different, this difference can be used to detect cis / trans isomerase activities. So it is z. B is capable of determining the rate of cis / trans isomerase-catalyzed equilibration of previously separated cis / trans isomers from different mobilities by capillary electrophoresis such as e.g. by Brandsch et al. (J. Biol. Chem. 1998, 273, 3861-3864). Disadvantage of this method is the low throughput of analyzes per unit time.
  • Isomer-specific proteolysis The cis / trans-isomer-specific substrate hydrolysis by means of cis / trans isomer-specific proteases makes it possible to deduce from the peculiarity of the proteolysis kinetics the presence or the effectuation of present cis / trans isomerases, as described e.g. by G. Fisher et al. (Biomed.Biochim, Acta 43, 1101-1111 (1984)). The disadvantage here is that highly concentrated proteases must be used and that the half-life of the measuring signal is relatively short. In addition, this test is unsuitable for cis / trans isomerase substrates which are resistant to proteases or which are not degraded configuration specific.
  • Disadvantages here are the relatively complicated spectroscopic methods or the sensitivity of such assays to substances which influence the renaturation of the observed protein in a manner which does not influence the isomerase activity.
  • Another disadvantage is the high cis / trans isomerase concentrations necessary for the method, which can prevent a quantitative determination of high affinity inhibitors.
  • Another disadvantage of the listed methods is furthermore the relatively large amount of solvent to be used, which is necessary in order to shift the natural equilibrium between cis and trans isomer before the measurement.
  • Auxiliary substances used for the deflection of the equilibrium such as trifluoroethanol, are capable of effecting cis / trans isomerases or necessary proenzymes in a manner which is detrimental to the assay.
  • the major drawback of all of the foregoing methods is that they operate with deflected equilibria of cis to trans isomers. The activation energies required to adjust the equilibrium are relatively low, so that the equilibration can take place without the addition of cis / trans isomerases.
  • the difference between whether quantitative or qualitative changes can be observed by adding a catalyst is in usually associated with the activation energy necessary for the respective reaction. While in the abovementioned, previously known detection method of cis / trans isomerase activity, the activation energy required for the course of the reaction is so low that the chemical / biochemical reaction proceeds even under normal conditions even without the addition of a cis / trans isomerase Complex biological systems by the incomprehensible interaction of individual molecules or their functional groups at the molecular level in detail to reactions that can occur only in the presence of active cis / trans isomerases.
  • cis / trans isomerases In addition to cis / trans isomerase activity, which is directed to cis / trans isomerization of peptide bonds as described above, cis / trans isomerases have surprisingly been found to have protease activity toward certain substrates.
  • the method according to the invention has the advantage that effectors which inhibit or activate the protease activity of cis / trans isomerases can be found by means of the same in a procedurally simple and cost-effective manner.
  • the method according to the invention has the advantage that by means of it the cis / trans isomerase inhibiting / activating effect of corresponding effectors procedurally simple and thus can be quantified cost-effectively and reliably.
  • the method according to the invention has the advantage that effectors can be carried out by means of the same high throughput screening for cis / trans isomerase.
  • the method according to the invention thus makes it possible to find effectors for inhibiting or activating cis / trans isomerases either by proteolytic cleavage of a substrate molecule by the cis / trans isomerase or by means of the proenzyme activated by the cis / trans isomerase.
  • the determination according to step d) and optionally according to step e) takes place by means of the substrate molecule and / or by means of at least one substrate molecule fragment which is generated by the cis / trans isomerase caused proteolytic cleavage of the substrate molecule.
  • the determination of the protease activity by means of the substrate molecule and / or by means of hydrolysis products of the substrate molecule requires only a small amount of substrate molecule or fragments and is procedurally simple and therefore particularly inexpensive to carry out.
  • step d) If the determination according to step d) leads to the result that the candidate substance is an effector, quantification of the inhibitory / activating effect of the effector on the Protease activity take place.
  • the quantification of the inhibiting / activating effect can likewise be effected by means of the substrate molecule and / or by means of at least one substrate molecule fragment or else by means of other parameters or methods. The same applies analogously to substrates (auxiliary molecule) which have been reacted by the activated proenzyme.
  • the substrate molecule is a molecule whose proteolytic cleavage can be detected by spectroscopy.
  • the substrate molecule or substrate reacted with the activated proenzyme, when this is a pro-protease is a fluorescently labeled oligo- or polypeptide that alters its fluorescence properties upon proteolytic cleavage.
