Use of compound bis-(3-hydroxy-4-(hydroxymethyl)-2,5-dimethylpyridinium) fumarate as activator of transcription factor nrf2 in biological and medical applications

Abstract

FIELD: biology; medicine.SUBSTANCE: invention relates to the use of the compound bis-(3-hydroxy-4-(hydroxymethyl)-2,5-dimethylpyridinium) fumarate as an activator of the transcription factor Nrf2 in biological and medical applications.EFFECT: use of the compound bis-(3-hydroxy-4-(hydroxymethyl)-2,5-dimethylpyridinium) fumarate as an activator of the transcription factor Nrf2.1 cl, 3 dwg, 2 ex

Description

[0001] The invention relates to the field of medicine, in particular to the development of new antioxidant compounds and the creation of a new type of drugs based on activators of the transcription factor Nrf2.

[0002] Drugs with antioxidant activity can be used to prevent the development of diseases associated with oxidative stress [DOI: 10.1146 / annurev-pharmtox-011112-140320]. One of the modern approaches to increasing the resistance of normal cells and tissues to oxidative damage is the activation of Nrf2-dependent protective mechanisms [DOI: 10.1089 / ars.2017.7342]. The transcription factor Nrf2 plays a critical role in maintaining cellular homeostasis by regulating the transcription of cytoprotective genes [DOI: 10.1016 / j.biochi.2013.05.004]. With an increase in the level of intracellular reactive oxygen species, Nrf2 is translocated to the nucleus and activates cytoprotective ARE genes. It has been shown that Nrf2 is activated by the action of not only oxidative stress inducers, but also some physiologically active compounds, thereby ensuring their therapeutic effectiveness [DOI: 10.1016 / j.mrfmmm.2004.06.041].Pharmacological activation of Nrf2 has been proposed as a key strategy in the prevention of neurodegenerative, inflammatory, and cardiovascular diseases, as well as carcinogenesis [DOI: 10.1016 / j.phrs.2008.09.003]. From a clinical perspective, it is crucial that Nrf2 can be pharmacologically targeted in diseases caused by oxidative stress and inflammation.

[0003] In most chronic disease models, enhancing homeostasis with Nrf2 activators provides beneficial therapeutic effects. The most commercially successful Nrf2 activator to date is fumaric acid dimethyl ester (DMF) (BG-12 or Tecfidera, Biogen), which was approved in 2013 for the treatment of multiple sclerosis. DMF is converted to monomethyl fumarate (MMF) by intestinal esterases. A monomethyl fumarate derivative, diroximel fumarate (2-(2,5-dioxo-1-pyrrolidinyl)ethyl ester; ALKS-8700; Alkermes), is currently in preclinical development [DOI: 10.3389 / fheur.2018.00005].

[0004] Vitamin B6 (pyridoxine) is a natural compound with a broad spectrum of antioxidant activity [DOI: 10.1016 / j.freeradbiomed.2003.09.012]. Pyridoxine is widely used in the Russian Federation in the complex treatment and prevention of various cardiovascular diseases, including coronary heart disease, grade II-III angina, myocardial infarction, and ischemic stroke (for example, the drug "Vitabs® cardio"). In a model of neurodegenerative diseases, it was shown that pyridoxine can activate the Nrf2 / HO-1 pathway, which increases the activity of the antioxidant system and the regenerative potential of cells [DOI: 10.1038 / s41467-020-14788-x].

