RU2602306C2 - Agent, activating transcription factor hif - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: group of inventions relates to pharmaceutics and medicine and concerns using ethyl ether of N-phenyl acetyl-L-prolylglycine for activating transcription factor HIF (hypoxia induced factor), as well as method of HIF activating using said factor and method of treating diseases, associated with HIF transcription factor deficiency.

EFFECT: group of inventions provides higher body resistance to oxygen supply deficit, which is effective for treatment of hypoxic states of various genesis.

3 cl, 1 tbl, 2 ex, 2 dwg

Description

Field of Invention

The invention relates to medicine, in particular to pharmacology, and for the use of the new activator HIF-1α.

State of the art

Due to the fact that hypoxia is the most important pathogenetic factor in a wide range of pathological conditions, the search for means that increase the body's resistance to oxygen supply deficiency is a task of a high degree of relevance. The high sensitivity of the brain to hypoxia is manifested in the fact that although the brain mass is only 2% of the mass of the whole organism, the brain consumes 20% of the amount of oxygen absorbed by the body. The system that plays a major role in realizing the homeostatic response of brain tissue to hypoxic effects is the hypoxia-induced transcription factor (Hypoxia Induced Factor, HIF-1α). HIF-1 is a heterodimer consisting of two subunits - the oxygen-sensitive subunit of HIF-1α and constitutively expressed HIF-1β. Hypoxia promotes an increase in the level of HIF-1α, its dimerization with HIF-1β, mobilization of co-activators (p300 / CBP) and the binding of this complex to the HRE sequence (hypoxia-response element) in the regulatory regions of target genes. Under normoxic conditions, the hydroxylation of proline residues of the HIF-1α protein by prolyl hydroxylases (PHD, prolyl hydroxylase-domain protein; EC 1.1411.29) is a prerequisite for binding of the ubiquitin protein ligase E3 component - von Hippel-Lindau protein (VHL). The presence of oxygen also causes hydroxylation of the asparagine residue of the C-terminal transactivation domain (C-TAD) of HIF-1α by the asparagine hydroxylase enzyme (FIH1, factor inhibiting HIF-1), which blocks its interaction with the p300 / CBP transcription co-activator.

If, under normal oxygen supply to tissues, the PHD and FIH enzymes inactivate HIF-1α, under conditions of hypoxia, the activity of PHD and FIH decreases, which leads to a decrease in the degradation of HIF-1α, its accumulation, and, as a consequence, the launch of HIF-1α-dependent genes [Semenza G. // Cell. 2012. V. 148. No. 3. P. 399-408]. Shown [Zheng K, Fridkin M., Youdim M. // Persp. Med. Chem. 2015. V. 7. P. 1-8] that HIF-1α activates a total of up to 100 genes, including genes involved in angiogenesis due to activation of vascular endothelial growth factor, increased synthesis of erythropoietin, activation of glucose transport systems through membranes, cytoprotection by neurotrophic factors, normalization of the cell cycle and metabolism at the level of mitochondria, including activation of antioxidant enzymes - superoxide dismutase and catalase. Thus, by regulating the expression of genes involved in energy metabolism, angiogenesis, erythropoiesis, cell proliferation, HIF determines the implementation of an adaptive response to hypoxic effects.

Along with this, HIF-1 affects the state of many neurotransmitter systems - it activates a protein that controls GABA receptors (GABARBP) [Park S.H., Kim B.R., Lee J.H. et al // Cell Signal. 2014. V. 26. No. 7. P. 1506-7573], increases the activity of tyrosine hydroxylase [Schnell P. O., Ignacak M.L., Bauer A.L., et al., // J. Neurochem. 2003 V. 85. No. 2. P. 483-491]. Close relationships of HIF-1α with cholinergic receptors have been described [Hirota K., Fukuda R., Takabuchi S. Et al. // J. Biol. Chemistry. 2004. V. 279. No. 40. P. 41521-41528].

