CA2708021A1 - Method for finding effectors of the protease activity of cis/trans isomerases - Google Patents

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 corresponding 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 which can catalyze the cis/trans isomerization of peptide bonds are called cis/trans isomerases. The cis/trans isomerases (EC 5.2.1.8) include the peptidyl prolyl cis/trans isomerases (PPlases) and the secondary amide peptide bond cis/trans isomerases (APlases). The assignment of enzymes to the class of the cis/trans isomerases takes place mostly via amino acid sequence identity or homology comparisons of the enzymes to be assigned with the amino acid sequences of known cis/trans isomerases, via the determination of the catalysis properties of the enzymes to be assigned as well as the effectuation of these catalysis properties with different 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 effect the specific properties of the cis/trans isomerases.

Independently of the nomenclature of the cis/trans isomerase families described below, names are occasionally found in the literature for certain cis/trans isomerases which are attributable to particular properties of these enzymes. Thus e.g. some cis/trans isomerases are grouped separately under the name "immunophilins", as these are clearly target molecules for immunosuppressive medicaments in human medicine (e.g.: Powell JD. Zheng Y.: Current Opinion in Investigational Drugs.
7(11):1002-1007, 2006; Bell A. et al.: International Journal for Parasitology. 36(3):261-276, 2006; He ZY. et al.: Plant Physiology. 134(4):1248-1267, 2004; Dugave C. : Current Organic

- 2 -Chemistry. 6(15):1397-1431, 2002). Other cis/trans isomerases on the other hand, such as e.g. FKBP38, are grouped under the name CaMAPs, as they can be activated by calmodulin (e.g.: Edlich F.
et al.: Journal of Biological Chemistry. 281(21):14961-14970, 2006; Edlich F. et al.: EMBO Journal. 24(14):2688-2699, 2005).
But, regardless of these separate names, all cis/trans isomerases can be assigned either to the APlases or to the PPlases and in this respect in turn to the cyclophilins, the FKBPs or the parvulins.

APlases differ from PPlases in that their cis/trans isomerase activity is directed towards the cis/trans isomerization of secondary amide peptide bonds (Schiene-Fischer C. et al.: Nature Structural Biology. 9(6):419-424, 2002; Schiene-Fischer C. et al.: Biological Chemistry. 383(12):1865-1873, 2002). APlases and the inhibition of their APIase activity are very important in human and veterinary medicine (e.g.: US 2006100130).

The PPlases include the families of the cyclophilins (Galat A.:
Eur. J. Biochem. 216(1993)689-707; Hacker J. & Fischer G.: Mol.
Microbiol 10(1993)445-456), the FKBPs and the parvulins.

A particular property of the cyclophilins is that their PPIase activity can be inhibited by cyclosporin A (e.g.: DD 281659).
Within the framework of the present invention, by "cyclophilins"
is meant enzymes which display a PPIase activity and which for example can be ascertained by means of customary methods of sequence comparison with known cyclophilins. Such methods of sequence comparison are described at length in the literature (e.g.: A. Galat: Arch. Bioch. & Biophys. 371(1999)149-162; I. T.
Chou & C.S. Gasser: Plant Mol. Biol. 35(1997)873-892; D. Roy et al.: Biochemical & Biophysical Research Communications 307(2003)422-429; J.W. Montague et al.: Journal of Biological Chemistry 272(1997)6677-6684). The term "cyclophilins" is also to include enzymes which, in addition to homologies to known

- 3 -cyclophilins, also display homologies to other PPlases, such as e.g. the recently found enzymes called FCBs (B. Adams et al.:
Journal of Biological Chemistry 280(2005)24308-24314).

A particular property of cis/trans isomerases of the FKBP family is that their cis/trans isomerase activity can be inhibited by means of FK506. Just like the cyclophilins, the FKBPs and the inhibition of their cis/trans isomerase activity are also very important in human and veterinary medicine as well as with regard to biochemical and bioengineering questions, as stated for example in numerous publications (e.g.: KR 2002024377, 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 cannot be significantly inhibited either with cyclosporin A or with FK506 at an inhibitor concentration of < 1 pM with 10-fold molar excess of inhibitor. A specific irreversible inhibition by the natural substance juglone (5-hydroxy-1,4-naphthoquinone) was able to be demonstrated for different representatives of the parvulin family, namely parvulin from Escherichia coli, ESS1/PTF1 from Saccharomyces cerevisiae and human Pint (L. Hennig et al.: Biochemistry 37(1998)5953-5960). Juglone is a natural substance isolated from the walnut with both bacteriostatic and fungicidal as well as with cytotoxic properties vis-a-vis eukaryotic cells (e.g.: T.J.
Monks et al.: Toxicol. Appl. Pharmacol. 112(1992)2-16; N. Didry et al.: Pharmazie 49(1994)681-683).

Within the parvulin family, a distinction is drawn between two groups of enzymes which differ from each other in respect of their substrate specificity. The first group comprises all the eukaryotic enzymes with a specificity for substrates with (P03H2) Ser-/ (PO3H2) Thr residues before the amino acid residue proline. These include among others human Pint (hPinl) and

4 -ESS1/PTF1 from yeast (e.g.: K.P. Lu et al.: Nature 380(1996)544-547; S.D. 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 as well as some of the eukaryotic isomerases are not specific for phosphorylated substrates. They are included in the second group. Parvulins as well as the inhibition of their PPIase activity are also the subject of discussions in the literature concerning their suitability as a biochemical or bioengineering tool (e.g.:
US6030826, US3798129). The literature contains extensive knowledge regarding the use of parvulin inhibitors as active ingredients in human and veterinary medicine (e.g.: WO 03093258, wo 03074001, US 2003096387, JP 2001316289).

The reversible phosphorylation of Ser/Thr residues plays a central role in the regulation of fundamental cellular processes. The regulation of the eukaryotic cell cycle is governed e.g. by the principle of a temporally very precise succession of activations of different 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 in proteins and thus regulates their biological activity, for example in respect of their stability, enzymatic activity or also their binding affinity vis-a-vis other proteins (E.A. Nigg Bioessays 17(1995)471-480).

The finding and the use of cis/trans isomerase effectors as inhibitors or activators is, apart from the medical interest, also of great importance in the scientific literature (e.g.:
J.F. Guichou et al.: J. Med. Chem. 49(2006)900-910; Y.Q. Yu et al.: J. Med. Chem. 46(2003)1112-1115) and the subject of many patent publications (e.g.: US 2004204340, US 2003013645, US
2003068321, US 6270957, WO 2006033409, WO 2006005580, WO
2005097164, WO 2005021028).

- 5 -The inhibition of the phosphatase activity of the protein phosphatase calcineurin by a complex of cyclosporin A and cyclophilin 18 is assumed for the immunosuppressive effect of cyclosporin A (e.g.: Rusnak F. & Mertz P.: Physiological Reviews 80(2000)1483-1521). This mechanism also led in the scientific literature to the term "immunophilins", which describes PPlases which display immunosuppressive effects together with a PPIase inhibitor (e.g.: Dugave C.: Current Organic Chemistry

6(2002)1397-1431; Jorgensen K.A. et al.: Scandinavian Journal of Immunology 57(2003)93-98). The discovery of the suppressive effect of the cyclophilin/cyclosporin A complex on the human immune system led to a series of pharmaceutically important applications in human medicine (e.g.: Pollard S. et al.:
Clinical Therapeutics 25(2003)1654-1669).

