DE10023743A1 - Peptide substrates for the direct measurement of enzymatic activities - Google Patents

Abstract

The present invention relates to peptide derivatives used to measure enzyme activities in homogenous reaction solutions by means of signals that are as direct and continuous as possible. Said invention also relates to kits, a method used to determine enzyme activities and to identify enzyme inhibitors or activators, and a method for selecting and using said peptide derivatives.

Description

The present invention relates to peptide derivatives which allow the enzymatic Track activity of various enzymes directly, as well as kits that contain the peptide derivatives contain. The invention further relates to methods for determining Enzyme activities, for the identification of inhibitors or activators of enzymes, Screening method and the use of peptide derivatives.

Given the increasing automation of biological and biochemical Test systems today can handle large amounts of samples for certain properties investigate. A sub-area of such mass search tests (high throughput screening) relates to systems for determining enzymatic activities. This serves on the one hand the determination of enzyme activities in samples whose activity is unknown and on the other hand to identify effector connections that a certain Inhibit or activate enzyme activity.

Such methods suitable for mass search tests preferably work with substrates in which the course of the reaction can be measured by an optical signal. Such optical signals, such as. B. color change or increase or decrease in fluorescence, are well suited to quickly and precisely in automated spectroscopic data acquisition systems to track the time course of the implementation of a substrate. The methods can be differentiated according to certain criteria with regard to the detection method. In addition to the type of signal (for example optical, fluorescence or color change), it is primarily signal directness and signal continuity that determine the quality of a mass search test procedure for determining the activity of enzymes. The greatest possible signal directness is desirable. This means that signals which indicate the reaction to be observed as directly as possible are advantageous in order to keep disturbances caused by possible influencing factors to a minimum. Often, however, signals that originate directly from the reaction to be observed are difficult to detect. In order nevertheless to achieve detection, auxiliary reactions are used which are coupled in a suitable manner to the reaction to be detected and generate detectable signals. With the associated lower signal directness, interference is accepted, which can significantly impair the measurement. For example, attempts have been made to improve the activity measurement of peptidyl prolyl cis / trans isomerase (PPlase) by coupling with isomer-specific proteolysis (Fischer, Biochim. Biophys. Acta 43 (1984), 1101; Fischer, Nature 337 (1989), 476) . A large number of suitable substrates are available to the person skilled in the art for the measurement of protease activity, which enable measurement, for example, by releasing a dye (Barrett, Meth. Enzymol. 80 (1981), 561; Carter, Science 237 (1987), 394; Tanaka , Biochemistry 24 (1985), 2040; Horsthemke, Biochemistry 19 (1980), 2867; Jacotot, Eur. J. Biochem. 239 (1996), 248; Heinemeyer, EMBO J. 10 (1991), 555; Thornberry, Nature 356 (1992), 768; Walker, Cell 78 (1994), 343; Xiang, Proc. Natl. Acad. Sci. USA 93 (1996), 14559). The sensitivity increased by the auxiliary reaction goes hand in hand with the disadvantages of the reduced signal directness. Using the PPlase mentioned above, this can have the following effects on the measurement of enzyme activity:

• - Effectors whose influence on the PPlase should be determined can have an inhibitory effect on the protease;
• The protease can adversely affect the activity of the PPlase; or
• - The protease attacks the effectors contained in the reaction.

The above example illustrates how the increased complexity of the Reactions multiply the potential interference. Attempts have been made to more direct measurements of PPlase activity. A process that one isomer-specific chemical shift when using magnetic Nuclear magnetic resonance spectroscopy (NMR) uses (Kern, Biochemistry 34 (1995), 13598; Reimer, Biochemical J. 326 (1997), 181), proved to be insufficiently sensitive. Better Results were provided by a method in which low isomer-specific Spectroscopic differences measured from suitable PPlase substrates (Janowski, Analytical Biochemistry 252 (1997), 299; Garcia-Echeverria, Biochem. Biophys. Res. Commun. 191 (1993); Garcia-Echeverria, J. Am. Chem. Soc. 114: 2758 (1992). However, this procedure could also be used for broad Do not enforce applications as it is only ideal under experimental Conditions works. For example, minor impairments  spectroscopic interference, such as when using biological samples are almost inevitable, the activity measurement strong.

The second criterion by which the quality of activity measurement of enzymes signal continuity is crucial. This affects both accuracy the measurement data as well as the practical applicability of a method in Mass search tests. While for example in the above Protease measurement with the help of a color reaction, the continuous recording of measured values is not a problem, one often has to switch to discontinuous enzymes Dodge procedure. Aliquots are continuously added to a reaction batch removed and a measurable signal in a downstream detection reaction generated. Such a procedure is currently used for Activity measurements of protein phosphatases and protein kinases necessary. In addition to the limited availability of measuring points per unit of time, which in results in a larger measurement error, discontinuous methods mean one comparatively high costs and materials compared to continuous Process and therefore a limited applicability for mass search tests.

In many procedures for measuring activity of enzymes also represents creation a sufficiently homogeneous mixture in the reaction mixture is a problem To be able to achieve reproducible data, enzyme, substrate and optionally, effector compound (s) must be homogeneously mixed. Mostly Pre-incubation solutions made that have all the necessary components except for the Substrate included. The reaction is started by adding the substrate. Here it often proves to be problematic to homogenize the substrate in the shortest possible time to mix with the pre-incubation approach. Naturally, this is only possible in Desirably if the substrate has a high solubility in the final volume owns. Substrates whose concentration in the final volume is close to that Solubility product, dissolve poorly within a short time. Out for this reason, such substrates are largely excluded from the Use in enzyme tests.

The present invention is therefore based on the object of substrates To provide the continuous measurement of the activity of the largest possible  Enabling a wide range of enzymes with the greatest possible signal directness, the Measurement takes place in homogeneous enzyme-substrate mixtures.

This object is achieved by the provision of the Claims characterized embodiments solved.

The present invention thus relates to peptide derivatives with the general formula:

A-B1-C1-C2-C3-B2-D;

• - wherein C1, C2 and C3 are compounds selected from the group consisting of from amino acids, N-alkyl amino acids, peptides, peptide derivatives, peptoids and compounds that can be incorporated into peptide chains;
• - wherein B1 and B2 are compounds that form a chemical bond with one another can train;
• - With the formation of the bond between B1 and B2 the peptide derivative in is in a tight state;
• - The bond between B1 and B2 can be easily loosened and the tensioned State then changes into an untensioned state; and
• - where A and D are groupings that measure the transition from clamped in the unstressed state or the direct measurement of a Allow enzyme reaction due to the change in an optical signal.

Peptide derivatives of the present invention are particularly suitable as substrates for measuring enzyme activities. The special mode of operation of these peptide derivatives is due to a change in conformation, which takes place independently of the enzyme reaction to be measured. In the context of the present invention, this change in conformation is referred to as a transition from a tensioned to a non-tensioned state. The peptide derivatives according to the invention are initially in a tense state which is stabilized by ring formation by means of a bond between the compounds B1 and B2. In the context of the present invention, the term “braced” means a conformation of the compound (s) C1, C2 and / or C3, which represents an energetically unfavorable situation and is unstable. In the "strained" state, the peptide derivative is preferably no or at least a poor substrate for an enzyme whose activity is to be measured. For the purposes of the present invention, the access of the enzyme to the substrate can also be blocked solely by the B1-B2 bond. When using the peptide derivative according to the invention, the enzyme reaction is started by releasing the bond between B1 and B2. The peptide derivative then automatically changes into a thermodynamically more favorable, untensioned state. In the context of the present invention, “not braced” means a lower-energy conformation than the braced state. Preferably, the peptide derivative in the "untensioned" state is readily accessible to the enzyme to be investigated and is therefore a good substrate for the reaction catalyzed by the enzyme. The non-tense state preferably has new binding properties for proteins compared to the tense state. The transition from the tensioned to the non-tensioned state can affect, for example, a change in conformation from a cis to a trans-isomeric state within the peptide derivative according to the invention. For such cis / trans isomers, the peptide bond between the amino group of a proline residue (or derivative thereof) and the carboxy group of the adjacent amino acid residue is suitable. Peptide derivatives which correspondingly contain a proline residue or a derivative thereof for one of the compounds C ₁ to C ₃ were used in Examples 1 to 3. According to the invention, further protein chemical effects can be used for the development of a tense state, which can change into a non-tense state, which are independent of the special properties of proline peptide bonds. In general, the tensioning of peptide substrates leads to properties that differ from those of unstrained ones. These differences can be so serious that enzymes such. B. Proteases, the strained substrate form can cleave proteolytically much worse than the unstrained form (Kazmaier, Organic Letters 1 (1999), 1763; Iwai, FEBS letters 459 (1999), 166-172; Bogdanowich-Knipp, J. Peptide Res. 53 (1999), 530-541; Gudmundsson, Pharm. Res. 16 (1999), 16-23).

The compounds C1, C2 and / or C3 determine the chemical properties the tensioned and the non-tensioned state of the invention Peptide derivative. They preferably also deliver the substrate for the investigating enzyme. The design of the compounds C1 to C3 determines mainly the transition speed from tense to not  tense condition. In the embodiment of the invention Peptide derivatives can be C1, C2 and C3 amino acids, among which amino acid all naturally occurring amino acids, modifications thereof and artificial synthesizable amino acids are understood. Examples of amino acids are the natural L and D amino acids or amino acids that are on the side chain or are substituted on the α-amino or α-carboxy group. examples for Modified amino acids that can be used are norleucine, hydroxyproline, hydroxylysine, γ-carboxyglutamic acid, etc. This includes, for example, N- Alkyl amino acids, d. H. Amino acids on the α-amino group with an alkyl radical were substituted. N-alkyl amino acids can also be in other positions be modified or substituted. Under "alkyl" are, for example, branched or to understand unbranched alkyl radicals with variable chain lengths. Furthermore the amino acids can be prepared by methods known to those skilled in the art be modified. Compounds C1, C2 and C3 can also contain peptides be, which in turn consist of amino acids as defined above. The in the Peptides according to the invention containing peptide derivatives preferably have a maximum length of three amino acids, particularly preferably not more than two Amino acids. Instead of peptides, peptide derivatives or peptoids can also be used be included. Peptoids differ chemically from peptides in that in addition to amino acids, they also contain other compounds that are covalently terminal to the amino and / or carboxy group and / or to one or more side chains are bound. Peptide derivatives are understood to mean peptides which are considered Part of such derivatizations of amino acids contain both the chemical substitution of individual atoms, functional groups as well Add whole chemical molecules by covalent attachment to the May affect amino acid backbone. The term peptide derivatives includes thus all chemical changes and additions to amino acids / peptides can be made. Furthermore C1, C2 and / or C3 can also Peptide mimetics, aminobenzoic acids or heterocyclic compounds with Amino and / or carboxyl groups are substituted.