It is known that acute deficiency of oxygen and macroerg delivery during ischemic stroke leads to the inclusion of endogenous protective mechanisms, the key of which is the HIF system (Shi, 2009; Xin et al, 2011). In mice knocked out for HIF-1α, ligation of the middle cerebral artery was accompanied by an increase in mortality and the development of a larger lesion than in normal animals (Baranova et al., 2007).

Chronic hypoxia plays an important role in the progression of neurodegenerative diseases. So, already in the early phases of Alzheimer's disease, there is a decrease in HIF with a concomitant slowdown in the utilization of glucose in the brain tissue (Zhang Z., Yan J., Chang Y., ShiDu Yan S., Shi H. // Curr. Med. Chem. 2011. V. 18. No. 28. P. 4335-4343). A decrease in the activity of the HIF system with concomitant inhibition of tyrosine hydroxylase and the degeneration of dopaminergic neurons was detected in patients with Parkinson's disease (Borghammer P., Cumming P., Ostergaard K. et al. J. Neurol. Sc. 2012, 313, (1-2), 123-128).

There is evidence of a role for HIF deficiency in pancreatic beta cells in the pathogenesis of diabetes. Thus, it is reported about the ability of insulin to disrupt the formation of HIF-1α and the role of deficiency of this factor in the development of both type 1 diabetes (absolute insulin deficiency) and type 2 diabetes (relative insulin deficiency), as well as their complications [Cheng K., But K, Stokes R. et al, // J. Clin. Invest. 2010. V. 120. No. 6. P. 2171-2183]. Dysfunction of glucose transport systems GLUT1 and GLUT3 across cell barriers, which occurs with HIF-1α deficiency, contributes to the development of insulin resistance in both Alzheimer's disease and diabetes [Liu Y., Liu F., Iqbal K., et al, / / FEBS Lett. 2008. V. 582. P. 359-364]. The participation of HIF-1α in the expression of incretins, the most important factors for the cytoprotection of pancreatic beta cells [Van de Velde S., Hogan M.F., Montminy M. // PNAS. 2011. V. 108. No. 41. P. 16876-16882].

The use of substances that activate the HIF system can be effective in a wide range of pathological conditions listed above.

Activators of the HIF system are divided into 2 groups: substances that enhance the transcription and translation of this transcription factor, and substances that inhibit its deactivation.

A multifunctional iron chelator M30 (5- [N-methyl-N-propargylaminomethyl] -8-hydroxyquinoline) is described, which activates HIF-1α and has neuroprotective activity in vitro and in vivo experiments (N. Zheng et al., Bioorganic and Medicinal Chemistry, 2005, vol. 13, No. 3, pp. 773-783; Y. Avramovich-Tirosh et al, Curr. Alzheimer Res. 2010. V. 7, No. 4, p. 300-306).

US Pat. No. 8,524,699 describes substituted dihydropyrazolones as inhibitors of HIF-prolyl-4-hydroxylase of the general formula:

These compounds are HIF activators and may be useful for the treatment of anemia, chronic kidney disease, myocardial infarction, atherosclerosis.

US Pat. No. 8,541,455 B2 describes 2-pyridin-2-yl-pyrazol-3 (2H) -one derivatives as activators of the transcription factor HIF of the general formula:

It was shown that these substances enhance the secretion of vascular endothelial growth factor (VEGF) and erythropoietin (EPO), two of the main markers of HIF-1α activation in hepatocytes.

N- (2-mercaptopropionyl) -glycine is described as an activator of HIF-1α due to inhibition of HIF-prolyl hydroxylase-2 (S. Yum et al., Biochem. Biophys. Res. Comm. 2014. V. 443, p. 1008-1013 ) It was shown that this compound enhances the secretion of vascular endothelial growth factor (VEGF) and has a protective effect in experimental colitis.

It has been shown (Y. Liu et al., Cell Physiol Biochem 2014; 33: 1036-1046) that the known iron chelator, deferoxamine, used in diseases characterized by an excess of iron, causes increased expression of HIF-1α and is involved in a signaling cascade that includes activation ERK, which indicates its invasive potential.

