Дослідження активації mTORC1 та його субстракту p70S6K, залучених при цукровому діабеті 2 типу та онкогенетичних процесах

Вступ. Новітні дослідження довели вплив патогенетичних факторів цукрового діабету на активність внутрішньоклітинних сигнальних шляхів регуляції онкогенезу іметаболізму, одним з яких є PI3K/Akt/mTORC1. Макрофаги та лімфоцити беруть участь у патогенезі діабету та раку. Надмірна активація компонентів та субстратів PI3K/Akt/mTORC1 в цих клітинах може вказувати на необхідність додаткової корекції метаболічних процесів у хворих на діабет 2 типу з точки зору профілактики онкологічних захворювань. Мета: вивчити активацію mTORC1 шляхом визначення фосфорилювання PRAS40 та p70S6K1 у лейкоцитах хворих на діабет 2-го типу та рак. Матеріали та методи.Жінки, залучені у дослідженні, були поділені на групи: контрольна група, пацієнти з діабетом 2 типу, хворі на рак та пацієнти з поєднанням раку і діабету. Вміст фосфорильованих PRAS40 (фосфо-T246) та p70S6K1 (фосфо-T389) визначали імуноферментним методом, використовуючи лабораторні набори ELISA КНО0421 таELISA 85-86053 фірми Invitrogen (США). Концентрацію білка в лізаті визначали за допомогою набору для аналізу протеїнів BCA Novagen (США). Вимірювання проводилися намікропланшетному зчитувачі (Bio-tek Instruments, США) при довжині хвилі 450 нм.Результати. Виявлений достовірно підвищений вміст фосфорильованих PRAS40 та p70S6K1 у лейкоцитах хворих на цукровий діабет 2-го типу та рак. Кількість позитивних фосфо-PRAS40 проб у хворих на діабет становила 83,3%, а у хворих на рак -66,7%. Виявлений знижений вміст фосфо-PRAS40 в лейкоцитах пацієнтів з поєднанням діабету і раку. Висновки.Підвищений вміст фосфорильованих PRAS40 та p70S6K1 доводить активацію досліджуваного сигнального шляху при цукровому діабеті 2 типу. Зниження їх активаціїпри поєднанні раку та діабету можна пояснити можливими конкуруючими ефектами білків, які впливають на регулятори цих кіназ або на них безпосередньо.ntroduction.Pathogenetic factors of diabetes may affect the activity of intracellular systems of oncogenesis andmetabolismregulation, one of which is PI3K/Akt/mTORC1. Macrophages and lymphocytes are involved in the pathogenesis of diabetes and cancer. Detection of excessive activation of PI3K/Akt/mTORC1 components and substrates in these cells may indicate the need for additional correction of metabolic processes in patients with type 2 diabetes from the point of prevention of cancer. The aim:to study the activation of mTORC1 by determining the phosphorylation of PRAS40 and p70S6K1 in the leukocytes of patients with type 2 diabetes and cancer.Materials and methods. The study included women from the following groups: control group, patients with type 2 diabetes, cancer patients, patients with both diseases. The content of phosphorylated PRAS40 (phospho-T246) and p70S6K1 (phospho-T389) was determined using laboratory kits ELISA KNO0421 and ELISA 85-86053 of Invitrogen (USA). The protein concentration in the lysate was determined using a BCA Novagen protein assay kit (USA). Measurements were performed on a microplate reader (Bio-tek Instruments, USA) at a wavelength of 450 nm. Results.Significantly increased content of phosphorylated PRAS40 and p70S6K1 inleukocytes of patients with type 2 diabetes mellitus and cancer was detected. The number of positive phospho-PRAS40 tests in patients with diabetes was 83.3%, and in cancer patients -66.7%. Was revealedthe reduced content of phospho-PRAS40 in leukocytesof patients with a combination of diabetes and cancer.Conclusions.The increased amount of phosphorylated PRAS40 and p70S6K1 proves the activation of the studied signaling pathway by diabetes mellitus type 2. Its decrease by cancer and diabetes can be explained by the possible competing effects of the proteins that affect upstream regulators of these kinases or them directly

Directed search for diuretics among 6-substituted pteridine-2,4,7(1H,3H,8H)-triones

