DE102005029920A1 - Fluorescence procedure used to determine activity of phosphatases, phosphodiesterases or kinases, employs trivalent lanthanide ion with organic ligand - Google Patents

Abstract

The enzyme-catalyzed reaction is allowed to proceed using any natural or synthetic enzyme substrate. The following stages determine reaction conversion. Before, during or after reaction, the solution is mixed with a reagent comprising a complex, i.e. a trivalent lanthanide ion with an organic ligand. The ligand is not the enzyme substrate. The reagent is excited to photoluminescence by irradiation with light. Intensity or decay time of this photoluminescence is determined. Alteration of the fluorescence characteristics of the reagent resulting from the reaction, is used for the detection and determination of the respective enzyme. The enzyme is a phosphatase and the enzyme substrate a phosphate ester of glycerin, glucose, ribose or deoxyribose, a nucleoside, of serins, threonin, farnesol, creatinin, or of any other synthetic or bio-organic alcohol or phenol. The enzyme is a phosphatase and the phosphate ester a diphosphate or a triphosphate. The enzyme is a phosphodiesterase and the phosphate ester is a cyclic mono nucleotide e.g. the cyclic adenosin monophosphate or in the case of nucleases, a polynucleotide. The enzyme is a kinase and the enzyme substrate, if appropriate, is protein-bound serin, threonin or tyrosin, creatinin, glycerin, glucose, ribose or deoxyribose, a nucleoside or another synthetic or organic mono- or polyalcohol, or a phenol. The reagent general chemical structure contains the trivalent ion of europium, terbium, samarium, lanthanum, dysoprosium or holmium, with a chelator of the central ion. This has an absorption maximum in the waveband 300-450 nm. The luminescence of the ion is not extinguished. Neither substrate nor product are involved in the reaction. The ratio of ion to chelate is in the range 10:1 to 1:3. The lanthanide ion is a europium (III) or terbium (III) ion. The chelator is a hydroxylized heterocyclic or a chelatizing beta diketone (or its enol). The chelator is optionally a cumarin, chromon or chinolon-3-carbonic acid or tetracyclin or one of its derivatives. It is used to determine the effectiveness of enzyme inhibitors. It is used to determine the activity of an enzyme which is bound to a fixed substrate, on a biological membrane, another protein, e.g. another antibody, or on a natural or synthetic polynucleotide.

Description

1. Summary

The The invention relates to fluorescent optical methods for the determination the activity of phosphorylating or dephosphorylating enzymes. It concerns also methods for the determination of inhibitors of these enzymes. The proceedings are based on the use of fluorescent probes from the group lanthanide chelate complexes (LCK). The chelate ligands in such LCK are not the enzyme substrate. The LCK are able to do that in a enzymatic reaction formed or spent phosphate demonstrated. The LCK are using light of wavelengths between 300 and 450 nm, and the resulting fluorescence intensity or decay time can simplify the determination of enzymes, but also their inhibitors serve.

2. The biochemical meaning of phosphatases, phosphodiesterases and kinases

Phosphatases are enzymes from the group of hydrolases that are able to cleave organic phosphates. In the Enzyme Catalog they are identified in the group of hydrolases. They catalyze the hydrolysis of organic esters of phosphoric acid according to the pH of the solution according to the following equations (1) or (2):

phosphatases can but also diphosphates and triphosphates dephosphorylate, z. B. the adenosine diphosphate to adenosine monophosphate.

phosphodiesterases such as For example, the (deoxy) ribonucleases split phosphophores into two Fragments, while cyclic Phosphatases cyclic esters such. B. the cyclo-adenosine monophosphate in the cleave corresponding open-chain monoester. The most popular Example is the cleavage of the cyclo-adenosine monophosphate (c-AMP) into the open adenosine monophosphate (AMP).

kinases are phosphate ester-forming, ie phosphorylating enzymes. she are summarized in the Enzyme Catalog under the group of transferases. Put simply, they catalyze those caused by phosphatases Conversions in the opposite direction. Above all, kinases phosphorylate the hydroxyamino acids Serine, threonine and tyrosine in proteins and require a high-energy phosphate such as As the adenosine triphosphate (ATP).