Methods for producing the peptide derivatives according to the invention are general known to the person skilled in the art and described in the literature. Manufacturing process for  Homomeric or heteromeric peptides can be, for example, Jakubke (peptides. Chemistry and biology, SPECTRUM ACADEMIC VLG, 1196 VIII, ISBN: 3-8274-0000-7); Bodanzsky (Peptide Chemistry: A Practical Textbook, Springer Verlag, ISBN: 0387566759) or Gutte (Editor, Peptides: Synthesis, Structures, and Applications (1995), Academic Press, ISBN: 0123109205). The invention Peptide derivatives can also be prepared as described in the examples.

The compounds B1 and B2 can form a chemical bond with one another form. This chemical bond is preferably covalent in nature. The Connections B1 or B2 can be the same or different. In a preferred embodiment of the invention is both B1 and B2 cysteine. The Ring formation of the peptide derivative, which generates the strained form, takes place here through a disulfide bond between the cysteines. Generation techniques such disulfide bonds within peptides are, for example, in Maruyama (Peptides 20 (7) (1999), 881-884) and Kiso (Brazilian Journal of Medical and Biological Research 27 (12) (1994), 2733-2744). The one after these or similar methods produced strained peptide derivatives are via a can be stored for a long time.

In the embodiment of the peptide derivatives according to the invention, the binding is easily detachable between B1 and B2. The bond can be released by physical or chemical influences. Are under physical influences for example light such. B. a brief flash of light or others to call electromagnetic radiation. To the bond between B1 and B2 To make it vulnerable to such influences, it must be photosensitive. Examples of such photosensitive bonds are aryl disulfides, which are for example between compounds such as those containing 4-mercapto-phenylalanine Form peptides (Lu, J. Am. Chem. Soc. 119 (1997), 71-73). Laser induced Photolysis generates tolylthiyl radicals within a few picoseconds (Volk, J. Phys. Chem. B, 101 (1997), 8607). For example, under chemical influences Changes in the reaction solution that affect the pH, a transition from an oxidative environment in a reducing environment or vice versa, the add acids, alkalis, reducing agents or oxidizing agents let it be accomplished. Further chemical influences are within the meaning of the invention  Reagents that, when added to the reaction solution, bind between B1 and Attack B2 directly. Examples of such reagents are trialkylphosphines. Preferably have added between B1 and B2 to release the bond Chemicals have no or only negligible effects on the measuring reaction or on detection.

Thus, in a preferred embodiment of the invention Peptide derivative is the process of breaking the bond between B1 and B2 Use of a flash of light or other electromagnetic radiation or the addition of a chemical reagent.

In a particularly preferred embodiment of the invention, the solving of the Binding between B1 and B2 achieved by adding a reducing agent. For example, the reducing agent causes the reduction of disulfide bridges that exist between two cysteine residues. Examples of reducing agents used in Dithiothreitol (DTT), DTE, Tri-n-butyl-phosphine and tri-carboxyethyl-phosphine.

The peptide derivatives according to the invention contain two in the general formula A and D designated groups that measure the transition from clamped in the unstressed state or the direct measurement of a Allow enzyme reaction due to a change in an optical signal.

For the purposes of the present invention, these groupings contain a dye. The groupings may also contain other chemical compounds, such as an amino acid residue covalently linked to the dye are. Such another connection is preferably between the dye and the neighboring connection B1 or B2. Group A preferably exists and / or D exclusively from the dye. In the sense of the present Dyes contained in A and D, react to irradiation with light a certain wavelength or a certain spectrum with the emission of Light that is detectably different from the incident light. The Wavelengths or spectra of light can be in the infrared, visible and / or ultraviolet wavelength range. This applies to both Radiation as well as for emission. In addition to radiation, the dyes  in A and D of the peptide derivative according to the invention also by other physical or stimulated by chemical or biological energy sources to emit light become. For the purposes of the invention, other physical sources can be used for example electromagnetic radiation other than light, electric fields, etc. his. Chemical or biological light emission can, for example, under Exploitation of an effect such as chemo-, bioluminescence or phosphorescence respectively.

Those skilled in the art are dyes that have the properties mentioned above correspond and can be used in the groups A and D, known. A Large selection of such compounds are described in the prior art and For example, the "Handbook of Fluorescent Probes and Research Chemicals "(R. P. Haugland, ISBN 0-9652240-0-7). Furthermore, Process known with which peptides or peptide derivatives with N- or C-terminal the groups A and D can be coupled, such as described in Jean (Biochem. J. 307 (1995), 689), Spetzler (J. Peptide Sci. 4 (1998), 128), Wang (Bioorg. Med. Chem. Lett. 2 (1992), 1665), Wang (Tetrahedron Lett. 31 (1990), 6493), Meldal & Svendsen (J. Chem. Soc. Perkin Trans. L (1995), 1591) or Bratovanova & Petkov (Anal. Biochem. 162 (1987), 213).

In a preferred embodiment, the invention relates to peptide derivatives, where A and D are groupings which contain dyes which, when excited with light, have a fluorescent at a suitable wavelength, or groupings that are dyes included that show a color change. The most popular types of signals used for Detection of enzymatic reactions are changes that are can be detected using optical methods. The big advantage of such Methods is the presence of various spectroscopic devices that make it possible to increase the enzymatic activity based on the color change describe. Among the light-optical signal changes are particularly those of Advantage that lead to a change in fluorescence. The biggest advantage of such Fluorescence methods lie in their large, known to those skilled in the art Sensitivity. Such methods are particularly sensitive here, in those triggered by the enzymatic reaction, the fluorescence signal increases. The high sensitivity of fluorescence methods is mainly due to  on the generally known higher detection sensitivity of Fluorescent dyes compared to non-fluorescent dyes.

Among groups that contain dyes that show a color change preferably to understand compounds that are themselves a substrate for the represent measuring enzyme and in which the color change by the enzymatic catalysis is caused. In the absence of one In this case, however, enzymatic activity will not change the optical Signal detected. Examples known to the person skilled in the art are e.g. B. substrates for Proteases in which after adding the protease to a suitable substrate a dye is released by the onset of proteolysis (Barrett, Meth. Enzymol. 1981, 80: 561; Carter, Science 237 (1987) 394; Tanaka, Biochemistry 24 (1985), 2040; Horsthemke, Biochemistry 19 (1980), 2867; Jacotot, Eur. J. Biochem. 239 (1996), 248; Heinemeyer, EMBO J. 10, (1991) 555; Thornberry, Nature 356, (992) 768; Walker, Cell 78 (1994), 343; Xiang, Proc. Natl. Acad. Sci. USA 93 (1996), 14559). As a measure of the enzymatic activity of this proteolytic activity, the released amount of dye per unit of time can be used.

In a particularly preferred embodiment, the present invention relates Peptide derivatives, the fluorescence of those contained in the groupings fluorescent dyes in the strained state of the peptide derivative by a Quench effect cancels or diminishes.

The spatial proximity between A and D in the strained peptide derivative causes one Quench effect, which means that the light emission is one of the two Groupings reduced or completely by the effect of the other grouping will be annulled. "Quench effect" is used in connection with the Present invention understood a physico-chemical effect, in which the Excitation energy, which can lead to light emission of the fluorescent dye, is also absorbed by the spatial proximity of the quencher, and so the Light emission of the fluorescent dye is reduced. The definition of Dyes, the peptide derivative according to the invention or the grouping A or D can therefore also include fluorescent dyes which serve as quenchers. With increased distance between A and D in the unstressed state of the Peptide derivative, this quench effect is reduced or completely eliminated, see above  that the light emission of the previously quenched compound is now greater than in strained peptide derivative.

For the sake of simplicity, A will be referred to below as the grouping defined whose optical signal is measured, and D as the grouping that A quericht. The embodiments of the present presented below Invention are to be understood so that A and D are interchangeable, so that D can be quenched by A. Pairs of fluorescent are known to those skilled in the art Connections where one connection quenches the other known.

Peptide derivatives of those described above are particularly preferred Embodiments of the invention, wherein A is a grouping selected from the Group consisting of: 2-aminobenzoic acid, D, L-2-amino-3- (7-methoxy-4- cumaryl) propanoic acid, coumarin, quinolinone, 4- (4'- Dimethylaminobenzenazo) benzoic acid (dabsyl) and 5- (2'- Aminoethylamino) naphthalene sulfonic acid (EDANS); and D is a grouping selected from the group consisting of: 4-nitroaniline (= para-nitroaniline), Aminoacyl-4-nitroaniline, 3-nitrotyrosine (meta-nitrotyrosine), aminoacyl-3-nitrotyrosine, Tryptophan and aminoacyl tryptophan. The fluorescent listed here Connections and their use are described in the prior art, for example in Jureus, Neuropeptides 32 (1998), 453-460 or White, Anal. Biochem. 268: 245-251 (1999).

The compounds used for A, B1-B2, C1-C3 and D of the invention Peptide derivatives can be prepared using those known to those skilled in the art Modifications are largely changed.

An essential property of the peptide derivatives according to the invention is that Conformation, which in connection with the invention described here as "Tense state" is referred to, whereby the peptide derivative is able (after Disconnect the connection between B1 and B2), autonomously in the not tense State to pass. This transition takes place with a certain one Speed, hereinafter referred to as the transition speed. The Transition speed is characteristic of every peptide derivative and below same reaction conditions always the same size.  

In essence, the transition speed is determined by the execution of the Constituent C1-C2-C3 determined within the peptide derivative according to the invention. Already atomic substitutions on one or more of these compounds can change the transition speed. The transition speed is an inherent feature of any peptide derivative according to the invention.

Measuring enzyme activity is based on one of the following two effects that an enzyme can have on the peptide derivative:

• 1. The enzyme to be measured has an activity that makes the transition from tensioned accelerated to the untensioned state. The acceleration is coming either because the enzyme changes the conformation catalyzes or that the enzyme catalyzes a reaction that facilitated one Transition from the tensioned to the non-tensioned state.
• 2. The enzyme to be measured does not have the activity mentioned under (1.), but attacks the unstrained peptide derivative or already the braced Peptide derivative after loosening the bond between B1 and B2, the catalyzed reaction to a chemical separation of A and D or to a Separation of the dye contained in A or D from the rest of the peptide derivative leads.