Directed search for biologically active compounds among heterocycles still remains a relevant area of medical chemistry. Among the significant number of heterocyclic compounds, pteridines deserve special attention. Among the above-mentioned ones the drugs with antitumor, antimicrobial, antiviral, diuretic and other types of biological action are known. Nevertheless, 6-substituted pteridine-2,4,7(1H,3H,8H)-triones, which are structurally similar to triamterene (6-phenylpteridine-2,4,7-triamine) – a diuretic with potassium-sparing action are interesting objects for search for diuretics. All the more, they are characterized by prototropic tautomerism, able to form hydrogen and donor-acceptor bonds with various ligands, and it is likely that these structural features will provide their diuretic effect. The aim of the study is the directed search for diuretics among 6-substituted pteridine2,4,7(1H,3H,8H)-triones using in silico and in vivo methodology and elucidation of the probable mechanism of action. 1-methyl-3-R-6- (2-oxo-2-aryl- (hetaryl-) ethyl) pteridine-2,4,7(1H,3H,8H)-triones were selected to study the effect on renal excretory function. and 1-methyl-3-R-6- (2-hydroxy-2-aryl- (hetaryl-) ethyl) pteridine-2,4,7(1H,3H,8H)-triones. Directed search for compounds that affect the excretory function of the kidneys of rats was conducted by the conventional method of E.B. Berkhin with water load. The content of creatinine, sodium, potassium and chlorides in blood and urine plasma was determined by biochemical methods using standard test kits of NPV "Philisit-Diagnostics" (Ukraine) and calculations were performed according to generally accepted methods. Research of the probable mechanism was conducted by flexible molecular docking, as an approach of finding molecules with affinity to a specific biological target. Macromolecular data were downloaded from the Protein Data Bank (PDB) namely, the crystal structures of Human carbonic anhydrase II (PDB ID – 3HS4) and epithelial sodium channel (ENaC) (PDB ID – 4NTX). Studies of the effect of the synthesized compounds on the excretory function of the kidneys of rats showed that 1-methyl-3-R-6- (2-oxo-2-aryl- (hetaryl-) ethyl) pteridine-2,4,7(1H,3H,8H)-triones containing 4-fluorophenyl, 2,4-difluorophenyl, 4-chlorophenyl fragments in the molecule increase diuresis by the second hour by 27.3-70.1% compared with the control group. According to the results of the impact on daily diuresis, it was found that the most active was 1-methyl-6- (2-oxo-2-phenyl) ethyl) pteridine-2,4,7(1H,3H,8H)-triones, which increased daily diuresis by 168.1%, exceeding the effect of Hydrochlorothiazide (41.8%) and Triamterene (49.1%). However, substituted 1-methyl-3-R-6- (2-hydroxy-2-aryl- (hetaryl-) ethyl) pteridine-2,4,7(1H,3H,8H)-triones are inactive compounds. In-depth studies using biological tests and molecular docking have suggested that 1-methyl-6- (2-oxo-2-aryl) ethyl) pteridine-2,4,7(1H,3H,8H)-triones 2.1, 2.5 and 2.6) probable mechanisms of diuretic action are disruption of sodium transport in the distal convoluted tubules, causing sodium excretion and water loss and possibly inhibition of epithelial sodium channels that promote sodium uptake and potassium secretion in the distal convolutions and tubules, which implements potassium-sparing action. A well-founded and developed strategy for the search for diuretics among 6-substituted pteridine-2,4,7(1H,3H,8H)-triones has identified a number of effective compounds that by diuretic effect are superior to the reference drugs "Hydrochlorothiazide" and "Triamterene". Importantly, the results of molecular docking suggested a mechanism of action of the compounds under study, similar to thiazide diuretics. This action may be related to the tautomerism of these compounds and, as a consequence, their ability to form coordination bonds with the zinc cation and the additional interaction of halogens in the active site of CA II. It was possible to detect the presence of potassium-sparing action, probably due to the ability to inhibit epithelial sodium channels (ENaC). The obtained results substantiate the further purposeful search for potential diuretics among this class of compound

In silico and in vivo screening of triamterene synthetic analogues as promising diuretics

