Peroxymonocarbonate as oxidative inhibitor of protein PTEN and use thereof

Abstract

The present invention relates to a PTEN oxidative inhibitor and a use thereof. More specifically, the present invention relates to a PTEN oxidative inhibitor comprising peroxymonocarbonate (HCO4-) as an active ingredient, and a use thereof, for example, a use in a pharmaceutical composition for treating a disease state regulated by the PI3K / AKT pathway.

Description

Peroxymonocarbonate, an oxidative inhibitor of protein PTEN, and uses thereof The present invention relates to PTEN oxidative inhibitors and uses thereof. More particularly, the present invention relates to PTEN oxidative inhibitors comprising peroxymonocarbonate (HCO4-) as an active ingredient, and uses thereof, for example, in pharmaceutical compositions for the treatment of disease states regulated by the AKT signaling pathway mediated by PTEN (PTEN / AKT). When cells are stimulated by growth factors and cytokines such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and interleukin-3 (IL-3), hydrogen peroxide (H2O2) is generated through the activity of NADPH oxidase (NOX) [1]. Activation of NOX generates superoxide (O2-), which is converted to H2O2 by the activity of superoxide dismutase (SOD) [2]. This physiological H2O2 influences various intracellular signaling pathways. Its ability to oxidize cysteine residues in some proteins, such as protein tyrosine phosphatases (PTPs), leads to functional modifications that significantly impair their activity [3]. PTPs play a pivotal role in cellular processes related to cell growth, proliferation, and differentiation. Within their structural framework, all PTPs contain a cysteine residue in their active site [5]. Exposure to H2O2 oxidizes the cysteine residue to form cysteine-sulfenic acid (Cys-SOH), thereby inhibiting the enzymatic activity of PTPs [6]. Phosphatase and tensin homolog (PTEN), a member of the PTP family, is structurally composed of a short N-terminal phosphatidylinositol (PtdIns)(4,5)P2-binding domain (PBD), a catalytic phosphatase domain, a C2 lipid / membrane-binding domain, a C-terminal tail containing Pro, Glu, Ser, and Thr (PEST) sequences, and a class I PDZ-binding (PDZ-BD) motif. PTEN interacts with phospholipid membranes through the C2 domain located at its C-terminal end. The strong connection between the C2 and phosphatase domains not only helps the C2 domain bind PTEN to the membrane but also optimizes the orientation of the catalytic domain relative to membrane-bound substrates. Moreover, the phosphatase domain of PTEN has an extended active site to accommodate the membrane-bound phosphatidylinositol-3,4,5-triphosphate (PIP3), a phosphoinositide substrate [7][8]. Therefore, PTEN is considered a tumor suppressor due to its ability to dephosphorylate PIP3 and consequently negatively regulate the PI3K / AKT signaling pathway, which is pivotal in regulating cell survival, growth, and proliferation [9].

[0010] . Beyond cancer research, PTEN inhibition has proven to be a promising therapeutic intervention for neurodegenerative diseases, ischemia, infections, and insulin-resistant metabolic disorders.

[0011] . Like other members of the PTP family, PTEN contains a cysteine residue in the active site of its phosphatase domain, making it vulnerable to oxidation inhibition by ROS, particularly H2O2 [5]. Lee et al. first demonstrated the reversible inactivation of PTEN by H2O2 through oxidation of the Cys124 catalytic residue in the active site and formation of an intramolecular disulfide bond with Cys71. This inactivation is reversible because oxidized PTEN can be converted back to a functional reduced form by cellular reductants such as the Trx / TrxR (Thioredoxin / Thioredoxin Reductase) system.

[0012] . Additionally, peroxiredoxins (Prx), thiol-specific antioxidants of the peroxidase family within cells, can function as regulators of H2O2-induced phosphorylation signaling due to their ability to sense and scavenge superoxide.