If the measurement of the enzyme activity is based on the effect described under (1.), the detection takes place in that the optical signal changes due to the transition from the strained to the unstrained state of the peptide derivative of the invention. Fluorescent dyes with a quench effect are used for groupings A and D. The fluorescence of group A is quenched by group D in the strained state of the peptide derivative, preferably with closed binding of B1 and B2. This quench effect is due to the spatial proximity of A and D. In the unstressed state, the distance between A and D is greater, which leads to a reduction in the quench effect and, consequently, to an increase in the fluorescence of A. However, A can also be partially quenched by D in the untensioned state. After the start of the reaction by releasing the bond between B1 and B2, in the absence of an enzyme, the peptide derivative automatically changes from the tensioned state to the non-tensioned state. This process follows stochastic principles and, depending on the structure of the peptide derivative molecule, becomes saturated after a certain time. This saturation kinetics can be represented by continuous measurement of the increase in fluorescence in a saturation curve in which absolute or relative fluorescence is plotted against time (see FIG. 1).

In a reaction batch that contains an enzyme that triggers the transition reaction accelerated, but is otherwise identical to the approach without enzyme, the Transition from tensioned to non-tensioned condition with a larger one Speed. This affects the kinetics in that the increase turns out steeper and saturation is reached earlier. Enzymes that control the transition of the Catalyst peptide derivative from the tensioned to the non-tensioned state or catalyze a reaction that facilitates the transition are within the meaning of the invention preferably isomerases, particularly preferably peptidyl isomerases. Furthermore, the influence of inhibitors or activators on such enzymes Reaction approaches can be determined according to (1.). When measuring inhibitors of an enzyme used is a slower speed of Fluorescence increase observed at the saturation level. In contrast to increases when observers of these enzymes are measured Response curve faster to the saturation level.

As already mentioned above, the transition speed essentially depends on the chemical properties of the component C1-C2-C3 of the peptide derivative. In addition, the reaction conditions, especially the temperature, Influence on the transition speed. For activity measurements according to the below (1.) Defined aspect should be the peptide derivative specific transition rate (without enzyme) be relatively small in order to obtain a favorable measuring range. If this basic transition speed is too high, the difference of Transition speeds between the measurement with and without the enzyme less significant. As a result, the measuring range is about to detect the Influence of inhibitors smaller. The catalyzed should preferably Transition speed must be at least twice as high as that catalyzed reaction.

In addition to enzymes, which, as described above, make the transition from tensioned to can accelerate the unstressed state, as also listed under (2.) other enzymes can be investigated with the aid of the peptide derivative according to the invention. This presupposes that the enzymes catalyze a reaction which leads to  a chemical separation of A and D or a separation of the in A or D. contained dye leads from the rest of the peptide derivative.

When using fluorescent dyes for A and D that have a quench effect, three levels of fluorescence to be measured can be distinguished:

• a) Initial state in the strained peptide derivative, in which A maximally of D is quenched;
• b) Fluorescence of A when the peptide derivative is in the untensioned state is present and A is partially quenched by D; and
• c) maximum fluorescence of A when cleaved within the Peptide derivatives A and D or the dyes contained therein are separated and D has no quench effect on A.

On the one hand, the activity measurement measures the fluorescence behavior of the peptide derivative in the reaction solution without enzyme. The fluorescence approaches (i) from level (ii). On the other hand, the fluorescence increases when measuring with enzyme from (i) to level (iii). The measurable difference in fluorescence between (ii) and (iii) is used to determine enzymatic reactions. This preferably relates to enzymes that cleave a covalent bond between A and D or within a group A or D, such as proteases. In measurement of the enzyme-catalyzed reaction (i) or (ii) to (iii) (depending on whether the enzyme to the solution of B ₁ - B ₂ bond already the strained substrate (i) or only the non-strained substrate ( ii) attacks), a slower rate of increase in fluorescence to level (iii) is observed when measuring inhibitors of an enzyme used. In contrast, when activators of these enzymes are measured, the observed response curve increases faster and also goes against level (iii).

For activity measurements, especially according to (2.), the Transition speed generally through the addition of an enzyme such as for example a peptidyl prolyl isomerase, in the approaches to be compared increase. This increase affects the transition from level (i) to (ii).

Thus, a particular embodiment of the invention relates to peptide derivatives, where the optical signal emanating from A and D, in addition to the tense  and unstressed state of the peptide derivative from the state which is when the chemical bond between A and D or between a dye contained in A or D and the rest of the peptide derivative enzymatic catalysis was separated.

Activity measurements according to (2.), in which essentially the difference in fluorescence between the untensioned state and the state in which A and D or a dye contained in A or D and the rest of the peptide derivative are chemically separated, are measured are relative high basic transition speeds (for the peptide derivative without enzyme) is an advantage. This applies in particular to the case where the enzyme utilizes the peptide derivative as a substrate both in the tensioned and in the non-tensioned state after the release of the bond between B1 and B2. If the transition rate is high compared to the enzymatic reaction rate, the majority of the peptide derivatives first go into the untensioned state and are catalyzed by the enzyme in the second step. As a result, the enzyme kinetics are linked to the basic transition kinetics and the two kinetics (reaction with and without enzyme) can be compared well. On the other hand, if the reaction rate of the enzyme is high compared to the basic transition rate, the enzyme kinetics are independent of the transition kinetics, because a large part of the peptide derivative molecules used directly from the strained state to the state of separation of A and D or the separation of a dye contained in A or D. from the rest of the peptide derivative, ie the maximum fluorescence. Favorable differences in the transition rates, expressed as the reaction rate constant k, exist between the reactions according to (1) (k _i-ii ) and (2) (k _ii-iii ) if k _{ii-iii is} at least twice as large as k _{i- ii} . This requirement can easily be met by adding peptidylprolyl cis / trans isomerases to the reaction mixture, since these enzymes only catalyze reactions according to (1). In the event that only the untensioned state is a usable substrate for the enzyme to be examined, the same applies as for case (1). The kinetics of the enzyme-catalyzed reaction depend on the basic transition kinetics, since the enzymatic reaction depends on the provision of the non-strained peptide derivative. If the enzymatic reaction rate is high compared to the basic transition rate, then the majority of the resulting non-strained peptide derivative is quickly converted enzymatically. This achieves a maximum difference between the fluorescence kinetics with and without enzyme. The consequence of this is that a maximum measuring range is available for measuring the influence of added inhibitors. At an enzymatic reaction rate that is relatively slow compared to the basic transition rate (without enzyme), only a small part of the unrestrained peptide derivative is first reacted enzymatically and the saturation of the products, in which A and D or the dyes contained therein are present, only occurs achieved long after the saturation of the transition from the tensioned to the non-tensioned state. In the case of activity measurements which proceed according to the effect listed under (2.), color reactions, that is to say differences in the optical properties of the reaction solution which can be perceived by the human eye, can also be used. In this case, A and / or D contain dyes which, for example, change their optical properties by proteolytic cleavage from the peptide derivative.

With the aid of the peptide derivatives according to the invention, the activities are all complete Enzymes measurable for which the peptide derivatives are a substrate, as long as they are Activities have an impact on the changes to the optical signal and at the same time this activity through the closed bond between B1 and B2, preferably also due to the tensioned condition of the Peptide derivative is blocked.

This preferably relates to enzymes whose activity is substrate-specific for Peptide structures. This particularly includes activities that stand out Peptide bonds are directed. All other bonds are also preferred may be present in a peptide (or peptide derivative), for example within the side chain of one or more amino acids or bonds that Connect substituents with amino acids or within such substituents lie. However, the peptide derivatives can also be used in the context of the invention To study enzymes that are specific to other classes of molecules, for example for nucleotides, oligonucleotides, lipids, carbohydrates or others organic molecules. According to the embodiment of the peptide derivative corresponding substrates for C1, C2 and / or C3 built into the peptide derivative become. For example, it is possible for C1, C2 or C3 connections use that are phosphorylierbar / dephosphorylierbar. So in the frame  of the present invention with one representative of a kinase and one Phosphatase are shown to be only the unrestrained form of such a executed peptide derivative according to the invention can be phosphorylated or is dephosphorylierbar.

Peptide-specific enzymes can preferably be obtained using the peptide derivative investigate, particularly preferably proteases, peptidylprolyl cis / trans isomerases, Kinases, phosphatases, glycosidases or lipases / esterases. In particular proteases and peptidylprolyl cis / trans isomerases are preferred.

One of the main advantages over those in the prior art The substrates described and their use in enzyme tests is the special one Property of the peptide derivative according to the invention that the start of the Enzyme reaction can be induced by an exogenous signal. As above described, this physical or chemical signal leads to the solution of the Binding between B1 and B2 in the peptide derivative. This enables the mix of all for necessary components of the enzyme reaction, including the enzyme, the Substrate and optionally an effector to be examined without the enzymatic reaction gets underway.

A common feature of the most diverse enzyme assays is the need for Creation of a homogeneous mixture, at least from substrate and enzyme consists. Creating a homogeneous mix is essential Precondition for reproducible enzymatic activity determinations to be able to perform. Typically, enzymatic reactions that for example, to assess enzyme effectors, by adding the substrate to a mixture of enzyme and added effector started. This procedure has the advantage that slow-acting effectors that the Those skilled in the art, for example, are known as "slow binding" inhibitors, their effect can unfold on the enzyme. To the by adding the substrate Concentrations of substances in the mixture, such as enzyme and effector The added volumes of the substrate only do not change significantly little to the resulting total volume of the reaction solution. The problem often consists in the substrate dissolved in a small volume in as much as possible to mix homogeneously with the pre-incubation mixture for a short time. This succeeds naturally only in the desired manner if the solubility of the substrate in  resulting final volume is relatively high. Substrates, the concentration of which Final volumes are close to their solubility product, are difficult solve homogeneously within a short time. For this reason they are Enzyme substrates previously unsuitable for use in routine assays for Detection of enzymatic reactions.

In the prior art, for example, to detect the activity of Proteases have already described some methods of calling for a Substrate, which is an activity measurement with great signal directness and Enable signal continuity according to the present patent application, very close come. So z. B. described substrates that are constructed so that by the direct effect of the protease on the substrate releases a dye. There the color change of the proteolytic reaction is directly proportional to this Change used as a measure of activity of the protease. When examining the effect of effectors on the catalysis of the proteolytic reaction is mostly the Protease preincubated with the effector for some time, the pre-incubation period. This enables the mixture of effector and enzyme, accordingly to interact with one another based on their chemical conditions. Inevitably must now the actual reaction, which is used to determine activity, by Addition of the substrate to the effector / enzyme mixture can be started. This is just then successful if it succeeds in using the substrate homogeneously within a short time to mix the effector / enzyme mixture. Unfortunately, there are many Known peptide substrates that have poor water solubility. Such Substrates are usually in suitable solvents, such as. B. DMSO, in high concentrations are then dissolved together with this solvent to be mixed with the effector / enzyme mixture.