The modification of lead-compound aimed to the increasing of activity, decrement of toxicity or improvement of selectivity is one of the most important methods used for elaboration of novel medications. Natural compounds, approved or investigational drugs or just compounds with proved biological activity could be the lead-compound. Often the chemical modification of lead compounds is directed at the enhancement of ligand-biological target interactions. Abovementioned approach, namely structural modification of known drug triamterene was used for purposeful search for novel diuretics. The preliminary prognostication of ligand-target interactions and affinity levels allow to reduce quantity of experimental animals, synthesis, and pharmacological studies costs. Conducted studies revealed the series of promising 6,7-disubstituted pteridine-2,4(1H,3H)-diones with diuretic activity that comparable with pharmacological effect of triamterene. Aim – purposeful search for promising diuretics among structural analogues of triamterene that includes preliminary in silico studies, synthesis and in vivo screening of novel compounds for diuretic activity. Methods used: organic synthesis, physicochemical methods of analysis of organic compounds (NMR 1 H-spectroscopy, chromato-mass spectrometry, elemental analysis). Prediction of affinity for a biological target, prediction of toxicity and lipophilicity of the combinatorial library, which was created on the basis of the drug triamterene, was carried out using computer services. Studies of compounds that affect the excretory function of the kidneys of rats were performed according to the generally accepted method of E.B. Berkhin with water load. Research of the probable mechanism was conducted by flexible molecular docking, as an approach of finding molecules with affinity to a specific biological target. Macromolecular data were downloaded from the Protein Data Bank (PDB) namely, the crystal structures of epithelial sodium channel (ENaC) ((PDB ID – 6WTH). The substantiation of potential diuretics design was conducted by in silico methods (prediction of affinity, ligandenzyme interactions and pharmacokinetic characteristics). The structural modification of triamterene molecule was carried out by replacing of amino-group in positions 2, 4 and 7 by others “pharmacophore” fragments. Abovementioned transformation is aimed at the changing of ligand-enzyme interactions in active site, lipophility and toxicity. Synthesis of 6,7- disubstituted pteridine-2,4(1H,3H)-diones was conducted by condensation 5,6-diamino-2-oxo-(thioxo-)-2,3-dihydropyrimidin4(1H)-ones with carbonyl-containing compounds or oxocarboxylic acids. The further modification of obtained compounds was performed by alkylation, hydrazinolysis and nucleophilic addition/elimination. The structure of obtained compounds was proven by elemental analysis, chromato-mass and 1 H NMR-spectral analysis. The studies of synthesized compounds effect on excretion function of kidneys allowed to detect series of promising structural analogues of triamterene that exceed it in pharmacological activity by 27.3-99.0%. The “structure-biological activity” relationship was discussed and perspective of the further search of diuretics among abovementioned compounds were shown. The design of new biologically active compounds with diuretic activity was performed using in silico methodologies and realized by structural modification of the well-known diuretic triamterene. Traditional organic synthesis was used for preparation of target compounds, in vivo experiments wereused to detect compounds with significant biological activity. Several effective compounds were identified among pteridines, which exceed the reference drug triamterene in terms of daily diuresis. The obtained results substantiate further purposeful search, in-depth research on experimental pathologies and study of the mechanism of action of potential diuretics among this class of compound

Effect of combined treatment with insulin and metformin on 5′AMPactivated protein kinase activity in lymphocytes of diabetic patients

The activity of 5′adenosine monophosphate-activated protein kinase (AMPK), controlling the energy balance of a cell, in blood cells at the combined treatment of patients with type 2 diabetes with metformin and insulin was determined by enzyme immunoassay. It has been shown that metformin increases threefold the AMPK activity in lymphocytes of patients with type 2 diabetes, compared with patients before treatment. Insulin and its analog reduced the activity of the protein kinase stimulated by metformin in the blood cells, acting as metformin antagonists. The mechanisms of drug interaction and the consequences of their antagonism are discussed.Методом імуноферментного аналізу визначали активність 5′аденозинмонофосфатактивованої протеїнкінази (AMPK), що контролює енергетичний баланс клітини, в клітинах крові при комбінованому лікуванні хворих на діабет 2-го типу метформіном і інсуліном. Показано, що метформін утричі підвищує активність AMPK у лімфоцитах хворих на діабет 2-го типу в порівнянні з пацієнтами до лікування. Інсулін і його аналог знижували стимульовану метформіном активність протеїнкінази в клітинах крові, виступаючи антагоністами метформіну. Обговорюються механізми взаємодії препаратів та наслідки їх антагонізму.Методом иммуноферментного анализа определяли активность 5′аденозинмонофосфатактивируемой протеинкиназы (АМРК), контролирующей энергетический баланс клетки, в клетках крови при комбинированном лечении больных диабетом 2-го типа метформином и инсулином. Показано, что метформин в три раза повышает активность АМРК в лимфоцитах больных диабетом 2-го типа по сравнению с пациентами до лечения. Инсулин и его аналог снижали стимулируемую метформином активность протеинкиназы в клетках крови, выступая антагонистами метформина. Обсуждаются механизмы взаимодействия препаратов и последствия их антагонизма