[0013]

[0014]

[0015] . Therefore, the mechanism of PTEN oxidation by transient H2O2, a signal transducer generated in response to growth factor receptor stimulation, raises the question of how small amounts of physiological H2O2 oxidize PTEN in a cellular environment rich in H2O2 scavengers such as catalase, glutathione peroxidase, and Prx, together with the presence of the ubiquitous Trx / TrxR reducing system. Prior art literature Non-patent literature 1. Rhee, SG; Bae, Y.S.; Lee, S.-R.; Kwon, J. Hydrogen peroxide: a key messenger that modulates protein phosphorylation through cysteine oxidation. Science's STKE 2000, 2000, pe1-pe1. 비특허문헌 2. Forman, H.J.; Maiorino, M.; Ursini, F. Signaling functions of reactive oxygen species. Biochemistry 2010, 49, 835-842. 비특허문헌 3. Rhee, S.G. H2O2, a necessary evil for cell signaling. Science 2006, 312, 1882-1883. 비특허문헌 4. van der Geer, P.; Hunter, T.; Lindberg, R.A. Receptor protein-tyrosine kinases and their signal transduction pathways. Annual review of cell biology 1994, 10, 251-337. 비특허문헌 5. Denu, J.M.; Dixon, J.E. Protein tyrosine phosphatases: mechanisms of catalysis and regulation. Curr Opin Chem Biol 1998, 2, 633-641, doi:10.1016 / s1367-5931(98)80095-1. 비특허문헌 6. Denu, J.M.; Tanner, K.G. Specific and reversible inactivation of protein tyrosine phosphatases by hydrogen peroxide: evidence for a sulfenic acid intermediate and implications for redox regulation. Biochemistry 1998, 37, 5633-5642. [CrossRef] 7. Lee, Y.-R.; Chen , M. ; Pandolfi, PP The functions and regulation of the PTEN tumor suppressor: new modes and prospects. Nature reviews Molecular cell biology 2018, 19, 547–562. [CrossRef] [PubMed] 8. Lee, J.-O.; Yang, H.; Georgescu, M.-M.; Di Cristofano, A.; Maehama, T.; Shi, Y.; Dixon , JE ; Pandolfi, P.; Pavletich, NP Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association. Cell 1999, 99, 323–334. 9. Stambolic, V.; Suzuki, A.; De La Pump, JL; Brothers, GM; Mirtsos , C. ; Sasaki, T.; Ruland, J.; Penninger , JM ; Siderovski, DP; Mak, TW Negative regulation of PKB / Akt-dependent cell survival by the tumor suppressor PTEN. Cell 1998, 95, 29–39. 10. Leslie, NR; Bennett , D. ; Lindsay, HE; Stewart , H. ; Gray , A. ; Downes, CP Redox regulation of PI 3-kinase signaling via inactivation of PTEN. The EMBO Journal 2003, 22, 5501–5510. 비특허문헌 11. Pulido, R. PTEN inhibition in human disease therapy. Molecules 2018, 23, 285. 비특허문헌 12. Lee, S.-R.; Yang, K.-S.; Kwon, J.; Lee, C.; Jeong, W.; Rhee, S.G. Reversible inactivation of the tumor suppressor PTEN by H2O2. Journal of Biological Chemistry 2002, 277, 20336-20342. 비특허문헌 13. Rhee, S.G.; Chae, H.Z.; Kim, K. Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free radical biology and Medicine 2005, 38, 1543-1552. 비특허문헌 14. Rhee, S.G.; Woo, H.A.; Kil, I.S.; Bae, S.H. Peroxiredoxin functions as a peroxidase and a regulator and sensor of local peroxides. Journal of Biological Chemistry 2012, 287, 4403-4410. 비특허문헌 15. Perkins, A.; Nelson, K.J.; Parsonage, D.; Poole, L.B.; Karplus, P.A. Peroxiredoxins: guardians against oxidative stress and modulators of peroxide signaling. Trends in biochemical sciences 2015, 40, 435-445. As a result of our efforts to develop a PTEN oxidative inhibitor, After confirming that the oxidation rate of PTEN and the phosphorylation rate of AKT can be increased when peroxymonocarbonate (HCO4-) is formed by combining bicarbonate (HCO3-) and hydrogen peroxide (H2O2), the present invention was completed. Accordingly, the present invention aims to provide a PTEN oxidation inhibitor containing peroxymonocarbonate (HCO4-) as an active ingredient. The present invention also aims to provide a pharmaceutical composition for inhibiting PTEN oxidation, which comprises peroxymonocarbonate (HCO4-) as an active ingredient. However, the problems that the present invention seeks to solve are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below. The present invention provides a PTEN oxidative inhibitor comprising peroxymonocarbonate (HCO4-) as an active ingredient and its use, for example, use in a pharmaceutical composition. According to the first implementation example, A PTEN oxidative inhibitor containing peroxymonocarbonate (HCO4-) as an active ingredient is disclosed. In the present invention, the peroxymonocarbonate is produced by a reaction between bicarbonate and hydrogen peroxide (H2O2), and thus the oxidation rate of PTEN can be increased. In the present invention, the PTEN oxidative inhibitor can be used in the manufacture of a pharmaceutical composition for treating a disease state regulated by the PTEN / AKT pathway. In the present invention, the PTEN oxidative inhibitor can be used to regulate cardiovascular disease, immune response, nervous system disease, or insulin-related metabolism. For example, the PTEN oxidative inhibitor can be used to regulate cardiovascular disease including myocardial