The Detection of new inhibitors of phosphatases, phosphodiesterases or kinases is one of the central tasks in development new drugs, and the test for kinase inhibitors thus one of the most common Tests in the so-called high-throughput screening for new lead structures for pharmaceuticals. Previous methods use either optical methods Help of synthetic enzyme substrates, or immunological methods.

3. Known optical determination methods

The most important optical method for the determination of phosphatases in that one approximates colorless synthetic enzyme substrate (usually the nitrophenyl phosphate; see the chemical formula in the upper box) by a phosphatase converted into its yellow hydrolysis product (the nitrophenolate). The Rate of formation of the yellow phenolate is a measure of the activity of the phosphatase.

The Determination of the phosphatase but z. B. according to DOS 3248043 also be done by using a non-fluorescent synthetic Substrate (a coumarin phosphate) of the chemical shown in the lower box Structure by a phosphatase in her strongly blue fluorescent Product (the free hydroxycoumarin) transferred.

Out the US Appl. 2003/0228646 (Whateley) are phosphoric acid esters Acidrone and quinacridone are known to be cleaved by phosphatases and thereby a change the fluorescent color and decay time. Unfortunately, own The acridones and quinacridones have a low solubility in water and therefore can be used only in very low concentration. In the US Appl. 2003/0036106 (Burke et al.) Are labeled with organic dyes reporter molecules described to phosphorylating or dephosphorylating enzymes and as a result of the binding process, a change in the polarization of the Undergo fluorescence. This procedure suffers from the circumstance that one for Each enzyme must have a specific binder available, given that the large number of usable enzymes means a considerable effort.

The DOS 10239005 (Mayer-Almes) describes a method for determination of kinases, phosphatases and phosphodiesterases in which a fluorescently labeled Substrate is reacted by a corresponding enzyme. In present a polycationic polymer undergoes a change the fluorescence intensity and decay time, which was used to quantify enzyme activity can be. A summary of older methods for determination These and many other enzymes can be found in the book Enzymatic Methods of Analysis by G.G. Guilbault, Pergamon Press, 1970. They all are not based on the use of lanthanide chelate complexes.

The use of lanthanide chelate complexes (LCK) in bioanalytics is comparatively new. The UP-PS 5854008 (Diamandis) describes chelators which bind to Eu ³⁺ and Tb ³⁺ ions and are converted by enzymes. This results in a change in the fluorescent properties of the LCK, and this can be used to quantify the enzyme. The process, referred to as EALL (enzyme-amplified lanthanide luminescence), is also well described in Evangelista et al .; Analytical Biochemistry 197 (1991) 213-224. In contrast to the method newly described here, the enzyme substrate must be fluorogenic in EALL, ie converted by the enzyme into a fluorescent product. This severely restricts the available enzyme substrates, requires considerable synthetic effort, and, because of the non-natural nature of the enzyme substrates, often results in very slow conversion rates. In addition, they require the presence of EDTA (ethylenediamine tetraacetate). In a very similar embodiment (US Pat. No. 5,312,922; Diamandis), 4-methylcumarinyl-7-phosphate is used as the enzyme substrate and detected by a change in the fluorescence of an LCK complex in the presence of EDTA. The method is also applicable to other enzymatic determinations, e.g. B. of glucose (Weber & Karst, Analyst 125 (2000) 1537-1538) transferable.

such Enzyme determination procedures must are delimited from those in which the LCK as fluorescent Markers for Proteins are used. This is mainly used in enzyme immunoassays use (Diamandis et al., J. Immunolog. Methods 112 (1988) 43-52).

All of the above methods using LCK require (a) the presence of EDTA, which is disadvantageous in biological systems, because EDTA has many divalent ions essential for enzyme function (e.g., Ca ²⁺ , Mg ²⁺ , Zn ^{2 +} ) binds; (b) the use of a synthetic and thus often difficult to access fluorogenic substrate as a ligand, and (c) hydrolysis of the substrate at the ternary LCK, which often proceeds very slowly. In addition, they require an excitation wavelength of about 330 nm, which gives rise to significant background fluorescence, since this is particularly strong in the UV. The search for suitable ligands (= substrates) is difficult, and improved ligands are the subject of U.S. Patent No. 5,854,008 (Diamandis).

