Method of identifying catechol o-methyltransferase modulators

FIELD: chemistry.

SUBSTANCE: invention relates to medicine and describes method of identifying modulators of enzyme catechol-O-methyltransferase (COMT) activity, including a) obtaining 4-nitrocatechol, covalently bound with Alexa Fluor® 488, b) bringing molecule from stage a) in contact with enzyme catechol-O-methyltransferase (COMT), S-adenosylmethionine (SAM) and candidate compound and c) measurement of indices of fluorescence of mixture from stage b), in which changed indices of fluorescence in presence of candidate compound in comparison with the control serve as sign of presence of modulator of enzyme catechol-O-methyltransferase (COMT). Also described is method of identifying substrate of enzyme catechol-O-methyltransferase (COMT).

EFFECT: invention can be used for identification of compounds, inhibiting enzyme COMT, to determine COMT activity in samples of animal tissues.

10 cl, 3 ex, 7 dwg

The present invention relates to a method of identifying modulators of the activity of the enzyme catechol-O-methyltransferase.

Catechol-O-methyltransferase (COMT) catalyzes the O-methylation of substrates that have catahoula part. The methyl donor in methylation, which performs COMT is S-adenosylmethionine (SAM). COMT plays an important role in the catabolism of endogenous catecholamine neurotransmitters, catecholestrogens and xenobiotics molecules. Inhibition of COMT is an important approach in the development of new therapeutic methods for the treatment of Parkinson's disease.

W. F. Herblin (Analytical Biochemistry 51, 19-22, 1973) describes a colorimetric assay for assessing the activity of COMT. In this analysis, as the methyl acceptor for somt used nitrocatechol. Nitrocatechol exists in the form of a yellow solution in water at acidic pH with a maximum absorption at a wavelength of 350 nm. When a small alkaline ionization pH para-hydroxyl change the solution on orange (λmax = 430 nm). When a stronger alkaline ionization meta-hydroxyl change the solution to cherry red (λmax = 520 nm). The analysis is based on the observation that nitrocatechol metiliruetsa using COMT and that methylated nitrocatechol shows not cherry-red color in the second ionization. In this assay substrate (nitrocatechol) and SAM should be in µm is oncentrated in the range which is at or above Km, which limits the sensitivity.

G. Zurcher and M. Da Prada (Journal of Neurochemistry, Vol.38, No. 1, 1982) describes a one-step radiochemical analysis for assessing the activity of COMT. In this analysis catechol turned into guaiacol a tritium labeled compound with a very low polarity, by incubation of COMT with [³H]methyl SAM, Mg²⁺and adenoidectomies. Guaiacol extracted using a medium of low polarity such as toluene, and counted in scintillation counters.

The above analyses are not intended for automated screening large numbers of compounds to assess their modulating effect on the activity of COMT due to limited sensitivity (colorimetric analysis) or in connection with the structure analysis (phase extraction in radiochemical analysis).

Thus, there is a need for sensitive homogeneous method of analysis suitable for screening large numbers of compounds in terms of their modulation of the activity of COMT.

First and foremost, the present invention provides a method of identifying modulation of the activity of the enzyme catechol-O-methyltransferase (COMT) that includes the following steps:

a) obtaining a substrate somt with covalently bound fluorescent dye,

b) interaction of the molecule of step (a) with fermentopathy-O-methyltransferase (COMT), S-adenosylmethionine (SAM) and the connection candidate and

C) measuring the fluorescence of the mixture from step b) in which the changed rates of fluorescence in the presence of the connection candidate compared to the control is indicative of a modulator of the enzyme catechol-O-methyltransferase (COMT).

In the preferred embodiment of this method is a method for identifying inhibitors of COMT in which low fluorescence in stage C), in comparison with the control, is indicative of an inhibitor of COMT.

In yet another preferred embodiment of the substrate for COMT is a 4-nitrocatechol.

In yet another preferred embodiment of a fluorescent dye is Alexa Fluor ® 488.

In yet another preferred embodiment of the fluorescence indicators in stage C) are the kinetic parameters.

In yet another preferred embodiment of COMT is human COMT.

In yet another preferred embodiment of the method is high-throughput screening method.