infarction or heart failure; immune response including influenza infection, hepatitis B virus (HBV) infection, human immunodeficiency virus (HIV) infection, acute infection including pneumonia, meningitis, or tuberculin; sepsis or cancer; or nervous system disease including spinal cord paralysis, cerebral ischemia, stroke, cranial nerve paralysis, diabetic neuropathy, peripheral neuropathy, or Alzheimer's; insulin-related metabolism including type 2 diabetes, diabetic ketoacidosis, or hyperosmolar hyperglycemia. According to the second implementation example, A pharmaceutical composition for inhibiting PTEN oxidation comprising peroxymonocarbonate (HCO4-) as an active ingredient is disclosed. In the present invention, the pharmaceutical composition may be for the treatment of a disease state regulated by the PTEN / AKT pathway. In the present invention, the PTEN oxidative inhibitor can be used to control cardiovascular diseases including myocardial infarction or heart failure; acute infections including influenza infection, hepatitis B virus (HBV) infection, human immunodeficiency virus (HIV) infection, pneumonia, meningitis or tuberculin; immune responses including sepsis or cancer; neurological diseases including spinal cord paralysis, cerebral ischemia, stroke, cranial nerve paralysis, diabetic neuropathy, peripheral neuropathy or Alzheimer's; or insulin-related metabolism including type 2 diabetes, diabetic ketoacidosis or hyperosmolar hyperglycemia. In the present invention, the pharmaceutical composition can be administered orally, intravenously, subcutaneously, intramuscularly, intraperitoneally, intradermally, topically, intranasally, intravaginally, intrapulmonary, or rectally. The PTEN inhibitor according to the present invention can significantly increase the oxidation rate of PTEN by peroxymonocarbonate (HCO4-) formed by combining bicarbonate and hydrogen peroxide (H2O2), thereby increasing the phosphorylation rate of AKT. Meanwhile, the scope of the present invention is not limited by the effects described above. Figure 1 demonstrates that HCO3- can enhance PTEN oxidation by H2O2. HepG2 cells were cultured to 90% confluency, washed with PBS, and transferred to DMEM (L-glutamine, 1X penicillin-streptomycin, 0.1% FBS, and 25 mM HEPES) with or without 44 mM sodium bicarbonate. Cells were incubated for 4 h at 37°C with 0.1% CO2 and treated with stimulation medium containing 44 mM HCO3- alone, 1 mM H2O2 alone, or a combination of 1 mM H2O2 and 22 mM HCO3-. After 10 min, the reaction was stopped by adding lysis buffer containing protease inhibitors and 10 mM NEM. Cell lysates were then collected and used for SDS-PAGE and Western blot analysis. (A) Results obtained using PTEN antibody. (B) Quantitative results: PTEN oxidation was significantly higher in the presence of 22 mM or 44 mM HCO3- than in the presence of H2O2 alone, while 44 mM HCO3- alone did not show any oxidation effect. Figure 2 shows that HCO3- can accelerate the redox regulation of PTEN by H2O2. HepG2 cells were cultured until 90% confluent, washed with PBS, and transferred to DMEM (L-glutamine, 1X penicillin-streptomycin, 0.1% FBS, and 25 mM HEPES) in the presence or absence of 44 mM sodium bicarbonate. Cells were incubated for 4 h at 37°C with 0.1% CO2 and treated with stimulation medium containing 0.5 mM H2O2. After 5, 10, 15, 30, 60, and 120 min, the reaction was stopped by adding lysis buffer containing protease inhibitors and 10 mM NEM. Cell lysates were then collected and used for SDS-PAGE and Western blot analysis. (A) Results obtained using PTEN and β-actin antibodies are shown. (B) Quantitative results: In the absence of HCO3-, the PTEN oxidation rate is reduced and the reduction period is prolonged. (C) Linear regression trend lines of PTEN oxidation and reduction rates: Indicating that the presence of HCO3- accelerates the H2O2-mediated redox regulation of PTEN. Figure 3 shows that HCO3- and H2O2 can activate the AKT pathway through PTEN oxidation. (A) Results obtained using PTEN and pAKT antibodies are shown. (B) Quantitative results: In the absence of HCO3-, the phosphorylation rate of AKT was decreased, whereas in the presence of HCO3-, the phosphorylation rate of AKT was increased. Hereinafter, an improved PTEN oxidative inhibitor and its use according to specific embodiments of the invention will be described in detail. However, this is presented as one example of the invention, and the scope of the invention is not limited thereby, and it will be apparent to those skilled in the art that various modifications to the embodiments are possible within the scope of the invention. Unless otherwise specified, throughout this specification, "include" or "containing" refers to including a certain component (or component) without any particular limitation, and cannot be interpreted as excluding the addition of other components (or components). The