The proline-arginine-enriched peptide PR-39 was found to have cardioprotective activity due to the stabilizing properties against HIF-1α (E.D. Muinck et al., Antioxid. Redox Signal. 2007; 9 (4): 437-445).

X. Ma et al., PLOS One, 2014. V. 9, No. 4, describes two proline compounds — N-benzyloxycarbonylproline (PA2) and bis- (prolyl-o-phenylenediamine) (PA1) as prolyl hydroxylase-3 inhibitors possessing an activating effect on the HIF-1α signaling pathway, including enhancing the synthesis of VEGF and key glycolysis modulators (pyruvate kinase isozymes M1 / M2 and alpha-enolase-1).

In the article J.-J. Min et al., Scientific Reports, 2014. V. 4. P. 1-5 describes D, L-3-n-butylphthalide, which enhanced HIF-1α expression and improved learning and memory in rats under conditions of chronic hypoxia. This compound also enhanced the expression of BDNF and phosphorylation of CREB.

A known prolyl hydroxylase inhibitor GSK360A, a quinoline derivative of the formula:

When administered orally, GSK360A has a cardioprotective effect in heart failure after myocardial infarction and exerts a protective effect on models of ischemic reperfusion injuries in mice (S.S. Banerjee et al., Toxicology Mechanisms and Methods, 2012; 22 (5): 347-358).

However, the above references do not open or suggest that the substituted di-peptide N-phenylacetyl-L-prolylglycine ethyl ester (Dipeptide I) has an activating effect on the transcription factor HIF-1α.

SUMMARY OF THE INVENTION

The objective of the invention was the search for a method of activation of the transcription factor HIF-1α. This goal was achieved using the dipeptide of ethyl ester of N-phenylacetyl-L-prolylglycine (RF Patent 2119496).

Description of the invention

The invention is illustrated by the following examples.

Example 1

The cultivation of cells. The use of cell lines stably expressing luciferase reporter constructs with binding sites for specific TFs is one of the most effective screening approaches for searching for compounds that affect the activity of transcription factors (TFs), [Phippard D., Manning AM (2003) Methods Mol Biol., 225, 19-23]. We used HEK293 line cells (transplantable cells of a human embryo kidney; Russian Collection of Cell Cultures, Institute of Cytology RAS, St. Petersburg). Cells were cultured at 37 ° C at 5% CO ₂ in DMEM (Biolot, Russia) containing 10% fetal calf serum (Sigma, USA), 2 mM L-glutamine, 50 μg / ml gentamicin sulfate, 2.5 μg / ml amphotericin B (PanEco, Russia).

Reporter vector designs. The luciferase reporter vectors contain the consensus nucleotide sequences for the corresponding TFs, and the reporter gene (luciferase gene) is under their control. The binding of TF to the recognition site leads to the expression of the luciferase gene and bioluminescence detected when a luciferin substrate is introduced. The level of bioluminescence is directly proportional to the amount of enzyme and the binding activity of a transcription factor. The principle of in vitro test systems for biological screening of compounds for their effect on a particular TF is based on the fact that if the test compound stimulates / inhibits the activity of TF, then there will be an increase / decrease in the level of luminescence (due to a change in the DNA-binding activity of TF ) Thus, it becomes possible to select compounds whose targets are transcription factors and to study the mechanisms of action of both new and already used drugs. CREB, NFAT, NF-kV, p53, STAT1, VDR, HSF1 and HIF1, and determining transcription activation on the minimum HSVtk promoter (the minimum herpes simplex thymidine kinase promoter), were specific responsive elements that recognize and bind transcription factors (TFs) The following sequences were used (Table 1). Oligonucleotides (Synthol, Russia) containing several copies of the sequences corresponding to the binding sites of individual TFs were cloned into the pTL-Luc plasmid vector (Panomics, USA; carries the Photinus pyralis luciferase gene) at the NheI restriction sites (5′-G ↓ CTAGC-3 ′) and BglII (5′-A ↓ GATCT-3 ′) ("Fermentas", Lithuania). This reporter luciferase vector contains a minimal herpes simplex virus thymidine kinase promoter, the activity of which depends on the overlying consensus sequence capable of binding a specific TF. In turn, luciferase gene expression is controlled by this model promoter. The presence and orientation of the inserts was confirmed by sequencing according to the ABI PRISM BigDye ™ Terminator Cycle Sequencing Ready Reaction Kit protocol (Applied Biosystems, USA). Isolation and purification of plasmid DNA was performed using the EndoFree Plasmid Maxi Kit (Qiagen, USA) [Salimgareeva MX, Sadovnikov SV, Farafontova EI, Zaynullina LF, Vakhitov VA, Vakhitova Yu .AT. Cellular test systems for searching for modulators of transcription factor activity. Applied Biochemistry and Microbiology, 2014. - No. 2. - S. 219-225].