The problem here is that the desired peptide substrate is in would solve the final volume of the reaction batch, but due to local Inhomogeneities mostly fail and only come back into solution later. The An activity determination can extend the mixing time through such processes impossible with poorly soluble substrates.

The problem of creating a homogeneous mixture in the approach of the enzyme Assays are solved by providing the substrate according to the invention. The for Determination of the necessary reagents, such as enzyme and substrate or desirable additives such as effectors to be investigated  can be of any length of time before the actual reaction starts incubate with each other, since the actual substrate only by loosening the bond of B1 and B2 is provided in the peptide derivative according to the invention. The Peptide derivative can with closed B1-B2 binding in such concentrations be used, the maximum solubility of the substrate among the selected test conditions come very close.

The B1-B2 bond is released by adding a small amount Volume of a suitable chemical or without volume change by means of Flash of light triggered. As a result, when the reaction starts, even with poorly soluble ones The concentrations of the pre-incubation approach and the actual Measurement approach changed little or not at all. For the purposes of the invention however, be sure that the start signal is effective and at the same time the Peptide derivatives achieved in a homogeneous mixture, otherwise a new one Would result in inhomogeneity problem. This should be when using a physical signal such as a flash of light with a corresponding Design of the experimental equipment may be given. When using a Starter chemical should be noted that it is in the given reaction mixture is easily soluble. In general, low molecular weight is preferred for this purpose To use compounds such as that used in Examples 1-3 DTT.

The peptide derivatives of the present invention provide substrates for enzymatic Reactions that can be detected with the greatest possible signal directness. Signal directness is an important criterion for the quality of an enzyme test. For some Enzymes are not suitable substrates with which the Reliably measure substrate product turnover directly. In some cases exist techniques that allow direct measurement of substrate product sales, however, these are often expensive in terms of equipment or only under ideal conditions applicable so that they are not for routine use, for example in Mass search tests are suitable. To escape such limitations, you had to hitherto assisted with auxiliary reactions with which the product quantity with the help of a additional enzymatic reaction shown in detectable signal strengths becomes. Such auxiliary reactions run synchronously with and in the primary reaction Reaction approach like this. The crucial disadvantage of such auxiliary reactions  is the lower signal directness, which is an increase in potential, more disturbing Influencing factors. This applies, for example, to the activity measurement of Peptidylprolyl-cis / trans isomerases (PPlases) generated by an auxiliary reaction coupled isomer-specific proteolysis can be detected (Fischer, Biochim. Biophys. Acta 43: 1101; Fischer, Nature 337 (1989), 476).

Firstly, influencing factors that affect the coupled reaction or its Components act, make the interpretation of the measurement signals difficult or impossible do. In the example of PPlase catalysis using coupled isomer-specific Proteolysis would be the inhibitory influence of effectors on the enzymatic Activity of the auxiliary protease used make it difficult or impossible to make one To recognize the effect of this protease inhibitor on PPlase catalysis. In order to would be used in the context of a Search tests for active substances that effect PPlase activity, all of them Substances cannot be tested which make the auxiliary protease undesirable inhibit. Another disadvantage of too low a signal directness can also be that Effect of the components necessary for the coupled reaction on the Primary reaction itself or to other components to be detected. So can the auxiliary protease necessary in this example has an adverse effect on the Have activity of the PPlase to be detected. You could also find them Do not detect PPlase effectors that are not protease stable.

Thanks to the present invention it is now possible to determine the activity of enzymes for who have so far had no suitable substrates, such as B. PPlasen with measure the greatest possible signal directness, as shown in Example 1. For the activity measurement of PPlases, however, there have been others recently Methods have been described that do not require auxiliary reactions. A procedure, that is particularly close to the invention uses the low isomer-specific spectroscopic differences from suitable PPlase substrates (Janowski, Analytical Biochemistry 252 (1997), 299; Garcia-Echeverria, Biochem. Biophys. Res. Commun. 191 (1993); Garcia-Echeverria, Am. Chem. Soc. 114: 2758 (1992). However, these differences are so small that this method is very high quality spectroscopic detectors needed. Another disadvantage of this minor Spectroscopic differences lie in the limitation to activity measurements under ideal conditions where low spectroscopic interference such as that  activity measurements in real biological fluids are unavoidable, do not occur.

An additional disadvantage of this method lies in the way it is provided Substrates. To detect isomer-specific spectroscopic differences must, the starting substrate in a difficult to handle solution Lithium chloride and trifluoroethanol are dissolved. The enzymatic reaction will then started by adding this substrate solution to the reaction mixture. So that is this method is disadvantageous for use under a Mass search tests.

In contrast, PPlase substrates according to the invention can be used as in Example 1 described, for a long time in easy-to-use solvents such as aqueous Buffers, or suitable organic solvents such as DMSO or ethanol stored and used in the assay. Your addition to the reaction mixture can be done well in advance. The actual PPlase reaction only starts when when using photosensitive tension caused by a flash of light, or at Use of other functionalities by adding a suitable chemical, which remove the tension, as described in Example 1.

In addition to the homogeneous mixture and the Signal directness is the third criterion, signal continuity as a quality characteristic for Activity measurements of enzymes. While with some substrates, as described above for protease assays, is relatively light, continuous throughout to obtain measured values of the enzymatic reaction, for example by measurement of a proteolytically released dye, this is for numerous enzymes and their Inadequate substrates possible. In order to describe the enzymatic reaction to obtain necessary data has so far been discontinuous procedures applied.

The common thing of these discontinuous processes is that the actually too evaluating continuous enzymatic reaction in time and space runs independently of a second reaction that the detection of the Substrate turnover enabled.

For example, to detect the activity of protein phosphatases A wide variety of assays have been described, in which during the phosphatase reaction Aliquots are continuously withdrawn from the reaction batch to this Subsequently being able to detect aliquots using a second assay (Cohen,  Methods in Enzymology 201: 389-391 (1991); Pinna, Biochim. Biophys. Acta 1222 (1994), 415-431; Ruzzene, Eur. J. Biochem. 211: 289-295 (1993); Harder, Biochem. J. 298 (1994), 395-401). These phosphatase activity processes have in common that the phosphate released by the protein phosphatase in this second assay is determined.

So z. B. by means of suitable phosphatase substrates, such as. B. by means of the sequence Abz-Cys-Ala-Ser (phosphorylated) -Pro-Cys-NHNp, the activity of a Protein phosphatase such as PP2a can be determined continuously. In this special case is exploited that a protease added as an auxiliary enzyme only can proteolytically cleave the non-phosphorylated form of the substrate. A time-dependent increase in fluorescence is then proportional in this approach Phosphatase activity.

Similar batch processes are described for protein kinases (Pin, Analytical Biochemistry 275 (2) (1999), 156-161; Seya, Analytical Biochemistry 272 (2) (1999), 243-249; Peterson, Analytical Biochemistry 271 (2) (1999), 131-136). The In addition to the above, the most serious disadvantage of this discontinuous process is discussed possible errors when working with auxiliary reactions, in the Compared to continuous processes, greater material and time expenditure to see, which is necessary to get such a set of signals with their Help it is possible to describe the enzymatic reaction.

With the help of the peptide derivative provided by the present invention, it is now possible to limit the activity of many enzymes that were previously only possible with the help of discontinuous detection methods were recorded continuously measure up. This applies in particular to protein phosphatases and protein kinases.

A preferred embodiment of the invention relates to peptide derivatives which thereby are characterized in that the strained state at least the cis conformation is and is not a chemical bond within compounds C1 to C3 strained state is the trans conformation at the bond.

Cis and trans conformations are familiar to the person skilled in the art. As part of the In the present invention, "cis" and "trans" mean different bond angles a peptide bond as z. B. in Fischer (Ang. Chemie 106 (1994), 1479-1501) are described.  

The cis conformations contained in the peptide derivatives according to the invention after the B1-B2 bond is opened, the trans conformation changes automatically. In the context of this embodiment, the cis / trans isomerism relates to the Peptide derivatives according to the invention all chemical bonds for which cis / trans Conformations can be defined. Furthermore, the peptide derivatives are these Embodiment capable of changing the conformation in the sense of the invention, d. H. the transition from a tensioned to a non-tensioned state, to execute. Preferably the bond that causes the cis / trans isomerism is within the component C1-C2-C3 of the peptide derivative, more precisely, between the connections C1 and C2 or C2 and C3 or within one of these Links. Such a cis / trans isomer is particularly preferably one Binding that is in the peptide backbone of the peptide derivative according to the invention, particularly preferably a peptide bond. The expression "trans conformation" in the present embodiment also includes the possibility that after Course of the reaction, for example when a state of equilibrium is reached, a fraction of the peptide derivatives used is in the cis conformation.

The peptide derivatives used in Examples 1 to 3 correspond to this embodiment. As can further be seen from the examples, peptide derivatives in which the transition from the strained to the unstrained state is based on a cis / trans isomerism can be used for both areas of application of the present invention, ie both

• 1. Activity measurements of enzymes that make the transition from strained to unstressed condition, d. H. in the case of the cis to the trans conformation catalyze (as in Example 1); for as well
• 2. Activity measurements of enzymes that show a chemical separation of A and D bring about contained dyes (as in Example 2).

In a particularly preferred embodiment of the invention Peptide derivative is the bond that causes cis / trans isomerism Peptide bond between the amino group of a proline residue or a derivative thereof and the carboxy group of the adjacent amino acid.

The peptide bond is a partial double bond between the carbon of the Carboxy group of the first amino acid and the nitrogen atom of the amino group of the subsequent amino acid (Stryer, Biochemie, German translation, 1990, page  25). Typically, in a peptide bond, the oxygen atom is the Carboxy group and the hydrogen atom of the amino group opposite, so in arranged trans. In contrast, the peptide bond in which the amino group or Imino group is contributed by a proline residue, in naturally occurring Proteins and peptides occur in the cis conformation. Depending on the Amino acid sequence is found in peptide derivatives according to the invention, in which for C1 to C3 contains at least one proline or a derivative thereof in aqueous Solutions in the untensioned state mix a mixture that is about 5-35% peptides contains which have a cis-prolyl peptide bond. In the strained peptides this percentage can be up to 100%. A good overview can be found at Fischer, Ang. Chemie 106 (1994), 1479-1501 and Reimer et al., J. Mol. Biol. 279 (1998), 449.