term “PTEN (Phosphatase and tensin homolog)” as used herein refers to a human phosphatase and tensin homolog enzyme that is a product of EntrezGene ID: 5728 and may have an amino acid sequence composition corresponding to UniProt Accession: P60484. Other non-human homologs are also readily identified using known methods, such as BLAST searches, and are considered within the scope of the present invention. The term "active ingredient" as used herein means an ingredient that exhibits the desired activity alone or can exhibit the activity together with a carrier that is inactive in itself. *The term “prevention” as used herein means any act of inhibiting or delaying the onset of a disease by administering a PTEN oxidative inhibitor according to the present invention. The term “treatment” as used herein means any action that improves or beneficially changes the symptoms of a subject suspected of having or developing a disease by administering the PTEN oxidative inhibitor. The term "improvement" in the present invention means any action that at least reduces the severity of symptoms, for example, a parameter related to the condition being treated by administration of the PTEN oxidative inhibitor. The term “patient” as used herein refers to an animal, including animals such as cows, monkeys, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs. For example, the patient may be a mammal, particularly a human. 1. PTEN oxidative inhibitor The present invention The present invention provides a PTEN oxidative inhibitor containing peroxymonocarbonate (HCO4-) as an active ingredient. In the PTEN oxidation inhibitor according to the present invention, the peroxymonocarbonate is produced by the reaction of bicarbonate and hydrogen peroxide (H2O2), and thus the oxidation rate of PTEN can be increased. In the PTEN oxidative inhibitor according to the present invention, the PTEN oxidative inhibitor can be used in the manufacture of a pharmaceutical composition for treating a disease state regulated by the PTEN / AKT pathway. In one specific example, the PTEN oxidative inhibitor may be used to control cardiovascular disease. For example, the cardiovascular disease may include, but is not limited to, heart failure or myocardial infarction. In one specific example, the PTEN oxidative inhibitor may be used to modulate an immune response. The immune response may include, but is not limited to, acute infection, sepsis, or cancer. For example, the acute infection may include influenza infection, hepatitis B virus (HBV) infection, human immunodeficiency virus (HIV) infection, pneumonia, meningitis, or tuberculosis. In one specific example, the PTEN oxidative inhibitor may be used to control a neurological disease. The neurological disease may include, but is not limited to, spinal cord paralysis, cerebral ischemia, stroke, cranial nerve palsy, diabetic neuropathy, peripheral neuropathy, and Alzheimer's disease. In one specific example, the PTEN oxidative inhibitor may be used to regulate insulin-related metabolism. For example, the insulin-related metabolism may include, but is not limited to, type 2 diabetes, diabetic ketoacidosis, or hyperosmolar hyperglycemia. 2. Uses of PTEN oxidation inhibitors The present invention The present invention provides a pharmaceutical composition for inhibiting PTEN oxidation, which comprises peroxymonocarbonate (HCO4-) as an active ingredient. In the pharmaceutical composition according to the present invention, the pharmaceutical composition can be used for the treatment of a disease state regulated by the PTEN / AKT pathway. In one specific example, the PTEN oxidative inhibitor may be used to control cardiovascular disease. For example, the cardiovascular disease may include, but is not limited to, heart failure or myocardial infarction. In one specific example, the PTEN oxidative inhibitor may be used to modulate an immune response. The immune response may include, but is not limited to, acute infection, sepsis, or cancer. For example, the acute infection may include influenza infection, hepatitis B virus (HBV) infection, human immunodeficiency virus (HIV) infection, pneumonia, meningitis, or tuberculosis. In one specific example, the PTEN oxidative inhibitor may be used to control a neurological disease. The neurological disease may include, but is not limited to, spinal cord paralysis, cerebral ischemia, stroke, cranial nerve palsy, diabetic neuropathy, peripheral neuropathy, and Alzheimer's disease. In one specific example, the PTEN oxidative inhibitor may be used to regulate insulin-related metabolism. For example, the insulin-related metabolism may include, but is not limited to, type 2 diabetes, diabetic ketoacidosis, or hyperosmolar hyperglycemia. In the pharmaceutical composition according to the present invention, the pharmaceutical composition may be administered orally or parenterally. In