WO2009071301A2

WO2009071301A2 - Method for finding effectors of the protease activity of cis/trans-isomerases

Info

Publication number: WO2009071301A2
Application number: PCT/EP2008/010303
Authority: WO
Inventors: Gunter Fischer; Cordula Schiene-Fischer; Tobias AUMÜLLER; Günther JAHREIS; Gerhard KÜLLERTZ
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date: 2007-12-04
Filing date: 2008-12-04
Publication date: 2009-06-11
Also published as: WO2009071301A3; CA2708021A1

Abstract

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 the respective effectors.

Description

Method for finding effectors of the protease activity of cis / trans isomerases

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 (EC 5.2.1.8) include the peptidyl prolyl cis / trans isomerases (PPIases) and the secondary amide peptide bond cis / trans isomerases (APIases). The assignment of enzymes to the class of cis / trans isomerases is usually via Aminosäuresequenzidentitäts- or -homologievergleiche the enzymes to be assigned with the amino acid sequences of known cis / trans isomerases, on the determination of Katalyseeigenschaften attributable enzymes and the effectuation of Katalyseeigenschaften with various effectors and / or by means of specific antibodies generated on the basis of known representatives of the cis / trans isomerases, which bind to epitopes which bring about the specific properties of the cis / trans isomerases.

Irrespective of the nomenclature of the cis / trans isomerase families described below, there are occasional names in the literature for certain cis / trans isomerases which are attributable to particular properties of these enzymes. For example, some cis / trans isomerases are summarized separately under the name "immunophilins", since these are obviously target molecules for immunosuppressive medicaments in human medicine (eg: Powell JD Zheng Y: Current Opinion in Investigational Drugs 7 (II): 1002-1007, 2006; Bell A. et al.: International Journal for Parasitology. 36 (3): 261-276, 2006; Hey ZY. et al. : Plant Physiology. 134 (4): 1248-1267, 2004; Dugave C.: Current Organic Chemistry. 6 (15): 1397-1431, 2002). In turn, other cis / trans isomerases, such as FKBP38, are grouped under the name CaMAP because they are activatable by calmodulin (eg Edlich F. et al., Journal of Biological Chemistry, 281 (21): 14961-14970 , 2006, Edlich F. et al .: EMBO Journal 24 (14): 2688-2699, 2005). Independently of these separate names, however, all cis / trans isomerases can be assigned either to the APIases or to the PPIases and in turn either to the cyclophilins, the FKBPs or the parvulins.

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).

Among the 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.

A particular property of cyclophilins is that their PPIase activity can be inhibited by cyclosporin A (eg: DD 281659). In the context of the present invention, the term "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. Roy et al .: Biochemical & Biophysical Research Communications 307 (2003) 422-429; JW Montague et al.: Journal of Biological Chemistry 272 (1997) 6677-6684). The term "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. Like the cyclophilins, 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 μM with a 10-fold molar excess of inhibitor. For various members of the parvulin family, namely the Escherichia coli parvulin, the Saccharomyces cerevisiae ESS1 / PTF1 and the human Pinl, 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).

Within the parvulin family, a distinction is made between two groups of enzymes that differ in their substrate specificity. 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). On the other hand, previously known prokaryotic cis / trans isomerases and some of the eukaryotic isomerases are not specific for phosphorylated substrates. They are summarized in the second group. Parvulins as well as the inhibition of their PPIase activity are the subject of discussions in the literature regarding their suitability as a biochemical or biotechnological tool (eg: US6030826, US3798129). Extensive knowledge exists regarding the use of parvulin inhibitors as active substances in human and veterinary medicine in the literature (eg: WO 03093258, WO 03074001, US 2003096387, JP 2001316289).

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).

The discovery and use of cis / trans isomerase effectors such as inhibitors or activators is of great importance in the scientific literature as well as the medical interest (eg: JF Guichou et al .: J. Med. Chem. 49 (2006) 900- YQ Yu et al .: J. Med. Chem. 46 (2003) 1112-1115) and subject matter of many patent publications (eg: US 2004204340, US 2003013645, US 2003068321, US 6270957, WO 2006033409, WO 2006005580, WO 2005097164, WO 2005021028).

For the immunosuppressive effect of cyclosporin A, the inhibition of the phosphatase activity of the protein phosphatase calcineurin by a complex of cyclosporin A and cyclophilin 18 (e.g., Rusnak F. & Mertz P .: Physiological Reviews 80 (2000) 1483-1521) is believed. This mechanism also led to the term "immunophilins" in the scientific literature, which identifies PPIases which show immunosuppressive effects together with a PPIase inhibitor (eg: Dugave C: Current Organic Chemistry 6 (2002) 1397-1431; Jorgensen KA et Scandinavia Journal of Immunology 57 (2003) 93-98.) The discovery of the suppressive effect of the cyclophilin / cyclosporin A complex on the human immune system has led to a number of pharmaceutically important applications in human medicine (eg: Pollard S et al .: Clinical Therapeutics 25 (2003) 1654-1669).