In the context of this embodiment, “proline residue or derivative thereof” is a natural proteinogenic L-proline, a D-proline as well as all possible to the expert to understand accessible modifications of the amino acid as long as the C-N The basic structure of the proline residue is preserved.

In a further embodiment, the invention additionally relates to peptide derivatives, which is also a substrate in the tensioned state for an enzyme activity to be tested represent.

In addition to the embodiments of the invention described so far, in which always only the non-braced peptide derivative or at least the peptide derivative with a released B1-B2 bond the functional substrate for the measurement of a Represents enzyme activity, the peptide derivatives represent this embodiment of the Invention even in the tense state, due to the closed B1-B2 binding a substrate is stabilized.

As described in Example 3, enzymes of this type can be used with such peptide derivatives select, whose activity consists, for example, of the B1-B2 binding solve or A or D or dyes contained in A and / or D from the peptide derivative to separate.

However, in this embodiment of the invention there is a particular advantage of not to wear the aforementioned embodiments, d. H. essentially that the Start of enzymatic catalysis cannot be induced from the outside. The means that in this embodiment of the peptide derivatives the reaction as in  many disadvantageous methods from the prior art Mixing the necessary components is started. However, with Using peptide derivatives of this embodiment isolate new enzymes whose Activity was not yet known.

The invention further relates to a kit comprising the peptide derivative according to one of the Embodiments characterized above, a device or a reagent to release the connection between B1 and B2 in the peptide derivative, one or more Buffer solutions and / or one or more enzymes for which the peptide derivative is a Represents substrate.

For practical reasons and for the purpose of ease of use and The peptide derivative according to the invention can be marketed together with for Use in enzyme assays required components, or a partial selection of that, summarize in kits. Depending on the intended application, the kit includes one or more peptide derivatives of the present invention, wherein the Peptide derivatives represent a substrate for one or more enzyme activities. In a preferred embodiment of the kit is contained therein Peptide derivatives in the closed bond between B1 and B2 stabilized tense form. Depending on the solubility of the peptide or derivatives suitable solvents should be selected for storage, such as DMSO, Ethanol, etc. As a further component, such a kit can be a device or contain a reagent to break the bond between B1 and B2. Act the peptide derivative (s) contained in the kit are versions with a photosensitive bond between B1 and B2, the kit should, for example a device for reliable generation of a flash of light included in the Is able to release the B1-B2 bond. If the B1-B2 bond is due to chemical Influence is closed, the kit can open a corresponding reagent contain. With a disulfide bridge bond between B1 and B2 there is one select reducing connection such. B. DTT, preferably in a direct usable concentration, such as B. 250mM DTT in 50mM Hepes buffer, pH 7.5. Furthermore, the kit can already include enzymes for which the contained Peptide derivatives represent substrates. The enzymes can be standards, for example display with defined activity units for quantitative comparison in enzyme Assays. On the other hand, enzymes contained in the kit for assays can be used for  Determination of the activity of certain effectors can be used. Furthermore In addition to the enzyme (s), effector compounds can also be included with Defined inhibitory or activating activity, which is the standard for those to be tested Effectors can serve.

In addition, a kit according to the invention may contain further components which Be helpful for the use of peptide derivatives in enzyme assays can, such as B. buffer solutions or stock solutions thereof for reaction solutions, for storage, dilution or modification of one or more Components of the kit. For example, if the kit contains enzymes, their activity is dependent on cofactors, these can also be included in the kit. There it is biochemical compounds / molecules contained in the kit we recommend packaging for the kit that allows cooling. Farther should be checked for opacity when packaging the kit, at least for the peptide derivatives that contain dyes.

Furthermore, the embodiment includes all kits in addition to the Peptide derivatives according to the invention in addition to or instead of the other Components mentioned here contain other or further components that the Specialist for a specific application of the peptide derivative necessary or appear useful.

A particular embodiment of the invention relates to methods for determining the activity of an enzyme for which the peptide derivatives according to the invention or parts thereof represent a substrate, the peptide derivatives preferably not or at least a poor substrate consisting of the steps when the bond between B1 and B2 is closed

• a) Mixing a suitable amount of the peptide derivative in one for each Activity of the enzyme suitable reaction solution with and without the enzyme;
• b) Start the reaction by breaking the bond between B1 and B2 in Peptide derivative;
• c) measuring the optical signal as a function of time; and
• d) determining the activity of the enzyme by determining the difference in Velocity of change of the optical signal in the Reaction solutions with and without the enzyme.

For the method of this embodiment of the invention, the Peptide derivatives according to the invention used for the enzyme to be examined the tense state, but at least the peptide derivative with closed B1- B2 binding is no or at least a poor substrate. The expression In this context, "no substrate" means that when the Binding between B1 and B2 no color change is observable. This closes however, does not mean that the peptide derivatives are not at least to a lesser extent than Serve substrate, but this is not observable because of the possible conformations to be in balance. Peptide derivatives are preferred for this method excluded in one of the aforementioned embodiments of the invention a substrate even when tensioned or when the B1-B2 bond is closed represent for an enzyme. Enzymes that are examined with this method can have already been defined above as enzymes, whose activity with the help of peptide derivative of the invention provided for this method are measurable.

In the embodiment described here, “enzyme” includes both enzymes understand that are homogeneously cleaned, as well as biological samples in which the investigating enzyme activity is suspected. When using such biological Samples have the particular advantage of the method or the invention Peptide derivatives over some of the methods described in the prior art to ensure that biological samples, such as crude extracts, Body fluids, culture supernatants, etc. that are relatively unpurified are used can be.

"Biological samples" in the sense of the method can also be more than one contain enzymatically active molecule. That is, comes in such biological samples the sum of the molecular species to bear, which corresponds to the total activity of the sample contribute both positively and negatively. This means that the biological Sample a mixture of one or more enzymes and one or more Molecules that can inhibit or activate the enzyme or enzymes, may include.

Under "enzyme" is in connection with the present method besides Proteins or proteins with non-proteinogenic cofactors (enzymes in the narrow Other organic macromolecules, the enzymatic Can catalyze reactions. "Enzymatic" means in the context in contrast to chemical catalysis, the catalysis of a reaction in the for  Enzymes typical way. This preferably means that the substrate specificity includes the stereochemical properties of substrates. Among other organic macromolecules "are, for example, ribozymes or synthetic ones Understand macromolecules with enzymatic activity, so-called synzymes. The The amount of enzyme used varies depending on the specific activity of the sample. Those skilled in the art will be able to do so by diluting or concentrating a sample Prepare series of different concentrations to provide the sample, provided it contains an activity to bring in a measuring range that is a comparison of the Kinetics of the transition from the tense to the untensed state of the Peptide derivative with and without enzyme allowed.

A preferred embodiment of the method relates to the use of Standard enzymes, such as are commercially available, the one have defined enzyme activity. With the help of such standard enzymes it is Specialist possible by making a series of dilutions and measuring their Activities using the procedure described here to obtain standard kinetic curves which allow a comparison with the enzyme samples to be examined in order to to obtain quantitative statements about the measured enzyme activity.

In step (a) of the method according to the invention described here, two reaction batches are produced, which differ from one another only in that one batch contains the enzyme sample to be investigated, the other does not. As a minimum requirement, these reaction solutions contain the following in suitable amounts for the enzyme reaction:

• - A peptide derivative according to the invention provided for this method as Substrate,
• - Water,
• - Buffer components and if necessary
• - cofactors or other components.

The enzyme sample is also added to one of the two reaction batches. In the other approach can be the one created by the missing enzyme sample Volume difference can be compensated by adding water or buffer.

The components contained in the reaction solution after mixing are preferably in dissolved form. To prepare the reaction batches in suitable reaction vessels are the components as large as possible  Add mixing. The mixture can already be made by suitable Pipetting techniques familiar to those skilled in the art can be achieved. In addition, by mechanical action, for example by vortexing, swirling or shaking, the mixing effect can be improved.

The method according to the invention preferably provides that after mixing all necessary components, including the enzyme in the approach with enzyme, generated reaction batches over a certain time, for example 5, 10, 20 or Incubate 30 minutes before starting the reaction. Such Pre-incubation is used to optimize the mixture, preferably by one to achieve a homogeneous mixture.

During or after the mixing of the reaction batches, these are applied to the Reaction conditions set under which the reaction should take place. This especially affects the temperature. The above is also here Pre-incubation makes sense to start the reaction in the reaction batches to achieve uniform and reproducible reaction conditions.

In step (b) the reaction is accomplished by releasing the bond between B1 and B2 in the peptide derivative according to the invention started. The one in this step started reaction in both approaches includes the transition of the one used Peptide derivative from the strained to the unstrained state. In addition the reaction in the approach with enzyme comprises the enzymatic to be measured Reaction. The B1-B2 bond is released by one of the aforementioned Measures achieved. This measure is preferably carried out both in Reaction batch carried out with enzyme as well as in that without enzyme possible identical reaction conditions in the two reaction solutions guarantee.

In step (c), the one used during the ongoing reaction Optical signal emitted by peptide derivatives was measured.

The measurement is carried out according to spectroscopic methods and with suitable ones Devices known to the person skilled in the art and described in the literature, for example in the "Handbook of Fluorescent Probes and Research Chemicals", R. P. Haugland, ISBN 0-9652240-0-7. The optical signals are at the Measurement converted into electrical signals, with analog electrical signals preferably digitized for the purpose of better evaluation.  

If the peptide derivative used carries fluorescent dyes (A and D), then either or both of the dyes are excited by light of a suitable wavelength. The emitted long-wave light is detected, using suitable ones Filter systems are separated from the stimulating light. Contain the groupings A and D in the peptide derivative used are dyes which are enzyme-dependent Color change show, the detection by measuring the absorption of the light emitted by a suitable light source after passing through the Reaction solutions take place.

The optical signal is preferably measured from the start of the reaction (Step b) or shortly before, until either or both reactions have a certain Have reached saturation. The measurement can also be stopped beforehand if, for example, there is no activity for the measurement data beforehand show investigating enzyme. The saturation of a reaction can then recognize that the kinetic curve reaches a plateau phase. As before As described above, the kinetics of the reaction depend essentially on that endogenous transition rate of the peptide derivative used. In addition, the reaction temperature is included in the reaction rate. On average, the duration of an enzyme test until saturation is between one and twenty minutes.

