RU2443710C1 - Cyclic nonapeptide showing ability to inhibit myosin light chain kinase - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to biologically active peptides able to inhibit myosin light chain kinase and thereby regulate the variability of vascular endothelial permeability. What is offered is a cyclic nonapeptide of formula cyclo[-Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-].

EFFECT: peptide can find application as a decongestant in various fields of medicine.

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Description

The present invention relates to biologically active peptides that can inhibit the kinase of light chains of myosin and thereby prevent an increase in the permeability of the vascular endothelium, and can be used in medicine as a decongestant.

Edema of vital organs (lungs, brain, etc.) is a formidable complication of various pathological conditions that cardiologists, infectious disease specialists, toxicologists, neurosurgeons, specialists in the field of field medicine and disaster medicine often encounter. The development of pulmonary edema is an indication for premature withdrawal of aggressive therapy in some groups of cancer patients. Tissue edema is a negative side effect of therapeutic angiogenesis with the help of vascular endothelial growth factor, leading to the formation of the so-called "Current" capillaries.

Despite advances in medical technology, mortality from acute pulmonary edema in Russia and the developed countries of the world remains high, which is associated, in particular, with the lack of effective decongestants. Today, the doctor’s arsenal is limited to diuretics - substances that improve the excretion of fluid from the body through the kidneys, crystalloids that increase intravascular osmotic pressure, and steroid hormones. These compounds do not affect the main pathogenetic link in the development of edema - the endothelial lining of microvessels.

According to modern concepts, in order to effectively combat the development of acute edema and postischemic tissue damage, it is necessary to weaken the contractile response of the microvascular endothelium, affecting the critical links of this process, namely, the non-muscular type II myosin and / or its activators, the main motor endothelial protein. A key activator of type II myosin in endothelial and other cells is the kinase of myosin light chains (MLC, KF 2.7.11.17). MLCK is involved in the regulation of various motor reactions, such as contraction of smooth muscles, mobility of neutrophils, fibroblasts, and permeability of endothelial and epithelial monolayers. MLCK inhibitors reduce cell motility and therefore can be used in medicine in a number of pathological conditions, in particular as a means to reduce the increased permeability of the microvascular endothelium and prevent acute edema of vital organs [1].

However, most of the previously synthesized MLCK inhibitors are not suitable for use in humans due to low specificity, which can lead to serious side effects. In this regard, the search for highly specific MLCK inhibitors is very relevant.

One of these compounds is a small molecule inhibitor based on aminopyradazine [2-4]. In recent years, peptide MLCK inhibitors have been developed in the world that affect the permeability of intestinal epithelium in the experiment and do not have pronounced toxicity [5]. The known nonapeptide is a derivative of the sequence of the autoinhibitory site of the MLCK H-Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH ₂ (L-PIK) [6, 7]. This peptide in vitro is able to influence the permeability of cells of the intestinal epithelium [7]. However, due to the peculiarities of its chemical structure, it is extremely susceptible to the action of proteolytic enzymes and therefore is hardly able to be active in vivo and cannot be used in practical medicine. Also known is the nonapeptide amide of the formula H- (N ^α -Me) -Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH ₂ [8, 9] (selected as a prototype), capable of preventing acute increase in vitro permeability of vascular endothelium. This peptide is more resistant to the action of proteolytic enzymes than L-PIK, since it contains the residue of N ^α -methylarginine in the N-terminal part, which increases its resistance to aminopeptidases. However, it should be noted that the synthesis of N-methylated analogues of amino acids in general, and arginine derivatives in particular, is a multi-stage, time-consuming and expensive process, which significantly increases the cost of such compounds [10].

The objective of the invention is the search for compounds that would have a pronounced ability to inhibit the kinase of light chains of myosin and thereby regulate the change in the permeability of the vascular endothelium, as well as technologically more accessible, which in the future, with a greater probability, will provide domestic practical medicine with effective anti-edematous drugs.

The problem is solved by synthesizing a cyclic nonapeptide of the formula

cyclo [-Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-].