the case of parenteral administration, the pharmaceutical composition may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intravaginal administration, intrapulmonary administration, and rectal administration. In the case of oral administration, for example, the pharmaceutical composition may be formulated as a tablet, or the active agent may be coated or formulated to protect it from degradation in the stomach. In addition, the composition may be administered by any device that allows the active agent to travel to the target cell. The route of administration may vary depending on the general condition and age of the subject to be treated, the nature of the treatment condition, and the active ingredient selected. In the pharmaceutical composition according to the present invention, the appropriate dosage of the pharmaceutical composition varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a generally skilled physician can easily determine and prescribe a dosage effective for the desired treatment or prevention. For example, the pharmaceutical composition may be administered in one or multiple doses, and may be administered once to four times a day. For example, the pharmaceutical composition may contain 0.01 mg / kg to 10 mg / kg, preferably 0.02 mg / kg to 9 mg / kg, and more preferably 0.03 mg / kg to 8 mg / kg per adult. In the pharmaceutical composition according to the present invention, the pharmaceutical composition may be prepared in a unit dose form or may be prepared by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may additionally include a dispersing agent or stabilizer. In addition, the pharmaceutical composition may be administered in the form of a suppository, spray, ointment, cream, gel, inhalant or skin patch. In addition, the pharmaceutical composition may be prepared for administration to mammals, more preferably for administration to humans. In the pharmaceutical composition according to the present invention, the pharmaceutically acceptable carrier may be a solid or a liquid, and may be at least one selected from excipients, antioxidants, buffers, bacteriostatic agents, dispersants, adsorbents, surfactants, binders, preservatives, disintegrants, sweeteners, flavoring agents, lubricants, release-controlling agents, wetting agents, stabilizers, suspending agents, and lubricants. In addition, the pharmaceutically acceptable carrier may be selected from saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof. In one embodiment, suitable fillers include, but are not limited to, sugars (e.g., dextrose, sucrose, maltose, and lactose), starches (e.g., corn starch), sugar-alcohols (e.g., mannitol, sorbitol, maltitol, erythritol, and xylitol), starch hydrolysates (e.g., dextrin and maltodextrin), cellulose or cellulose derivatives (e.g., microcrystalline cellulose), or mixtures thereof. In one specific embodiment, suitable binders include, but are not limited to, povidone, copovidone, methylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, gelatin, gums, sucrose, starch, or mixtures thereof. In one specific example, suitable preservatives include, but are not limited to, benzoic acid, sodium benzoate, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, chlorbutol, gallate, hydroxybenzoate, EDTA or mixtures thereof. In one specific example, suitable disintegrants include, but are not limited to, sodium starch glycolate, cross-linked polyvinyl pyrrolidone, cross-linked carboxymethylcellulose, starch, microcrystalline cellulose, or mixtures thereof. In one embodiment, suitable sweeteners include, but are not limited to, sucralose, saccharin, sodium or potassium or calcium saccharin, acesulfame potassium or sodium cyclamate, mannitol, fructose, sucrose, maltose or mixtures thereof. In one specific example, suitable glidants include, but are not limited to, silica, colloidal silicon dioxide, talc, and the like. In one specific embodiment, suitable lubricants include, but are not limited to, long-chain fatty acids and their salts, such as magnesium stearate and stearic acid, talc, glyceride waxes, or mixtures thereof. Below, various examples are presented to aid understanding of the invention. These examples are provided solely to facilitate understanding of the invention and are not intended to limit the scope of protection of the invention. Materials and Methods 1. Materials Dulbecco's modified Eagle's medium (DMEM) D5648 and N-Ethylmaleimide (NEM) were purchased from Sigma-Aldrich, 1 M HEPES from Enzynomics, 100X Penicillin / Streptomycin from Capricorn Scientific, fetal bovine serum (FBS) from Welgene, 3% hydrogen peroxide from Samchun, sodium bicarbonate from Amresco, Pro-prep Protein Extraction Solution from iNtRON Biotechnology, and BCA Protein Assay Kit from Thermo Scientific. Antibodies used for Western blotting consisted of primary PTEN antibody, β antibody, and anti-rabbit immunoglobulin G horseradish peroxidase-conjugated secondary antibody. 