One of the particular advantages of the present invention is the provision of one continuous measuring process for enzyme activities. In principle, it is therefore possible record an unlimited number of measured values per unit of time. However, should be noted be that the amount of data recorded is reasonable stand by their informative value and the required size of the cost-intensive Data storage space. Preferred time intervals are for the individual measuring points between 300 and 6 measurements per minute, so that depending on the duration of the Enzyme tests approx. 100-1000 data points per measurement can be obtained.

The result of the measurement described in step (c) is preferably a curve in which the strength of the optical signal is plotted against time. If peptide derivatives are used in the present method in which the transition from the strained to the unstrained state can be detected, both curves show an increase in the optical signal for the reaction batches with and without enzyme. If the enzyme to be measured has an activity, the curve for the reaction mixture with enzyme shows a greater increase than the curve for the reaction mixture without enzyme (see curves 1 and 4 in FIGS. 1 and 2). In step (d), the activity of the enzyme under investigation is determined by determining the difference in the rates of change of the optical signal in the reaction solutions with and without the enzyme. The change in the optical signal is preferably an increase in the optical signal. The optical signal is preferably a fluorescence or a color signal.

When determining the enzyme activity on the basis of the measured kinetic curves, two cases have to be distinguished as in step (c) (above):

• a) If the change in the optical signal is based on different speeds of the transition from the tensioned to the non-tensioned state in the reaction solutions with and without enzyme, then the enzyme activity can be determined from the difference of these transition rates as follows. For some applications, a qualitative statement of whether there is enzymatic activity or not is sufficient. Such statements can already be made by visually comparing the curves. Quantitative statements can be made, for example, by exact, preferably computer-aided, analysis of the kinetic curves. The transition speed is expressed in the curve by its slope:
  In order to largely rule out the saturation effect when calculating the transition speeds, the gradient should be calculated at a point on the curve which is in a region that is as linear as possible. This linear range is preferably as close as possible to the starting point of the reaction, such as. B. in the range below 10% of the maximum optical signal. The current transition speed can be expressed by the slope of the curve at a certain point. The difference between two curves can be determined by subtracting the transition speed at comparable points of the two curves, for example measuring points at the same time or at the same height of the optical signal. The same level of the optical signal should preferably be used for the comparison of transition speeds. By correlating the maximum optical signal with the amount of substance of the substrate that has been completely converted (for example, as a non-strained peptide derivative) and the optical signal at the start of the reaction with the amount of substance 0 of the converted substrate, the relative indication of the optical signal can be in Convert absolute amounts of substance. As a result, the transition speeds can also be expressed as absolute instantaneous conversion rates (unit: mol / sec). By subtracting two instantaneous turnover rates at comparable measuring points of the two curves, preferably at the same level of fluorescence, the activity of the enzyme results as the difference in the transition rates.
  In addition to the above-mentioned possibility of linearizing the increases (below 10% of the maximum optical signal), the data of the entire response time or curve can preferably be used to calculate the substrate turnover and thus the transition speed. Corresponding evaluation software for the calculation of "first-order kinetics" which is familiar to a person skilled in the art can be used for this purpose. So the concentration-time law of a first order reaction is used. To calculate the reaction constants, data collection rates are used which correspond to the following time law:
  Dsz = In (1 - A) / k with A = (1 - exp ^-k.gZ ) / Dp.
  It means:
  Dsz = time at which a measuring point is used for the calculation;
  k = kinetic constant of a first order reaction;
  gZ = total reaction time over which the reaction is observed; and
  Dp = number of all data points with which the reaction is described. Using these data points, the exact kinetic constants can now be calculated using conventional algorithms.
• b) If the groups A and D of the peptide derivative used dyes contain, which show an enzyme-dependent color change, results in the enzymatic activity directly from the slope of the curve of the Reaction solution with enzyme, since the curve to the reaction solution without Ideally, the enzyme has no slope. The transition speed and thus preferably the enzyme activity can be according to one of the above  calculate the described procedure. For experimental reasons the Curve to the reaction solution without enzyme from the x-axis parallel may differ, for example because the dye is not 100% stable you use this curve to correct the enzyme-measuring curve, for example, by taking the measured values at the same times were taken from each other.

The method of this embodiment of the invention is excellent for Use in mass search tests, so-called high-throughput screenings, suitable. Techniques for the automated manipulation of the reaction solutions in Reaction vessels by robots, automated detection and evaluation are familiar to the person skilled in the art and are described, for example, in "Drug Metabolism: Databases and High-Throughput Testing During Drug Design and Development "; P. W. Erhardt (Editor) (1999), Blackwell Science Inc. ISBN: 0632053429; "Cost Effective Strategies for Automated and Accelerated High-Throughput Screening " (Ibcs Biomedical Library Series) (1996), IBC United States Conferences, ISBN: 1579360025; "High-Throughput Screening; The Discovery of Bioactive Substances" (1997), ISBN 0-8247-0067-8; "New Challenges Arising from High Throughput Screening", Dixon, G. K .; Major, J. S .; Rice, M.J. (1995), ISBN 1-85996-111-8.

A further embodiment of the invention relates to a method for determining whether an effector is an inhibitor or an activator of an enzyme for which the peptide derivatives according to the invention or parts thereof represent a substrate, the peptide derivatives preferably having no or at least one when the bond between B1 and B2 is closed represent poor substrate consisting of the steps

• a) Mixing appropriate amounts of the peptide derivative and the enzyme in each a reaction solution suitable for the activity of the enzyme with and without the effector;
• b) Start the reaction by breaking the bond between B1 and B2 in Peptide derivative;
• c) measuring the optical signal as a function of time; and
• d) Determine that the effector
  • a) is an inhibitor if the rate of change of the optical signal in the reaction solution with the effector is less than in the reaction solution without the effector; or
  • b) is an activator if the rate of change of the optical signal in the reaction solution with the effector is less than in the reaction solution without the effector.

"Effectors" are defined in the present embodiment as molecules that are based on a certain enzymatic activity has an influence as an inhibitor or activator to have. "Inhibitors" are defined as molecules that have a particular enzymatic Inhibit activity. "Activators" are defined as molecules that a certain Enhance enzymatic activity. According to the present procedure Analyzable effectors belong to all possible classes of substances that are are suitable for the process due to their physicochemical properties. These properties primarily affect the solubility of the effector. To the analyzable effectors include both inorganic and organic Molecules, but preferably organic molecules. The effectors can by chemical synthesis or by isolation from natural sources, such as for example, living beings are provided. It is preferably the Effectors around nucleic acids, lipids, carbohydrates, (poly) peptides or Combinations of these groups of molecules. It is particularly preferred in the analyzable effectors around peptides or polypeptides or derivatives thereof. The effectors particularly preferably comprise molecules which are known to coexist Enzymes interact, such as peptide inhibitors, antibodies, lectins or Fragments or derivatives thereof, their inhibitory or activating activity in the present method is to be tested.

The analyzable effectors are preferably capable of the enzyme and / or Bind peptide derivative. Such bonds are preferably made to the active one Center of the enzyme or other accessible sites of the enzyme, where the latter can lead to allosteric effects on enzyme activity.

The method shown in the present embodiment corresponds to essential to the description of the aforementioned method for determining the Activity of an enzyme. The main difference concerns the fact that the two reaction solutions prepared in step (a) for comparison purposes are not with or without enzyme, but with and without effector. Except of which are the execution descriptions for step (a), (b) and (c) of the aforementioned method also on the method of determining whether an effector an inhibitor or an activator is applicable.  

The kinetic obtainable by step (c) by measuring the optical signal Curves are evaluated in step (d) and enable the determination of whether a the effector used is an inhibitor or an activator. The procedure for Comparison of the curves corresponds to the procedure in the aforementioned Embodiment. The change in the optical signal is based on the transition from the tensioned to the non-tensioned state of the peptide derivative used, can also change the transition rate of the respective reaction by the Calculation of the rate constants according to a concentration-time law a first order reaction. The relative difference between The measured transition speeds provide information about the activity of the Effector. The transition speed of the approach with effector is lower of the approach without an effector, the effector is one Inhibitor of the enzyme. In the opposite case, if the transition speed in Approach with effector over the transition speed of the approach without effector the effector is an activator.

The third possibility in this embodiment includes the possibility that the effector used has no inhibitory or activating activity possesses the enzyme under investigation. In this case, the kinetics of the Response with and without effector not significant. There are significant differences for example, when the transition speeds, for example expressed by rate constants, more than double distinguish statistical errors of the underlying.

The embodiment of the invention described here includes the possibility of combining the method for determining whether an effector is an inhibitor or an activator and the method for determining the activity of an enzyme. This means that a third is added to the two reaction solutions in step (a), so that in the process of the present embodiment, in addition to two enzyme-containing reaction solutions (with and without effector), a reaction solution without enzyme also takes part. In this way, in addition to the curves for the reaction solutions with and without an effector, a zero value curve is obtained for the kinetics of the peptide derivative without enzyme. This combination of the two methods is particularly suitable for cases in which the change in the optical signal is based on the transition from the tensioned to the non-tensioned state. The zero value curve makes it possible to relate the difference between the curves with effector and without effector to the difference between the zero value curve and the curve without effector, i.e. the curve that shows the enzyme-specific activity. In this way, a relative quantification of the activating or inhibitory effect of an effector is possible. Examples of such curves are shown in FIGS. 1 and 2.

In a further preferred embodiment, the invention relates to a method for screening an inhibitor or an activator of an enzyme for which the peptide derivatives according to the invention or parts thereof represent a substrate, the peptide derivatives preferably not or at least a poor substrate when the bond between B1 and B2 is closed , consisting of the steps

• a) Mixing appropriate amounts of the peptide derivative and the enzyme in each a reaction solution suitable for the activity of the enzyme with and without a sample containing a single or a multitude of compounds, who are candidates for an inhibitor or activator;
• b) Start the reaction by breaking the bond between B1 and B2 in Peptide derivative;
• c) measuring the optical signal as a function of time, and
• d) Determine that the sample
  • a) has inhibitory activity if the rate of change of the optical signal in the reaction solution with the sample is less than in the reaction solution without the sample; or
  • b) has activating activity if the rate of change of the optical signal in the reaction solution with the sample is greater than in the reaction solution without the sample.

In addition, as a preferred embodiment, the invention relates to a method which comprises the aforementioned steps (a) to (d) and additionally the step:

• a) Subdivide the sample for which in step (d) inhibitory or activating activity was determined and repeating the steps (a) to (d) until the inhibitor or activator contained in the sample is present cleaned.

"Screening" means in connection with the present invention Screening a variety of samples, a single or a variety of Contain compounds containing candidates for inhibitors or activators of represent given enzyme, with the aim of inhibitors or activators of To identify enzyme. Generally, "screening" means a process in which which a large number of samples are examined for a specific property, you don't know how to respond to the property being tested.