Since there is no clear correlation between structure and activity in the series of peptide analogues, the ability of the selected compound to inhibit the kinase of myosin light chains is not obvious. The inventive peptide was obtained by solid-phase peptide synthesis using Fmoc technology, then it was cyclized by amide, head-to-tail, N- and C-terminal amino acids by the method described in Example 1 below.

Example 1. Synthesis of the claimed peptide

cyclo [-Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-] (I)

List of abbreviations:

AcOH - acetic acid;

Boc is tert-butyloxycarbonyl;

BOP - O- (benzotriazol-1-yl) -tris-dimethylamino) phosphonium hexafluorophosphate;

Bu ^t is tert-butyl;

DIC - N, N'-diisopropylcarbodiimide;

DIPEA - N, N-diisopropylethylamine;

DCM - dichloromethane;

Fmoc is 9-fluorenylmethoxycarbonyl;

HOBt - 1-hydroxybenzotriazole;

Me is methyl;

Mops - 3- [N-morpholino] propanesulfonic acid;

NMP - N-methylpyrrolidone;

Pip - piperidine;

Pmc - 2,2,5,7,8-pentamethylchroman-6-sulfonyl;

TIBS - triisobutylsilane;

TFA - trifluoroacetic acid;

HPLC - high performance liquid chromatography;

DTT - dithiothreitol;

KLCM - kinase of light chains of myosin;

RLC - regulatory myosin light chains;

EGTA - ethylene glycol bis (2-aminoethyl ether) N, N, N ', N'-tetraacetic acid.

Derivatives of L-amino acids (Bachem, Switzerland), DIC, DIPEA, HOBt, TIBS (Fluka, Switzerland) were used in the work. For the synthesis, N-methylpyrrolidone, dichloromethane, piperidine, methanol and TFA (Applied Biosystems GmbH, Germany) were used. Analytical HPLC was performed on a chromatograph (Gilson, France), a Nucleosil 100 C18 column, 5 μm (4.6 × 250 mm) (Sigma, USA) was used, buffer A — 0.1% TFA, buffer B — 80% acetonitrile in buffer was used as eluents A, elution with a concentration gradient of buffer B in buffer A from 0% to 60% in 30 minutes A flow rate of 1 ml / min, detection at 220 nm. For thin layer chromatography, Merck silica gel plastics (Germany) and chloroform – MeOH – AcOH (90: 10: 5) chromatographic systems (system 1) and chloroform – MeOH — 32% AcOH (15: 4: 1) (system 2) were used. . For the development of chromatograms, a ninhydrin solution and a chlorobenzidine reagent were used. The structure of the obtained peptides was proved by ¹ H-NMR spectra and mass spectrometry data. Mass spectra were obtained on a VISION 2000 time-of-flight mass spectrometer (Termobioanalsis corp., Finnigan, USA) equipped with a Class 3B pulsed nitrogen laser with a radiation wavelength of 337 nm, using the MALDI method using a 2,4,6-trihydroxyacetophenone matrix.

The semi-protected linear precursor of the nonapeptide H-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Tyr (Bu ^t ) -Lys (Boc) -Tyr (Bu ^t ) -Arg (Pmc) -Arg (Pmc) -Lys ( Boc) -OH (II) was obtained by the automatic solid-phase method. For solid-phase synthesis, a copolymer of styrene with 1% divinylbenzene with 2-chlorotrityl chloride-anchor group (2-Clorotrityl chloride resin) from Iris Biothech (Germany) was used, intended for the preparation of protected peptides with a free carboxyl group containing 1.56 mmol Cl / g polymer . The starting amino acid, Fmoc-Lys (Boc) -OH, was attached to the polymer carrier by double treatment (2 times for 1 h) with the corresponding diisopropylethylammonium salt of the amino acid in dichloromethane [11]. The residual chlorine was cleaved off by treating the aminoacyl polymer with a 10% solution of DIPEA in methanol (2 times 1 min), then the aminoacyl polymer was washed successively with DMF (3 × 1 min), i-PrOH (3 × 1 min), DMF (3 × 1 min), MeOH (3 × 1 min), DCM (3 × 1 min) and dried. The degree of substitution of the carrier polymer with the starting amino acid, determined spectrophotometrically [12], was 0.42 mmol of the amino acid / g of the aminoacyl polymer. The nonapeptide was synthesized from the C-terminus, stepwise (adding one amino acid each), starting from 0.43 g (0.25 mmol) of the Fmoc-Lys (Boc) polymer. The next 8 cycles of synthesis were performed automatically on an Applied Biosystems 431A peptide synthesizer according to the standard program for a single condensation of Fmoc amino acids. Pmc protection was used to block guanidine groups of arginine residues, Boc protection for ε-amino groups of lysine, and tert-butyl protection for tyrosine hydroxyl groups. A 25% solution of 4-methylpiperidine in NMP was used to cleave Fmoc protections (See Solid-State Synthesis Protocol).