In addition to the immunosuppression used in transplantation medicine by inhibition of the PPIase activity of cyclophilin with cyclosporin A, further effects brought about by inhibition of cis/trans isomerases have also been observed, such as e.g.
increased hair growth (e.g.: 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), an influence on hair graying (e.g.: Rebora J. et al.: International Journal of Dermatology 38(1999)229-230), a widely applicable effect on mammalian parasites, such as e.g. on the Plasmodium falciparum that causes malaria (e.g.: R. Kumar et al.: Molecular &
Biochemical Parasitology 141(2005)29-37; A. Bell et al.:
Biochemical Pharmacology 48(1994)495-503) and on Trypanosoma (e.g.: J. Bua et al.: Bioorganic & Medicinal Chemistry Letters 14(2004)4633-4637), an effect on Leishmania major parasites (e.g.: Meissner U. et al.: Parasitology Research 89(2003)221-227), an effect on Echinococcus (e.g.: Colebrook A.L. et al.:
Parasitology 125(2002)485-493) or on nematodes (such as e.g.: Ma U. et al.: Journal of Biological Chemistry 277(2002)14925-14932), an effect on Chlamydia and pneumococci, an effect on viruses, such as e.g. on the Hepatitis C virus (HCV) or the Dengue virus that causes Dengue fever and retroviruses such as the HIV virus (e.g. Boss V. et al.: Molecular Pharmacology 54(1998)264-72), an effect on psoriasis (e.g.: Zachariae H. &
Olsen T.S.: Clinical Nephrology 43(1995)154-8) and an effect on the nephrotic syndrome (e.g.: Noyan A. et al.: Nephron 70(1995)410-15). Therapeutically significant effects on nerve cells (e.g.: Wong A., Cortopassi G.: Biochemical & Biophysical Research Communications 239(1997)139-45), on pulmonary tissue (e.g.: J.W. Eckstein & J. Fung: Expert Opinion on Investigational Drugs 12(2003)647-653) and on tumours (e.g.:
T.Z. Wang et al.: Analytical Chemistry 76(2004)4343-4348; K.
Kawano et al.: Cancer Research 60(2000)3550-3558) were also able to be observed. A large number of these effects were discovered through the application of the active ingredient cyclosporin A
in transplantation medicine and observed there as a side-effect (e.g.: David-Neto E. et al.: Journal American Society Nephrology 11 (2000) 343-9) .

Particularly serious side-effects during a prolonged therapy with cyclosporin A are kidney damage, such as e.g.
nephrotoxicitiy, influencing of glomerular filtration or irreversible interstitial fibrosation (e.g.: Kopp et al: Journal American Society Nephrology 1(1991)162-12), neurological changes, such as e.g. the occurrence of a tremor (e.g.: De Groen et al.: New England Journal of Medicine 317(1987)861-74), vascular hypertension (e.g.: Kahan K. et al.: New England Journal of Medicine 321(1989)1725-33), the formation of tumours (e.g.: Kauffman L. et al.: Transplantation 80(2005)883-889) and complications associated with this damage. Liver damage or enlargements of the periodontium have also become known (e.g.:
Tosti A. et al.: Drug Safety 10(1994)310-17; Borel J.F. et al.:
Advances in Pharmacology 35(1996)79-114).

- 7 -Closer examinations of the cyclosporin A effects at 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 on condition that the spatial structure of the cyclosporin A/cyclophilin/calcineurin complex is largely preserved (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 produce cyclophilin inhibitors which can inhibit the PPIase activity of Cypl8, wherein however the Cypl8/inhibitor complex no longer inhibits the protein phosphatase calcineurin (e.g.: Zhang Y.X. et al.: J.
of Biological Chemistry 280(2005)4842-4850). Such Cyp18 inhibitors that do not inhibit the protein phosphatase calcineurin in the complex with Cyp18 are often called "non-immunosuppressive" cyclophilin inhibitors (non-immunosuppressive cyclophilin inhibitor compounds) in the literature (e.g.: Carry J.C. et al.: Synlett 2(2004)316-20; Evers M. et al.: Bioorganic & Medicinal Chemistry Letters 13(2003)4415-4419).

However, the literature contains numerous examples of low-molecular active ingredients which contribute to immunosuppression even without an inhibition of the protein phosphatase calcineurin. Thus e.g. PPIase inhibitors such as Sirolimus or Everolimus show comparable effects in transplantation immunology to cyclosporin A, without these active ingredients inhibiting the protein phosphatase calcineurin in vitro (e.g. Lisk W. et al.: Transplantation Proceedings 38(2006)69-73). Moreover, these active ingredients do not have the observed cancer-causing effect like cyclosporin A (e.g.: Kauffman et al.: Transplantation 80(2005)883-889).
While the inhibition of calcineurin leads, in combination with an increase in TGF-B, to cancer progression, it was at least able to be shown in animal tests that PPIase inhibitors which are applied therapeutically in transplantation immunology and do not inhibit calcineurin show an inhibiting influence on tumour

8 -growth and tumorangionesis (e.g.: J. Andrassy et al.:
Transplantation 80(2005)171-174; S.H. Kim et al.: Am 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 essays on the benefits of cyclophilin inhibitors in human and veterinary medicine or as a biochemical or bioengineering tool, there are also a large number of patent publications which prove the great economic interest in cyclophilins and their effectors (e.g.: 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).
As cis/trans isomerases as well as their effectuation are connected with a large number of diseases as well as cosmetic problems, there is a need for methods with the help of which the presence of cis/trans isomerases can be detected or with the help of which effectors of these enzymes which have the potential to be used as pharmaceuticals can be found.

Known assays which detect the acceleration of the cis/trans isomerization of peptide bonds by cis/trans isomerases can be divided into direct and indirect methods. Direct assays (a-c) make use of physical measurable parameters the size of which is directly coupled to the isomerization of cis/trans bonds.
Indirect tests (d, e) make use of either the condition, changed by the isomerization, of a substance used for detection or use isomer-specific reaction principles.

a) Isomer-specific mobility: if the electrophoretic mobility of suitable cis/trans isomerase substrates for cis and trans isomers is different, this difference can be used to detect cis/trans isomerase activities. Thus it is e.g. possible to

9 -analyse the rate of the establishment of equilibrium catalyzed by cis/trans isomerases of previously separated cis/trans isomers using different mobilities by means of capillary electrophoretic investigations, as described e.g. by M. Brandsch et al. (J. Biol. Chem. 1998, 273, 3861-3864). A disadvantage of this method is the small throughput of analyses per unit of time.

b) Isomer-specific spectroscopic differences: some cis and trans isomerase substrates display spectroscopic properties that differ from one another depending on configuration. A cis/trans isomerase effects a more rapid establishment of the natural equilibrium of a deflected equilibrium of cis and trans isomers.
The establishment of the natural equilibrium can be spectroscopically tracked in such a case, as described e.g. by B. Janowski et al. (Analytical Biochemistry 1997, 252, 299-307).
Disadvantages of this method are the small spectral changes which necessitate highly sensitive and therefore expensive measuring devices for the determination of isomerase activity.
Another disadvantage is that it is very difficult to stop the establishment of the cis/trans equilibrium, with the result that end-point measurements are scarcely possible.

c) Isomer-specific chemical shift when using nuclear magnetic resonance (NMR) spectroscopy: certain cis/trans isomerase substrates display nuclear-resonance spectroscopic differences in the chemical shift of both configuration isomers. The effect of isomerases on the cis/trans isomerization rate of these substrates can be quantified by the methods used in NMR
spectroscopy such as magnetization transfer or line form analysis, as described e.g. by D. Kern et al. (Biochemistry 1995 34, 13594-13602). A disadvantage of this method is that the measurement requires high substrate concentrations.

- 10 -d) Isomer-specific proteolysis: cis/trans isomer-specific substrate hydrolysis by means of cis/trans isomer-specific proteases can be used to ascertain from the peculiar property of the proteolysis kinetics whether cis/trans isomerases are present or if there is effectuation of cis/trans isomerases present, as described e.g. by G.Fischer et al. (Biomed.Biochim.
Acta 43, 1101-1111(1984)). A disadvantage in this case is that highly concentrated proteases must be used and the half-life of the measurement signal is relatively short. Moreover, this test is unsuitable for cis/trans isomerase substrates which are resistant to proteases or which are not degraded in a configuration-specific way.

e) Renaturing of proteins: as cis/trans isomerases can accelerate the renaturing of denatured proteins in numerous described 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), the acceleration of this renaturing by cis/trans isomerases can be utilized to detect the activity of isomerases, as described e.g.
by D. Kern et al. (Biochemistry 1995 34, 13594-13602). A
disadvantage here is the relatively costly spectroscopic methods or the sensitivity of such assays to substances which influence the renaturing of the observed protein in a way that does not influence the isomerase activity. A further disadvantage is the high cis/trans isomerase concentrations needed for the method, which can prevent a quantitative determination of highly affine inhibitors.