2. Cell culture HepG2 cells were cultured in DMEM supplemented with 5% FBS and 1X Penicillin / Streptomycin. 3. Immunoblot analysis of H2O2-induced oxidation of PTEN To assess the redox state of PTEN, a mobility shift assay was performed in cells using N-Ethylmaleimide (NEM) as an alkylating agent. HepG2 cells were cultured until 90% confluency. The cells were washed with phosphate-buffered saline (PBS) and replaced with medium containing 0.1% FBS and 25 mM HEPES in the presence or absence of sodium bicarbonate, and pre-incubated at 37°C with 0.1% CO2. Serum-free stimulation medium containing 0.5–1 mM H2O2 with varying bicarbonate conditions was prepared prior to application to the cells. After 4 hours of pre-incubation, the medium was removed and the cells were incubated with the prepared stimulation medium. At various time points, the stimulation medium was removed, and the cells were washed twice with cold PBS. The reaction was stopped by adding Pro-prep lysis buffer containing 10 mM NEM. Cell lysates were collected, and protein concentrations were quantified using the BCA method. Next, the samples were subjected to non-reducing SDS-PAGE, followed by Western blotting using PTEN-specific antibodies and β-actin-specific antibodies. <Result> 1. Whether HCO3- can enhance PTEN oxidation by H2O2 HepG2 cells were pre-incubated for 4 h at 37°C in HCO3-free medium with 0.1% CO2. Various stimulation media were prepared before administration to the cells, and the percentage of oxidized PTEN was assessed after 10 min of treatment: 44 mM HCO3- alone, 1 mM H2O2 alone, a combination of 1 mM H2O2 and 22 mM HCO3-, and a combination of 1 mM H2O2 and 44 mM HCO3-. As a result, it was confirmed that the stimulation medium containing only 44 mM HCO3- did not show an oxidation effect, whereas when 22 mM or 44 mM HCO3- was present in the stimulation medium, the oxidation of PTEN was significantly increased after 10 minutes compared to cells treated with only H2O2 (Fig. 1). 2. Whether bicarbonate can accelerate the redox regulation of PTEN by H2O2. To investigate changes in PTEN oxidation, the percentage of oxidized PTEN was assessed at different time points. Treatment of HepG2 cells with H2O2 in the presence or absence of 44 mM HCO3- resulted in PTEN oxidation in a time-dependent manner for 5, 10, 15, 30, 60, and 120 min. In the first group, cells were pre-incubated and treated in a stimulation medium containing 44 mM HCO3-. In the second group, cells were treated under the same conditions as in the first group, except that the medium did not contain HCO3. As a result, in the HCO3- containing group, the PTEN oxidation rate reached a peak at 10 minutes and then gradually decreased, and almost all oxidized PTEN was reduced within 60 minutes and recovery was completed within 120 minutes, whereas in the HCO3- non-containing group, the oxidation rate increased at a slow rate, reaching a peak at 30 minutes, and at 60 minutes, a significant amount of unreduced oxidized PTEN was present, which was observed to be significantly higher than that of the HCO3- containing group. The linear regression trend line indicates that PTEN oxidation was faster in the HCO3- containing group than in the HCO3- non-containing group. Comparing the slopes between the trend lines (2.87 and 1.16), it was confirmed that the difference in oxidation rate was almost 2.5 times. In the absence of HCO3- during the recovery period, the reduction of oxidized PTEN was significantly impaired, and a comparison of the slopes showed that the difference in the deceleration rate was also almost 2.3 times. Therefore, it was demonstrated that HCO3- can accelerate the H2O2-mediated redox regulation of PTEN (Fig. 2). 3. Whether the AKT pathway can be activated through PTEN oxidation by bicarbonate and H2O2. To investigate whether PTEN oxidative inhibition affects the phosphorylation of AKT, SDS-PAGE was performed on cell lysates using antibodies specific for phosphorylated-serine473 AKT and total AKT using the same method as above. As a result, in the case of the HCO3-free group, phosphorylation of AKT slowly increased to a peak at 30 minutes after exposure to H2O2, whereas in the 44 mM HCO3-containing group according to the present invention, phosphorylation of AKT began to increase significantly after 10 minutes of exposure to H2O2, reached a maximum at 15 minutes, and then gradually decreased, and the ratio of phosphorylated AKT was found to be higher than that of the HCO3-free group at all time points (Fig. 3). While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred implementation examples and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents. It is expected that the PTEN inhibitor according to the present invention can be used for the treatment or prevention of diseases regulated by the PTEN / AKT pathway.