Solid Phase Synthesis Protocol No. Operation Reagent Time of processing Cycles 1-8 one Flushing 5 × NMP 3 min 2 The release of b-amino groups 25% 4-MePip / NMP 10 min 3 Flushing 5 × NMP 3 min four Activation 1 mmol Fmoc-AA + 1 mmol HOBt + 1 mmol DIC in NMP 20 minutes 5 Condensation 1 mmol activated derivative Fmoc-AA in NMP 90 min 6 Flushing 5 × NMP 3 min

To control the quality of the synthesized linear nonapeptide precursor, the peptidyl polymer sample (50 mg) was completely unlocked by sequential treatment of 1) 25% 4-MePip / NMP solution to remove the Fmoc group, 2) TFA with scavengers to remove all other protections and the polymer carrier. The obtained product, H-Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-OH, judging by analytical HPLC, contained 94.6% of the target substance (R _t - 8.7 min, Kromasil column 4.6 × 25 mm, 5 μm, detection at 220 nm; buffer A - 0.1% aqueous TFA, buffer B - 80% acetonitrile; gradient - from 10 to 70% buffer B in 30 min).

To obtain the protected nonapeptide Fmoc-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Tyr (Bu ^t ) -Lys (Boc) -Tyr (Bu ^t ) -Arg (Pmc) -Arg (Pmc) -Lys ( Boc) -OH (III) 0.74 g (0.14 mmol) of nonapeptidylpolymer was treated with a mixture of 1 ml of acetic acid and 2 ml of trifluoroethanol in 7 ml of DCM for 1 h. (Under these conditions, all the protection of the amino acid side chains are stable, the most acid labile peptide bond is cleaved - polymer.) The polymer was filtered off, washed with a de-blocking mixture and DCM, the filtrate was evaporated, the product was precipitated with diethyl ether. The precipitate was filtered off, washed on the filter with ether and dried. Received 0.33 g (82%) of the protected nonapeptide (III). R _f 0.48 (system 1); 0.9 (system 2).

To obtain the linear precursor of the nonapeptide H-Arg (Pmc) -Lys (Boc) -Lys (Boc) -Tyr (Bu ^t ) -Lys (Boc) -Tyr (Bu ^t ) -Arg (Pmc) -Arg (Pmc) -Lys (Boc) -OH (II) 0.33 protected peptide (III) was treated with 5 ml of a 50% solution of morpholine in DMF for 1 h, the reaction mixture was evaporated, the product was precipitated with ether. The precipitate was filtered off, washed on the filter with ether and dried. Obtained 0.29 g (80% of the starting amino acid attached to the polymer). R _f 0.31 (system 1).

To cyclize, 0.093 g (0.035 mmol) of the linear nonapeptide (II) was dissolved in 100 ml of DMF, 0.011 g (0.07 mmol) of HOBt, 0.031 g (0.07 mmol) of Castro-BOP reagent [13] and 0.03 ml (0.15 mmol) of DIPEA were added. TLC was used to control the occurring transformations. After 1 h, the reaction mixture was evaporated, the product was precipitated with ether. The precipitate was filtered off, washed on the filter with ether and dried. Received 80 mg (87%) of a protected cyclic nonapeptide, R _f 0.50 (system 1).

