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Development of effective anti-inflammatory drug candidates among novel thiazolopyridines

T. I. Chaban1*, V. S. Matiychuk2, V. V. Ogurtsov1, I. G. Chaban1, I. A. Nektegayev1

1Danylo Halytsky Lviv National Medical University, Ukraine;
2Ivan Franko National University of Lviv, Lviv, Ukraine;

Received: 26 December 2019; Accepted: 27 March 2020

In an effort to develop novel anti-inflammatory agents, a series of thiazolo[4,5-b]pyridines were synthesized and modified at the N3 position. The structures of the obtained compounds were confirmed by 1H NMR spectroscopy and elemental analysis. The synthesized substances were preselected via molecular docking­ to be tested for their anti-inflammatory activity in vitro. Evaluation of compounds using the carrageenan-induced rat paw edema method showed strong anti-inflammatory action of some compounds (1, 2, 8) which exceeded that of ibuprofen.

Effects of thiazole derivatives on intracellular structure and functions in murine lymphoma cells

V. P. Hreniukh1, N. S. Finiuk1,2, Ya. R. Shalai1, B. O. Manko1,
B. V. Manko1, Yu. V. Ostapiuk1, O. R. Kulachkovskyy1,
M. D. Obushak1, R. S. Stoika1,2, A. M. Babsky1*

1Ivan Franko National University of Lviv, Ukraine;
2Institute of Cell Biology, Nationl Academy of Sciences of Ukraine, Lviv;

Received: 22 December 2019; Accepted: 27 March 2020

Thiazole derivatives have cytotoxic effects towards tumor cells, such as glioblastoma, melanoma, leukemia and lymphoma. However, the intracellular mechanism of this action is not clear. The aim of our study was to investigate the action of N-(5-benzyl-1,3-thiazol-2-yl)-3,5-dimethyl-1-benzofuran-2-carboxamide (BF1) and 7-benzyl-8-methyl-2-propylpyrazolo[4,3-e]thiazolo[3,2-a]pyrimidin-4(2H)-one (PP2) on cellular structure, and bioenergetic functions of mitochondria in Nemeth-Kellner lymphoma cells (NK/Ly). The structure of treated NK/Ly cells and their mitochondria was examined using electron microscopy. The rate of oxygen uptake by isolated mitochondria was recorded by a polarographic method using a Clark electrode. The mitochondrial potential relative values were registered using fluorescence dye rhodamine 123. In the short-term (15 min), incubation with BF1 and PP2 in 10 and 50 µM concentrations induced apoptotic and necrotic changes in the structure of NK/Ly cells, such as fragmentation and disintegration of the nucleus, destruction of the plasma membrane, and an increase in numbers of lysosomes and mitochondria. A polarographic method did not show significant metabolic shifts in lymphoma mitochondria, in either in vitro or ex vivo actions of the thiazole derivatives. However, fluorescent microscopy showed a significant decrease in mitochondria potential, following a 15 min incubation of cells with 50 µM of PP2. Thus, the electron and fluorescent microscopy data suggest that mitochondria are involved in the mechanism of cytotoxic action of the studied thiazole derivatives.

Synthesis and anti-leukemic activity of pyrrolidinedione-thiazolidinone hybrids

A. Kryshchyshyn1*, D. Kaminskyy1, O. Roman1, R. Kralovics2, O. Karpenko3, R. Lesyk1

1Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv National Medical University, Ukraine;
2Research Center for Molecular Medicine, Austrian Academy of Sciences, Vienna, Austria;
3Enamine Ltd., Kyiv, Ukraine;

Received: 22 December 2019; Accepted: 27 March 2020

A series of novel 2-(5-ylidene-4-oxo-2-thioxo-thiazolidin-3-yl)-succinimides and 5-ylidene-3-(1-arylpyrrolidine-2,5-dione)-thiazolidine-2,4-diones were synthesized. An efficient simple protocol for rhodanine-pyrrolidinedione hybrids synthesis which allows avoiding the step of anhydride formation was proposed. Following the previous data on antileukemic properties of related thiazolidinone derivatives, the activity of 19 target compounds was investigated towards four leukemia cell lines: Dami, HL-60, Jurkat, and K562. Among the tested compounds, 3-[5-(4-chloro-benzylidene)-4-oxo-2-thioxo-thiazolidin-3-yl]-1-phenyl-pyrrolidine-2,5-dione (Compound 1) possessed good and selective antiproliferative action against Dami and HL-60 cell lines and satisfactory toxicity level (acute toxicity evaluated in vivo in mice).