Of the above disadvantages, the process described here is free: (a) the presence of EDTA not required (yes, even detrimental); (b) the chelator of the LCK used is independent of the enzyme table boundary conditions freely selectable; (c) the ligand used in the LCK is not the enzyme substrate; (d) the organic product formed by enzymatic reaction is also not the ligand (as in EALL). In the method described here, enzyme substrate and LCK form two separate and chemically defined compounds. Also in contrast to the methods described, the reagent (the LCK) can be added before, during, but also at the end of the enzymatic reaction. Addition of the LCK at the end of the reaction has the advantage that the reagent can not adversely affect the course of the enzymatic reaction. Finally, because of its novel detection scheme, the method described here (in contrast to the methods described so far) is applicable not only to dephosphorylating but also to phosphorylating enzymes (eg kinases) and to phosphodiesterases and the excitation wavelengths can be up to be extended to 430 nm.

4. The lanthanide chelate complexes of the invention (LCK)

fluorimetrisch nachweisen kann. Hier steht

Ln³⁺

für ein 3-wertiges Ion der Elemente Europium, Terbium, Samarium, Lanthan, Dysprosium oder Holmium,

Lig

für einen beliebigen Liganden oder Chelator, der (a) ein Absorptionsmaximum zwischen 330 und 450 nm aufweist, (b) die Lumineszenz des Ln(III)-Ions nicht löscht, und (c) weder Substrat noch Produkt einer enzymatischen phosphorylierenden oder dephosphorylierenden Reaktion ist,

wobei das Verhältnis x:y zwischen 10:1 und 1:3 liegen kann.The method is based on the new finding that inorganic or organic phosphate by the addition of lanthanide chelate complexes (LCK) of the following general chemical structure

can detect fluorimetrically. It says

Ln ³⁺

for a trivalent ion of the elements europium, terbium, samarium, lanthanum, dysprosium or holmium,

Lig

for any ligand or chelator that (a) has an absorption maximum between 330 and 450 nm, (b) does not quench the luminescence of the Ln (III) ion, and (c) is neither substrate nor product of an enzymatic phosphorylating or dephosphorylating reaction .

where the ratio x: y can be between 10: 1 and 1: 3.

The Photoluminescence of the LCK becomes common excited at 330 to 450 nm and at wavelengths between 450 and 800 nm measured. The inventive method are not based on chemiluminescence. The fluorescence of such LCK is enhanced by phosphates and phosphate esters, the average cooldown usually extended. Throughout this document the term fluorescence is used, although in the literature the issue of LCK is occasionally referred to as Phosphorescence or photoluminescence is called.

preferred Lanthanide ions are those of europium and terbium. preferred Ligands are β-diketones such as the benzoyl trifluoroacetone, thenoyl trifluoroacetone, heterocyclic -carboxylic acids (ortho-hydroxy) including of 4-quinolone-3-carboxylic acids and their derivatives, aromatic or heterocyclic ortho-hydroxyketones and their derivatives, hydroxyquinones and partially hydrogenated and substituted Hydroxyquinone-type compounds including tetracycline and its derivatives, as well as 2,2'-dipyridyls or 1,10-phenanthrolines, as well as 8-hydroxyquinolines. Preference is furthermore given to complexes, their fluorescence at over 360 nm can be excited and their decay times are over 10 microseconds.

The Emission bands of the LCK according to the invention are customary very narrow and their maxima are usually between 450 and 780 nm. For example, the emission of the Eu (III) -tetracycline complex shows a characteristic narrow emission with a maximum at 616 nm. The terbium complex with the N-methyl-4-quinolone-3-carboxylic acid shows the typical terbium emission with a narrow peak at 545 nm. The luminescence of these complexes is most effective with light wavelength between 320 and 420 nm are excited. This happens in the claimed Method preferably by means of a light emitting diode or a laser diode.