A further disadvantage of the listed methods is moreover the relatively large quantity of solvent to be used which is necessary to shift the natural equilibrium between cis and trans isomer prior to the measurement. Adjuvants used to deflect the equilibrium, such as e.g. trifluoroethanol, are capable of

- 11 -effectuating cis/trans isomerases or necessary proenzymes in a way that is disadvantageous for the assay. However, the greatest disadvantage of all the above-named methods is that they operate with deflected equilibria of cis to trans isomers. The activation energies needed to establish the equilibrium are relatively small, with the result that the establishment of equilibrium can also proceed without the addition of cis/trans isomerases. In order to be able to measure the activity of cis/trans isomerases, operation is therefore often at temperatures below room temperature. The recognition of cis/trans isomerase activity is often possible only if the temporary differences in the reaction kinetics with and without active cis/trans isomerase have been ascertained. A
determination of the cis/trans isomerase activity by means of the end-point method, that is after the chemical reaction has run its course, has hitherto been possible only if the kinetic differences of the reaction with and without cis/trans isomerase activity have been frozen by suitable means, such as e.g. by reducing the temperature or - as stated above - by removing the substrate or product molecules converted by the cis/trans catalysis.

Unlike the methods described above for detecting cis/trans isomerase activities which observe quantitative changes brought about by chemical reactions, it has also long been known that cis/trans isomerase activity can bring about qualitative changes in complex biological systems. Thus the removal of cis/trans isomerases that is possible by means of genetic engineering methods leads to changed phenotypes of different living organisms (e.g.: 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;

- 12 -Fanghanel J. et al.: FEBS Letters. 580(13):3237-3245, 2006;
Ardon 0. et al.: Journal of Virology. 80(8):3694-3700, 2006). In a few cases it was able to be shown that qualitative effects can also be demonstrated by inhibition of the cis/trans isomerase activity of cis/trans isomerases which can be assigned to spatially delimitable cellular regions, such as e.g. ion channels, (e.g.: Nakagawa T. et al.: Nature. 434(7033):652-658, 2005; Hansson MJ. et al: Journal of Bioenergetics &
Biomembranes. 36(4):407-413, 2004; 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 as to whether it is possible to observe quantitative or qualitative changes through the addition of a catalyst is normally connected with the activation energy needed for the reaction in question. While, in the case of the above-listed, previously known detection methods for cis/trans isomerase activity, the activation energy necessary for the reaction to proceed is so small that the chemical/biochemical reaction already proceeds under normal conditions even without the addition of a cis/trans isomerase, in the mentioned complex biological systems, because of cooperation of individual molecules or their functional groups that is still not understood in detail at molecular level, reactions occur which can proceed only in the presence of active cis/trans isomerases.
In addition to the cis/trans isomerase activity which - as described above - is directed towards the cis/trans isomerization of peptide bonds, cis/trans isomerases, as was surprisingly found, also have protease activity vis-a-vis certain substrates.

Since, as was already stated above, cis/trans isomerases are connected with a large number of diseases and cosmetic problems, there is now also a need for methods with the help of which

- 13 -effectors that influence the protease activity of cis/trans isomerases can be found in a way that is simple in terms of process engineering and favourable in terms of cost, and a need for methods by means of which the protease activity inhibiting/activating effect of corresponding effectors can be quantified.

The object of the present invention is therefore to provide a method by means of which effectors which influence the protease activity of cis/trans isomerases can be found in a way that is simple in terms of process engineering and favourable in terms of cost, and by means of which the protease activity inhibiting/activating effect of corresponding effectors can be quantified.

This object is achieved by a method comprising the steps of:
a) providing a cis/trans isomerase;

b) bringing the cis/trans isomerase into contact 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) bringing the cis/trans isomerase into contact with an effector candidate substance;

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

and optionally

- 14 -e) determining the extent of the inhibition or activation of the cis/trans isomerase effected by the effector candidate substance.

The method according to the invention has the advantage that, by means of same, effectors which inhibit or activate the protease activity of cis/trans isomerases can be found in a way that is simple in terms of process engineering and favourable in terms of cost.

Moreover, the method according to the invention has the advantage that, by means of same, the cis/trans isomerase inhibiting/activating effect of corresponding effectors can be reliably quantified in a way that is simple in terms of process engineering and thus favourable in terms of cost.

The method according to the invention has the further advantage that, by means of same, high-throughput screenings for cis/trans isomerase effectors can be carried out.

It was also able to be shown that the substances effectuating the protease activity of cis/trans isomerases (effectors) 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 using the proteolytic cleavage of a substrate molecule by the cis/trans isomerase or by means of the proenzyme that is activated by the cis/trans isomerase.

If the method is carried out using 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

- 15 -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 proteolytic cleavage of the substrate molecule effected by the cis/trans isomerase. 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 quantity of substrate molecule or fragments and is simple in terms of process engineering and can thus be carried out particularly favourably in terms of cost. If the determination according to step d) shows that the candidate substance is an effector, then - if desired - there can be a quantification of the inhibiting/activating effect of the effector on protease activity. The quantification of the inhibiting/activating effect can likewise take place by means of the substrate molecule and/or by means of at least one substrate molecule fragment or also by means of other parameters or methods. The same applies analogously for substrates (auxiliary molecule) which have been converted by the activated proenzyme.
The determinations according to method steps d) and e) can in this case be carried out by means of evaluation of different or identical parameters or applying different or identical methods.
According to a preferred embodiment it is provided that the substrate molecule is a molecule, the occurrence of the proteolytic cleavage of which can be spectroscopically detected.
In this connection it is particularly preferred that the substrate molecule or the substrate that is converted by the activated proenzyme - if this is a pro-protease - is a fluorescence-labelled oligo- or polypeptide which changes its fluorescence properties after proteolytic cleavage. The substrate molecule is preferably a fluorescence-labelled oligopeptide with up to 12 amino acid residues.

16 -The cis/trans isomerase with protease activity that is to be used is preferably substrate-specific as regards the substrate molecule used.

The hitherto found 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 is reduced only relatively little by the cis/trans isomerase, with the result that the proteolytic cleavage of peptides that is catalyzed by cis/trans isomerases proceeds relatively slowly. Accordingly it is provided according to a further preferred embodiment of the method according to the invention that the cis/trans isomerase is brought into contact with an auxiliary molecule which increases the protease activity of the cis/trans isomerase. The auxiliary molecule is preferably a protein or a peptide or an organic molecule, wherein the organic molecule has a mass less than/equal to 2,000 Da. A faster and thus more cost-favourable screening for effector substances of the protease activity of cis/trans isomerases is made possible by this method. There can be used as auxiliary molecule, for example, effectors found by means of the method according to the invention activating the protease activity of cis/trans isomerases, such as peptides, proteins or organic molecules displaying a mass less than/equal to 2,000 Da. By "peptide" is meant in this case a polypeptide with fewer than 12 amino acid residues. From 12 amino acid residues upwards, the term proteins is used within the framework 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;

- 17 -b) bringing the cis/trans isomerase into contact with a substrate molecule candidate substance;

c) determining whether the substrate molecule candidate substance is proteolytically cleaved.

By means of this method, substrate molecules which are proteolytically cleaved by cis/trans isomerases can be found in a way that is simple in terms of process engineering.

The method according to the invention further makes it possible to find effectors which inhibit or activate the protease activity of cis/trans isomerases, and for quantifying the inhibiting or activating effect of corresponding effectors on the protease activity of cis/trans isomerases by bringing the cis/trans isomerase into contact with a proenzyme which interacts with the cis/trans isomerase.