Transfection. For transient transfections, HEK293 cells were transplanted in an amount of 45 × 10 ³ cells per well in 96-well plates in 100 μl of antibiotic-free medium (DMEM, 10% fetal calf serum, 2 mM L-glutamine). After 24 hours, transfection was performed with these constructs (Table 1) using Lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer's protocol. 6 h after transfection, the medium was replaced with an antibiotic-containing medium (DMEM, 10% fetal calf serum, 2 mM L-glutamine, 50 μg / ml gentamicin, 2.5 μg / ml amphotericin B) and the studied drugs were added 18 hours later (Dipeptide I at a concentration 10 μM; Piracetam at a concentration of 1 mm). Cells were incubated in the presence of Dipeptide (N-phenylacetyl-L-prolylglycine ethyl ester, I) or Piracetam for another 24 hours. Luciferase activity in cell lysates was detected using the Dual Luciferase Reporter Assay System kit (Promega, USA) on a 2300 plate analyzer EnSpire® Multimode Plate Reader (Perkin Elmer, USA). Co-transfection with plasmid pRL-TK (Promega, USA) encoding the Renilla reniformis luciferase gene was used as an internal control of transfections. The luminescence values for Photinus pyralis were normalized by the luminescence for Renilla reniformis in each dimension.

The arithmetic average of the values obtained in two repetitions in each experiment in three independent experiments, and the standard error of the mean were calculated using the program Statistica 6.1 (StatSoft. Inc., USA). Comparison of experimental groups was performed using paired t-student test for dependent samples.

The data presented in FIG. 1A, indicate that Dipeptide I, when incubated for 24 hours at a concentration of 10 μM, increased the DNA-binding activity of HIF-1α by 43% and did not affect the DNA-binding activity of factors CREB, NFAT, NF-kB, p 53, STAT1, GAS, VDR and HSF1. As follows from the data presented in FIG. 2 (left - the effect on basal activity), the severity of the HIF-positive effect of Dipeptide I depended on its concentration.

Experiments with the comparison drug showed that Piracetam neither in equimolar (10 μM, data not shown) nor in a higher concentration (1 mm) causes statistically significant changes in the DNA-binding activity of the studied transcription factors (Fig. 1B).

EXAMPLE 2

The experimental modeling of hypoxia was carried out using CoCl ₂ , which causes stabilization of HIF-1α and is a pharmacological mimetic of hypoxia [Piret JP, Mottet D, Raes M, Michiels C. CoCl ₂ , a chemical inducer of hypoxia-inducible factor-1, and hypoxia reduce apoptotic cell death in hepatoma cell line HepG2. Ann NY Acad Sci. 2002; 973: 443-7]. CoCl ₂ The mechanism of action is due to its ability to substitute for Fe ²⁺ in the active site of prolyl, inactivate, thereby inhibiting the hydroxylation enzyme and HIF-1α [Epstein, AC1, Gleadle JM, McNeill LA, Hewitson KS, O'Rourke J, Mole DR, Mukherji M, Metzen E, Wilson MI, Dhanda A, Tian YM, Masson N, Hamilton DL, Jaakkola P, Barstead R, Hodgkin J, Maxwell PH, Pugh CW, Schofield CJ, Ratcliffe PJ. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell. 2001; 107 (1): 43-54].