In addition to the immunosuppression used in transplantation medicine by inhibiting the PPIase activity of cyclophilin with cyclosporin A, other effects brought about by inhibition of cis / trans isomerases were also observed, such as, for example, B. Enhanced hair growth (eg: Gafter-Gvili A. et al .: Archives of Dermatological Research 296 (2004) 265-9; Shirai A. et al.: Journal of Dermatological Science 27 (2001) 7-13) Influence on the hair graying (eg: Rebora J. et al.: International Journal of Dermatology 38 (1999) 229-230), a broadly applicable effect on mammalian parasites, such as on the malaria-causing Plasmodium falciparum (eg: R. Kumar et al Molecular & Biochemical Parasitology 141 (2005) 29-37; A. Bell et al.: Biochemical Pharmacology 48 (1994) 495-503) and on trypanosomes (eg: J. Bua et al .: Bioorganic & Medicinal Chemistry Letters 14 (2004) 4633-4637), an effect on Leishmania major parasites (eg: Meissner U. et al.: Parasitology Research 89 (2003) 221-227), an effect on echinococci (eg: Colebrook AL et al .: Parasitology 125 (2002) 485-493) or on nematodes (such as: Ma U. et al .: Journal of Biological Chemistry 277 (2002) 14925-14932), an effect on chlamydia and pneumococci, an effect on viruses such as the Hepatitis C virus (HCV) or dengue fever inducing dengue virus and retroviruses such as the HIV virus (eg Boss V. et al .: Molecular Pharmacology 54 (1998) 264-72), an effect on psoriasis (eg: Zachariae H. & Olsen TS: Clinical Nephrology 43 (1995) 154-8) and an effect on the nephrotic syndrome (eg: Noyan A. et al. : Nephron 70 (1995) 410-15). In addition, therapeutically significant effects on nerve cells (eg: Wong A., Cortopassi G .: Biochemical & Biophysical Research Communications 239 (1997) 139-45), on lung tissue (eg: JW Eckstein & J. Fung: Expert Opinion on Investigational Drugs 12 (2003) 647-653) and on tumors (eg: TZ Wang et al .: Analytical Chemistry 76 (2004) 4343-4348; K. Kawano et al.: Cancer Research 60 (2000) 3550-3558) , A large number of these effects have been discovered by the use of the drug cyclosporin A in transplantation medicine and observed there as a side effect (eg: David-Neto E. et al .: Journal American Society Nephrology 11 (2000) 343-9).

Particularly serious side effects with a lasting therapy with Cyclosporin A are damages of the kidneys, like eg nephrotoxicity, influencing the glomerular filtration or irreversible interstitial fibrosis (eg: Kopp et al: Journal American Society Nephrology 1 (1991) 162-12), neurological changes, such as the occurrence of a tremor (eg: De Groen et al.: New England Journal of Medicine 317 (1987) 861-74), vascular hypertension (eg: Kahan K. et al .: New England Journal of Medicine 321 (1989) 1725-33), the formation of tumors (eg: Kauffman L. et al.-Transplantation 80 (2005) 883-889) and complications associated with these lesions. Damage to the liver or enlargements of the peridontium have also become known (for example: Tosti A. et al .: Drug Safety 10 (1994) 310-17; Borel JF et al.: Advances in Pharmacology 35 (1996) 79-114).

Further investigations of cyclosporin A effects at the molecular level showed that part of the observed effects can be attributed to the inhibition of the protein phosphatase calcineurin. By derivatization of the cyclosporin with the provision of largely maintaining the spatial structure of the cyclosporin A / cyclophilin / calcineurin complex (Jin L. & Harrison S.C .: Proceedings of the National Academy of Sciences of the United States of America 99 (2002) 13522 6) it was possible to prepare cyclophilin inhibitors which can inhibit the PPIase activity of Cypl8, but the Cypl8 / inhibitor complex no longer inhibits the protein phosphatase calcineurin (for example: Zhang YX et al.: J. of Biological Chemistry 280 (FIG. 2005) 4842-4850). Such, the protein phosphatase calcineurin in complex with Cypl8 non-inhibiting Cypl8 inhibitors are often referred to in the literature as "non-immunosuppressive" cyclophilin inhibitors (non-immunosuppressive cyclophilin inhibitor compounds) (eg: Carry JC et al - Synlett. 2004) 316-20; Evers M. et al.: Bioorganic & Medicinal Chemistry Letters 13 (2003) 4415-4419). However, there are numerous examples in the literature of low-molecular-weight active substances which contribute to imraunosuppression even without inhibition of the protein phosphatase calcineurin. For example, 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). While the inhibition of calcineurin in combination with an increase in TGF-ß leads to cancer progression, at least in animal experiments it was shown that 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). In addition to a large number of scientific papers on the benefits of cyclophilin inhibitors in human and veterinary medicine or as a biochemical or biotechnological tool, there are also a large number of patent publications, which demonstrates the great economic interest in cyclophilins and their effectors (eg: KR 20040041458 , CA 2541497, AU 2002259217, US 7041297, US 2006094646, WO 2006033409, JP 2006042803, CN 1698880, US 2005214317, US 2004157919, MXPA 03006666, US 2004053840, US 2003232815, MXPA 03004821, US 2004204340, US 2003013645, US 2003068787, CN 1379092, MXPA 02002578, US 2002165275, CN 1428348, WO 0248178).

Since cis / trans isomerases and their effect are associated with a variety of diseases and cosmetic problems, there is a need for methods by which the presence of cis / trans isomerases can be detected or with their help can be found effectors of these enzymes, which have the potential to be used as pharmaceuticals.

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.

a) 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.

b) Isomer-specific spectroscopic differences: Some cis and trans isomerase substrates have different spectroscopic properties depending on the configuration. A cis / trans isomerase causes a faster adjustment of the natural equilibrium of a deflected equilibrium of cis and trans isomers. The adjustment of the natural equilibrium can be followed spectroscopically in such a case, as described, for example, by B. Janowski et al. (Analytical Biochemistry 1997, 252, 299-307). Disadvantages of this method are the low Spectral changes that require highly sensitive and therefore expensive isomers for isomerase activity determination. Another disadvantage is that it is very difficult to stop the setting of the cis / trans equilibrium so that end point measurements are hardly possible.

c) Isomer-specific chemical shift when using nuclear magnetic resonance (NMR): Certain cis / trans isomerase substrates show nuclear resonance spectroscopic differences in the chemical shift of both configurational isomers. By the methods used in NMR spectroscopy, such as magnetization transfer or line analysis, the effect of isomerases on the cis / trans isomerization rate of these substrates can be quantified, e.g. by D. Kern et al. (Biochemistry 1995 34, 13594-13602). Disadvantages of this method are the high substrate concentrations necessary for the measurement.

d) 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.

e) Renaturation of Proteins: Since cis / trans isomerases have been described in numerous methods (Liu CP, Zhou JM, Biochemical & Biophysical Research Communications, 313 (3): 509-515, 2004, Ow WB. et al. : Protein Science. 10 (11): 2346-2353, 2001; Ideno A. et al .: Biochemical Journal. 357 (Part 2): 465-471, 2001) can accelerate the renaturation of denatured proteins, the acceleration of this renaturation by cis / trans isomerases can be used to detect the activity of isomerases, as described, for example, by D. Kern et al. (Biochemistry 1995 34, 13594-13602). 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, however, 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. In order to be able to measure the activity of cis / trans isomerases, work is therefore often carried out at temperatures below room temperature. The recognition of cis / trans isomerase activity is often only possible if it is possible to record the temporary differences in the reaction kinetics with and without active cis / trans isomerase. A determination of cis / trans isomerase activity by endpoint method, ie after expiration The chemical reaction has hitherto been possible only if it is possible to freeze the kinetic differences of the reaction with and without cis / trans isomerase activity by suitable means, such as by lowering the temperature or by removal of the by the cis / trans catalysis of reacted substrate or product molecules.