[0005] The pyridoxine molecule may be a promising precursor for the creation of new compounds with potential antioxidant activity [DOI: 10.1079 / NRR200125]. The best-known representative of mitochondrial cytoprotectors - derivatives of pyridoxine is ethylmethylhydroxypyridine succinate (Mexicor). The advantage of this drug is the unique combination of antihypoxic and antioxidant effects of its components. Mexicor contains a metabolite of the tricarboxylic acid cycle - succinate, which is able to maintain the activity of the succinate oxidase link during hypoxia [Lukyanova L.D., 1990, Polumiskov V.Yu. et al. 2006].Previously, we synthesized and studied a pyridoxine derivative, di(3-hydroxy-4,5-bis(hydroxymethyl)-2-methylpyridinium) nitroxysuccinate [RU 2712914 C2], which has low cytotoxicity, iron-chelating activity, the ability to reduce lipid peroxidation, increase intracellular nitric oxide levels [DOI: 10.3390 / antiox10091451], and activate Nrf2-dependent processes in vitro [DOI: 10.47056 / 1814-3490-2023-4-265-275]. Despite the large number of scientific papers devoted to the study of pyridoxine derivatives in the Russian Federation, the use of such compounds as Nrf2 activators has not previously been considered. Given the positive results in the study of the biological properties of the nitroxysuccinic acid derivative and pyridoxine, we propose combining in one molecule the properties of fumarate as an Nrf2 activator and pyridoxine as an antioxidant, which will enhance the physiological effect of the compound.

[0006] Thus, the object of the invention is to provide a new chemical compound as an activator of the transcription factor Nrf2.

[0007] The problem is solved by using bis-(3-hydroxy-4-(hydroxymethyl)-2,5-dimethylpyridinium) fumarate 1 as an activator of the transcription factor Nrf2 in cells.

[0008]

[0009] Figure 1 shows the data on immunofluorescence staining of FetMSC cells with antibodies to the Nrf2 protein after 24 hours of exposure to 1 at a concentration of 10 and 40 μM.

[0010] Figure 2 shows the quantitative analysis of the immunofluorescence results of FetMSC cells with antibodies to the Nrf2 protein after 24 hours of exposure to 1 at a concentration of 10 and 40 μM. The significance of differences from the control is shown as *** P < 0.001.

[0011] Figure 3 shows the effect of compound 1 on the expression level of antioxidant target genes Nrf2 and the NFE2L2 gene in FetMSC cells. The significance of differences from the control is shown as *P<0.05.

[0012] The essence of the proposed invention is characterized by the examples given below.

[0013] Example 1

[0014] The ability of bis-(3-hydroxy-4-(hydroxymethyl)-2,5-dimethylpyridinium) fumarate (1) to enhance intracellular accumulation and nuclear translocation of the transcription factor Nrf2 was studied. Immunofluorescence staining of cells followed by microscopy was used for this purpose. FetMSC cells (human mesenchymal stem cells) were seeded at a concentration of 2×10 5Cells / ml were seeded onto coverslips in 6-well plates in 2 ml of standard incubation medium. Test compound 1 was added to the culture medium at a concentration of 10 or 40 μM 24 h after seeding. After 24 h of incubation, the cells were washed three times with PBS (pH 7.4) and fixed with a 4% paraformaldehyde solution (Acros Organics, Germany) in PBS for 30 min at RT. Permeabilization was performed in 2 ml of a 0.5% Triton® X-100 solution (Amresco, USA) in PBS for 10 minutes. Next, the samples were incubated in a solution containing 5% bovine serum albumin (Amresco, USA) in PBS for 1 hour. Then, the samples were incubated with antibodies to the Nrf2 protein (1:500; #PAL947Hu01, Cloud-Clone Corp., USA) for 12 hours at +4°C. The samples were washed three times with PBS and incubated in a solution of Alexa Fluor™568 secondary antibodies (1:100; #A-11011, Invitrogen, Thermo Fisher Scientific, USA) for 1 hour.Cell nuclei were stained using the fluorescent dye 4,6-diamidino-2-phenylindole dihydrochloride (DAPI, Serva, Germany) in PBS for 15 minutes. Samples were visualized using an Axio Scope.A1 microscope (Zeiss, Germany) and an A-Plan 40× / 0.65 objective using the following fluorescence filter sets: Fs 49 DAPI (EX G 365, EM BP 445 / 50), Fs 45 HQ TexasRed (EX BP 560 / 40, EM BP 630 / 75). Fluorescence intensity was analyzed in 5 fields of view containing 100 cells. Images of samples were acquired using an AxioCam MRc5 camera and processed using ZEN 2 lite software; quantitative fluorescence measurements were performed using ImageJ (NIH).