  • the substrate molecule is a fluorescently labeled oligopeptide having up to 12 amino acid residues.
  • the cis / trans isomerase with protease activity to be used is preferably substrate-specific with respect to the substrate molecule used.
  • the helper molecule is a protein or a peptide or an organic molecule, wherein the organic molecule has a mass of less than or equal to 2,000 Da.
  • the organic molecule has a mass of less than or equal to 2,000 Da.
  • auxiliary molecule it is possible, for example, to use organic molecules found by the method according to the invention and having activating the protease activity of cis / trans isomerases, such as peptides, proteins or a mass of less than or equal to 2,000 Da.
  • peptide is understood to mean a polypeptide having fewer than 12 amino acid residues, and from 12 amino acid residues, the term “protein” is used in the context of the present invention.
  • the present invention further relates in this connection to a method for finding substrate molecules which are proteolytically cleaved by cis / trans isomerases, comprising the steps of
  • substrate molecules candidate substance is proteolytically cleaved.
  • the method according to the invention furthermore makes it possible to find effectors which inhibit or activate the protease activity of cis / trans isomerases and to quantify the inhibiting or activating effect of corresponding effectors on the protease activity of cis / trans isomerases by contacting the cis / trans isomerase a proenzyme that interacts with the cis / trans isomerase.
  • EctIC reactions are preferably distinguished, for example, from the "conventional", ie known cis / trans isomerase catalyzed reactions, by comparison of the ratio of uncatalyzed and catalysed reaction rate of the reaction measured with the proenzyme
  • the physical / chemical / biochemical parameters are optimized which lead to the maximum reaction rate of the proenzyme when adding a suitable amount of cis / trans isomerase Conditions determined only without cis / trans isomerase addition If, under the conditions determined, the ratio of catalysed reaction rate to uncatalyzed reaction rate is greater than 10 and can be determined by addition of cis / trans isomerase inhibitors obtained a reaction rate with respect to the proenzyme, which corresponds to the no cis / trans isomerase addition, so it is an EctIC reaction in the context of the present invention.
  • Nonlinear turnover curve of a typical cis / trans isomerase activity determination can be measured and calculated according to non-linear evaluation models in order to quantify cis / trans isomerase activities.
  • EctIC reactions can circumvent these limitations. Both endpoint determinations and linear kinetics - known to the skilled person as "initial velocity measurement" - are possible by means of the EctIC reaction for the determination of cis / trans isomerase activities.
  • EctIC reactions The molecular basis of EctIC reactions is still largely unknown and may include, for example, both the cis / trans catalysis of a peptide bond of the proenzyme and the specific binding to the proenzyme.
  • cis / trans isomerization of one or more peptide bonds of the proenzyme such flexibility of chemical functionalities of the protein may occur that the proenzyme changes its properties so that it is suitable as a proenzyme for detecting an EctIC reaction.
  • the catalytic activity of the cis / trans isomerase on the proenzyme can also be the cis / trans isomerization of the enzyme substrate bound to the proenzyme or of the substrate interacting with the proenzyme in general, with bound enzyme substrate in the context of the present invention also all being obtained by catalysis of the proenzyme to the product leading intermediates of the enzyme substrate including cleaving residues or the product itself are to be understood.
  • the molecular basis of influencing the proenzyme may be in a change in the three-dimensional structure of the complex To find proenzyme and cis / trans isomerase be.
  • Such proteinaceous changing qualitative properties of one of the binding partners have long been described for numerous protein interactions and are detailed below.
  • a cis / trans isomerase interacts with a proenzyme to qualitatively alter its properties so that an EctlC reaction can be observed and that this change in the proenzyme by the cis / trans isomerase is mediated by the cis / trans isomerase Activity -effective effectors can be changed so that a state of the proenzyme is achieved, which corresponds to that of the proenzyme without effectuation by the cis / trans isomerase.
  • determining whether the proenzyme has the property or that determining the extent of the property of the proenzyme by means of an auxiliary molecule, which with the proenzyme depending on the extent of the property in Interaction occurs. It has been found that the above-mentioned determinations can be carried out more simply by means of an auxiliary molecule compared to a direct determination on the proenzyme.
  • the auxiliary molecule is a substance by means of which a successful interaction with the proenzyme can be detected spectroscopically.
  • the helper molecule is a fluorogenic molecule which, when interacting with the proenzyme, alters its spectroscopic properties.