In contrast to the above-described detection methods of cis / trans isomerase activities, which observe quantitative changes caused by chemical reactions, it has also been known for a long time that cis / trans isomerase activity can cause qualitative changes in complex biological systems. Thus, the removal of cis / trans isomerases, which is possible by means of genetic engineering, leads to altered phenotypes of the most diverse organisms (for example: Geisler M. et al.: Molecular Biology of the Cell, 14 (10): 4238-4249, 2003, Ansari H. et al Molecular & Cellular Biology 22 (20): 6993-7003, 2002; Bernhardt TG et al.: Molecular Microbiology 45 (1): 99-108, 2002; Metzner M. et al.: Journal of Biological Chemistry 276 (17): 13524-13529, 2001; DebRoy S. et al .: Infection & Immunity 74 (9): 5152-5160, 2006, Fanghanel J. et al .: FEBS Letters 580 (13): 3237 -3245, 2006; Ardon O. et al.: Journal of Virology 80 (8): 3694-3700, 2006). In a few cases, it has been shown that by inhibiting the cis / trans isomerase activity of cis / trans isomerases, which can be assigned to spatially delineate cellular regions, e.g. Ion channels, also detect qualitative effects (eg: Nakagawa T. et al .: Nature 434 (7033): 652-658, 2005; Hansson MJ et al: Journal of Bioenergetics & Biomembranes 36 (4): 407-413 Waldmeier PC, et al .: Current Medicinal Chemistry 10 (16): 1485-1506, 2003, Lin DT et al., Journal of Biological Chemistry 277 (34): 31134-31141, 2002).

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.

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.

Since, as already stated above, cis / trans isomerases are associated with a large number of diseases and cosmetic problems, there is now also a need for methods by means of which it is possible to find effectors which are simple in terms of process engineering and have a cost effective effect on the protease activity of cis / trans isomerases, and a need for methods by which the protease activity inhibiting / activating effect of corresponding effectors can be quantified.

It is therefore an object of the present invention to provide a method by means of which effectors can be found in a simple and cost-effective manner in terms of process engineering, which influence the protease activity of cis / trans isomerases, and by means of which the protease activity can be quantified inhibitory / activating effect of corresponding effectors.

This object is achieved by a method comprising the steps of:

a) providing a cis / trans isomerase;

b) contacting the cis / trans isomerase with a substrate molecule which is proteolytically cleaved by the cis / trans isomerase or a proenzyme which is activated by the cis / trans isomerase,

c) contacting the cis / trans isomerase with an effector candidate substance;

d) determining whether the effector candidate substance inhibits or activates cis / trans isomerase activity;

and optionally

e) Determining the extent of inhibition or activation of the cis / trans isomerase caused by the effector candidate substance.

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.

In addition, 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.

Furthermore, 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.

It has also been shown that the substances (effectors) which effect the protease activity of cis / trans isomerases can also influence the cis / trans isomerase activity of the isomerases.

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.

If the method is carried out on the basis of the proteolytic cleavage of a substrate molecule, it is provided according to a first preferred embodiment of the method according to the invention that 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. 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. In this case, 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 determinations according to method steps d) and e) can be carried out here by means of evaluation of different or identical parameters or using different or identical methods.

According to a preferred embodiment, it is provided that the substrate molecule is a molecule whose proteolytic cleavage can be detected by spectroscopy. In this context, it is particularly preferred that 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. Preferably, 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 previously discovered pairs of cis / trans isomerase and substrate molecule, which is proteolytically cleaved by the associated cis / trans isomerase, show that the activation energy for the proteolytic cleavage of a peptide bond by the cis / trans isomerase is reduced only relatively little, so that by cis / trans isomerases catalyzed proteolytic cleavage of peptides proceeds relatively slowly. Accordingly, according to a further preferred embodiment of the method according to the invention, it is provided that the cis / trans isomerase is brought into contact with an auxiliary molecule which increases the protease activity of the cis / trans isomerase. Preferably, 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. By this measure, a faster and thus more cost-effective screening for effector substances of the protease activity of cis / trans isomerases is made possible. As an 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. The term "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

a) providing a cis / trans isomerase;

b) contacting the cis / trans isomerase with a substrate molecule candidate substance;

c) determining if the substrate molecule candidate substance is proteolytically cleaved. By means of this method, substrate molecules can be found which are proteolytically cleaved by cis / trans isomerases in a process-technically simple manner.

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.

The reactions known in the prior art, which are catalyzed by cis / trans isomerases, all have such a low activation energy that they can proceed at room temperature even without added isomerase. The cis / trans isomerase therefore serves only as a reaction accelerator in these reactions. Surprisingly, it has now been shown that cis / trans isomerases also have reactions which can take place only with the addition of cis / trans isomerases at a significant reaction rate and which are inhibited again by inhibiting / activating the cis / trans isomerase activity by means of suitable effectors or continue to activate.

For example, it has been shown that the proteolytic activity of AvrRpt2 against corresponding substrates can only be significantly generated and maintained by the presence of certain cis / trans isomerases with a suitable cis / trans isomerase activity, whereby this generation of proteolytic activity by the cis / trans isomerase activity directed inhibitors significantly and quantifiable effect. To separate the language from the previously known chemical / biochemical reactions, With the help of which cis / trans isomerases can be detected, these newly discovered reactions, which take place only by addition of non-inhibited cis / trans isomerases with a significant reaction rate, are called "essential cis / trans isomerase catalysis" (EctIC).