"Sample" means all natural or artificial samples that Contain candidates for inhibitors or activators of the given enzyme and can be tested in the method according to the invention. This includes both homogeneous solutions of one molecule and mixtures of several molecules. Among molecules that can be screened in the process are as Effectors labeled molecules of the aforementioned embodiment locked in. The samples can be taken from natural sources or be synthetically manufactured. For example, the samples can be molecular libraries can be removed, such as for oligopeptides or natural products exist. Furthermore, samples can also be digested by biological Material, for example living material or formerly living material, are removed or culture supernatants from microorganism cultures come from. The samples can be in the form of a crude extract or supernatant or can be in a cleaning form of your choice. Can be used for cleaning the extracts or supernatants are fractionated. The specialist is responsible for this numerous techniques available, such as differential precipitation, Gradient centrifugation, chromatography techniques, etc. The biological material encompasses all areas of the organism, the material either cultivated or can be taken from nature.

The method described in the present embodiment includes the Execution descriptions for steps (a) to (b) of the method for Determine whether an effector is an inhibitor or an activator with a a sample is examined instead of an effector. This also leaves Process a combination with the process described above Determination of the activity of an enzyme so that in addition to the  kinetic curves reflecting the enzyme measurement with and oh 17659 00070 552 001000280000000200012000285911754800040 0002010023743 00004 17540ne sample, a Measures without enzyme, which gives a zero value curve.

The screening process extended by step (e) affects applications in which are the starting samples mixtures of different molecules that Represent candidates for inhibitors or activators.

It relates to samples for which an inhibitory or activating step (d) Activity was detected. Such samples are then according to a person skilled in the art known technology divided into chemical or biological fractions. The Fractionation takes place according to one or more physical, chemical or biological properties such as solubility, molecular mass, electrical Charge, affinity, hydrophobicity, etc. These fractions are in turn the subjected to screening processes characterized by steps (a) to (d). This sequence, consisting of subdivisions and screening, can be repeated several times until a level of purity specified by the user is reached. On Such a degree of purity can be determined by an enrichment level of the specific express inhibitory or activating activity. The degree of purity can also by means of qualitative ones suitable for the respective molecular classes which the sample contains or quantitative detection methods can be determined. Examples of such Detection methods are HPLC, gas chromatography, mass spectrometry, Thin layer chromatography, gel chromatography, blotting, etc. Dem Such detection methods are familiar to a person skilled in the art.

A particular embodiment of the invention relates to the aforementioned methods, wherein the change in the optical signal is at least partially on the transition is based on the tensioned in the non-tensioned state of the peptide derivative.

As already described in detail for the respective procedure, the change can of the optical signal on the transition from the tensioned to the non-tensioned State of the peptide derivative according to the invention provided for the methods are based.

"Partially" here means that in the reaction solutions containing an enzyme included, the optical signal additionally changed by the enzymatic activity can be, for example, if the enzymatic activity is a separation of the Groupings A and D of the peptide derivative or a separation of the in A or D. contained dye catalyzed by the rest of the peptide derivative.  

This special case is supported by a further special embodiment of the Invention detected, which relates to the aforementioned methods, the change in optical signal in enzyme reactions with enzyme at least partially on the Separation of the chemical bond between groups A and D of Peptide derivative or on the separation of the dye contained in A or D from remaining peptide derivative is based.

As already described for some embodiments of the present invention, part or all of the detection of the enzymatic activity can be based on the separation of the chemical bond between the groups A and D of the peptide derivative or on the separation of the dye contained in A or D from the rest of the peptide derivative . In any event, the activity of the enzyme being tested or the sample containing such enzyme activity will effect this separation. As has already happened several times, the description of this preferred embodiment is to be considered separately according to the principles on which the detection is based:

• - If the change in the optical signal on an enzyme-dependent Color change based, the enzymatic activity mostly affects directly Dye. This can result in a separation of A and / or D or one in A. and / or D contained dye come from the peptide derivative. This can be seen from of example 2 are illustrated. Chymotrypsin turns p-nitroaniline (NHNp) split off. According to the invention, this leads to the separation of a dye in D from the rest of the peptide derivative. The release of p-nitroaniline can be as Color changes can be observed at 390 nm. In example 2, NHNp functions however, as a quencher and the fluorescence from Abz is detected.
• - If the change in the optical signal is at least partially on the transition of the peptide derivative used according to the invention from the strained in the unstressed state, and this through a quench effect is detected, the chemical separation of those contained in A and D can Dyes in the present preferred embodiment to increase the fluorescence compared to the untensioned state of the peptide derivative to lead. The spatial separation of the dyes contained in A and D by Removal of the chemical compound causes a maximum reduction in the  Quench effect. The particular advantage of this embodiment is that the signal indicating the enzymatic activity is opposite the signal stands out for the reaction without enzyme. You can do this from one Speak amplification effect.

Another embodiment of the invention relates to the aforementioned methods, wherein the reaction solution is mixed to homogeneity in step (a).

As already described above, an excellent property affects the Invention the fact that all for a test approach to measure a Necessary components, including the substrate, before starting the enzyme Reaction can be mixed together. Furthermore, it has already been stated that the mixing defined in step (a) of the method according to the invention preferably leads to a homogeneous mixture of the components if necessary by observing a pre-incubation period.

"Homogeneous mixture" means in connection with the present invention a solution, the constituents of which are uniform (homogeneous) within the Volume that the solution occupies are distributed.

In a further preferred embodiment, the present invention relates to aforementioned method, wherein the enzyme is a protease, peptidylprolyl-cis / trans Isomerase, kinase, phosphatase, glycosidase or lipase / esterase.

In a further preferred embodiment, the present invention relates to a Kit comprising the peptide derivative according to the invention, a device or a Reagent for releasing the bond between B1 and B2 in the peptide derivative, one or several buffer solutions, one or more enzymes for which the peptide derivative Representing substrate, and / or instructions for performing one or more of the procedures described above.

The kit includes all components contained in the kit, the further was described above, and in addition instructions for performing an or several of the aforementioned methods.

Such instructions are contained in the present description of the invention contained execution descriptions, which allow the user, the or to use the method (s) according to the invention. In addition, the instructions  Contain information from the prior art, which the user Facilitate certain techniques.

Another particular embodiment of the invention relates to the use of the Peptide derivative according to the invention or the kit according to the invention for Determination of the activity of enzymes or for the identification of inhibitors or Activators of enzymes.

These and other embodiments are disclosed to those skilled in the art obvious and are embraced by the description and examples of the present invention. Further literature can be found in one of the above the methods, means and uses mentioned within the meaning of the present Invention can be applied, can be found in the prior art, e.g. B. from public libraries under z. B. the use of electronic Aids. For this purpose, there are, among other things, public databases like the "Medline" that are available on the Internet, e.g. B. at the address http: / / www.ncbi.nlm.nhi.gov/PubMed/medline.html. Other databases and Addresses are familiar to the expert and can be found on the Internet be, e.g. B. at the address http: / / www.lycos.com. An overview of sources and information on patents and patent applications in biotechnology is in Berks, TIBTECH 12 (1994), 352-364.

The figures show:

Fig. 1 recording curves showing the transition from the strained in the non-tightened state of the peptide derivative used in Example 1 in the reaction solutions without enzyme (4) with enzyme (1) and two different effectors (2, 3) have an inhibitory effect on the enzymatic activity . The peptidylprolyl cis / trans isomerase PIN 1 used as an enzyme catalyzes the transition from the strained to the untensioned state, which is based on the cis / trans isomerization of the phosphoseryl-prolyl-peptide bond in the peptide derivative used.

Fig. 2 shows various curves registration kinetics of a protease (α- chymotrypsin) whose activity was measured using the peptide derivative of Example 2. The increase in fluorescence in the reaction solution without enzyme (4) is due exclusively to the transition from the strained to the unstrained state, which is due to the cis / trans isomerization of the alanyl prolyl peptide bond in the peptide derivative used. In the reaction solutions with enzyme, the increase in fluorescence, in addition to the transition from the strained to the unstrained state, is due to the proteolytic cleavage of p-nitroaniline (NHNp) from the peptide derivative (1 to 3). Compared to the reaction solution with enzyme and without effector (1), the reactions with added inhibitory effectors show significantly lower transition speeds (2 and 3).

The following examples illustrate the invention:

Methodological background

The peptide derivatives used in the following examples were in essentially using the solid phase peptide synthesis method of fluorenyloxycarbonyl-protected amino acid derivatives with a automatic peptide synthesizer. Served as starting products commercially available amino acids or fluorochromes, which are on the side groups are protected. The amino acids were added with an efficiency of 95-98% per addition step. The protecting groups were then replaced by 96% Trifluoroacetic acid / 2% water / 2% tributylsilane / 2% phenol removed. The purification the peptide derivatives were carried out by high pressure liquid chromatography (HPLC) using an acetonitrile / water gradient system. The purity of the Peptide derivatives obtained were determined by analytical HPLC and by capillary Electrophoresis determined. The identity of the connections was established by  mass spectrometric methods tested. To generate the braced The disulfide bond in the purified peptide derivatives became state between the cysteines contained for B1 and B2, as in Albericio (Int. J. Peptide Prot. Res. 37 (1991), 402).

example 1 Determination of the effect of effectors on a peptidyl prolyl cis / trans isomerase

The following solutions are made:
Substrate solution: 2 mg / ml Abz-Cys-Ser (PO ₃ ) -Pro-Ala-Cys-Phe-NHNp in DMSO (Abz: aminobenzoic acid; NHNp: para-nitroaniline)
Enzyme solution: 5 mM solution of human PIN 1 in 50 mM Hepes buffer, pH 7.5
Effector solutions: 0.1 mg / ml Juglon substance in DMSO
Starter solution: 250 mM DTT in 50 mM Hepes buffer, pH 7.5

2 µl per chamber are placed in a standard titer plate with 384 reaction chambers Pipette substrate solution, 1 µl effector solution and 20 µl enzyme solution. In the Control approaches with and without enzyme, both without effector, are the Enzyme and effector solutions using appropriate volumes of 50 mM Hepes buffer, pH 7.5 or DMSO replaced. It can be beneficial to reduce pipetting errors be one from the appropriate amounts of enzyme and substrate solution Generate mixture that when pipetting 22 µl of this mixture per chamber the same concentrations can be reached as when pipetting the Individual volumes. The plate is then at 6 ° C for a pre-incubation period of 20 Minutes so that each of the reaction chambers after 20 minutes Has temperature of 6 ° C. The actual reaction is done by adding 20 µl starter solution each started.