Every one of the reactions known in the state of the art which are catalyzed by cis/trans isomerases have such a small activation energy that they can proceed at room temperature even without isomerase addition. In these reactions, the cis/trans isomerase therefore serves only as a reaction accelerator.
Surprisingly it was now able to be shown that reactions also exist for cis/trans isomerases which can proceed at a significant reaction rate only through the addition of cis/trans isomerases and which can be stopped again or further activated by inhibition/activation of the cis/trans isomerase activity by means of suitable effectors.

Thus for example it was able to be shown that the proteolytic activity of AvrRpt2 vis-a-vis corresponding substrates can be significantly generated and maintained only through the presence of certain cis/trans isomerases with a suitable cis/trans isomerase activity, wherein this generation of the proteolytic

18 -activity can be significantly and quantifiably effectuated by inhibitors directed against the cis/trans isomerase activity. To distinguish them linguistically from the hitherto known chemical/biochemical reactions with the help of which cis/trans isomerases can be detected, these newly found reactions, which proceed at a significant reaction rate only through the addition of non-inhibited cis/trans isomerases, are called "essential cis/trans isomerase catalysis" (EctIC).

EctIC reactions are preferably distinguished for example from the "conventional", i.e. known reactions that can be catalyzed by cis/trans isomerases, by comparing the ratio of uncatalyzed to catalyzed reaction rate of the reaction measured with the proenzyme. By reaction rate is meant the reaction rate which can be observed under ascertainable best conditions. For this, in a first step the physical/chemical/biochemical parameters are optimized which lead to the maximum reaction rate at the proenzyme when a suitable quantity of cis/trans isomerase is added. In a second step the reaction rate is determined under the same conditions, only without added cis/trans isomerase. If, under the ascertained conditions, the ratio of catalyzed reaction rate to uncatalyzed reaction rate is greater than 10 and by adding cis/trans isomerase inhibitors a reaction rate as regards the proenzyme can be obtained which corresponds to that without the addition of cis/trans isomerase, it is an EctIC
reaction within the meaning of the present invention. Previously known kinetic detection methods possess only a very small time window of a few seconds in respect of cis/trans isomerases, because of the rapidly proceeding uncatalyzed reaction, e.g. for the observation of the catalysis of cis/trans isomerizations in peptidyl prolyl peptide bonds, with rate constants for the cis-trans or trans-cis reaction of >10-3 s-1 at room temperature in order to detect the inhibition of an enzymatically catalyzed cis/trans isomerization. Often the whole non-linear conversion curve of a typical cis/trans isomerase activity determination

- 19 -must be measured and reckoned up according to non-linear evaluation models in order to be able to quantify cis/trans isomerase activities. On the other hand, EctIC reactions can circumvent these limitations, as is documented by means of the examples. Both end-point determinations and linear kinetics -known as "initial rate measurement" to a person skilled in the art - are possible by means of EctIC reaction to determine cis/trans isomerase activities.

The molecular bases of EctIC reactions are still largely unknown and can for example include both the cis/trans catalysis of a peptide bond of the proenzyme and the specific binding to the proenzyme. Thus the result of cis/trans isomerization of one or more peptide bonds of the proenzyme can be such a flexibility of chemical functionalities of the protein that the proenzyme changes its properties in such a way that is suitable as proenzyme for detecting an EctIC reaction. However, the catalytic activity of the cis/trans isomerase at the proenzyme may also be the cis/trans isomerization of the enzyme substrate bound to the proenzyme or of the substrate interacting quite generally with the proenzyme, wherein by bound enzyme substrate is also meant within the meaning of the present invention all the intermediate stages of the enzyme substrate leading to the product through the catalysis of the proenzyme including residues that split off or the product itself.

If the binding of the cis/trans isomerase to the proenzyme itself and not the catalysis of the cis/trans isomerization at the proenzyme or bound peptide substrate is the cause of an EctIC reaction, the molecular bases of the influencing of the proenzyme may be found in changes in the three-dimensional structure of the complex between proenzyme and cis/trans isomerase. Such qualitative properties, changing due to protein interaction, of one of the bonding partners have for some time been described for numerous protein interactions and are listed

- 20 -in more detail below. But it has never previously been observed that due to interaction with a proenzyme a cis/trans isomerase qualitatively changes the latter's properties in such a way that an EctIC reaction can be observed and that this change in the proenzyme that is triggered by the cis/trans isomerase can be changed by the effectors effectuating the cis/trans isomerase activity such 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 the determination as to whether the proenzyme displays the property or the determination of the extent of the property of the proenzyme takes place by means of an auxiliary molecule which interacts with the proenzyme depending on the extent of the property. It was found that the named determinations can be carried out more easily in terms of process engineering by means of an auxiliary molecule compared with a direct determination at 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 it can be spectroscopically detected that an interaction with the proenzyme has taken place. It is particularly preferred in this connection if the auxiliary molecule is a fluorogenic molecule which changes its spectroscopic properties when interacting with the proeznyme.

As an enzymatic activity of the proenzyme is brought about by the interaction of the proenzyme with the cis/trans isomerase, it is guaranteed that the determinations according to steps d) and e) of the method according to the invention can be carried out in a way that is simple in terms of process engineering by means of an activity determination of the activated enzyme.

- 21 -If the method 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 of advantage 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 was found that by means of the proenzyme AvrRpt2 EctIC reactions can be carried out which react particularly sensitively to effectors.

In the method according to the invention it can be provided that the determination according to step d) or e) takes place using the enzyme substrate and/or using the substrate converted by the generated enzyme.

Within the framework 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, as proteolytic activity can be relatively easily established and quantified using the enzyme substrate or the fragments resulting therefrom.

It is particularly preferred that the property which is generated by the cis/trans isomerase in the proenzyme is 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 takes place by means of fluorescence measurement.

- 22 -Both inhibiting and activating effectors can be found with the method according to the invention. Accordingly it is preferred that the effector is an inhibitor or an activator.

As stated above, the proenzyme is converted into the active form by a cis/trans isomerase.

The found EctIC reactions can also be used to measure PPIase or APIase activities, e.g. in biological fluids such as blood serum, blood plasma, liquor, urine or tissue homogenates. For this, a method is particularly preferred comprising the steps of:

a) providing a proenzyme that can interact with a cis/trans isomerase and, through the interaction in the proenzyme, a property is triggered the extent of which depends on the activity of the cis/trans isomerase;

b) bringing the proenzyme into contact with a sample which is to be examined in order to ascertain whether it contains one or more cis/trans isomerases;

c) determining whether the proenzyme displays the property;
or d) determining the extent of the property of the proenzyme.
The enzymatic effect of the proenzyme is significantly influenced by the interaction with the cis/trans isomerase. By significant influencing is preferably meant the reduction of the molar catalytic constant, measured under the conditions suitable for this catalysis, by a factor of 10.

- 23 -It has been known for some time that cis/trans isomerases can form bonds with a second protein with formation of a heterodimer or heteromeric complexes with several different proteins and smaller ligands can also form (e.g.: Cell. 87(7):1157-1159, 1996; Journal of Biological Chemistry. 282(47):34148-34158, 2007; 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). There are numerous descriptions of comprehensive examinations of interactions and methods of examining interactions, such as 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 through the addition of other enzymes or the action of the same enzyme (autocatalysis e.g. in the case of trypsin) or certain chemical compounds (such as e.g. HC1 in the case of pepsinogen) is the proenzyme converted into the active form.

There are also numerous examples in the literature which prove that through the interaction of the most varied proteins with one another, including the binding to further ligands, properties of these complexes can be detected which differ from the properties of the individual constituents of such complexes and also do not represent the sum total of the properties of the individual constituents. However, despite the numerous previously discovered protein complexes which contain cis/trans isomerases, it was not possible hitherto to find a mention of an EctIC reaction. Published examples of interactions between cis/trans isomerases and other proteins are e.g.:

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

= the interactions between heat-shock proteins and the most varied cis/trans isomerases such as Cyp40, FKBP51 and FKBP52 (Carrello A. et al.: Cell Stress & Chaperones.
9(2):167-181, 2004).