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 In a first step, 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. Previously known kinetic detection methods with respect to cis / trans isomerases have, due to the fast-running uncatalyzed reaction, eg for the observation of the catalysis of cis / trans isomerizations in peptidyl-prolyl peptide bonds, with rate constants for the cis to trans or trans - After cis reaction of> 10 ^"3 s ^{" 1} at room temperature only a very small time window of a few seconds to detect the inhibition of an enzymatically catalyzed cis / trans isomerization. Often the entire 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. In contrast, as documented by the examples, 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.

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. Thus, by 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. However, 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.

If the binding of cis / trans isomerase to the proenzyme itself and not the catalysis of cis / trans isomerization on the proenzyme or bound peptide substrate is the cause of an EctIC reaction, 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. However, it has never previously been observed that 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.

According to a preferred embodiment of the method according to the invention, it is provided that 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.

According to a further preferred embodiment of the method according to the invention, it is provided that the auxiliary molecule is a substance by means of which a successful interaction with the proenzyme can be detected spectroscopically. In this context, it is particularly preferred if the helper molecule is a fluorogenic molecule which, when interacting with the proenzyme, alters its spectroscopic properties.

Since by interaction of the proenzyme with the cis / trans isomerase an enzymatic activity of the proenzyme is caused, it is ensured that the determinations according to steps d) and e) of the method according to the invention can be carried out in a procedurally simple manner by means of an activity determination of the activated enzyme.

If the process according to the invention is carried out via the interaction of a cis / trans isomerase and a proenzyme, it is preferred if the auxiliary molecule is a corresponding enzyme substrate for the enzyme generated from the proenzyme. For a particularly accurate determination according to steps d) and e) of the method according to the invention, it is advantageous here if the enzyme substrate used is specific for the enzyme generated from the proenzyme.

According to a particularly preferred embodiment of the method according to the invention, 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.

In the method according to the invention it can be provided that the determination according to 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.

In the context of the method according to the invention, it is particularly preferred if 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

It is particularly preferred that the property generated by the cis / trans isomerase in the proenzyme proteolytic activity, the auxiliary molecule 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.

With the method according to the invention, both inhibiting and activating effectors can be found. Accordingly, it is preferred that the effector is an inhibitor or an activator.

As stated above, 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. For this purpose, a method is particularly preferred, comprising the steps of:

a) providing a proenzyme which is capable of interacting with a cis / trans isomerase and the interaction with the proenzyme produces a property the extent of which depends on the activity of the cis / trans isomerase;

b) contacting the proenzyme with a sample to be examined for the presence of one or more cis / trans isomerases;

c) determining if the proenzyme has the property;

or

d) determining the extent of the property of the proenzyme. Interaction with the cis / trans isomerase significantly influences the enzymatic action of the proenzyme. As a significant influence is preferably the reduction of the molar catalytic constant, measured under the conditions suitable for this catalysis, by a factor of 10 understood.

It has been known for some time that cis / trans isomerases can form bonds with a second protein to form a heterodimer or that heteromeric complexes with several different proteins and smaller ligands can also be formed (eg: Cell 87 (7): 1157-1159, 1996 Biochemistry, 46 (26): 7832-7843, 2007; Analytical Biochemistry, 359 (2): 285-287, 2006; Biochemical & Biophysical Research Communications. 321 (3): 638-647, 2004; Endocrine 20 (1-2): 83-89, 2003). Extensive investigations of interactions and methods for studying interactions have been extensively described, e.g. e.g. Parrish JR. et al. in Current Opinion in Biotechnology. 17 (2006): 387-393; Ito T. et al. Trends in Biotechnology. 19 (10 Suppl S): S23-S27, 2001) The term proenzyme generally describes inactive enzyme precursors. Such 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.

Likewise, there are numerous examples in the literature which show that the interaction of a great variety of proteins with one another, including the binding to other ligands, makes it possible to detect properties of these complexes which depend on the properties of the individual constituents Complex and not represent the summation of the properties of the individual components. However, despite the numerous previously discovered protein complexes that contain cis / trans isomerases, no evidence for an EctIC reaction has yet been found. Published examples of interactions between cis / trans isomerases and other proteins include:

• the SR-cyclophilin interaction to pinine (Lin CL et al., Biochemical & Biophysical Research Communications, 321 (3) -638-647, 2004).

• the interactions between heat shock proteins and various cis / trans isomerases such as Cyp40, FKBP51 and FKBP52 (Carrello A. et al .: Cell Stress & Chaperones.

9 (2): 167-181, 2004).

• binding of Cypl8 to HIV virion (BonHomme M. et al .: Biophysical Chemistry, 105 (1): 67-77, 2003.

The interaction of CypH and spliceosome (Ingelfinger D. et al: Nucleic Acids Research. 31 (16): 4791-4796, 2003).

The binding between plO5Rb (riboblastoma gene product) and Cypl (Cui Y. et al .: Journal of Cellular Biochemistry, 86 (4): 630-641, 2002;).

• binding of 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).

The binding of a plant cyclophilin (ROC7) to the protein phosphatase PP2A (Jackson K. Soll D .: Molecular & General Genetics 262 (4-5): 830-838, 1999); Binding of Cypl8 on calreticulin (Reddy PA, Atreya CD: International Journal of Biological Macromolecules 25 (4): 345-351, 1999).

• the interaction between CypH and the adenine nucleotide translocase (Biochemical Journal, 336 {Part 2): 287-290, 1998).

The binding of FKBP12.6 to the ryanodine receptor (Huang FN et al.: Proceedings of the National Academy of Sciences of the United States of America 103 (9): 3456-3461, 2006).

• the interaction of presenilins with FKBP38 (Wang HQ et al .: Human Molecular Genetics, 14 (13): 1889-1902, 2005).