Novel approach for discrimination of eosinophilic granulocytes and evaluation of their surface receptors in a multicolor fluorescent histological assessment

G. Bila1, M. Schneider1, S. Peshkova1, B. Krajnik2, L. Besh3, A. Lutsyk1, O. Matsyura3*, R. Bilyy1*

1Department of Histology, Cytology and Embryology, Danylo Halytsky Lviv National Medical University, Ukraine;
2Faculty of Fundamental Problems of Technology, Wroclaw University of Science and Technology, Poland;
3Department of Pediatrics 2, Danylo Halytsky Lviv National Medical University, Ukraine;

Received: 09 January 2020; Accepted: 27 March 2020

Eosinophilic granulocytes mediate immune responses against multicellular parasites and are also the main contributor to such pathological conditions as allergy and asthma. Eosinophils are discriminated with eosin staining in conventional histology using light microscopy. However, molecular detection of antigens and the widely introduced automated analyzers usually require fluorescent markers to allow quantitative determination. Surprisingly, there is no selective CD marker to differentiate eosinophils and basophils. Recently reported analogs to replace hematoxylin and eosin staining for immune-histochemical applications such as DRAQ5 & eosin are also unsuitable due to wide fluorescent spectra. Different combinations of fluorescent dyes were tested using fluorescent microscopy aimed to develop a simple and specific method for detecting eosinophilic granules, DNA and surface receptors, the approach was used for evaluation of IgE levels (total and specific to casein) on cells of patients suffering from cow milk allergy. We were able to achieve selective visualization of eosinophil granules using aniline blue dye by modifying the method of Berretty & Cormane (1978) and detecting emission at 440nm; this allowed simultaneous staining of blood smears with anti-IgE-FITC (emission at 520 nm) and casein-FITC, detection of DNA with propidium iodide (em. 590 nm), and also provided specific metachromatic signal of eosinophils in the NIR region (em. ~700 nm) with subsequent quantification of fluorescent signal. Application of this approach to clinical cases revealed increased IgE levels and increased casein-binding targets on eosinophils in 3 patients with cow milk allergy compared to 2 healthy donors, demonstrating the general usefulness of the approach.

Exogenous hydrogen sulfide for the treatment of mesenteric damage associated with fructose-induced malfunctions via inhibition of oxidative stress

O. Revenko1*, N. Zaichko2, J. Wallace3, O. Zayachkivska1

1Department of Physiology, Danylo Halytskyy Lviv National Medical University, Ukraine;
2Department of Biochemistry and General Chemistry,
National Pirogov Memorial Medical University, Vinnytsia, Ukraine;
3Department of Physiology and Pharmacology, University of Calgary, Canada;

Received: 30 December 2019; Accepted: 27 March 2020

Remodeling of adipocytes in mesentery (AM) associated with nutritional overload from high fructose diet (HFD) is a source of several comorbidities. However, its pathogenesis is still unclear and there are no specific effective drugs for AM remodeling. Recently hydrogen sulfide (H2S) demonstrated potent cytoprotective actions. The purpose of this study was to investigate the effects and underlying mechanisms of AM remodeling in rats fed HFD and with H2S pre-treatment. Adult male rats on standard diet (SD, control group) or HFD that underwent acute water-immersion restraint stress (WIS) were evaluated for subcellular AM adaptive responses by electron microscopy. The effects on AM of exogenous sodium hydrosulfide (NaHS, 5.6 mg/kg/day for 9 days) and the Н2S-releasing aspirin (ASA) derivative (H2S-ASA [ATB-340], 17.5 mg/kg/day) vs conventional ASA (10 mg/kg/day) vs vehicle were investigated. Serum glucose level, thiobarbituric acid reactive substances (TBARS), and activities of cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) were examined biochemically using spectrophotometry. In the HFD groups, treatment with NaHS protected AM, as mesenteric microvascular endothelial and sub-endothelial structures were observed vs the vehicle-treated group that had signs of endothelial dysfunction, AM damage and dysfunctional mitochondria. The effect of H2S-ASA was characterized by protection of AM against HFD and WIS-induced injury, with lower TBARS blood level and increased CSE and CBS activities. Carbohydrate overload for 4 weeks is sufficient to cause AM oxidative damage, mitochondrial dysfunction and endothelial changes. H2S plays an important role in mesenteric adipocyte cellular survival against HFD-induced oxidative stress by decreasing overproduction of TBARS and mitochondrial dysfunction. The use of H2S could lead to a novel approach for anti-obesity treatment.