The final release of the cyclic nonapeptide was carried out by treating the corresponding protected product with a mixture of 10 ml of TFA, 0.25 ml of deionized water and 0.25 ml of TIBS for 16 hours. Then the polymer was filtered off, washed with 2 × 2 ml of the unlocking mixture, the filtrate was evaporated and dry ether was added to the residue. The precipitate was filtered off, washed with DCM (3 × 3 ml), ether (3 × 5 ml), dried in a vacuum desiccator. The crude solid-phase synthesis product (0.036 g) with a basic substance content of 86% was purified using preparative HPLC on a Beckman instrument (United States) using a Diasorb - C16 130T column (2.5 × 250 mm), sorbent particle size 10 μm. Buffer A — 0.1% TFA solution and buffer B — 80% acetonitrile in water were used as eluents. Elution was performed with a gradient of 0.5% per minute of buffer B in buffer A from 100% of buffer A at a rate of 10 ml / min. Peptides were detected at a wavelength of 220 nm. Fractions containing the desired product were combined, acetonitrile was evaporated and lyophilized. As a result, 0.026 g (56.8%) of cyclic nonapeptide (I) was obtained. Product homogeneity determined by analytical HPLC was 93.1%. Mass spectrum: 1307.5 [M + H] ⁺ , 1329.5 [M + Na] ⁺ , 1345.5 [M + K] ⁺ , calculated for C ₆₀ H ₁₀₃ N ₂₃ O ₁₀ : 1306.6.

Example 2. Phosphorylation of regulatory KLMM myosin light chains in the presence of peptide I in vitro.

The effect of peptide I on phosphorylation of regulatory light chains (RLCs) of smooth muscle myosin in vitro was tested by analyzing the dynamics of phosphate incorporation into serine-19 RLCs by the intensity of binding of RLCs to the corresponding phosphospecific antibodies using the dot blot method (Figs. 1A, 2A). MLCK and RLC were isolated from the muscular stomach of chickens, and calmodulin protein was isolated from the brain of a bull. Phosphorylation was carried out in a reaction mixture of the following composition: 100 mM NaCl, 1 mM dithiothreitol, 1 mM MgCl ₂ , 0.5 mM CaCl ₂ , 0.3 μM calmodulin, 5 nM MLCK, 20 μM RLC, 10 mM MOPS at a temperature of 30 ° C. Peptides were used at a concentration of 5 μM. The reaction was initiated by introducing a Mg ²⁺ -ATP solution into the sample to a final concentration of 0.5 mM. Samples were taken within 20 min after the start of the reaction. The reaction was stopped by making a sample in a 15 mM EGTA solution. Radar phosphorylation level was determined by dot blot method. Samples with the studied proteins (2 μl) were applied onto a nitrocellulose membrane (Millipore, USA) in the form of a drop, fixed for 10 minutes with 0.25% glutaraldehyde, and incubated in a 5% skim milk solution in TBS for 40 minutes. Then, membranes with mouse antibodies against a monophosphorylated form of RLC (Cell Signaling Technology, United States) were diluted 1: 1200 in a membrane and then 1 hour with secondary antibodies against mouse IgG conjugated with peroxidase at a dilution of 1: 25000. After each stage, the membranes were washed in a TBST solution 3 times for 5 minutes. The resulting immune complexes were identified by ECL using solutions from Pierce (USA). In preliminary experiments, by staining the membranes with antibodies to RLC (Proteintech Group, Inc., USA), the same load of all samples in the total number of RLC was confirmed. The obtained image on an X-ray film Retina (Germany) was scanned and counted in the ImageJ program (USA). The data were processed in Excel and plotted the dependence of the signal magnitude (rel. Units) on the reaction time. Comparison of the inhibitory effects of peptides was carried out according to the values of the signals at the tenth minute of the reaction.