  • the auxiliary molecule is a corresponding enzyme substrate for the enzyme generated from the proenzyme.
  • the enzyme substrate used is specific for the enzyme generated from the proenzyme.
  • the proenzyme is AvrRpt2. It has been found that the proenzyme AvrRpt2 can be used to carry out Ectlc reactions that are particularly sensitive to effectors.
  • step d) or e) takes place on the basis of the enzyme substrate and / or on the basis of the substrate reacted by the generated enzyme.
  • the property of the proenzyme generated by the cis / trans isomerase is proteolytic activity, since proteolytic activity can be determined and quantified relatively simply on the basis of the enzyme substrate or the fragments resulting therefrom
  • the property generated by the cis / trans isomerase in the proenzyme proteolytic activity is a fluorogenic oligopeptide, which changes its fluorescence properties after proteolytic cleavage, and the determination according to step d) or e) of the method according to the invention by means of fluorescence measurement.
  • the effector is an inhibitor or an activator.
  • the proenzyme is converted to the active form by a cis / trans isomerase.
  • the found EctIC responses can also be used to measure PPIase or APIase activities, e.g. in biological fluids such as blood serum, blood plasma, cerebrospinal fluid, urine or tissue homogenates.
  • biological fluids such as blood serum, blood plasma, cerebrospinal fluid, urine or tissue homogenates.
  • a method is particularly preferred, comprising the steps of:
  • proenzyme generally describes inactive enzyme precursors.
  • inactive enzyme precursors are e.g. known for pepsinogen or chymotrypsinogen. Only by the addition of other enzymes or the action of the same enzyme (autocatalysis, for example, in trypsin) or certain chemical compounds (such as HCl in pepsinogen), the proenzyme is converted into the active form.
  • plO5Rb riboblastoma gene product
  • Cypl Cui Y. et al .: Journal of Cellular Biochemistry, 86 (4): 630-641, 2002;).
  • glycosaminoglycan (GAGS) to CypB (Allain F. et al .: Proceedings of the National Academy of Sciences of the United States of America 99 (5): 2714-2719, 2002).
  • ATFKBP12 The binding of ATFKBP12 to ATFIP37, an Arabidopsis protein (Faure JD Plant Journal 15 (6): 783-789, 1998).
  • proenzyme and cis / trans isomerase are necessary.
  • proenzyme and cis / trans isomerase may be advantageous to use proenzyme and cis / trans isomerase as separate, non-coupled molecules.
  • the proenzyme N-terminal and the cis / trans Isomerase C-terminal or the proenzyme C-terminal and the cis / trans isomerase N-terminal linkage may also be advantageous to combine one or more molecules of the proenzyme with one or more molecules of cis / trans isomerase in a wide variety of sequences.
  • the linking of these molecules can be done directly to each other, but also over the skilled person under the term "linker" summed connecting structures.
  • proenzyme and cis / trans isomerase may also be advantageous to use the proenzyme or cis / trans isomerase or the above-mentioned proenzyme and cis / trans isomerase construct on a water-soluble or insoluble support to bind.
  • a water-soluble or insoluble support As carriers, molecules with a molecular weight> 1000, but also surfaces, such as. Cuvettes or surfaces of the wells of titer plates often used for screening serve. Chemical coupling to such surfaces may be via covalent bonds, as will be apparent to those skilled in the art in a variety of coupling techniques (via linkers such as carbodiimide or disulfide bond).
  • the coupling can also be carried out by means of non-covalent methods, which are also known to the person skilled in the art in a broad spectrum of diverse methods (for example: Myszka DG, Energetics of Biological Macromolecules, pt c, 323 pg.
  • Preferred cis / trans isomerases for carrying out the process according to the invention via the interaction with a proenzyme are those cis / trans isomerases which, together with a suitable proenzyme, interact to such an extent that an EctIC reaction can be detected.
  • Suitable cis / trans isomerases should also be understood as meaning those molecules whose peptide sequence has been modified by a very wide variety of methods known to the person skilled in the art in such a way that neither by means of their molar mass nor of their peptide sequence does one react correspond to natural gene-encoded cis / trans isomerase, which, however, have cis / trans isomerase activity as a safe marker for a cis / trans isomerase under suitably optimized conditions.
  • Peptide sequence is to be understood as meaning the amino acid sequence which results from the chemical or biochemical assembly of natural or non-natural amino acids or their derivatives and which has a cis / trans isomerase activity in the correct three-dimensional conformation under optimal conditions.