Aggregation of platelets, proliferation of endothelial cells and motility of cancer cells are mediated by the Bβ1(15)-42 residue of fibrin(ogen)

Y. M. Stohnii1, M. V. Ryzhykova1, A. V. Rebriev1,
M. D. Kuchma2, R. Y. Marunych1, V. O. Chernyshenko1*,
V. A. Shablii2, N. M. Lypova3, O. Yu. Slominskyi1,
L. V. Garmanchuk4, T. M. Platonova1, S. V. Komisarenko1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine;
2Institute of Cell Therapy, Kyiv, Ukraine;
3University of Louisville, USA;
4ESC “Institute of Biology and Medicine”, Taras Shevchenko National University of Kyiv, Ukraine;

Received: 23 December 2019; Accepted: 27 March 2020

The fibrinogen molecule contains multiple binding motifs for different types of cellular receptors, acting as a molecular link between coagulation and cell adhesion. In this study we generated a truncated form of the fibrinogen molecule lacking the Bβ1-42 sequence by site-specific proteolysis and evaluated the role of the fragment in adhesive capabilities of platelets, endothelial and cancer cells. Fibrinogen with the removed Bβ1-42 sequence and fibrin without the Bβ15-42 fragment (desβ1-42 fibrinogen and desABβ15-42 fibrin) were obtained by proteolysis using the specific protease from the venom of Echis multisquamatis. The cleaved fragment was purified by HPLC and was identified using MALDI-TOF. ADP- and collagen-induced aggregation of washed platelets in the presence of fibrinogen desBβ1-42 was studied using an aggregometer. Proliferation of mice aortic endothelial cells (MAEC) and human umbilical vein endothelial cells (HUVEC) was studied using the fibrin desABβ15-42 as the scaffold. Cell viability was quantified by the MTT test (MAEC). Generation time was calculated for the estimation of proliferative activity of HUVEC. Lung cancer cell line Н1299 was used to evaluate cancer cell motility in vitro using the scratch assay. Direct comparison of cellular behavior in the presence of truncated vs native forms demonstrated attenuated cell adhesion in the presence of fibrinogen desBβ1-42 and fibrin desBβ15-42. The platelet aggregation rate was only slightly decreased in the presence of fibrinogen desBβ1-42 but resulted in 15-20% disaggregation of adhered platelets. We also observed the substantial decrease of generation time of HUVEC and inhibition of viability of MAEC cells grown on scaffolds of a desABβ15-42 matrix. Finally, desBβ1-42 modulated the motility of H1299 cells in vitro and suppressed the wound healing by 20% compared to the full-length fibrinogen. We postulate that fragment 1-42 of the BβN-domain of fibrinogen is not sufficient for platelet aggregation, however it may contribute to platelet clot formation in later stages. At the same time, this fragment may be important for establishing proper cell-to-cell contacts and cell viability of endothelial cells. Also, 1-42 amino acid fragment of the BβN-domain supported the migration of cancer cells suggesting that interactions of fibrinogen with cancer cells could be a target for anticancer therapy. The Bβ1-42 fragment of fibrinogen contributes to efficient intracellular interactions of different types of cells, including platelets, endothelial cells and cancer cells.