5. The determination according to the invention the activity of phosphatases

To determine the activity of a phosphatase, the phosphatase-containing solution is mixed with any natural or synthetic phosphate ester. The following reaction takes place afterwards:

Here is phosphate ester (ie the enzyme substrate) for any organic phosphate, eg. For phenylphosphate or glucose phosphate, while the product is dephosphorylated by the enzyme Ester (ie, for example, phenol or glucose) is. P _anorg stands for the (inorganic) phosphate ion. Inorganic phosphates form weak complexes with the LCK reagents according to the invention whose fluorescence-optical properties differ measurably from those of the LCK.

Of the Addition of the LCK may occur before, during or after the enzymatic reaction. Then you determine the change occurred the intensity or the cooldown of the LCK emission at wavelengths between 450 and 800 nm. Typical reaction times are between 5 and 30 Minutes.

The Procedure can of course also for Determination of the efficacy of inhibitors of phosphatases (P-asen) be applied. This is when screening for potential drugs really important. When screening for inhibitors of P-asen goes in itself in the same way as in determining the activity of P-asen before, but you now determine the change the absorption or fluorescence of the reagent compared to a Solution, although a certain amount of P-Ase, but no inhibitor added had been. Naturally, the product is formed in greater concentration, the less the activity of the Phosphatase is inhibited.

6. The determination according to the invention the activity of kinases

The indicators of the invention can also be used to determine the activity of kinases. To this end, the kinase-containing solution is mixed with any natural or synthetic enzyme substrate, typically a protein with a hydroxy amino acid (eg, serine, threonine or tyrosine). A typical kinase reaction is shown schematically below:

to Determination of the conversion is added to the sample before, during, or after the reaction time with an LCK according to the invention. Then you determine the change occurred the fluorescence properties of the LCK at wavelengths between 450 and 800 nm. Typical reaction times are between 5 and 60 minutes.

The cited Procedures may also serve to increase the effectiveness of inhibitors of To detect kinases. For this purpose, the above reaction solution before the addition of the natural or synthetic enzyme substrate additionally mixed with a substance, be tested for its effectiveness as inhibitor of kinase should. It is no longer determined the change in the fluorescence of the LCK compared with a solution, the no kinase activity owns, but the change in comparison with a solution, Although a certain amount of kinase, but no inhibitor added had been.

Example: Determination of activity alkaline phosphatase

The invention will be explained with reference to the following example. Therein, the Eu ³⁺ / tetracycline / EuTc) is used as a fluorescent phosphate probe, which allows to detect the phosphate released by the alkaline phosphatase via the increase in the fluorescence intensity or the change in the decay time:

Reaction Principle:

• (1) phenyl phosphate == (alk-P-ase) ===> phenol + phosphate
• (2) Phosphate + EuTc ==> EuTc-phosphate complex

Used solutions

• MOPS Buffer (Solution A): 3.00 g of MOPS Na salt are dissolved in 990 mL bidistilled water, adjusted to pH 8 with 72% HClO ₄ (pA) and made up to 1000 mL.
• EuTc stock solution (solution B): 2.11 mg tetracycline × HCl and 4.83 mg EuCl ₃ .6H ₂ O are dissolved in solution A and made up to 50 mL.
• Alkaline Phosphatase stock solution (solution C): 5.21 mg alkaline Phosphatase are in solution A dissolved and made up to 25 mL (alkaline phosphatase 24 units = 1 mg)
• Alkaline phosphatase solution (Solution D): 125 μL solution C are made up to 10 mL.
• Phenylphosphatlösung (Solution E): One dissolves 6.35 mg phenylphosphate in 25 mL solution A.
• magnesium acetate (Solution F): 53.6 mg of magnesium acetate tetrahydrate are dissolved in solution A and dissolved 50 mL refilled.

protocol

A microplate is successively filled with solutions D (64 μL), E (20 μL), A (6 μL) and F (60 μL) and incubated for 10 minutes at 25 ° C. Then solution B (50 μL) is added and the fluorescence intensity is measured after 10 min at 25 ° C. The fluorescence is excited at 398 nm and the emission is measured at 617 nm. The linear range is in the range of 1.2-10 mU / mL. The calibration curve ( 1 ) shows a dynamic range of 0-10 milli-units / mL.