Binding of FKBP51 to calcineurin (Li TK et al., Journal of Cellular Biochemistry, 84 (3): 460-471, 2002).

The binding of Hsp90 to FKBP52 (Galigniana MD, et al.: Journal of Biological Chemistry 276 (18): 14884-14889, 2001).

The interaction of FAP48 with FKBP12 and FKBP52 (Neye H. Regulatory Peptides, 97 (2-3): 147-152, 2000).

The binding of ATFKBP12 to ATFIP37, an Arabidopsis protein (Faure JD Plant Journal 15 (6): 783-789, 1998).

The binding of FKBP12 to TGF (Okadome T. et al .: Journal of Biological Chemistry 271 (36): 21687-21690, 1996).

The interaction between Pinl and Nek6 (Chen J. et al.: Biochemical & Biophysical Research Communications 341 (4): 1059-1065, 2006). The interaction between the protein kinase CK2 and Pinl (Messenger MM et al.: Journal of Biological Chemistry, 277 (25) = 23054-23064, 2002).

In none of the previously published and summarized in excerpts examples so far, a reaction which corresponds to an EctIC reaction, can be observed. It is often even reported that the binding of a protein to a cis / trans isomerase apparently conceals its active site in such a way that high-affine effectors can no longer bind to the active site for cis / trans isomerases (eg: Journal of Cellular Biochemistry (4): 630-641, 2002; Li TK et al.: Journal of Cellular Biochemistry 84 (3): 460-471, 2002). In turn, an effectuation of the binding between a cis / trans isomerase and another protein is observed by a cis / trans isomerase inhibitor, e.g. in the binding of CypH to the adenine nucleotide translocase (Biochemical Journal 336 (Part 2): 287-290, 1998) or the binding of an FKBP to a vegetable protein (Faure JD Plant Journal, 15 (6): 783-789 , 1998), an effectivation of the potential proenzyme (here the translocase) is not observed.

In the context of the present invention, the interaction between proenzyme and cis / trans isomerase is necessary. To perform an assay, it may be advantageous to use proenzyme and cis / trans isomerase as separate, non-coupled molecules. However, it may also be advantageous to simplify test systems for example, to combine proenzyme and cis / trans isomerase by means of chemical linkers or to prepare genetic engineering methods known to the biochemist as a fusion protein in which proenzyme and cis / trans isomerase are linked to one another. It may be advantageous, the proenzyme N-terminal and the cis / trans Isomerase C-terminal or the proenzyme C-terminal and the cis / trans isomerase N-terminal linkage. However, it 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.

In addition to linking proenzyme and cis / trans isomerase by chemical or genetic engineering techniques, it 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. 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). However, 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.

By interacting with the cis / trans isomerase, 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.

If the proenzyme is, for example, a precursor of a protease, 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. 387 (8): 1063-1074, 2006; Sparidans RW et al .: Biomedical Chromatography 20 (8): 671-673, 2006; De Vries L. et al: Biochemical Pharmacology 71 (10): 1449-1458, 2006; van Maarseveen NM, et al.: Journal of Virological Methods, 133 (2).-185-194, 2006; Guarise C. et al., Proceedings of the National Academy of Sciences of the United States of America 103 (11): 3978 Van Laethem et al .: Journal of Virological Methods, 132 (1-2): 181-186, 2006; Cottier V. et al.: Antimicrobial Agents & Chemotherapy. 50 (2): 565-571, 2006; Sparidans RW et al.: Biomedical Chromatography 20 (l): 72-76, 2006; Tsongalis GJ et al.: Journal of Clinical Virology 34 (4): 268-271, 2005; Kainmuller EK et al .: Chemical Communications (43): 5459-5461, 2005; Vega Y. et al. : Journal of Clinical Microbiology. 43 (10): 5301-5304, 2005; Zhang ZD. et al. : Acta Oceanologica Sinica. 24 (4): 155-161, 2005; Snoeck J. et al. : Journal of Virological Methods. 128 (1-2): 47-53, 2005; Hu K. et al .: Journal of Virological Methods. 128 (1-2): 93-103, 2005; Yoshida A. et al. : Microbes & Infection. 7 (5-6): 820-824, 2005; Tribute O. et al. : Therapeutic Drug Monitoring. 27 (3): 265-269, 2005; Menotti J. et al. : Antimicrobial Agents & Chemotherapy. 49 (6): 2362-2366, 2005, Pelerin H. et al. : Analytical Technologies in the Biomedical & Life Sciences. 819 (1): 47-57, 2005, Debrah O. et al. : Bioconjugate Chemistry. 15 (6): 1322-1333, 2004; Shan YF. et al .: Biochemical & Biophysical Research Communications 324 (2): 579-583, 2004; Kao RY. et al. : FEBS Letters. 576 (3): 325-330, 2004) and in numerous patents (eg: WO2005097818, WO03065004, US2006240503A1, US2006183177A1, US2006183177A1, US2003170770A1, US6821744B2, US6306619B1, US5891661A1, US3607859A1, EP0168738A2, CA2522795A1). Most of these methods are based on the Detection of spectroscopic differences between substrate and product molecules. If the proenzyme is a generated exopeptidase, protease activity detection is often done by using a chromogenic substrate whose chromogen is released by the protease reaction.

Apart from 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. For example, by 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.

In general, one can distinguish between 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.

Like kinetic protease assays, a variety of endpoint methods are known. Roughly, the methods can be differentiated according to how the reaction product to be analyzed or the remaining amount of starting material is quantified.

All of the 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. Thus, buffering agents may be necessary to obtain a suitable proton concentration. The addition of 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.

Further detection methods are 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. 75 (2000) 203-242; Takizawa T. and Robinson JM. : Histology & Histopathology. 15 (2000) 515-522). The tissues 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. For example, 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.

In the context of the present invention, it is particularly preferred that the determination according to step d) and optionally according to step e) is carried out by means of an electrochemical, calorimetric, fluorimetric or luminescence method.

It is particularly preferred that 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.

The determination according to 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.

In the context of the present invention, however, 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. By 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.