Protective action of N-stearoylethanolamine on blood coagulation and arterial changes in spontaneously hypertensive rats fed cholesterol-rich diet

O. S. Tkachenko1, Ie. A. Hudz1*, H. V. Kosiakova1,
P. P. Klymenko2, Y. M. Stohnii1, V. A. Didkivskyi1,
T. M. Chernyshenko1, V. O. Chernyshenko1, T. M. Platonova1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2SI “D. F. Chebotarev Institute of Gerontology of the National Academy of Medical Sciences of Ukraine”, Kyiv;

Received: 24 December 2019; Accepted: 27 March 2020

In this work we aimed to test the atherosclerotic changes in the aortic wall and pro-coagulant response of the blood coagulation system of spontaneously hypertensive rats (SHR) fed cholesterol-rich diet (CRD) and to study the effect of the anti-inflammatory agent N-stearoylethanolamine (NSE) on the development of atherosclerosis in this model. Female rats (n = 30) with genetically determined hypertension proven by direct measurement of blood pressure were fed CRD (5% cholesterol) for 2 months. Control group of SHR (n = 10) received standard pellet diet, 10 were fed CRD and 10 received CRD with daily per os application of NSE at a dose of 50 mg/kg of body weight. Histological analysis detected swelling and detachment of endothelial cells, huge edema of the subendothelial layer and a disruption of the middle shell integrity. CRD rats had higher fibrinogen concentration, increased rate of platelet aggregation and decreased level of anticoagulant PC. Platelet aggregation speed increased in CRD-fed rats (52.5±4.1%/min) was slightly normalized under the action of NSE (40±8.3 vs 35±9%/min in controls). Fibrinogen concentration was slightly increased in CRD-fed rats (2.75±0.7 vs 1.9±0.5 mg/ml in controls). However, the level of anticoagulant PC that was decreased in CRD-fed rats (65±16 vs 100±11% in controls) was normalized under the action of NSE (92±17%). NSE also influenced the aorta architecture, however normalizing the thickness of the aorticwall did not change the cholesterol-induced inclusions within aorta media. NSE anti-inflammatory action changes the atherogenic processes in CRD-fed rats mainly protecting PC from consumption during the inflammatory process and reducing edema of the aorta. However hematological parameters (including clotting time in the APTT test and fibrinogen concentration) changed independently on NSE application. Anti-aggregatory action of NSE on platelets can be a result of direct action on platelets or the consequence of its anti-inflammatory action. During atherogenesis induced by CRD in the model, NSE demonstrated valuable anti-inflammatory action protecting the organism during atherogenesis, however it cannot be assumed as an antithrombotic or antiatherogenic agent because it is unable to influence hemostasis directly.

The effect of N-stearoylethanolamine on the lipid composition of the rat testes and testosterone level during the early stages of streptozotocin-іnduced diabetes

O. V. Onopchenko*, T. M. Horid’ko, H. V. Kosiakova,
A. G. Berdyshev, V. M. Klimashevsky, N. M. Hula

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;

Received: 23 December 2019; Accepted: 27 March 2020

Diabetes is a metabolic disorder with multiorgan complications, including reproductive system dysfunction where lipid imbalance of germ cells play an important role. N-stearoylethanolamine (NSE) shows a modulatory effect on the lipid composition under different pathologies. Therefore, the aim of our study was to investigate the NSE effect on the testes lipid composition and testosterone level in plasma of diabetic rats. Diabetes was induced in Sprague-Dawley rats by a single streptozotocin injection (50 mg/kg). Animals with glucose levels of 8-12 mmol/l were further selected. NSE was administrated to rats (50 mg/kg) for 10 days at 1.5 months after the streptozotocin injection. The rat testes were used for lipid analysis, namely, phospholipid level, fatty acid methyl esters and plasma testosterone estimation. NSE administration to diabetic rats triggered normalization of total and individual phospholipid content, as well as composition of free and phospholipids fatty acids in the rat testes. In addition, the testosterone content showed a slight increase under the action of NSE. Our results showed that the early stages of diabetes caused destructive changes in rat testes that may induce a decrease in future testicular function. NSE administration to diabetic rats normalized the lipid content of rat testes and was correlated with an increased testosterone level. NSE induced the restoration of testes structure and function during the early stages of streptozotocin-іnduced diabetes in rats.