Effect of combined treatment with insulin, metformin, and gliclazide on the expression and activity of Akt, mTOR, and p70S6K protein kinases in lymphocytes of diabetic patients

The expression of the main effector protein kinase Akt of the PI3K signaling pathway and the activities of mTOR and p70S6K protein kinases in lymphocytes of patients with diabetes mellitus are determined at the combination treatment with insulin and hypoglycemic drugs. It is shown that the Akt expression is reduced in blood mononuclear cells of patients with diabetes of the 2nd and, to a less extent, type 1. The activities of mTOR and p70S6K are also reduced in lymphocytes of patients with type 2 diabetes. Possible mechanisms of inhibiting the expression and activity of protein kinases are discussed.Визначали експресію основної ефекторної протеїнкінази сигнального шляху PI3K Akt та активність протеїнкіназ mTOR і p70S6K в лімфоцитах хворих на цукровий діабет при комбінованому лікуванні інсуліном і цукрознижувальними препаратами. Показано, що експресія Akt знижена в мононуклеарах крові хворих на діабет 2-го і, меншою мірою, 1-го типу. Активність mTOR і p70S6K також знижена в лімфоцитах хворих на діабет 2-го типу. Обговорюються можливі механізми пригнічення експресії і активності протеїнкіназ.Определяли экспрессию основной эффекторной протеинкиназы сигнального пути PI3K Akt и активность протеинкиназ mTOR и p70S6K в лимфоцитах больных сахарным диабетом при комбинированном лечении инсулином и сахароснижающими препаратами. Показано, что экспрессия Akt снижена в мононуклеарах крови больных диабетом 2-го и, в меньшей степени, 1-го типа. Активность mTOR и p70S6K также снижена в лимфоцитах больных диабетом 2-го типа. Обсуждаются возможные механизмы подавления экспрессии и активности протеинкиназ

Association of 5’AMP-activated protein kinase activity with disease duration and HbA1c content in leukocytes in diabetic patients

Background. 5’AMP-activated protein kinase (AMPK) is an enzyme that controls the cell energy ba­lance. With energetic stress in the cell and an increase in the AMP concentration, ATP is replaced by AMP in the exchange centers, resulting in the allosteric activation of AMPK by phosphorylation of 172 threonine within alpha subunit of LKB1 complex in response to changes in cell energy or CAMKKβ, which activates intracellular Ca2+. The purpose was to study the activity of the main energy sensor of cells — AMPK in leukocytes in patients taking insulin preparations, metformin, and other hypoglycemic drugs in association with disease duration and glycated hemoglobin content. Materials and methods. The diabetic patients receiving single-drug or combined therapy with insulin and its analogues, metformin, dapagliflozin and sulfonylureas were randomized into 5 groups: the first group — with an HbA1c level close to the norm — 6.9–7.6 %; the second group — 7.6–9.0 %; the third group — > 9 %; the fourth group > 10 %; the fifth group — > 11 %. To determine the amount of phospho-AMPK (p-Thr172), ELISA kits were used. To get the calibration curve for the AMPK determination, a kidney cell culture HEK293T of the human embryonic kidney was used, which is recommended by manufacturer as a positive control. Results. It was shown that with increase of blood HbA1c, the level of AMPK activity in leukocytes gradually decreased. With increase of blood HbA1c, the level of AMPK activity in leukocytes gradually decreased. The activity of AMPK in leukocytes of patients with HbA1c > 11 % was more than 3.5-fold lower compared to the group with 6.9–7.6 % of HbA1c; AMPK activity in leukocytes in patients with disease duration of 20 years was 3-fold lower. Thus, the AMPK activity in leukocytes may be an indicator of diabetic compensation in diabetic patients. Conclusions. With increase of blood HbA1c, the level of p-AMPK in leukocytes gradually decreased. AMPK activity in leukocytes in diabetes patients with disease duration of 20 years was 3-fold lower than in patients with 10-year experience

Design and search for prospective diuretics (CA II Inhibitors) among aroylhydrazones of esters quinone oxime using in silico and in vivo methodology