  • Peptide sequence should also be understood as meaning the sequences in which one or more functional groups of amino acids have been altered by means of chemical or biochemical methods.
  • Assays for detecting cis / trans isomerase activity are known in the art and have been listed in part above. Suitable optimal conditions may require the addition of a wide variety of substances, such as suitable buffer substances, salts, chelators, emulsifiers, activators, inactivators, sugars and other substances, but also suitable physical parameters, such as a suitable reaction temperature.
  • the enzymatic action of the proenzyme can be significantly influenced and the influence can be reversed by inhibiting the cis / trans isomerase activity of the cis / trans isomerase.
  • the detection method of the EctIC reaction can be carried out here by quantification of the enzymatic reaction of the proenzyme. Detection methods for enzyme reactions are widely known to the person skilled in the art and are continuously available in the specialist press.
  • the detection of the EctIC reaction with the activated protease can take place by means of the protease reaction.
  • Suitable protease substrates are those substrates which allow the proteolytic To identify the activity of a protease activated by cis / trans isomerase activity by means of suitable detection methods. Detection methods of proteolytic activities have long been known and extensively published (eg: AU Kim JH et al .: Analytica Chimica Acta 577 (2): 171-177, 2006; Hamill et al .: Biological Chemistry.
  • protease detection methods with specific substrates, optimized for the proteolytic activity to be investigated, a whole series of methods are also described with which the proteolytic activity of numerous different proteases can be detected.
  • Kim J.H. et al. Fluorescence polarization assay described in Analytica Chimica Acta 577 (2006) 171-177
  • as a protease substrate a tetramethylrhodamine conjugated casein.
  • kinetic and endpoint methods in the above-described methods. Both types of methods can be used to detect an EctIC reaction when the proenzyme is a (pro-) protease, for example.
  • the kinetic method records changing parameters that correlate with the proteolytic reaction during the proteolytic reaction, depending on the reaction time before the end of the reaction. It then results in the application of values of the measurement signals obtained a relation to the course of the protease reaction.
  • An EctIC reaction for example, of a protease activated by the cis / trans isomerase activity can be recognized by the inhibition of the protease reaction after addition of an active substance which inhibits cis / trans isomerase activity.
  • Endpoint methods are usually characterized in that the enzymatic reaction is interrupted at a certain point in time and the reaction products formed up to this time or not yet up to this time fully reacted substrate of the enzymatic reaction is analyzed / quantified. For example, with knowledge of the course of the reaction, it is possible to conclude the reaction rate of the catalysis by means of calibration curves or calculation methods known to the person skilled in the art from the quantitative analysis of reaction product or starting substrate.
  • An EctIC reaction of a protease activated by the cis / trans isomerase activity can be recognized by the inhibition of the course of the protease reaction after addition of an inhibitor of the cis / trans isomerase activity.
  • EctlC reactions listed in items 1 to 8 above may require the addition of a wide variety of helper molecules to optimize the particular EctIC reaction.
  • buffering agents may be necessary to obtain a suitable proton concentration.
  • sugar molecules may be necessary to improve the physical consistency of the solution of ready-made test ingredients. It may also be necessary to use organic solvents, e.g. Ethanol or DMSO, e.g. to improve the solubility of the cis / trans isomerase activity effectors.
  • cytochemical methods for example methods which serve for the cytochemical representation of proteins in tissue sections or cell effusions (for example: Bendayan M .: Microscopy Research & Technique 57 (2002) 327-349; Lopez-Garcia C. et al.: Microscopy Research & Technique 56 (2002) 318-331; Boonacker E. and Van Noorden CJF: Journal of Histochemistry & Cytochemistry 49 (2001) 1473-1486; Nagata T: International Review of Cytology - a Survey of Cell Biology, 211 (2001) 33-151; Bertoni-Freddari C. et al. : Micron. 32 (2001) 405-410; Bendayan M.: Biotechnic & Histochemistry.
  • tissue or cells are fixed with a variety of methods on a suitable object (such as a glass or plastic slide). Often here in tissues fixation by means of wax is made, which then allows the separation of fine tissue sections. Accordingly, tissue sections or cells after incubation with proenzyme and / or isomerase can be used to detect an EctlC reaction.
  • the incubation of cells with the proenzyme AvrRpt2 and a suitable fluorogenic substrate peptide such as Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr (NO 2 ) amide, for the cytochemical detection of Cyclophilin be used.