= the binding of Cyp18 to the HIV-1 virion (BonHomme M. et al.: Biophysical Chemistry. 105(l):67-77, 2003).

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

= the bond between pl05Rb (riboblastoma gene product) and Cypl (Cui Y. et al.: Journal of Cellular Biochemistry.
86(4):630-641, 2002).

= the binding of glycosaminoglycans (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 Cyp18 to calreticulin (Reddy PA. Atreya CD.: International Journal of Biological Macromolecules. 25(4):345-351, 1999).

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

- 25 -= 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).
= the binding of FKBP51 to calcineurin (Li TK. et al.:
Journal of Cellular Biochemistry. 84(3):460-471, 2002).
= the binding of Hsp9O 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 Pint and Nek6 (Chen J. et al.:
Biochemical & Biophysical Research Communications.
341(4):1059-1065, 2006).

= the interaction between the protein kinase CK2 and Pint (Messenger MM. et al.: Journal of Biological Chemistry.
277(25):23054-23064, 2002).

In none of the examples published hitherto and summarized in extract form above has it as yet been possible to observe a reaction which corresponds to an EctIC reaction. It is often

- 26 -even reported that because of the binding of a protein to a cis/trans isomerase its active centre is apparently masked such that effectors highly affine for cis/trans isomerases can no longer bind to the active centre (e.g.: Journal of Cellular Biochemistry. 86(4):630-641, 2002; Li TK. et al.: Journal of Cellular Biochemistry. 84(3):460-471, 2002). If, on the other hand, an effectuation of the bond between a cis/trans isomerase and a further protein through a cis/trans isomerase inhibitor is observed, such as e.g. with 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 plant protein (Faure JD. Plant Journal. 15(6):783-789, 1998), no effectuation of the potential proenzyme (here translocase) is to be observed.

Within the framework of the present invention the interaction between proenzyme and cis/trans isomerase is necessary. To carry out an assay it may be of advantage to use proenzyme and cis/trans isomerase as separate molecules not coupled to each other. But it may also be of advantage, in order for example to simplify test systems, to join proenzyme and cis/trans isomerase together by means of chemical linkers or produce them, by means of genetic engineering methods known to the biochemist, as fusion protein in which proenzyme and cis/trans isomerase are joined together. It may be of advantage in this case to couple the N-terminus of the proenzyme and the C-terminus of the cis/trans isomerase together or else the C-terminus of the proenzyme and the N-terminus of the cis/trans isomerase. But it may also be of advantage to couple one or more molecules of the proenzyme and one or more cis/trans isomerase molecules together in very different orders. These molecules can be coupled to one another directly, but also via connection structures collectively known to a person skilled in the art as "linkers".
In addition to the coupling of proenzyme and cis/trans isomerase by means of chemical or genetic engineering techniques, it may

- 27 -also be of advantage to bind the proenzyme or the cis/trans isomerase or the abovementioned construct of proenzyme and cis/trans isomerase to a water-soluble or insoluble support.
Molecules with a molecular weight >1000, but also surfaces such as e.g. cuvettes or surfaces often used for screening of the cavities of titre plates, can serve as support. The chemical coupling to such surfaces can take place via covalent bonds, a whole series of coupling methods (via linkers such as e.g. by means of carbodiimide or disulphide bridge bond) are known here to a person skilled in the art. But the coupling can also be carried out by means of non-covalent methods, here too a broad range of varied methods are known to a person skilled in the art (e.g.: Myszka DG. Energetics of Biological Macromolecules, pt c.
323 pg. 325-. 2000).

Preferred cis/trans isomerases for carrying out the method according to the invention via the interaction with a proenzyme are cis/trans isomerases which together with a suitable proenzyme participate in an interaction such that an EctIC
reaction can be detected. By suitable cis/trans isomerases is also meant molecules the peptide sequence of which has been changed by the most varied methods known to a person skilled in the art in such a way that they correspond, using neither their molar mass nor their peptide sequence, to a natural gene-coded cis/trans isomerase but which, as a reliable indication of a cis/trans isomerase, display cis/trans activity under suitable optimized conditions. By peptide sequence is meant the amino acid sequence which results from the chemical or biochemical splicing of natural or non-natural amino acids or their derivatives and which displays a cis/trans isomerase activity in the correct three-dimensional conformation under optimal conditions. By peptide sequence is also meant the sequences in which one or more functional groups of amino acids have been changed by means of chemical or biochemical methods. Assays for detecting cis/trans isomerase activity are known to a person

- 28 -skilled in the art and have been listed in part above. Suitable optimal conditions may require the addition of the most varied substances, such as e.g. suitable buffer substances, salts, chelating agents, emulsifiers, activators, inactivators, sugars and further substances, but also suitable physical parameters, such as e.g. a suitable reaction temperature.

The enzymatic effect of the proenzyme can be significantly influenced by the interaction with the cis/trans isomerase and the influencing removed again by inhibition of the cis/trans isomerase activity of the cis/trans isomerase. The detection method for the EctIC reaction can take place here using the quantification of the enzymatic reaction of the proenzyme.
Detection methods for enzyme reactions are extensively known to a person skilled in the art and constantly freshly available in the specialist press.

If the proenzyme is e.g. 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 substrates which make it possible to identify by means of suitable detection methods the proteolytic activity of a protease activated by cis/trans isomerase activity. Detection methods for proteolytic activities have long been known and published extensively (e.g.: 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):3978-3982, 2006; 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;

- 29 -Sparidans RW. et al.: Biomedical Chromatography. 20(1):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;
Tribut 0. 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 0. 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 described in numerous patent specifications (e.g.: W02005097818, W003065004, US2006240503A1, U52006183177A1, US2003170770A1, US6821744B2, US6306619B1, US5891661A1, US3607859A1, EP0168738A2, CA2522795A1). Most of these methods are based on the recognition of spectroscopic differences between substrate and product molecules. If the proenzyme is a generated exopeptidase, a chromogenic substrate, the chromogen of which is released by the protease reaction, is often used to detect protease activity.

In addition to protease detection methods with specific substrates, optimized for the proteolytic activity to be examined, a whole series of methods are also described with the help of which the proteolytic activity of numerous different proteases can be detected. Thus e.g. the fluorescence polarization assay described by Kim J.H. et al. (Analytica Chimica Acta 577 (2006)171-177) uses as protease substrate a casein conjugated with tetramethylrhodamine.

- 30 -In general, a distinction can be drawn between kinetic and end-point methods in the methods described above. Both method types can be used to detect an EctIC reaction if the proenzyme is for example a (pro-)protease. The kinetic method logs parameters that change during the proteolytic reaction and correlate to the proteolytic reaction depending on the reaction time before the end of the reaction. When the values of the obtained measurement signals are plotted a relationship to the course of the protease reaction then results. An EctIC reaction for example of a protease activated by cis/trans isomerase activity can be recognized from the fact that after addition of an active ingredient inhibiting cis/trans isomerase activity the protease reaction is inhibited.

End-point methods are as a rule characterized in that at a certain point in time the enzymatic reaction is interrupted and the reaction products that have formed up until this point in time or the substrate of the enzymatic reaction not yet completely converted up until this point in time are/is analyzed/quantified. For example if the course of the reaction is known a conclusion can be drawn as to the reaction rate of the catalysis by means of calibration curves or by computation methods known to a person skilled in the art from the quantitative analysis of reaction product or starting substrate.
An EctIC reaction of a protease activated by cis/trans isomerase activity is to be recognized by the fact that after addition of an effector inhibiting cis/trans isomerase activity the course of the protease reaction is inhibited.

Just like kinetic protease assays a large number of end-point methods are known. Roughly speaking the methods can be distinguished according to how the reaction product to be analyzed or the quantity of starting product still remaining is quantified.