The design and search for new selective inhibitors of CA II with a better pharmacological profile, which would cause minimal electrolyte disturbances in the body, remains an urgent problem of medical chemistry and pharmacology today. It is important that the discovered new classes of inhibitors do not always contain the main “pharmacophoric” function (sulfamide), which is characteristic of “classic” drugs (Acetazolamide, Methazolamide, Ethoxzolamide, Dorzolamide and others), but are derivatives of phenols, polyamines, coumarins/thiocoumarins, ureas, thioureas, hydroxamates, etc. These molecules also bind in the active site of the enzyme, but do not interact directly with the catalytic zinc ion or interact through zinc-coordinated water molecules/hydroxide ion. However, this leads to an increase in their selectivity and, as a result, pharmacological action. Continuing the search for compounds that affect urination, we were interested in aroylhydrazones of esters of quinone oxime. Firstly, they are characterized by certain structural features (dynamic and geometric isomerism); secondly, they exhibit redox properties; thirdly, the presence of aromatic fragments makes it possible to create a voluminous combinatorial library for analysis. These compounds are ligands in complexation reactions, and an additional increase in the number of hydrogen acceptors in the molecule due to structural modification will improve ligand-enzymatic interactions with carbonic anhydrase (CAII) and, as a result, reveal new promising diuretics. The aim – design and search for potential diuretics (CA II inhibitors) among aroylhydrazones of esters of quinone oxime using in silico, traditional synthesis and in vivo methodologies. Methods of organic synthesis, physico-chemical methods of analysis of organic compounds (NMR 1H-spectroscopy, elemental analysis). Prediction of affinity to the biological target, prediction of toxicity and lipophilicity of the combinatorial library of benzohydrazides O-aroyl esters of quinone oxime using computer services. The study of compounds affecting the excretory function of rat kidneys was carried out according to the generally accepted method of E.B.Berkhin with water load. The investigation of the probable mechanism was carried out using flexible molecular docking, as an approach to search for molecules that have affinity for human carbonic anhydrase type II (CA II). Macromolecular data of the crystal structure of CA II (PDB ID – 3HS4) were downloaded from the Protein Data Bank (PDB). The design was developed and the search for diuretic agents among benzohydrazides of O-aroyl esters of quinone oximes was developed using in silico methods (prediction of affinity, lipophilicity, toxicity and enzyme-ligand interactions), traditional organic synthesis, and in vivo methods (effect on excretory function of rat kidneys). The synthesis of benzohydrazides of O-aroyl esters of quinone oxime was carried out by the interaction of aroylhydrazines with 4-[(aroylimino)]cyclohexa-2,5-dien-1-ones. The structure of the synthesized compounds was confirmed by elemental analysis and 1H NMR spectra. Studies of the effect of synthesized compounds on the excretory function of rat kidneys allowed us to identify a number of promising compounds among aroylhydrazones of quinonexime esters, which increase daily diuresis by 54.2-352.8% compared to the control group. At the same time, it was established that the most active was N'-(4-[(2-chlorobenzoyloxy)imino]cyclohexa-2,5-dien-1-ylidene)-3-nitrobenzohydrazide, which increased daily diuresis by 352.8% in comparison with the control group, while exceeding the effect of “Hydrochlorothiazide” (170.8%). The developed and implemented strategy for the search for diuretics among benzohydrazides of O-aroylesters of quinone oxime allowed the identification of an effective compound, which in terms of diuretic effect exceeds the comparison drug “Hydrochlorothiazide”. Visualization of the molecular docking of the active compounds showed that their geometry makes it difficult to place them in the pocket of the active site of CA II, but the pronounced diuretic effect can also be associated with their ability to form coordination bonds with the zinc cation. The obtained results justify the further targeted search for potential diuretics among this class of compounds for a more detailed understanding and study of the mechanism of action

PALEOGEOGRAPHIC RESEARCHES OF LATE GLACIAL AND EARLY HOLOCENE IN THE NORTHWEST OF THE EAST EUROPEAN PLAIN

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APPLICATION PALEOGEOGRAPHIC DATABASE OF "Q-KOLA" IN THE STUDY OF THE PLEISTOCENE-HOLOCENE BOUNDARY

38-3

Investigation of plate-type barrier ozonizers with ac and pulse power supplies

In this paper the experimental results on the investigation of plate-type reactors operated on the base of barrier discharge have been presented. Different reactors with planar, strip, and trench electrodes were investigated. Such reactors operated under atmospheric pressure with ac and pulse power sources with voltage of up to 10 kV, frequency up to 12 kHz. Using atomized spectroscopy system the measurements of the main specifications of the reactors such as ozone yielding rate, the temperature in the reactor and the air flow rate were carried out