In another preferred embodiment the method is performed on the microplate.

In another preferred embodiment the final concentration of COMT is approximately 25 nm.

In another preferred embodiment the final concentration of substrate somt faced the t of approximately 200 nm.

In another preferred embodiment the final concentration of SAM is about 500 nm.

In the second place, the present invention provides a method of identifying a substrate for the enzyme catechol-O-methyltransferase (COMT) that includes the following steps:

a) obtaining a mixture containing the substrate for COMT, covalently linked to a fluorescent dye and S-adenosylmethionine (SAM),

b) contacting the mixture from step a) with different concentrations of compound candidate,

C) contacting the mixture from step b) with the enzyme catechol-O-methyltransferase (COMT) and

g) measurement of the kinetic parameters of the fluorescence of the mixture from stage b), in which the decrease of the plateau fluorescence as a function of increasing concentration of the compound candidate is indicative of the substrate of the enzyme catechol-O-methyltransferase (COMT).

In the preferred embodiment the substrate COMT is a 4-nitrocatechol.

In yet another preferred embodiment of a fluorescent dye is Alexa Fluor ® 488.

A brief description of the figures.

Fig.1 shows the chemical structure of Alexa Fluor ® 488, covalently linked with a 4-nitrocatechol;

Fig.2 shows a constant quenching stern-Volmer for nitrocatechol (blue), 2-methoxy-5-NITROPHENOL (red) and 1,2-dimethoxy-4-nitrobenzene (green); 20 nm free the aqueous Alexa Fluor 488 were mixed with high - up to 25 mm concentrations of nitrocatechol, 2-methoxy-5-NITROPHENOL and 1,2-dimethoxy-4-nitrobenzene, respectively. Only nitrocatechol observed change in fluorescence intensity (I₀/I) Alexa Fluor ® 488, methylated products do not affect the fluorescence intensity of Alexa Fluor ® 488.

Fig.3 shows the enzyme kinetics of methylation Alexa Fluor 488 - nitrocatechol catalyzed SAT, fluorescence analysis of the present invention;

Fig.4A shows the measurement of the kinetics of changes in fluorescence intensity in the presence of various concentrations of inhibitor of COMT Tolcapone;

Fig.4B shows the dose-dependent curve for Tolcapone calculated by the slope of the kinetic measurements in Fig.3A;

Fig.5 shows fluorescence in the presence of dopamine, a natural substrate of COMT. The lower plateau was achieved by increasing the concentration of dopamine. Since dopamine is a substrate, it metiliruetsa as the substrate Alexa Fluor ® 488-nitrocatechol. Availability limited SAM (500 nm), so that at high concentration of dopamine substrate Alexa Fluor ® 488-nitrocatechol can no longer be fully methylated.

Fig.6 shows the dose-dependent curves for the substrate with a low (500 nm) and high (200 μm) concentration of SAM, and for each concentration of SAM the presence and absence of pre-incubation with the organisations and SAM for one hour, prior to adding substrate Alexa Fluor ® 488-nitrocatechol. Pre-incubation with low concentrations of SAM shifts the curve to the lower IC₅₀because the connection uses all available SAM. At high concentrations SAM there is no difference between the presence and absence of pre-incubation, because SAM is not the limiting factor and the curve is shifted to larger IC₅₀compared to the low concentration of SAM due to the depletion of the connection.

Fig.7 shows the dose-dependent curves with a low (500 nm) and high (200 μm) concentration of SAM again with the presence and in the absence of a one hour pre-incubation of the compounds with SAM from SAM-competitive compounds. For SAM competitive compounds the presence and absence of pre-incubation does not affect the IC₅₀for each concentration of SAM, but at high concentrations SAM IC₅₀shifting to larger values.

Detailed description of the invention.

The analysis of the present invention based on the findings that a fluorescent dye covalently associated with the substrate for COMT, such as Alexa Fluor ® 488 covalently associated with nitrocatechols shows the decrease in fluorescence due to intramolecular quenching and that methylation of the substrate COMT in the complex substrate somt - fluorescent dye with the help of SAT from Estet quenching of fluorescence, i.e. methylation sustrate of COMT in the complex substrate somt - fluorescent dye leads to an increase in fluorescence compared to demetilirovanny complex.