With regard to the implementation of high throughput screening methods, according to a further preferred embodiment in that 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.

In order to determine, for example, laboratory-scale effectors, it may be provided according to a further preferred embodiment of the method according to the invention that the carrier surface is part of a detection strip. Under test strips are to be understood as test strips, as used for example for pH determination.

As an alternative to the immobilization of one or more of the mentioned molecules for carrying out the process according to the invention, it may also be provided that the process is carried out in homogeneous solution.

According to a further preferred embodiment of the method according to the invention, it is provided that 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.

Alternatively, the method according to the invention can also be carried out ex vivo, preferably by means of human body fluids.

According to a further preferred embodiment of the method according to the invention, the same by means of 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. In this case, 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. In this case, 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.

According to a preferred embodiment, it is provided that the device is designed as a detection strip.

In particular, the present invention relates to a method for finding effectors which inhibit or activate the protease activity of cis / trans isomerases

comprising the steps of a) providing a mixture of cyclophilin, especially hCyplδ, and AvrRPT2;

b) contacting the mixture with a substrate molecule which is proteolytically cleaved by the mixture;

c) contacting the mixture according to b) with an effector candidate substance;

d) determining whether the effector candidate substance inhibits or activates the activity of the cyclophilin;

and optionally

e) Determining the extent of inhibition or activation of cyclophilin caused by the effector candidate substance.

Examples and figures

The following examples and figures are used in conjunction with the drawings to explain the invention. Show it:

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 hCyplδ (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

Trp-Tyr (NO ₂ ) NH ₂ at different reaction times by proteolysis of Abz-Ile-Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp-Tyr (NO ₂ ) NH ₂ by a mixture of hCypl8 and AvrRpt2 in Absence and in the presence of the inhibitor cyclosporin A (filled circles: without inhibitor; triangles: with inhibitor). On the inside, the chromatograms obtained after 0, 10, 30, 60 and 180 minutes (with and without inhibitor) are shown; FIG. 5: change in the fluorescence intensity over time after A) presentation of the cis / trans isomerase hCyplδ, B) incubation of the isomerase with the proenzyme AvrRpt2, C) incubation of the isomerase / proenzyme mixture with the auxiliary molecule Abz-Ile-Glu Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr (NO2) -amide;

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

Pro-Ala-Phe-Gly-Gly-Trp-Gly-AEA-TAMRA in the presence of Cyclophilin (hCypl8) and AvrRpt2. The points correspond to individual measured values, the solid line was obtained by calculating the kinetic curve after a first-order reaction.

Example 1: Detection of protease activity of hCyplδ

Used as a substrate molecule was 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.

The following solutions were produced:

-cis / trans isomerase solution: 10 μM solution of hCyplδ 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).

For fluorescence measurement, 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 μM and that of hCypl8 being 2 μM. The protease activity of hCyplδ was confirmed by the registration of a fluorescence signal, which increased in intensity over time.

Example 2: Finding of natural cis / trans isomerase protease substrates

In the following, 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.

Leukocyte preparation

50 ml of BuffyCoat from a healthy blood donor were centrifuged at 1500 g for 10 min. For the removal of erythrocytes, the centrate was at a temperature of 0 ⁰ C with 50 ml of lysis buffer (155 mM ammonium chloride, 10 mM sodium carbonate, 0.1 mM EDTA) for 10 minutes. After repeated centrifugation at 1500 g and removal of the supernatant, the lysis step was repeated again. Subsequently, 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 μl 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 μl of the supernatant were pipetted into an Eppendorf tube 2 times in each case. The first vessel was treated with 20 μl of a 100 μM solution of dimethylcycloheximide (DMCHX, in 100 mM Tris buffer, pH 7.4) or the second vessel with 20 μl of corresponding Tris buffer. The solutions of both vessels were incubated at a temperature of 37 ⁰ C.

After 1, 3 and 8 hours each 20 .mu.l of sample were removed and incubated with 180 .mu.l of the above tris buffer at a temperature of 95 ⁰ C for 5 min. Each 8 μl of the solutions thus obtained were applied together with a molecular weight standard to a 17.5% SDS gel and electrophoresed by standard procedure and visualized by means of silver staining (Rabilloud T. et al.: Electrophoresis 9 (1988) 288-91) , FIG. 6 shows the influence of the cis / trans isomerase inhibitor DMCHX on the protease activity of inhibited cis / trans isomerases. Thus, with FKBP inhibition, the protein band indicated with A is more stable at about 38 kDa than without DMCHX inhibition. In contrast, without FKBP inhibition, protein bands in the region of <20 kDa, such as the band indicated with B, can be found to be increasingly amplified as potential degradation products of proteolysis by cis / trans isomerases.

Example 3: Protease activity of hCypl8 over one

Oligopeptide with and without inhibition of cis / trans isomerase activity

The 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.

When using different oligopeptide sequences, it is thus possible to obtain information both about the substrate specificity of the protease activity of cis / trans isomerases and about the catalytic constants of the protease activity of these enzymes with respect to the respective polypeptide sequences.

FIG. 7a shows the chromatographic separation of an incubation batch of hCyplδ with the substrate molecule without CsA as indicated below after 130 h. By means of mass spectrometry, the signals appearing at a retention time of about 24.8 min and 23 min could be assigned to the cis / trans isomerase hCyplδ 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. Since the area of the signals correlates directly with the respective peptide concentration, 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.

While the substrate molecule was completely hydrolyzed after about 290 hours under the chosen conditions (FIG. 2b, circles), the rate of hydrolysis can be slowed by complete inhibition of the cis / trans isomerase activity with a 1.6-fold CsA concentration in relation to hCyplδ in the reaction ⁽ Figure 2b, squares). Kinetic analysis of the increase in hydrolysis products (Figure 3) with (Figure 3a) and without (Figure 3b) inhibition of cis / trans isomerase activity demonstrates the effect of this inhibition on the substrate specificity of protease activity, as evidenced by the transient appearance of differential hydrolysis products.