Used devices

to Measurement of fluorescence intensity Tecan GENios + Microplate Reader (Tecan, Groedig, Austria, www.tecan.com) and the Fluostar Optima (BMG LabTechnologies, Germany) used. The excitation and emission filters of the Tecan GENios were at 405/612 nm, and that of the Fluostar Optima at 400-410 / 612 set. With both devices was a lag-time 40 μs and an integration time of 100 μs set. The microplates used were 96-well microplates from Greiner Bio-One GmbH (Frickenhausen, www.greiner-lab.com).

1 , Calibration curve for determining the activity of a phosphatase by determining the enzymatically liberated phosphate by means of the method according to the invention using a europium-chelate complex whose fluorescence is increased by phosphate. F ₀ is the intensity of the fluorescence before the reaction, F is the fluorescence after enzymatic reaction or after a 10-minute incubation period with the reagent EuTc. It can be seen that under the chosen conditions, the activity of the P-ase can be determined in the range of about 2 to 10 milli-units per milliliter. If an additional inhibitor of the enzyme has been added to the system, a correspondingly lower activity is found.

Claims (11)

Process for the fluorescence-optical determination of the activity of phosphatases, phosphodiesterases or kinases, characterized in that a reaction catalyzed by said enzymes is carried out using any natural or synthetic enzyme substrate and the conversion of the reaction is determined by (a) dissolving before, during or after the reaction with a reagent consisting of a complex of a 3-valent lanthanide ion with an organic ligand, which itself is not an enzyme substrate, (b) the reagent stimulates photoluminescence by irradiation with light, (c) the intensity or the cooldown of the photoluminescence of the reagent is determined, and (d) the change in fluorescence properties of the reagent due to the reaction is used to detect and determine the particular enzyme. Method according to claim (1), characterized that the enzyme is a phosphatase and the enzyme substrate is a phosphate ester glycerol, glucose, ribose or deoxyribose, a nucleoside, of serine, threonine, tyrosine or farnesol, creatinine, or any other synthetic or bioorganic Alcohol or phenol is. Method according to claim (1), characterized that the enzyme is a phosphatase and the phosphate ester is a diphosphate or a triphosphate. Process according to claim (1), characterized in that the enzyme is a phosphodiesterase and the phosphate ester is a cyclic mono-nucleotide such as e.g. B. the cyclic adenosine monophosphate or in the case of nucleases is a polynucleotide. Method according to claim (1), characterized that the enzyme is a kinase and the enzyme substrate optionally protein-bound serine, threonine or tyrosine, the creatinine, glycerol, the glucose, a ribose or deoxyribose, a nucleoside, or another synthetic or organic mono- or polyalcohol or a phenol. Process according to claim (1), characterized in that the reagent has the following general chemical structure:

wherein Ln ^{3+ is} a trivalent ion of the elements europium, terbium, samarium, lanthanum, dysprosium or holmium, and Chel is any chelator of the central ion having (a) an absorption maximum between 300 and 450 nm, (b) does not quench the luminescence of the Ln (III) ion, and (c) is neither the substrate nor the product of the particular enzymatic phosphorylating or dephosphorylating reaction, the ratio x: y being between 10: 1 and 1: 3 , Method according to claim (6), characterized the lanthanide ion is a europium (III) or terbium (III) ion is. Method according to claim (6), characterized that the chelator is a hydroxylated heterocycle or or chelatisierendesβ-diketone (or its enol) is, Method according to claim (8), characterized that the chelator is any coumarin, chromone or quinolone-3-carboxylic acid or which is tetracycline or one of its derivatives. Method according to claim (1), characterized that it is used to determine the effectiveness of enzyme inhibitors becomes. Method according to claim (1), characterized that with its help one determines the activity of an enzyme, the to a solid support, to a biological membrane, to another protein, e.g. B. to a Antibody, or a natural one or synthetic polynucleotide bound.