The luciferase construct for analyzing HIF-1 activity contains 4 copies of the 5 ′ ACGTG 3 ′ consensus sequence, which is the binding site for the HIF-1 protein (HIF-1-Luc construct). Cells transfected with the HIF-1-Luc plasmid vector were preincubated with Dipeptide I for 8 h (final concentrations of 1, 10, 100 μM; two times every 4 hours), then a CoCl ₂ hypoxia inducer was introduced at a working concentration of 50 μM and continued co-incubation of Dipeptide I and CoCl ₂ for 16 hours, after which luciferase activity was detected as described above. The arithmetic mean of the values obtained in two repetitions in each experiment in three independent experiments, and the standard error of the mean, were calculated using the program Statistica 6.1 (StatSoft. Inc., USA). Comparison of experimental groups was performed using paired t-student test for dependent samples.

Experiments studying the effect of Dipeptide I on the DNA-binding activity of HIF-1α in the presence of a pharmacological mimetic of hypoxia CoCl ₂ gave the following results. In full accordance with published data, in this experiment, against the background of the action of CoCl ₂ , an increase in the activity of HIF-1α is noted. For the first time, a concentration-dependent effect of Dipeptide I on the DNA-binding activity of HIF-1α was detected (Fig. 2): at a concentration of 1 μM, only an unreliable decrease in this parameter was noted, and at concentrations of 10 and 100 μM, Dipeptide I caused a corresponding increase in HIF-1- dependent luciferase activity.

Thus, it was found that Dipeptide I has the ability to increase both basal and hypoxia-induced pharmacological mimetic activity of HIF-1α in vitro.

The totality of the obtained data indicates that Dipeptide I causes a concentration-dependent increase in the basal DNA-binding activity of HIF-1α. It was shown that under stabilization conditions HIF-1α using the chemical inducer of this transcription factor CoCl ₂ . Dipeptide I causes an additional increase in the DNA-binding activity of HIF-1α. The effect on HIF-1α is specific for Dipeptide I :, the classic nootropic drug Piracetam does not affect the activity of this transcription factor. Dipeptide I increased the DNA-binding activity of only HIF-1α, while the activity of other studied transcription factors (CREB, NFAT, NF-κB, p 53, STAT1, GAS, VDR and HSF1) did not increase under the influence of Dipeptide I.

A brief description of the drawings.

In FIG. Figure 1 shows the effect of Dipeptide I a 10 μM (A) and the comparison drug Piracetam a 1 mM (B) on the basal DNA-binding activity of transcription factors NF κB, NFAT, STAT1, HIF-1, p 53, CREB, GAS, VDR, HSF1 in vitro. The significance of differences was assessed using paired t-student test for dependent samples (n = 3, * p <0.05).

In FIG. Figure 2 shows the effect of Dipeptide I on the basal and induced activity of HIF-1α. Group “control” - values of the basal activity of HIF-1α unstimulated cells; group “CoCl ₂ ” - activity values of HIF-1α CoCl ₂ -stimulated cells. The data are presented as arithmetic mean ± standard error of the mean (n = 3; * p <0.05 in relation to the control group; ^# p <0.05 in relation to the CoCl ₂ group).

Claims (3)

1. The use of N-phenylacetyl-L-prolylglycine ethyl ester to activate the transcription factor HIF (Hypoxia induced factor, hypoxia-induced factor). 2. A method of activating the transcription factor HIF, which is the introduction of a prophylactically or therapeutically effective amount of ethyl ester of N-phenylacetyl-L-prolylglycine. 3. A method of treating diseases associated with a deficiency of transcription factor HIF, which consists in the introduction of a prophylactically or therapeutically effective amount of ethyl ester of N-phenylacetyl-glycylproline, causing activation of transcription factor HIF.

Images