Figure 1A presents experimental data on the phosphorylation of regulatory MLCK myosin light chains in the presence of peptide I and L-PIK in comparison with two controls - phosphorylation of RLC in the absence of a peptide and in the presence of a mixture of amino acids that make up the above peptides taken in the corresponding molar concentrations. It is seen that in the control reactions, phosphorylated RLCs are detected by antibodies already at 1.5 minutes of the reaction, and by 5 minutes the reaction ends, since at a later date the color density of the deposited drops of the reaction mixture does not change. At the same time, in the presence of peptide I, staining develops starting from 5 min and does not reach the intensity of staining in the control by the end of the experiment (20 min). In the presence of L-PIK, membrane staining is not detected throughout the duration of the experiment. The digitized data of this experiment are presented in figure 1B, from which it follows that for 5 min, peptide I inhibits the phosphorylation of RLCC KLCM by 92.5%, for 10 min - 90% and for 20 min - 85%, and L-PIK inhibits KLCM 100% for the entire duration of the experiment. This experiment was repeated twice with a similar result - peptide I almost completely inhibited the catalytic activity of MLCK in vitro.

Figure 2A presents the results of exactly the same experiment as that shown in figure 1, where the prototype of the claimed peptide I - [N ^α MeArg ¹ ] -L-PIK and L-PIK was used as inhibitory peptides. In this experiment, the control reaction of MLCK RLC phosphorylation also ended by 5 min, and in the presence of [N ^α MeArg ¹ ] -L-PIK and L-PIK RLC phosphorylation did not occur within 20 min of the experiment. Thus, MLCK activity was completely inhibited. The digitized results of this experiment are presented in the form of a graph in figure 2B. It can be seen that in the presence of [N ^α MeArg ¹ ] -L-PIK and L-PIK, 95-100% inhibition of MLCK is achieved. The experiment shown in Figure 2B was repeated three times with a similar result: [N ^α MeArg ¹ ] -L-PIK and L-PIK almost completely suppressed the catalytic activity of MLCK in vitro.

Since both experiments were performed under the same conditions and in both cases one of the studied peptides was L-PIK, the results of these experiments can be compared with each other. From comparison it follows that the claimed peptide and prototype exhibit inhibitory properties almost equally. Therefore, in vitro and the claimed peptide (I), and L-PIK, and the prototype [N ^α MeArg ¹ ] -L-PIK have a high and equivalent inhibitory activity. Although in the described experiments L-PIK completely suppresses the activity of MLCK in vitro, it rapidly loses its inhibitory activity in vivo in the presence of proteolytic enzymes [14]. Peptide cyclization is a generally accepted method for limiting the steric mobility of a molecule and increasing its resistance to degradation by peptidases [15], but does not guarantee the conservation of biological activity inherent in the corresponding linear peptide. Since cyclic peptide I retains the activity of the linear precursor, it should be more effective than L-PIK and [N ^α MeArg ¹ ] -L-PIK, an MLCK inhibitor in biological systems where proteolytic enzymes are present, for example, when it is introduced into the bloodstream, gastrointestinal tract, lungs.

The inventive peptide is non-toxic, since it is an analogue of the endogenous fragment of the auto-inhibitory site of MLCK and consists of residues of natural amino acids that are usually present in the body.

Bibliography

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3. Rossi J.L., Velentza A.V., Steinhorn D.M., Watterson D.M., Wainwright M.S. MLCK210 gene knockout or kinase inhibition preserves lung function following endotoxin-induced lung injury in mice. Am. J. Physiol. Lung Cell Mol. Physiol. 2007.292 (6): L1327-1334.

4. Wainwright M.S., Rossi J., Schavocky J., Crawford S., Steinhorn D., Velentza A.V., Zasadzki M., Shirinsky V., Jia Y., Haiech J., Van Eldik L.J., Watterson D.M. Protein kinase involved in lung injury susceptibility: evidence from enzyme isoform genetic knockout and in vivo inhibitor treatment. Proc. Natl. Acad. Sci. USA 2003.100 (10): 6233-6238.

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Claims (1)

Cyclo peptide [-Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-].

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