  • a suitable fluorogenic substrate peptide such as Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr (NO 2 ) amide
  • step d) and optionally according to step e) is carried out by means of an electrochemical, calorimetric, fluorimetric or luminescence method.
  • the substrate molecule is a fluorogenic peptide which measurably changes its fluorescence properties after proteolytic cleavage, and the determination according to step d) and optionally according to step e) is carried out by means of fluorescence measurement.
  • step d) and optionally according to step e) of the method according to the invention is preferably carried out by means of an endpoint method or a kinetic method.
  • Cis / trans isomerases from the group consisting of APIases and PPIases can be used in the process according to the invention. It is particularly preferred if the cis / trans Isomerase is selected from the family of FKBP 's, the cyclophilins or the family of parvulins.
  • cis / trans isomerases should also be understood as meaning those molecules whose peptide sequence has been modified by a very wide variety of methods known to the person skilled in the art so that they do not show either a natural, gene-encoded cis / trans Isomerase would be assigned, but which have cis / trans isomerase activity as a safe indicator for a cis / trans isomerase under suitable optimized conditions.
  • peptide sequence is meant the amino acid sequence which results from the chemical or biochemical assembly of natural or non-natural amino acids or their derivatives and which in the correct three-dimensional conformation has cis / trans isomerase activity under suitable conditions.
  • Peptide sequence should also be understood as meaning those sequences in which one or more functional groups of amino acids have been altered by means of chemical or biochemical methods.
  • Assays for detecting cis / trans isomerase activity are known in the art and have been listed in part above. Suitable conditions may include the addition of a wide variety of substances, e.g. suitable buffering agents, salts, chelators, emulsifiers, activators, inactivators, sugars and other substances, but also suitable physical parameters, e.g. a suitable reaction temperature.
  • the inventive method can be carried out in any containers usually. With regard to spectroscopic evaluations, however, it is preferred that the method is carried out on a titer plate or in a cuvette.
  • the cis / trans isorase and / or the substrate molecule and, if appropriate, the auxiliary molecule are immobilized on a carrier surface. It is preferred that the molecule (s) is / are immobilized by adsorption, by means of an antibody, by a covalent bond or by binding to biotin, avidin or streptavidin on the support surface.
  • the carrier surface is part of a detection strip.
  • test strips are to be understood as test strips, as used for example for pH determination.
  • the method is carried out in vivo.
  • the process is preferably carried out in eukaryotes or prokaryotes, whereby the effectuation is carried out by means of endogenously present isomerases as well as substrate molecules and exogenously added effectors.
  • the method according to the invention can also be carried out ex vivo, preferably by means of human body fluids.
  • tissue homogenates, cell suspensions, cell smears or Tissue sections performed in which the protease activity of PPIases and APIases and their effect is carried out by means of endogenously present in the respective media isomerases and substrate molecules and exogenously added effectors.
  • the present invention further relates to a kit for carrying out the method according to the invention, comprising a cis / trans isomerase and a substrate molecule which can be proteolytically cleaved by the cis / trans isomerase.
  • a kit for carrying out the method according to the invention comprising a cis / trans isomerase and a substrate molecule which can be proteolytically cleaved by the cis / trans isomerase.
  • the cis / trans isomerase and the substrate molecule can be developed according to the preferred embodiments of the method according to the invention.
  • the present invention further relates to a device for finding effectors which inhibit or activate the protease activity of cis / trans isomerases.
  • the device comprises a carrier material on whose surface a cis / trans isomerase and a substrate molecule are immobilized, it being possible for the substrate molecule to be proteolytically cleaved by the cis / trans isomerase.
  • the cis / trans isomerase and the substrate molecule can be developed according to the preferred embodiments of the method according to the invention.
  • the device according to the invention can also comprise an auxiliary molecule as described above immobilized on the substrate surface.
  • the device is designed as a detection strip.
  • the present invention relates to a method for finding effectors which inhibit or activate the protease activity of cis / trans isomerases
  • FIG. 1 Change in the concentrations of the cleavage products H-Gly-Trp-Tyr (NO 2 ) NH 2 (curve A) and Abz-Ile-Glu-Leu-Pro-Ala-Phe-Gly-OH (curve B) and the concentration of the auxiliary molecule Abz-IIe-Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp-Tyr (NO 2 ) NH 2 (curve C) at different reaction times.