[0015] Figure 1 shows the data on immunofluorescence staining of FetMSC cells with antibodies to the Nrf2 protein after 24 hours of exposure to 1 at a concentration of 10 and 40 μM.

[0016] Figure 2 shows the quantitative analysis of the immunofluorescence results of FetMSC cells with antibodies to the Nrf2 protein after 24 hours of exposure to 1 at a concentration of 10 and 40 μM. The significance of differences from the control is shown as *** P < 0.001.

[0017] Compound 1 was found to increase intracellular accumulation of the transcription factor Nrf2 and its nuclear translocation. Compound 1 at a concentration of 10 μM increased Nrf2 levels in the cell cytosol by 5-fold and in the nuclei by 9-fold relative to the control. When using 1 at a concentration of 40 μM, an 8-fold increase in Nrf2 levels was observed in the cytosol and a 14-fold increase in the nuclei relative to the control. Thus, bis-(3-hydroxy-4-(hydroxymethyl)-2,5-dimethylpyridinium) fumarate is an activator of the transcription factor Nrf2.

[0018] Example 2

[0019] The ability of bis-(3-hydroxy-4-(hydroxymethyl)-2,5-dimethylpyridinium) fumarate (1) to increase the expression of antioxidant target genes of the transcription factor Nrf2 was studied. For this purpose, the real-time polymerase chain reaction method was used. FetMSC cells (human mesenchymal stem cells) were seeded at a concentration of 10 6cells / well in 6-well plates in 2 ml of standard incubation medium. Twenty-four hours after seeding, the test compound 1 was added to the culture medium at a concentration of 40 μM. After 24 hours of incubation, total RNA was isolated from the cells using the ExtractRNA reagent (Evrogen, Russia) according to the manufacturer's protocol. First-strand cDNA synthesis on an RNA template was performed using the MMLV RT kit (Evrogen, Russia). Quantitative real-time PCR was performed using the qPCRmix-HS SYBR reaction mixture (Evrogen, Russia) and a LightCycler® 96 Real-Time PCR System (Roche, Switzerland). The beta-actin gene (ACTB) was used as a reference gene to normalize expression results. For gene expression analysis, primer pairs to the following genes were used:

[0020] ASTB: 5'-AGCGGGAAATCGTGCGTGAC-3' and 5'-AGCAGCCGTGGCCATCTCTT-3';

[0021] НМОХ1: 5'-GACTGCGTTCCTGCTCAACA-3' and 5'-CGGGGGCAGAATCTTGCACT-3';

[0022] NQO1: 5'-TGCAGTGGTTTGGAGTCCCT-3' and 5'-CACTGCCTTCTTACTCCGGAAGG-3';

[0023] NFE2L2: 5'-AGGTTGCCCACATTCCCAAA-3' and 5'-ACGTAGCCGAAGAAACCCTCA-3';

[0024] TXN1: 5'-TTCAGGAAGCCTTGGACGCTG-3' and 5'-TTTTGCAAGGCCCACACCACG-3'.

[0025] The relative gene expression levels were calculated using LightCycler 96 Software 1.1 based on the results of three independent experiments.

[0026] Figure 3 shows the effect of compound 1 on the expression level of antioxidant target genes Nrf2 and the NFE2L2 gene in FetMSC cells. The significance of differences from the control is shown as *P<0.05.

[0027] It was determined that the introduction of 1 significantly increased the expression level of the main Nrf2 target genes - heme oxygenase 1 (HMOX1) and NADPH(H):quinone oxidoreductase 1 (NQO1) in cells. In addition, compound 1 enhances the expression level of the NFE2L2 gene, which encodes the Nrf2 transcription factor, that is, it can lead to an increase in the intracellular content of Nrf2 due to an increase in its de novo biosynthesis. Thus, bis-(3-hydroxy-4-(hydroxymethyl)-2,5-dimethylpyridinium) fumarate increases the expression level of antioxidant and cytoprotective genes, which indicates the activation of Nrf2-dependent pathways that protect cells from stress.

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