Incubation batch (with and without CsA): Total volume of the mixture: 400 μl

Substrate molecule ^• . Abz-Ile-Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp

Tyr (NO ₂ ) NH ₂ substrate molecule concentration in the reaction: 150 μM

cis / trans isomerase: hCyplδ hCyplδ concentration in the batch: 30 μM

hCyplδ inhibitor: Cyclosporin A (CsA) CsA concentration in the approach: 50 μM

Buffer: 35 mM Hepes pH 7.8

Incubation conditions: incubation temperature: 20 ^° C.

Chromatographic separation: 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

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.

The following solutions were produced:

-cis / trans isomerase solution: 10 μM solution of hCypl8 in 50 mM

Hepes buffer, pH 7.8.

Proenzyme (protease) solution: 100 μM 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).

For fluorescence measurement, 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 results of the fluorescence measurement are shown graphically in FIG. 1: Signal A) Submission of the cis / trans isomerase hCyplδ; Signal B) Incubation of isomerase with 10 μM AvrRpt2 (proenzyme); Signal C) start the reaction at time t = 0 by adding auxiliary molecule solution to the mixture of hCypl8 with AvrRpt2; the initial concentration of the auxiliary molecule was 10.5 μM, of hCyplδ 1 μM and of AvrRpt2 10 μM. Example 5 Detection of an Inactive PPIase by EctlC Reaction and Fluorogenic Substrate

While hCyplδ shows PPIase activity in standard PPIase activity assays, substitution of the amino acid arginine at sequence position 55 with the amino acid alanine (R55A) results in a PPIase inactive in the same assay. The present example demonstrates that the EctIC reaction can be used to detect inactive PPIases.

The following solutions were produced:

-cis / trans isomerase solution: 10 μM solution of hCyplδ (R55A) in 50 mM Hepes buffer, pH 7.8.

Proenzyme (protease) solution: 100 μM AvrRpt2 (mature AvrRpt2).

For fluorescence measurement, a fluorescence meter FluoroMax-2 (Horiba Jobin Yvσn 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.

The results of the fluorescence measurement are shown graphically in FIG. 2: signal A) start of the reaction at time t = 0 by addition of auxiliary molecule solution to a mixture of hCyplδ and AvrRpt2; the initial concentration of the auxiliary molecule was 10.5 μM, of hCyplδ 8, 3 μM and of AvrRpt2 10 μM. Signal B) start the reaction at time t = 0 by adding auxiliary molecule solution to a mixture of the hCyplδ mutant R55A with AvrRpt2; the initial concentration of Helper molecule was 10.5 μM, the hCypl8 mutant R55A 8.6 μM and the AvrRpt2 10 μM.

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. In the present example, the inhibition of PPIase activity of hCypl8 by the inhibitor cyclosporin A (CsA) is demonstrated by means of an EctIC reaction.

The following solutions were produced:

Proenzyme (protease) solution: 100 μM AvrRpt2 (mature AvrRpt2).

The results of the fluorescence measurement are shown graphically in FIG. 3: signal A) start of the reaction at time t = 0 by addition of auxiliary molecule solution to a mixture of hCypiS and AvrRpt2; the initial concentration of the helper molecule was 10.5 μM, hCyplδ was 8.3 μM and AvrRpt2 was 10 μM. Inset: At time t = 100 s will be added to the Reaction mixture 19.3 μM CsA mixed. During the mixing time of about 5 s, the fluorescence increase stops.

Example 7: Detection of an EctIC reaction by means of analytical HPLC

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 following solutions were produced:

hCyplδ: 216 μM in Hepes, AvrRpt2: 46 μM; CsA: 0.5 M in 50% EtOH / H ₂ O solution, auxiliary molecule: Abz-Ile-Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp-Tyr (NO ₂ ) NH ₂ 1, 71 mM in Hepes; Buffer: 35 mM Hepes pH 7.8; the auxiliary molecule solution contained 5% DMSO (photometric determination: ε _{3 Blm} = 2200 M ^-1 cm ^-1 ). The cell volume was 2.2 ml. The EctIC reaction was investigated without and in the presence of the inhibitor hCyplδ- CsA at a temperature of 10 ⁰ C.

Approach (A) Approach (B)

18 μl of HiIfMolekül solution 18 .mu.l Hilfsmolekül solution

18.3 μl hCyplδ 18.3 μl hCyplδ

9.6 μl AvrRpt2 9.6 μl AvrRpt2

2150 μl buffer 4 μl CsA

2146 μl buffer

The reaction was started by addition of hCyplδ. After 2, 4, 6, 10, 20, 30, 40, 50, 20, 60, 70, 80, 100, 120, 140, 160 and 180 min, 100 μl of sample were removed and mixed with 4 μl (0.5 M). CsA stopped and frozen in N ₂ i _q or measured immediately. After rapid thawing of the (inhibited) samples, 80 μl 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

Chromatographic curves) of the cleavage product H-Gly-Trp-Tyr (NO ₂ ) NH ₂ at different reaction times of the cleavage of 14 μM Abz-Ile-Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp-Tyr (NO ₂ ) NH ₂ by a mixture of 1.8 μM hCyplδ and 0, 2 μM AvrRpt2 at 10 ⁰ C without and in the presence of 0.9 μM CsA (filled circles: no inhibitor, triangles: with inhibitor). The inside shows the chromatograms obtained after 0, 10, 30, 60 and 180 minutes (with and without inhibitor).

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 μM. 0.2 μM AvrRpt2 were used as the proenzyme and 1.8 μM hCyplδ as the cis / trans isomerase.