  • FIG. 2 Change in fluorescence intensity over time after A) incubation of a hCypl8 / AvrRpt2 mixture with the excipient Abz-He-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr (NO2) -amide , B) incubation of a hCypl ⁇ (R55A) / AvrRpt2 mixture with the excipient Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr (NO2) -amide;
  • FIG. 3 Change in the fluorescence intensity over time after A) incubation of a hCypl8 / AvrRpt2 mixture with the excipient Abz-He-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr (NO2) -amide
  • Inset incubation of a hCypl8 / AvrRpt2 / Abz-He-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr (NO2) -amide mixture with the inhibitor cyclosporin A;
  • FIG. 4 Change in the concentration of the cleavage product H-Gly
  • Figure 6 H-Gly silver stained, 17.5% SDS gel.
  • Lane 1 molecular weight standards
  • Lane 2 sample before incubation at 37 0 C
  • Lanes 3,5 and 7 samples with FKBP inhibitor additive
  • Lanes 4,6 and 8 Samples without FKBP inhibitor additive. The samples were incubated for 1 h (lanes 3 and 4), 3 h (lanes 5 and 6) and 8 h (lanes 7 and 8) at 37 °.
  • FIG. 7 HPLC separation of a hydrolysis batch without CsA addition (a) after an incubation time of 130 h. Application of substrate removal during the incubation period with (b, squares) and without CsA addition (b, circles).
  • FIG. 8 Plot of product increase as area / time of the hydrolysis batch identified by HPLC and identified by MALDI with (a) and without (b) CsA addition. According to the HPLC / MALDI identification, square, circle or triangle represent fission products 1 to 3.
  • FIG. 9 Fluorescence spectrum of the substrate Atto425-Ile-Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp-Gly-AEA-TAMRA (starting product (broken line) and end product (solid line).
  • FIG. 10 Cleavage kinetics of the substrate Atto425-Ile-Glu-Leu
  • a protease substrate which contains a fluorescence excitable moiety, the donor, and another part of the molecule, the quencher, wherein the quencher suppresses the excitability of the donor in the protease substrate. If a chemical bond between the quencher and the donor is separated by a protease, the donor can be excited, which can be qualitatively and quantitatively determined by means of devices suitable for fluorescence measurement.
  • Suitable donor-quencher pairs e.g. Aminobenzoic acid (Abz) as a donor and nitro-tyrosine as a quencher are known in the art. Methods to find suitable new pairs of donor and quencher are also fully described in the art.
  • -cis / trans isomerase solution 10 ⁇ M solution of hCypl ⁇ in 50 mM Hepes buffer, pH 7.8.
  • Substrate Molecule Solution Dissolve 2 mg / ml of Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr (NO2) -amide in dimethylsulfoxide (DMSO).
  • a FluoroMax-2 fluorescence meter (Horiba Jobin Yvon Ine, USA) was used, which was excited to measure at a wavelength of 320 nm and measured at a wavelength of 418 nm. The measurement was carried out at a temperature of 20 ° C.
  • the cis / trans isomerase solution and the substrate molecule solution were mixed together with the initial concentration of the substrate molecule being 10 ⁇ M and that of hCypl8 being 2 ⁇ M.
  • the protease activity of hCypl ⁇ was confirmed by the registration of a fluorescence signal, which increased in intensity over time.
  • the example of the inhibition of the cis / trans isomerase activity of cis / trans isotnerases of the FKBP type shows how substrate molecules can be found that are proteolytically cleaved by these isomerases.
  • a basic prerequisite for finding corresponding substrate molecules according to this strategy is that the inhibition of the cis / trans isomerase activity of the isomerases also inhibits their protease activity.
  • Used for this purpose is a protein mixture, which is obtained in the homogenization of human blood cells and subsequent centrifugation.
  • lysis buffer 155 mM ammonium chloride, 10 mM sodium carbonate, 0.1 mM EDTA
  • the remaining blood cells were washed three times with 25 ml of isotonic saline solution at a temperature of 4 0 C, and then with 10 ml of 100 mM Tris buffer (pH 7.4) were mixed, aliquoted to each 500 ul at a temperature of -20 0 C stored. An aliquot of 500 ⁇ l was thawed, the cells contained therein were disrupted by ultrasound and the resulting suspension was then centrifuged for 5 minutes at 10,000 g. 180 ⁇ l of the supernatant were pipetted into an Eppendorf tube 2 times in each case.