With proper temperature constancy of 6 ° C during the measuring time and reaching a homogeneous mixing of the reaction solutions containing substrate, enzyme and effector solution with the starter solution, each can be excited with a UV lamp with an excitation spectral range between 250 and 330 nm when the fluorescence is excited single reaction chamber register the increase in visible light at 420 nm. Even the visual comparison of the registration curves obtained ( FIG. 1) enables the identification of effectors which, in the present example, have a clearly inhibitory effect on the enzyme PIN I.

Example 2 Determination of the effect of effectors on a protease

The following solutions are made:
Substrate solution: 2 mg / ml Abz-Cys-Ala-Pro-Ala-Cys-Phe-NHNp in DMSO
Enzyme solution: 1 nM solution of α-chymotrypsin in 50 mM Hepes buffer, pH 7.5
Effector solutions: 0.1 mg / ml Eglin C in DMSO
Starter solution: 250 mM DTT in 50 mM Hepes buffer, pH 7.5

2 µl per chamber are placed in a standard titer plate with 384 reaction chambers Pipette substrate solution, 1 µl effector solution and 20 µl enzyme solution. In the Control approaches with and without enzyme, both without effector, are the Enzyme and effector solutions using appropriate volumes of 50 mM Hepes buffer, pH 7.5 or DMSO replaced. It can be beneficial to reduce pipetting errors be one from the appropriate amounts of enzyme and substrate solution Generate mixture that when pipetting 22 ul of this mixture per chamber same concentrations can be achieved as when pipetting the individual volumes. The plate is then so at 30 ° C for a preincubation time of 20 minutes stored that each of the reaction chambers after 20 minutes a temperature of 30 ° C.  having. The actual reaction is carried out by adding 20 µl each Starter solution started.

With proper temperature constancy of 30 ° C during the measuring time and when homogeneous mixing of the reaction solutions is achieved. When the fluorescence is excited with a UV lamp with an excitation spectral range between 250 and 330 nm, substrate, enzyme and effector solutions with the starter solution register the increase in visible light at 420 nm in each individual reaction chamber. Even the visual comparison of the registration curves obtained ( FIG. 2) enables the identification of effectors which, in the present example, have a clearly inhibitory effect on α-chymotrypsin.

Example 3 Screening for enzyme activities with substrate specificity for Peptide derivatives, which in the by closed bond between B1 and B2 stabilized tensioned condition

It has been observed that, for example, proteases such as e.g. B. chymotrypsin, but in very high concentrations are able to attack peptide derivatives that are stabilized by the closed B1-B2 bond in the strained state. This finding prompted the search for enzymes that prefer such strained peptides. The following solutions were made for this:
Substrate solution I: 2 mg / ml Abz-Cys-Ala-Pro-Ala-Cys-Tyr (NO ₂ ) -NH ₂ in DMSO.
(Tyr (NO ₂ ): meta-nitrotyrosine)
Substrate solution II: 20 μl of substrate solution I are added to 100 ml of 35 mM HEPES buffer (pH 7.6).

Substrate solution II is stable for about 8 hours at room temperature. It will Fractions of biological solutions used in which an enzymatic activity is suspected, which is suitable for either breaking the bond between B1 and B2 or else cleave off at least one of the compounds A and D from the peptide derivative. 10 µl per chamber are placed in a standard titer plate with 384 reaction chambers pipetted biological solution. After equilibrating the titer plate with The reaction is carried out at room temperature for 20 min by adding 60 μl of substrate solution II started. If the solutions are mixed properly and homogeneously, when fluorescence is excited with a UV lamp with a Excitation spectral range between 250 and 330 nm in each one Reaction chamber register the increase in visible light at 420 nm. The Visual comparison of the registration curves obtained is used to find one Enzyme activity, the substrate of which is the strained substrate shape.  

SEQUENCE LOG

Claims (22)

1. Peptide derivative with the general formula
A-B1-C1-C2-C3-B2-D;
wherein C1, C2 and C3 are compounds selected from the group consisting of amino acids, N-alkyl amino acids, peptides, peptide derivatives, peptoids and compounds which can be incorporated into peptide chains;
where B1 and B2 are compounds which can form a chemical bond with one another;
the peptide derivative being in a tense state when the bond between B1 and B2 is formed;
the bond between B1 and B2 can easily be released and the tensioned state then changes to an untensioned state; and
where A and D are groupings which allow the measurement of the transition from the tensioned to the non-tensioned state or the direct measurement of an enzyme reaction due to the change in an optical signal. 2. A peptide derivative according to claim 1, wherein B1 and B2 are each cysteine. 3. Peptide derivative according to claim 1 or 2, wherein the method for solving the Binding between B1 and B2 using a flash of light or a other electromagnetic radiation or the addition of a chemical Reagent is. 4. A peptide derivative according to claim 3, wherein the chemical reagent is a reducing connection is. 5. Peptide derivative according to one of claims 1 to 4, wherein A and D Groupings are those that contain dyes that when excited with light  appropriate wavelength fluoresce, or are groupings that Contain dyes that show a change in color. 6. peptide derivative according to claim 5, wherein the fluorescence of the in Groupings contain fluorescent dyes in the strained Cancels the state of the peptide derivative by means of a quench effect or reduced. 7. A peptide derivative according to claim 6, wherein A is a group selected from the group consisting of: 2-aminobenzoic acid, D, L-2-amino-3- (7-methoxy- 4-cumaryl) propanoic acid, coumarin, quinolinone, 4- (4'- Dimethylaminobenzenazo) benzoic acid (dabsyl) and 5- (2'- Aminoethylamino) naphthalene sulfonic acid (EDANS); and D is a grouping selected from the group consisting of: 4-nitroaniline, aminoacyl-4- Nitroaniline, 3-nitrotyrosine, aminoacyl-3-nitrotyrosine, tryptophan and Aminoacyl tryptophan. 8. Peptide derivative according to one of claims 1 to 7, wherein the optical Signal emanating from A and D, in addition to the tense and not strained state of the peptide derivative differs from the state that is present when the chemical bond between A and D or between a dye contained in A or D and the rest of the peptide derivative was separated by enzymatic catalysis. 9. peptide derivative according to any one of claims 1 to 8, wherein the strained State the cis conformation of at least one of the chemical bonds is within the connections C1 to C3 and the untensioned state is the trans conformation of the bond. 10. A peptide derivative according to claim 9, wherein the binding is a peptide bond between the amino group of a proline residue or a derivative thereof and the carboxy group of the adjacent amino acid.   11. A peptide derivative according to any one of claims 1 to 10, wherein the peptide derivative a substrate even in the tensioned state for an enzyme activity to be tested represents. 12. Kit comprising the peptide derivative according to any one of claims 1 to 11, one Device or reagent for releasing the connection between B1 and B2 in the peptide derivative, one or more buffer solutions and / or one or several enzymes for which the peptide derivative is a substrate. 13. A method for determining the activity of an enzyme for which the peptide derivative according to any one of claims 1 to 10 or parts thereof is a substrate, consisting of the steps

• a) mixing a suitable amount of the peptide derivative according to any one of claims 1 to 10 in a reaction solution suitable for the activity of the enzyme with and without the enzyme;
• b) starting the reaction by breaking the bond between B1 and B2 in the peptide derivative;
• c) measuring the optical signal as a function of time; and
• d) determining the activity of the enzyme by determining the difference in the rates of change of the optical signal in the reaction solutions with and without the enzyme.

14. A method for determining whether an effector is an inhibitor or an activator of an enzyme for which the peptide derivative according to any one of claims 1 to 10 or parts thereof is a substrate, consisting of the steps

• a) mixing suitable amounts of the peptide derivative according to any one of claims 1 to 10 and the enzyme in a reaction solution suitable for the activity of the enzyme with and without the effector;
• b) starting the reaction by releasing the connection between B1 and B2 in the peptide derivative;
• c) measuring the optical signal as a function of time; and
• d) Determine that the effector
  • a) is an inhibitor if the rate of change of the optical signal in the reaction solution with the effector is less than in the reaction solution without the effector; or
  • b) is an activator if the rate of change of the optical signal in the reaction solution with the effector in the reaction solution with the effector is greater than in the reaction solution without the effector.

15. A method for screening an inhibitor or an activator of an enzyme for which the peptide derivative according to any one of claims 1 to 10 or parts thereof is a substrate, consisting of the steps

• a) Mixing appropriate amounts of the peptide derivative according to any one of claims 1 to 10 and the enzyme in a reaction solution suitable for the activity of the enzyme, with and without a sample containing a single or a plurality of compounds, the candidates for an inhibitor or activator are;
• b) starting the reaction by breaking the bond between B1 and B2 in the peptide derivative;
• c) measuring the optical signal as a function of time, and
• d) Determine that the sample
  • a) has inhibitory activity if the rate of change of the optical signal in the reaction solution with the effector in the reaction solution with the sample is less than in the reaction solution without the sample; or
  • b) has activating activity if the rate of change of the optical signal in the reaction solution with the effector of the reaction solution with the sample is greater than in the reaction solution without the sample.

16. The method according to claim 16, additionally comprising the step

• a) Subdivide the sample for which inhibitory or activating activity was determined in step (d), and repeat steps (a) to (d) until the inhibitor or activator contained in the sample is present in a purified form.

17. The method according to any one of claims 13 to 16, wherein the change of optical signal at least partially on the transition from tensioned to is based on the untensioned state of the peptide derivative. 18. The method according to any one of claims 13 to 16, wherein the change of optical signal in enzyme reactions with enzyme at least partially the separation of the chemical bond between compounds A and D of the peptide derivative or on the separation of that contained in A or D. Dye based on the rest of the peptide derivative. 19. The method according to any one of claims 13 to 18, wherein the reaction solution in Step (a) is mixed to homogeneity. 20. The method according to any one of claims 13 to 19, wherein the enzyme is a Protease, peptidylprolyl-cis / trans isomerase, kinase, phosphatase, Is glycosidase or lipase / esterase. 21. Kit comprising the peptide derivative according to one of claims 1 to 11, one Device or reagent for releasing the bond between B1 and B2 in the peptide derivative, one or more buffer solutions, one or more Enzymes for which the peptide derivative is a substrate and / or a Instructions for performing the procedure or procedures according to at least one of claims 13 to 20. 22. Use of the peptide derivative according to one of claims 1 to 11 or of Kits according to claim 12 or 21 for determining the activity of enzymes or to identify inhibitors or activators of enzymes.