Claims

1. PTEN oxidative inhibitor containing peroxymonocarbonate (HCO4-) as an active ingredient.

2. In paragraph 1, The above peroxymonocarbonate (HCO4-) is a PTEN oxidative inhibitor characterized in that it is produced by the reaction of bicarbonate and hydrogen peroxide (H2O2), thereby increasing the oxidation rate of PTEN.

3. In paragraph 1, A PTEN oxidative inhibitor characterized in that the above PTEN oxidative inhibitor is used in the manufacture of a pharmaceutical composition for treating a disease state regulated by the PTEN / AKT pathway.

4. In paragraph 1, A PTEN oxidative inhibitor characterized in that the above PETN oxidative inhibitor is used to regulate cardiovascular disease, immune response, nervous system disease or insulin-related metabolism.

5. In paragraph 4, The PTEN oxidative inhibitor is characterized in that it is used for controlling cardiovascular diseases including myocardial infarction or heart failure; acute infections including influenza infection, hepatitis B virus (HBV) infection, human immunodeficiency virus (HIV) infection, pneumonia, meningitis or tuberculin; immune responses including sepsis or cancer; neurological diseases including spinal cord paralysis, cerebral ischemia, stroke, cranial nerve paralysis, diabetic neuropathy, peripheral neuropathy or Alzheimer's; or insulin-related metabolism including type 2 diabetes, diabetic ketoacidosis or hyperosmolar hyperglycemia.

6. A pharmaceutical composition for inhibiting PTEN oxidation, comprising peroxymonocarbonate (HCO4-) as an active ingredient.

7. In paragraph 6, A pharmaceutical composition characterized in that the pharmaceutical composition is for the treatment of a disease state regulated by the PTEN / AKT pathway.

8. In paragraph 7, A pharmaceutical composition characterized in that the PTEN oxidative inhibitor is used for controlling cardiovascular diseases including myocardial infarction or heart failure; acute infections including influenza infection, hepatitis B virus (HBV) infection, human immunodeficiency virus (HIV) infection, pneumonia, meningitis or tuberculin; immune responses including sepsis or cancer; neurological diseases including spinal cord paralysis, cerebral ischemia, stroke, cranial nerve paralysis, diabetic neuropathy, peripheral neuropathy or Alzheimer's; or insulin-related metabolism including type 2 diabetes, diabetic ketoacidosis or hyperosmolar hyperglycemia.

9. In paragraph 6, A pharmaceutical composition characterized in that the pharmaceutical composition is administered orally, intravenously, subcutaneously, intramuscularly, intraperitoneally, intradermally, topically, intranasally, intravaginally, intrapulmonary, or rectally.

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