  • the first vessel was treated with 20 ⁇ l of a 100 ⁇ M solution of dimethylcycloheximide (DMCHX, in 100 mM Tris buffer, pH 7.4) or the second vessel with 20 ⁇ l of corresponding Tris buffer.
  • DMCHX dimethylcycloheximide
  • the solutions of both vessels were incubated at a temperature of 37 0 C.
  • FIG. 6 shows the influence of the cis / trans isomerase inhibitor DMCHX on the protease activity of inhibited cis / trans isomerases.
  • the protein band indicated with A is more stable at about 38 kDa than without DMCHX inhibition.
  • protein bands in the region of ⁇ 20 kDa can be found to be increasingly amplified as potential degradation products of proteolysis by cis / trans isomerases.
  • Oligopeptide with and without inhibition of cis / trans isomerase activity Oligopeptide with and without inhibition of cis / trans isomerase activity
  • protease activity of cis / trans isomerases towards oligopeptides can be assessed by analysis of aliquots of the composition of the incubation mixture over the incubation period with a combination of chromatographic separation of the batch and subsequent mass spectrometry of the separated components of the approach to be determined.
  • the chromatographic separation allows the quantitative detection of changes in concentration of substrate molecule and occurring hydrolysis products.
  • Mass spectrometry makes it possible to assign the separate components of the measurement batch by means of their molecular mass.
  • FIG. 7a shows the chromatographic separation of an incubation batch of hCypl ⁇ with the substrate molecule without CsA as indicated below after 130 h.
  • the signals appearing at a retention time of about 24.8 min and 23 min could be assigned to the cis / trans isomerase hCypl ⁇ used or to the substrate molecule.
  • the signals between 10 and 20 min retention time could be assigned to the hydrolysis products of the substrate molecule.
  • the decrease of the initial concentration of the substrate molecule and the increase of the hydrolysis products over time can be graphically represented by analysis and evaluation of the experimental batch at different incubation times by means of samples taken from the experimental batch and their chromatographic behavior ( Fig. 2b, Fig. 3) and evaluate.
  • hCypl ⁇ inhibitor Cyclosporin A (CsA) CsA concentration in the approach: 50 ⁇ M
  • Incubation conditions incubation temperature: 20 ° C.
  • HPLC separation material Vydac-C8 HPLC gradient: 5-55% acetonitrile / aqua (0.1% TFA)
  • Example 4 Detection of an EctIC reaction by means of a fluorogenic substrate
  • a helper molecule Used as a helper molecule was a protease substrate containing a fluorescence excitable moiety, the donor, and another moiety, the quencher, wherein the Quencher suppresses the excitability of the donor in the protease substrate. If a chemical bond between the quencher and the donor is separated by a protease, the donor can be excited, which can be qualitatively and quantitatively determined by means of devices suitable for fluorescence measurement.
  • Suitable donor-quencher pairs such as aminobenzoic acid (Abz) as donor and nitro-tyrosine as quencher, are known in the art. Methods to find suitable new pairs of donor and quencher are well described in the art.
  • Hepes buffer pH 7.8.
  • Proenzyme (protease) solution 100 ⁇ M AvrRpt2 (mature AvrRpt2).
  • Auxiliary (protease substrate) solution Dissolve 2 mg / ml Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr (NO2) -amide in dimethylsulfoxide (DMSO).
  • a fluorescence meter FluoroMax-2 (Horiba Jobin Yvon Ine, USA) was used; was excited at a wavelength of 320 nm; was measured at a wavelength of 418 nm and at a temperature of 20 0 C.
  • Example 5 Detection of an Inactive PPIase by EctlC Reaction and Fluorogenic Substrate
  • -cis / trans isomerase solution 10 ⁇ M solution of hCypl ⁇ (R55A) in 50 mM Hepes buffer, pH 7.8.
  • Proenzyme (protease) solution 100 ⁇ M AvrRpt2 (mature AvrRpt2).
  • Auxiliary (protease substrate) solution Dissolve 2 mg / ml Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr (NO2) -amide in dimethylsulfoxide (DMSO).
  • a fluorescence meter FluoroMax-2 (Horiba Jobin Yv ⁇ n Ine, USA) was used; was excited at a wavelength of 320 nm; was measured at a wavelength of 418 nm and at a temperature of 20 ° C.
  • Example 6 Detection of a PPIase inhibitor by means of EctlC reaction and fluorogenic substrate
  • the PPIase activity of hCypl8 can be specifically inhibited by numerous known inhibitors.