The term "SAT" is used herein to denote a natural sequence of somt from any animal, for example mammalian species, including humans, and variants of COMT (defined below). The polypeptides of COMT can be selected from a variety of sources, including types of human tissues or prepared by recombinant or synthetic methods.

Natural or recombinante-received AMT can be used in this analysis. "Recombinant protein" is a protein isolated, purified or identified in the force expression in heterologous cells, said cells were transpulmonary or transliterowany, either temporarily or consistently, using a recombinant expressing vector, designed for the control of expression of the protein in the cell host. Recombinant human COMT can be produced in prokaryotic cells, such as E. coli, yeast such as S. pombe, or in eukaryotic cells, for example, HEK 293 (human embryonic kidney transformed by the DNA of adenovirus), insect cells Sf9. Preferably, the cells are insect Sf9 used high expression of recombinant COMT. Somt used in the analysis, can be cleaned. The term "purified" as used herein refers to polypeptides that are removed from their natural environment or from a source of recombinant production, isolated or separated, at least 60% and more preferably at least 80% free from other components, such as membranes and microsomes, with which they are naturally associated.

"Natural sequence of somt" refers to a polypeptide having the same amino acid sequence as the polypeptide of COMT occurring in nature, regardless of its method of preparation. The natural sequence of COMT can be isolated from nature or produced using recombinant and/or synthetic methods. The term "natural sequence of somt" more accurately encompasses naturally occurring truncated or secreted forms, naturally occurring variants of forms (e.g., alternative splicing) and naturally occurring allelic variants of COMT. ID polypeptide of human COMT in the NCBI database is AAA (Seq. Id. No. 1).

The term "variant of COMT" refers to the variant amino acid sequences of the natural sequences of COMT containing one or more amino acid substitution, and/or d is leziy, and/or insert in natural sequence. Variants of the amino acid sequence, typically at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, most preferably at least about 95% identical to the sequence of the natural amino acid sequence of SAT.

The term "connection" is used herein in the context of a "test compound" or "drug compound candidate" described in connection with the analyses of the present invention. Essentially, these compounds contain organic or inorganic compounds, obtained synthetically or from natural sources. Compounds include inorganic or organic compounds, such as polynucleotide, lipids or analogues hormones, which are characterized by relatively low molecular weight. Other biopolymer organic test compounds include peptides containing from about 2 to about 40 amino acids, and larger polypeptides containing from about 40 to about 500 amino acids, such as antibodies or conjugates of antibodies.

The term "kinetic rate" refers to the difference between fluore the interest signal, measured at two specific points in time in the linear part of the enzymatic reaction. One measurement is made at the beginning of the enzymatic reaction (start point) and the second indicator is removed after the incubation period (the end point). The final signal is calculated in OEF / min (OEF (endpoint) - OE (start point)) / incubation period. (OEF: relative fluorescence units).

The method of the present invention can be used to identify compounds that inhibit the enzyme catechol-O-methyltransferase (COMT). Thus, inhibitors of COMT determined by the method of the present invention can be used in methods of treatment, prevention or control of diseases in which plays the role of decontamination extraneuronal of catecholamines at the expense of COMT, for example, for the prevention or control of depression. In this case, the compounds according to the invention can be used as individual compounds or in combination with other therapeutically active substances that have a positive impact on the disease. Compounds according to the invention can also be used as one of the drugs together with other therapeutically active substances.

The method of the present invention may be used to determine the activity of COMT in education is Zach animal tissues, which was introduced by the test compound. For example, the analysis is suitable for determining the activity of COMT in samples of brain and liver tissue from animals such as mice and rats that were treated with the test compound (modulator of somt).

The experimental part.

Synthesis of 4-nitrocatechol - Alexa Fluor ® 488

10 mm solution Aminoethyl-nitro-benzathin [1] in DMSO containing 1% of Triethylamine were mixed with 10 mm solution of Alexa Fluor ® 488 Succinimidyl ether carboxylic acids [2] (Invitrogen Corporation, 5791 Van Alien Way, Carlsbad, California 92008) in DMSO with 1% Triethylamine in a stoichiometric ratio of 1:1. The reaction mixture was gently stirred overnight at room temperature and was purified on an Akta Explorer 100 reverse-phase HPLC. Product liofilizirovanny and resuspendable in DMSO.