- 31 -All the EctIC reactions listed under the above points 1 to 8 may require the addition of the most varied auxiliary molecules in order to optimize the respective EctIC reaction. Thus buffer substances may be necessary in order to obtain a suitable concentration of protons. The addition of sugar molecules may be necessary in order to improve the physical consistency of the solution of prefabricated test constituents. It may further be necessary to add organic solvents, such as e.g. ethanol or DMSO, in order e.g. to improve the solubility of effectors effectuating cis/trans isomerase activity.

Further detection methods are cytochemical methods, for example methods which serve for the cytochemical presentation of proteins in tissue sections or cell smears (e.g.: 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 tissue or cells are fixed on a suitable object (such as e.g.
a glass or plastic microscope slide) by the most varied methods.
Here in the case of tissues the fixing is often carried out by means of wax which then makes it possible to separate fine tissue sections. Accordingly, tissue sections or cells can be used, after incubation with proenzyme and/or isomerase, to detect an EctIC reaction. Thus e.g. the incubation of cells with the proenzyme AvrRpt2 and a suitable fluorogenic substrate peptide, such as e.g. Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr(N02)amide, can be used for the cytochemical detection of cyclophilin.

- 32 -Within the framework of the present invention it is particularly preferred that the determination according to step d) and optionally according to step e) takes place 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 once pyrolytic cleavage has taken place, and the determination according to step d) and optionally according to step e) takes place by means of fluorescence measurement.

The determination according to step d) and optionally according to step e) of the method according to the invention preferably takes place by means of an end-point method or a kinetic method.
Cis/trans isomerases from the group consisting of APlases and PPlases can be used in the method according to the invention. It is particularly preferred in this case if the cis/trans isomerase is selected from the family of the FKBPs, the cyclophilins or the family of the parvulins.

By cis/trans isomerases are also meant however within the framework of the present invention molecules the peptide sequence of which has been changed by the most varied methods known to a person skilled in the art in such a way that they would not be assigned to a natural, gene-coded cis/trans isomerase using either their molar mass or their peptide sequence, but which, as a reliable indication of a cis/trans isomerase, display cis/trans activity under suitable optimized conditions. By peptide sequence is meant the amino acid sequence which results from the chemical or biochemical splicing of natural or non-natural amino acids or their derivatives and which displays a cis/trans isomerase activity in the correct three-dimensional conformation under suitable conditions. By

- 33 -peptide sequence is also meant in this case the sequences in which one or more functional groups of amino acids have been changed by means of chemical or biochemical methods. Assays for detecting cis/trans isomerase activity are known to a person skilled in the art and have been listed in part above. Suitable conditions may require the addition of the most varied substances, such as e.g. suitable buffer substances, salts, chelating agents, emulsifiers, activators, inactivators, sugars and further substances, but also suitable physical parameters, such as e.g. a suitable reaction temperature.

The method according to the invention can as a rule be carried out in any vessels. With regard to spectroscopic evaluations, it is however preferred that the method is carried out on a titre plate or in a cuvette.

With regard to carrying out high-throughput screening methods it can be provided according to a further preferred embodiment of the method according to the invention that the cis/trans isomerase and/or the substrate molecule and optionally the auxiliary molecule is/are immobilized on a support surface. In this case it is preferred that the molecule(s) is (are) immobilized at the support surface by adsorption, by means of an antibody, by a covalent bond or by binding to biotin, avidin or streptavidin.

In order for example to determine effectors on a laboratory scale, it can be provided according to a further preferred embodiment of the method according to the invention that the support surface is part of a detection strip. By detection strips are meant in this case test strips such as are used for example for pH determination.

As an alternative to the immobilization of one or more of the named molecules to carry out the method according to the

- 34 -invention it can also be provided that the method 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. In this case the method is preferably carried out in eukaryotes or prokaryotes, wherein the effectuation is carried out by means of endogenously present isomerases as well as substrate molecules and exogenously added effectors.

As an alternative to this 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 latter is carried out by means of tissue homogenates, cell suspensions, cell smears or tissue sections in which the protease activity of PPlases and APlases and their effectuation is carried out by means of isomerases endogenously present in the respective media as well as 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 embodiment 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 support material at the surface of which a cis/trans isomerase as well as a

- 35 -substrate molecule are immobilized, wherein the substrate molecule 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 embodiment of the method according to the invention.
The device according to the invention can also comprise an auxiliary molecule as described above immobilized at the surface of the support material.

According to a preferred embodiment it is provided that the device is formed as detection strips.

The present invention relates in particular 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, in particular hCypl8, and AvrRPT2;

b) bringing the mixture into contact with a substrate molecule which is proteolytically cleaved by the mixture;

c) bringing the mixture according to b) into contact 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 the inhibition or activation of the cyclophilin effected by the effector candidate substance.

- 36 -Examples and Figures The following examples and figures serve in conjunction with the drawing to explain the invention. There are shown in:

Figure 1: Change in the concentrations of the cleavage products H-Gly-Trp-Tyr(N02)NH2 (curve A) and Abz-Ile-Glu-Leu-Pro-Ala-Phe-Gly-OH (curve B) as well as the concentration of the auxiliary molecule Abz-Ile-Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp-Tyr(N02)NH2 (curve C) at different reaction times.

Figure 2: Change in the fluorescence intensity over time after A) incubation of an hCypl8/AvrRpt2 mixture with the adjuvant Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr(N02) amide, B) incubation of an hCypl8 (R55A)/AvrRpt2 mixture with the adjuvant Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr(N02) amide;

Figure 3: Change in the fluorescence intensity over time after A) incubation of an hCypl8/AvrRpt2 mixture with the adjuvant Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr(N02) amide, insertion: incubation of an hCypl8 /AvrRpt2/Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr(N02) amide mixture with the inhibitor cyclosporin A;
Figure 4: Change in the concentration of the cleavage product H-Gly-Trp-Tyr(N02)NH2 at different reaction times due to proteolysis of Abz-Ile-Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp-Tyr(NO2)NH2 by a mixture of hCypl8 and AvrRpt2 in the absence and in the presence of the inhibitor cyclosporin A (filled-in circles: without inhibitor;
triangles: with inhibitor). The chromatograms obtained

- 37 -after 0, 10, 30, 60 and 180 min (with and without inhibitor) are inset;

Figure 5: Change in the fluorescence intensity over time after A) introduction of the cis/trans isomerase hCypl8, 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, 17.5% SDS gel. Lane 1: molecular weight standards; lane 2: sample prior to incubation at 37 C;
lanes 3, 5 and 7: samples with added FKBP inhibitor;
lanes 4, 6 and 8: samples without FKBP inhibitor addition. The samples were incubated for 1 h (lanes 3 and 4), 3 h (lanes 5 and 6) and 8 h (lanes 7 and 8) respectively at 37 C.

Figure 7: HPLC separation of a hydrolysis batch without CsA
addition (a) after an incubation time of 130 h. Plot of the reduction in substrate during the incubation time with (b, squares) and without CsA addition (b, circles).

Figure 8: Plot of the product increase as area/time of the hydrolysis batch quantified by means of HPLC and identified by MALDI with (a) and without (b) CsA
addition. Corresponding to the HPLC/MALDI
identification, square, circle and triangle represent cleavage products 1 to 3 respectively.

Figure 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)).

- 38 -Figure 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 dots correspond to individual measured values, the solid line was obtained by calculating the kinetic curve after a first-order reaction.

Example 1: Detection of the protease activity of hCypl8 A protease substrate which contains a molecule part excitable by fluorescence, the donor, and a further molecule part, the quencher, was used as substrate molecule, wherein the quencher suppresses the excitability of the donor in the protease substrate. If a chemical bond between quencher and donor is broken by a protease, the donor can be excited, which can be ascertained qualitatively and quantitatively by means of apparatuses suitable for fluorescence measurement. Suitable donor-quencher pairs, such as e.g. aminobenzoic acid (Abz) as donor and nitrotyrosine as quencher, are known in the state of the art. Methods for finding suitable new pairs of donor and quencher are also described at length in the state of the art.
The following solutions were prepared:

-cis/trans isomerase solution: 10 pM solution of hCypl8 in 50 mM
HEPES buffer, pH 7.8.
-substrate molecule solution: 2 mg/ml Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr (N02) amide dissolved in dimethyl sulphoxide (DMSO).