Example 8: Detection of EctIC reaction by fluorogenic substrate

a) Synthesis of the substrate

The fluorogenic substrate Atto425-Ile-Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp-Gly-AEA-TAMRA containing the two dyes ATTO 425

(Fluorescent marker with coumarin structure (ATTO-TEC GmbH, Germany) and TAMRA (a rhodamine derivative) was synthesized on a DAE-chlorotrityl matrix by automated solid-phase synthesis

(Syro II, MultiSynTech, Witten, Germany) after a Fmoc Standard protocol synthesized. The fractions ATTO-425 and TAMRA were coupled as N-succinimide ester. The purification and isolation of the final product was carried out by preparative RP-HPLC, the analysis by means of MALDI mass spectroscopy. It was determined a molecular weight of 1884.16.

b) Evidence of the EctIC reaction:

The molecules ATTO 425 and TAMRA in the substrate Atto425-Ile-Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp-Gly-AEA-TAMRA form a fluorescence / quencher pair. After cleavage of the covalent bond between the two dyes, the fluorescence spectrum of the incubation mixture changes (FIG. 9). As a result, the cleavage of the substrate is accessible to a kinetic analysis. The time course of the hydrolysis of the substrate Atto425-Ile-Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp-Gly-AEA-TAMRA in the presence of 7.5 uM hCyplδ and 0.9 uM AvrRpt2 at 20 ⁰ C. in 35 mM HEPES buffer at pH 7, 8 a substrate concentration of 100 uM and a total concentration of 1% DMSO Figure 10 shows.

Claims

claims

1. A method for finding effectors, which the

Inhibit or activate protease activity of cis / trans isomerases, and for quantifying the inhibitory or activating effect of corresponding effectors on the protease activity of cis / trans isomerases,

comprising the steps of

a) providing a cis / trans isomerase;

b) contacting the cis / trans isomerase with a substrate molecule which is proteolytically cleaved by the cis / trans isomerase or with a proenzyme activated by the cis / trans isomerase;

c) contacting the cis / trans isomerase with an effector candidate substance;

2. The method according to claim 1, characterized in that the determination according to step d) and optionally according to step e) by means of the product of the proteolytic cleavage of the substrate molecule or by means of a product which has been reacted by the activated proenzyme.

3. The method according to claim 1 or 2, characterized in that the determination according to step d) and optionally according to step e) is carried out by means of a spectroscopic method.

4. The method according to claim 1 or 2, characterized in that the determination according to step d) and optionally according to step e) takes place by means of an electrochemical method.

5. The method according to claim 1 or 2, characterized in that the determination according to step d) and optionally according to step e) is carried out by means of a calorimetric method.

6. The method according to claim 1 or 2, characterized in that the determination according to step d) and optionally according to step e) takes place by means of a fluorimetric method.

7. The method according to claim 1 or 2, characterized in that the determination according to step d) and optionally according to step e) by means of a luminescence method.

8. The method according to any one of claims 1 or 2, characterized in that the determination according to step d) and optionally according to step e) is carried out by means of an endpoint method.

9. The method according to any one of claims 1 or 2, characterized in that the determination according to step d) and optionally according to step e) is carried out by means of a kinetic method.

10. The method according to any one of claims 1 to 9, characterized in that the cis / trans isomerase is an enzyme selected from the group consisting of the APIases and the PPIases.

11. The method according to any one of claims 1 to 9, characterized in that the cis / trans isomerase is an enzyme selected from the group consisting of members of the family Parvuline.

12. The method according to any one of claims 1 to 9, characterized in that the cis / trans isomerase is an enzyme selected from the group consisting of the members of the family of FKBPs.

13. The method according to any one of claims 1 to 9, characterized in that the cis / trans isomerase is an enzyme selected from the group consisting of the members of the family of cyclophilins.

14. The method according to any one of the preceding claims, characterized in that the method further comprises the addition of an auxiliary molecule, which is brought into contact with the cis / trans isomerase or is brought into contact with the activated proenzyme.

15. The method according to claim 14, characterized in that the auxiliary molecule is a molecule which increases the protease activity of the cis / trans isomerase.

16. The method according to claim 15, characterized in that the auxiliary molecule is a protein, a peptide or an organic molecule, wherein the organic molecule has a mass of less than or equal to 2,000 Da.

17. The method according to claim 14, characterized in that the auxiliary molecule is an enzyme substrate for the activated proenzyme.

18. The method according to any one of the preceding claims, characterized in that the method is carried out on a titer plate or in a cuvette.

19. The method according to any one of the preceding claims, characterized in that the method is carried out on a slide by means of a cell smear or a tissue section.

20. The method according to any one of the preceding claims, characterized in that the cis / trans isomerase and optionally the auxiliary molecule is immobilized on a support surface / are.

21. The method according to claim 20, characterized in that the cis / trans isomerase and optionally the auxiliary molecule is immobilized by adsorption on the support surface / are.

22. The method according to claim 20, characterized in that the cis / trans isomerase and optionally the auxiliary molecule is immobilized by means of an antibody on the support surface / are.

23. The method according to claim 20, characterized in that the cis / trans isomerase and optionally the auxiliary molecule is immobilized by means of covalent bonding to the support surface / are.

24. The method according to claim 20, characterized in that the cis / trans isomerase and optionally the auxiliary molecule is immobilized by means of a bond to biotin, avidin or streptavidin on the support surface / are.

25. The method according to any one of claims 20 to 24, characterized in that the carrier surface is part of a detection strip.

26. The method according to any one of claims 1 to 25, characterized in that the method is carried out in homogeneous solution.

27. The method according to any one of the preceding claims, characterized in that the effector is an inhibitor or an activator.

28. A kit for carrying out a method according to any one of the preceding claims, comprising a cis / trans isomerase and a substrate molecule which can be proteolytically cleaved by the cis / trans isomerase or a proenzyme which is activated by the cis / trans isimerase.

29. Kit according to claim 28, characterized in that the kit further comprises an auxiliary molecule as defined in claims 14 to 17.

30. Device for finding effectors which inhibit or activate the protease activity of cis / trans isomerases, characterized in that the device comprises a carrier material on whose surface a cis / trans isomerase and a substrate molecule or a proenzyme are immobilized, wherein the substrate molecule is cleaved by the cis / trans isomerase proteolytically or the proenzyme is activated by the cis / trans isomerase.

31. The device according to claim 30, characterized in that on the carrier material surface further a Hilfsmolekül as defined in claims 14 to 17 is immobilized.

32. Apparatus according to claim 30 or 31, characterized in that the device is designed as a detection strip.