4-Nitrocatechol-Alexa Fluor® 488

The Protocol for fluorescence analysis.

The following Protocol analysis, reagents and materials were used in examples of the present invention. The results of the examples described in figures 1-7.

Microplates:

384-hole microplate, Corning black with flat clear bottom, non-binding surface, polystyrene (ref. 3655)

Reagents and buffer solutions:

Buffer solutions:

- M phosphate buffer pH of 7.6 (Na₂HPO4 Fluka 71644, NaH₂PO4 Merck 6346.0500), stored at 4°C

- 580 mm MgCl₂(Merck 1.0833.0250), stores the ri room temperature

- 1 M CaCl₂stored at 4°C

- 65 mm DTT (Sigma D-0632), stored at -20°C

Recombinant human COMT: self-made, stored at -80°C

- 4-Nitrocatechol-Aeha Fluor 488: self-made, 1.3 mm in DMSO, stored at room temperature in the dark

- S-Adenosyl-methionine: 10 mm Nad (Sigma-Aldirch A2804), stored at -20°C

Reagents and buffer solutions:

Buffer for analysis (final concentration):

- 40 mm Phosphate buffer pH 7,6

- 2,88 mm MgCl₂

0.9 mm DTT

- 0.25 mm CaCl₂

- Cultivation connection: cultivation in 100% DMSO (Sigma 41640), and 6.25% final concentration of DMSO in the analysis

- Rivers. human COMT: 80 nm in buffer for analysis, 25 nm final concentration for analysis

- 4-Nitrocatechol-Alexa Fluor ® 488: 320 nm in the buffer for analysis, 200 nm final concentration for analysis

- S-Adenosyl-methionine: 800 nm in buffer for analysis, 500 nm final concentration for analysis

Method of analysis:

10 μl hCOMT (human COMT)

2 μl of the test compounds

1 min on a shaker

20 μl of a mixture of substrate-SAM

5 minutes on the shaker

Meter readings: kinetic measurements on the plateau: the TM image reader (absorption 475(40) nm, emission 535(45) nm), the intensity of 7.5%, the exposure time of 1 sec.

1. A method for identifying modulators of the activity of the enzyme catechol-O-acyltransferase (COMT), includes the following stages:
a) obtaining 4-nitrocatechol, covalently linked to Alexa Fluor® 488,
b) bringing into contact of the molecule of step (a) with the enzyme catechol-O-methyltransferase (COMT), S-adenosylmethionine (SAM) and the connection candidate and
C) measuring the fluorescence of the mixture from step b) in which the changed rates of fluorescence in the presence of the connection candidate compared to the control is indicative of a modulator of the enzyme catechol-O-methyltransferase (COMT).

2. The method according to p. 1, where the method is a method of identifying an inhibitor of COMT and the decrease of fluorescence on stage) compared to the control is indicative of an inhibitor of COMT.

3. The method according to p. 1, where the level of fluorescence on the stage) are the kinetic parameters.

4. The method according to p. 1, where COMT is a human COMT.

5. The method according to p. 1, where the method is by high-throughput screening method.

6. The method according to p. 1, where the method is performed on the microplate.

7. The method according to p. 1, where the final concentration COMT is approximately 25 nm.

8. The method according to p. 1, where the final concentration of substrate of COMT is approximately 200 nm.

9. The method according to any of paragraphs.1-10, where the final concentration of SAM is about 500 nm.

10. How to identify the substrate of the enzyme catego is-O-methyltransferase (COMT), includes the following stages:
a) obtaining a mixture containing 4-nitrocatechol associated with Alexa Fluor® 488, and S-adenosylmethionine (SAM),
b) bringing into contact the mixture from step a) with different concentrations of the connection candidate
C) bringing into contact the mixture from step b) with the enzyme catechol-O-methyltransferase (COMT) and
g) measurement of the kinetic parameters of the fluorescence of the mixture from stage b), where the decrease of the plateau fluorescence as a function of increasing concentration of the compound candidate is a sign of the presence of the substrate of the enzyme catechol-O-methyltransferase (COMT).