A FluoroMax-2 fluorescence measuring apparatus (Horiba Jobin Yvon Inc, USA) was used for the fluorescence measurement, wherein to carry out the measurement excitation was at a wavelength of 320 nm and measurement was at a wavelength of 418 nm. The measurement was carried out at a temperature of 20 C.

- 39 -The cis/trans isomerase solution and the substrate molecule solution were mixed together, wherein the initial concentration of the substrate molecule was 10 pM and that of the hCypl8 was 2 pM. The protease activity of hCypl8 was able to be demonstrated using the registration of a fluorescence signal which progressively increased in intensity over the course of time.
Example 2: Finding natural cis/trans isomerase protease substrates In the following, it is shown, taking as example the inhibition of the cis/trans isomerase activity of cis/trans isomerase of the FKBP type, how substrate molecules can be found which are proteolytically cleaved by these isomerases. A basic requirement for finding corresponding substrate molecules according to this strategy is that, by inhibiting the cis/trans isomerase activity of the isomerases, their protease activity is also inhibited. A
protein mixture which forms during the homogenization of human blood cells and subsequent centrifugation is used for this.
Preparation of leucocytes 50 ml buffy coat of a healthy blood donor was centrifuged at 1500 g for 10 min. To remove erythrocytes, the centrifuged material was incubated at a temperature of 0 C with 50 ml lysis buffer (155 mM ammonium chloride, 10 mM sodium carbonate, 0.1 mM
EDTA) for 10 min. After repeated centrifugation at 1500 g and removal of the supernatant the lysis step was repeated once again. The remaining blood cells were then washed three times with 25 ml isotonic saline solution each time at a temperature of 4 C and then mixed with 10 ml 100 mM tris buffer (pH 7.4), aliquoted to 500 pl in each case and stored at a temperature of -20 C.

- 40 -An aliquot of 500 pl was thawed, the cells contained therein were decomposed by means of ultrasound and the resulting suspension was then centrifuged for 5 min at 10,000 g. 2 times 180 pl of the supernatant were pipetted in each case into an Eppendorf tube. The first container was filled with 20 pl of a 100 pM solution of dimethyl cycloheximide (DMCHX; in 100 mM tris buffer, pH 7.4) and the second container was filled with only 20 pl of corresponding tris buffer. The solutions of both containers were incubated at a temperature of 37 C.

After 1, 3 and 8 hours, in each case 20 pl of sample was taken and incubated with 180 pl of aforementioned tris buffer at a temperature of 95 C for 5 min. In each case 8 pl of the thus-obtained solutions was deposited on a 17.5% SDS gel together with a molecular weight standard and electrophoretically separated according to a standard procedure and made visible by means of silver staining (Rabilloud T. et al.: Electrophoresis 9(1988)288-91). Figure 6 shows the influence of the cis/trans isomerase inhibitor DMCHX on the protease activity of inhibited cis/trans isomerases. Thus during FKBP inhibition the protein band labelled A is more stable at approximately 38 kDa than without DMCHX inhibition. On the other hand, without FKBP
inhibition protein bands in the range < 20 kDa, such as e.g. the band labelled B, are to be found more markedly as possible decomposition products of the proteolysis by cis/trans isomerases.

Example 3: Protease activity of hCypl8 vis-a-vis an oligopeptide with and without inhibition of the cis/trans isomerase activity The protease activity of cis/trans isomerases vis-a-vis oligopeptides can be determined by analysis of aliquots of the composition of the incubation batch over the incubation time with a combination of chromatographic separation of the batch = - 41 -and subsequent mass spectrometry of the separated constituents of the batch. Chromatographic separation makes it possible to quantitatively ascertain changes in concentration of substrate molecule and occurring hydrolysis products. Mass spectrometry makes possible the assignment of the separated constituents of the measurement batch by means of their molecular mass.

When different oligopeptide sequences are used, information can thus be obtained 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 vis-a-vis the respective polypeptide sequences.

Figure 7a shows the chromatographic separation of an incubation batch of hCypl8 with the substrate molecule without CsA as given below after 130 h. By means of mass spectrometry, the signals appearing at a retention time of approximately 24.8 min and 23 min were able to be allocated to the hCypl8 cis/trans isomerase used and the substrate molecule respectively. The signals between 10 and 20 min retention time were able to be allocated to the hydrolysis products of the substrate molecule. As the area of the signals correlates directly with the respective peptide concentration, the decrease in the starting concentration of the substrate molecule as well as the increase in the hydrolysis products over time can be represented graphically (Fig. 2b; Fig. 3) and evaluated by analysis and evaluation of the test batch at different incubation times by means of samples taken from the test batch and their chromatography behaviour.

While the substrate molecule was completely hydrolyzed (Fig. 2b, circles) after approx. 290 hours under the chosen conditions, the hydrolysis rate can be slowed down by complete inhibition of the cis/trans isomerase activity with a 1.6-fold CsA
concentration vis-a-vis hCyp18 in the batch (Fig. 2b, squares).

The kinetic analysis of the increase in the hydrolysis products (Fig. 3) with (Fig. 3a) and without (Fig. 3b) inhibition of the cis/trans isomerase activity demonstrates the influence of this inhibition on the substrate specificity of the protease activity, visible in the transient occurrence of different hydrolysis products.

Incubation batch (with and without CsA):
Total volume of the batch: 400 .l Substrate molecule: Abz-Ile-Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp-Tyr (NO2) NH2 Substrate molecule concentration in the batch: 150 }iM
cis/trans isomerase: hCyp18 hCypl8 concentration in the batch: 30 pM
hCypl8 inhibitor: cyclosporin A (CsA) CsA concentration in the batch: 50 uM
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 A protease substrate which contains a molecule part excitable by fluorescence, the donor, and a further molecule part, the quencher, was used as auxiliary molecule, wherein the quencher suppresses the excitability of the donor in the protease substrate. If a chemical bond between quencher and donor is broken by a protease, the donor can be excited, which can be ascertained qualitatively and quantitatively using apparatuses suitable for measuring fluorescence. Suitable donor-quencher pairs, such as e.g. aminobenzoic acid (Abz) as donor and nitrotyrosine as quencher, are known in the state of the art.
Methods for finding suitable new pairs of donor and quencher are described at length in the state of the art.

The following solutions were prepared:

-cis/trans isomerase solution: 10 pM solution of hCypl8 in 50 mM
HEPES buffer, pH 7.8.
-proenzyme (protease) solution: 100 pM AvrRpt2 (mature AvrRpt2).
-auxiliary molecule (protease substrate) solution: 2 mg/ml Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr(N02) amide dissolved in dimethyl sulphoxide (DMSO).

A FluoroMax-2 fluorescence measuring apparatus (Horiba Jobin Yvon Inc, USA) was used for the fluorescence measurement;
excition was at a wavelength of 320 nm; measurement was at a wavelength of 418 nm and at a temperature of 20 C.

The results of the fluorescence measurement are represented graphically in Figure 1: signal A) introduction of the cis/trans isomerase hCypl8; signal B) incubation of the isomerase with 10 pM AvrRpt2 (proenzyme); signal C) start of 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 pM, that of the hCypl8 was 1 pM and that of the AvrRpt2 was 10 pM.

Example 5: Detection of an inactive PPIase by means of EctIC
reaction and fluorogenic substrate While hCyp18 shows a PPIase activity in customary PPIase activity assays, the substitution of the amino acid arginine in sequence position 55 by the amino acid alanine (R55A) leads to a PPIase inactive in the same assay. The present example shows that the EctIC reaction can be used to detect inactive PPlases.
The following solutions were prepared:

-cis/trans isomerase solution: 10 pM solution of hCypl8(R55A) in 50 mM HEPES buffer, pH 7.8.