  • the inhibition of PPIase activity of hCypl8 by the inhibitor cyclosporin A (CsA) is demonstrated by means of an EctIC reaction.
  • -cis / trans isomerase solution 10 ⁇ M solution of hCypl ⁇ (R55A) in 50 mM Hepes buffer, pH 7.8.
  • Proenzyme (protease) solution 100 ⁇ M AvrRpt2 (mature AvrRpt2).
  • Auxiliary (protease substrate) solution Dissolve 2 mg / ml Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr (NO2) -amide in dimethylsulfoxide (DMSO).
  • a fluorescence meter FluoroMax-2 (Horiba Jobin Yvon Ine, USA) was used; was excited at a wavelength of 320 nm; was measured at a wavelength of 418 nm and at a temperature of 20 0 C.
  • the example is based on the ability to separate starting product and cleavage products of proteolysis by chromatography from each other and then to quantify.
  • the EctIC reaction was investigated without and in the presence of the inhibitor hCypl ⁇ - CsA at a temperature of 10 0 C.
  • the reaction was started by addition of hCypl ⁇ . After 2, 4, 6, 10, 20, 30, 40, 50, 20, 60, 70, 80, 100, 120, 140, 160 and 180 min, 100 ⁇ l of sample were removed and mixed with 4 ⁇ l (0.5 M). CsA stopped and frozen in N 2 i q or measured immediately. After rapid thawing of the (inhibited) samples, 80 ⁇ l were applied to a chromatography column (VydacCS) and washed with a water / HCN gradient of 15-60% (0.1% TFA) over 20 min chromatographed.
  • Figure 4 shows the detection of an EctlC reaction by product analysis by HPLC. Plotted are the concentrations (indicated as signal area of the
  • FIG. 5 shows evidence of an EctIC reaction by means of HPLC product analysis. Plotted are the concentrations (indicated as the signal area of the chromatographic curves) of the cleavage products H-Gly-Trp-Tyr (NO 2 ) NH 2 (curve A) and Abz-Ile-Glu-Leu-Pro-Ala-Phe-Gly-OH (curve B) as well as the concentrations of the starting substrate Abz-Ile-Glu-Leu-Pro-Phe AIA-Gly-Gly-Trp-Tyr (NO 2) 2 NH (curve C) at different reaction times at 10 0 C. the concentration of the auxiliary molecule in the measurement batch was 14 ⁇ M. 0.2 ⁇ M AvrRpt2 were used as the proenzyme and 1.8 ⁇ M hCypl ⁇ as the cis / trans isomerase.

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Abstract

L'invention concerne un procédé permettant de détecter des effecteurs de cis/trans isomérases et de quantifier l'effet inhibiteur ou activateur de cis/trans isomérases d'effecteurs correspondants.
PCT/EP2008/010303 2007-12-04 2008-12-04 Procédé permettant de détecter des effecteurs de l'activité protéase de cis/trans isomérases WO2009071301A2 (fr)

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WO2002060436A2 (fr) * 2000-12-22 2002-08-08 Pintex Pharmaceuticals, Inc. Methodes d'inhibition de pathologies associees a pin1 a l'aide d'un compose de fredericamycine a

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002060436A2 (fr) * 2000-12-22 2002-08-08 Pintex Pharmaceuticals, Inc. Methodes d'inhibition de pathologies associees a pin1 a l'aide d'un compose de fredericamycine a

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
COAKER GITTA ET AL: "Eukaryotic cyclophilin as a molecular switch for effector activation" MOLECULAR MICROBIOLOGY, Bd. 61, Nr. 6, September 2006 (2006-09), Seiten 1485-1496, XP002528760 ISSN: 0950-382X *
LYON WILLIAM R ET AL: "Trigger factor-mediated prolyl isomerization influences maturation of the Streptococcus pyogenes cysteine protease." JOURNAL OF BACTERIOLOGY JUN 2003, Bd. 185, Nr. 12, Juni 2003 (2003-06), Seiten 3661-3667, XP002528759 ISSN: 0021-9193 *
MCCORNACK M A ET AL: "HIV protease substrate conformation: modulation by cyclophilin A." FEBS LETTERS 1 SEP 1997, Bd. 414, Nr. 1, 1. September 1997 (1997-09-01), Seiten 84-88, XP002528758 ISSN: 0014-5793 *

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