-proenzyme (protease) solution: 100 pM AvrRpt2 (mature AvrRpt2).
-auxiliary molecule (protease substrate) solution: 2 mg/ml Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr(NO2) amide dissolved in dimethyl sulphoxide (DMSO).

A FluoroMax-2 fluorescence measuring apparatus (Horiba Jobin Yvon Inc, USA) was used for the fluorescence measurement;
excition was at a wavelength of 320 nm; measurement was at a wavelength of 418 nm and at a temperature of 20 C.

The results of the fluorescence measurement are represented graphically in Figure 2: signal A) start of the reaction at time t=0 by adding auxiliary molecule solution to a mixture of hCypl8 and AvrRpt2; the initial concentration of the auxiliary molecule was 10.5 pM, that of the hCypl8 was 8.3 pM and that of the AvrRpt2 was 10 pM. Signal B) start of the reaction at time t=0 by adding auxiliary molecule solution to a mixture of the hCypl8 mutant R55A with AvrRpt2; the initial concentration of the auxiliary molecule was 10.5 pM, that of the hCypl8 mutant R55A
was 8.6 pM and that of the AvrRpt2 was 10 pM.

Example 6: Detection of a PPIase inhibitor by means of EctIC
reaction and fluorogenic substrate The PPIase activity of hCyp18 can be specifically inhibited by numerous known inhibitors. In the present example, the inhibition of the PPIase activity of hCyp18 by the inhibitor cyclosporin A (CsA) is shown by means of an EctIC reaction.
The following solutions were prepared:

-cis/trans isomerase solution: 10 uM solution of hCypl8(R55A) in 50 mM HEPES buffer, pH 7.8.

-proenzyme (protease) solution: 100 }iM AvrRpt2 (mature AvrRpt2).
-auxiliary molecule (protease substrate) solution: 2 mg/ml Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr(NO2) amide dissolved in dimethyl sulphoxide (DMSO).

A FluoroMax-2 fluorescence measuring apparatus (Horiba Jobin Yvon Inc, USA) was used for the fluorescence measurement;
excition was at a wavelength of 320 nm; measurement was at a wavelength of 418 nm and at a temperature of 20 C.

The results of the fluorescence measurement are represented graphically in Figure 3: signal A) start of the reaction at time t=0 by adding auxiliary molecule solution to a mixture of hCyp18 and AvrRpt2; the initial concentration of the auxiliary molecule was 10.5 pM, that of the hCypl8 was 8.3 pM and that of the AvrRpt2 was 10 pM. Insertion: at time t=100 s, 19.3 pM CsA is added to the reaction mixture. The fluorescence increase stops within the mixing time of approx. 5 s.

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

The example is based on the possibility of separating and then quantifying starting product and cleavage products of the proteolysis by means of chromatography.

The following solutions were prepared:

hCypl8: 216 pM in HEPES; AvrRpt2: 46 pM; CsA: 0.5 M in 50%
EtOH/H20 solution; auxiliary molecule: Abz-Ile-Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp-Tyr(NO2)NH2 1.71 mM in HEPES; buffer: 35 mM HEPES
pH 7.8; the auxiliary molecule solution contained 5% DMSO
(photometric determination: E381nm 2200 M-1 cm 1) . The capacity of the cuvette was 2.2 ml. The EctIC reaction was examined without and in the presence of the hCyp18 inhibitor CsA at a temperature of 10 C.

Batch (A) Batch (B) 18 pl auxiliary molecule 18 pl auxiliary molecule solution solution 18.3 p1 hCypl8 18.3 pl hCypl8 9.6 pl AvrRpt2 9.6 pl AvrRpt2 2150 pl buffer 4 pl CsA
2146 pl buffer The reaction was started in each case by adding hCyp18. After 2, 4, 6, 10, 20, 30, 40, 50, 20, 60, 70, 80, 100, 120, 140, 160 and 180 min, 100 pl of sample was taken and stopped with 4 pl (0.5 M) CsA and frozen in N21q or measured immediately. After the rapid thaw of the (inhibited) samples, 80 p1 was applied to a chromatography column (VydacC8) and chromatographed with a water/HCN gradient of 15-60% (0.1% TFA) over 20 min. Figure 4 shows the detection of an EctIC reaction by product analysis by means of HPLC. The concentrations (given as signal area of the chromatography curves) of the cleavage product H-Gly-Trp-Tyr(N02)NH2 at different reaction times of the cleavage of 14 PM
Abz-Ile-Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp-Tyr(NO2)NH2 by a mixture of 1.8 }iM hCypl8 and 0.2 pM AvrRpt2 at 10 C without and in the presence of 0.9 pM CsA (filled-in circles: without inhibitor;
triangles: with inhibitor) are plotted. The chromatograms obtained after 0, 10, 30, 60 and 180 min (with and without inhibitor) are inset.

A detection of an EctIC reaction by means of HPLC product analysis is shown in Figure 5. The concentrations (given as signal area of the chromatography curves) of the cleavage products H-Gly-Trp-Tyr(N02)NH2 (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-Ala-Phe-Gly-Gly-Trp-Tyr(N02)NH2 (curve C) at different reaction times at 10 C are plotted. The concentration of the auxiliary molecule in the measurement batch was 14 rM. 0.2 pM AvrRpt2 was used as proenzyme, 1.8 pM hCypl8 as cis/trans isomerase.

Example 8: Detection of an EctIC reaction by means of 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 means of automated solid-phase synthesis (Syro II, MultiSynTech, Witten, Germany) according to an Fmoc standard protocol. The dyes ATTO-425 and TAMRA were coupled as N-succinimide ester. The cleaning and isolation of the end product took place by means of preparative RP-HPLC, the analysis by means of MALDI mass spectroscopy. A molar mass of 1884.16 was ascertained.

b) Detection 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 batch changes (Figure 9). The cleavage of the substrate is thereby available for a kinetic analysis. Figure 10 shows the temporal 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 hCypl8 and 0.9 uM AvrRpt2 at 20 C in 35 mM
HEPES buffer at pH 7.8, a substrate concentration of 100 uM and a total concentration of 1% DMSO.

Claims (32)

Claims

1. Method for finding effectors which inhibit or activate the protease activity of cis/trans isomerases, and for quantifying the inhibiting 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) bringing the cis/trans isomerase into contact with a substrate molecule which is proteolytically cleaved by the cis/trans isomerase or with a proenzyme which is activated by the cis/trans isomerase;

c) bringing the cis/trans isomerase into contact with an effector candidate substance;

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

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

3. 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 spectroscopic method.

4. 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. 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 calorimetric method.

6. 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. 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 luminescence method.

8. Method according to one of claims 1 or 2, characterized in that the determination according to step d) and optionally according to step e) takes place by means of an end-point method.

9. Method according to one of claims 1 or 2, characterized in that the determination according to step d) and optionally according to step e) takes place by means of a kinetic method.

10. Method according to 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 PPIases.

11. Method according to 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 the parvulins.

12. Method according to 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 the FKBPs.

13. Method according to 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 the cyclophilins.

14. Method according to one of the preceding claims, characterized in that the method furthermore 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. 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. 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 less than/equal to 2,000 Da.

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

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

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

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

21. Method according to claim 20, characterized in that the cis/trans isomerase and optionally the auxiliary molecule is/are immobilized at the support surface by means of adsorption.

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

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

24. Method according to claim 20, characterized in that the cis/trans isomerase and optionally the auxiliary molecule is/are immobilized at the support surface by means of binding to biotin, avidin or streptavidin.

25. Method according to one of claims 20 to 24, characterized in that the support surface is part of a detection strip.

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

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

28. Kit for carrying out a method according to 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 isomerase.

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 support material at the surface of which a cis/trans isomerase as well as a substrate molecule or a proenzyme are immobilized, wherein the substrate molecule is proteolytically cleaved by the cis/trans isomerase or the proenzyme is activated by the cis/trans isomerase.

31. Device according to claim 30, characterized in that at the surface of the support material an auxiliary molecule as defined in claims 14 to 17 is further immobilized.

32. Device according to claim 30 or 31, characterized in that the device is formed as detection strips.