[Characterization and biological activity of new 4-oxo-1,4-dihydrocinnoline-based inhibitors of the tyrosine phosphatase PTP1B and TCPTP]. / Kharakteristika i biologicheskaia aktivnost' novykh ingibitorov tirozinfosfataz PTP1B i TCPTP na osnove 4-okso-1,4-digidrotsinnolina.

Functional disorders in obesity are largely due to a decrease in tissue sensitivity to insulin and leptin. One of the ways to restore it is inhibition of protein phosphotyrosine phosphatase 1B (PTP1B) and T-cell protein phosphotyrosine phosphatase (TCPTP), negative regulators of the insulin and leptin signaling. Despite progress in the development of inhibitors of these phosphatases, commercial preparations based on them have not been developed yet, and the mechanisms of action are poorly understood. The aim of the work was to study the effect of new derivatives of 4-oxo-1,4-dihydrocinnoline (PI04, PI06, PI07) on the activity of PTP1B and TCPTP, as well as to study the effect of their five-day administration (i.p., 10 mg/kg/day) to Wistar rats with diet-induced obesity on body weight and fat, metabolic and hormonal parameters, and gene expression of phosphatase and insulin and leptin receptors in the liver. It has been shown that PI04 is a mild, low selective inhibitor of both phosphatases (PTP1B, IC50=3.42(2.60-4.51) µM; TCPTP, IC50=4.16(3.49-4.95) µM), while PI06 and PI07 preferentially inhibit PTP1B (IC50=3.55 (2.63-4.78) µM) and TCPTP (IC50=1.45(1.18-1.78) µM), respectively. PI04 significantly reduced food intake, body weight and fat, attenuated hyperglycemia, normalized glucose tolerance, basal and glucose-stimulated levels of insulin and leptin, and insulin resistance index. Despite the anorexigenic effect, PI06 and PI07 were less effective, having little effect on glucose homeostasis and insulin sensitivity. PI04 significantly increased the expression of the PTP1B and TCPTP genes and decreased the expression of the insulin and leptin receptor genes. PI06 and PI07 had little effect on these indicators. Thus, PI04, the inhibitor of PTP1B and TCPTP phosphatases, restored metabolic and hormonal parameters in obese rats with greater efficiency than inhibitors of PTP1B (PI06) and TCPTP (PI07). This indicates the prospect of creating mixed PTP1B/TCPTP inhibitors for correction of metabolic disorders.

Development of Low-Molecular-Weight Allosteric Agonist of Thyroid-Stimulating Hormone Receptor with Thyroidogenic Activity.

To normalize the thyroid status in hypothyroidism caused by resistance to thyroid-stimulating hormone (TSH), low-molecular-weight allosteric agonists of TSH receptor can be used. A new compound ethyl-2-(4-(4-(5-amino-6-(tert-butylcarbamoyl)-2-(methylthio)thieno[2,3-d]-pyrimidine-4-yl)phenyl)-1H-1,2,3-triazol-1-yl) acetate (TPY3m), which stimulated the production of thyroxine when administered to rats (25 mg/kg, i.p.) and also increased the expression of thyroidogenic genes in the cultured FRTL-5 thyrocytes (30 µM) and the rat thyroid gland. The in vitro and in vivo treatment with TPY3m did not lead to a decrease in the expression of the TSH receptor gene in thyrocytes, restoring it under the conditions of receptor hyperactivation by the hormone. This determines the retaining and, in some cases, potentiation of the thyroidogenic effects of TSH (FRTL-5) or thyroliberin (rats) when they are coadministered with TPY3m. TPY3m is a prototype drug for correcting thyroid system functions in subclinical hypothyroidism.

The Study of Biological Activity of a New Thieno[2,3-D]-Pyrimidine-Based Neutral Antagonist of Thyrotropin Receptor.

The development of low-molecular-weight antagonists of thyroid-stimulating hormone (TSH) receptor is a promising trend in the treatment of autoimmune hyperthyroidism. We studied the effect of thieno[2,3-d]-pyrimidine derivative TPY1 on TSH-stimulated synthesis of thyroid hormones in the culture of FRTL-5 thyrocytes and on thyroliberin-stimulated production of thyroid hormones in rat blood. Preincubation of FRTL-5 cells with TPY1 suppressed the stimulatory effect of TSH on the synthesis of thyroxine and triiodothyronine. Intraperitoneal injection of TPY1 in a dose of 25 mg/kg reduced thyroliberin-stimulated levels of thyroid hormones in the blood and inhibited the expression of genes encoding thyroid peroxidase, thyroglobulin, and Na+/I- cotransporter responsible for thyroxine synthesis. In the absence of thyroliberin stimulation, TPY1 did not affect the levels of thyroid hormones and expression of thyroidogenesis genes. Thus, a new TPY1 antagonist of TSH receptor can be a prototype of a drug for the treatment of autoimmune hyperthyroidism.

Comparative Study of the Restoring Effect of Metformin, Gonadotropin, and Allosteric Agonist of Luteinizing Hormone Receptor on Spermatogenesis in Male Rats with Streptozotocin-Induced Type 2 Diabetes Mellitus.

We compared the effectiveness of human chorionic gonadotropin (hCG; 5 days, 20 IU/rat/day), allosteric luteinizing hormone receptor agonist TP04 (5 days, 20 mg/kg/day), and metformin (28 days, 120 mg/kg/day) in restoring spermatogenesis in male rats with type 2 diabetes mellitus. hCG and TP04 increased the levels of testosterone and expression of the steroidogenic protein StAR, the number of spermatogenic cells, thickness of the seminal epithelium, and the number and motility of mature sperm that were reduced in diabetic rats, though they did not reduce the number of defective spermatozoa. Metformin had a weak effect on steroidogenesis, but was not inferior to luteinizing hormone receptor agonist by its restorative effect on spermatogenesis and also reduced the number of defective forms of spermatozoa. Thus, the spermatogenesis-restoring effect of metformin and luteinizing hormone receptor agonist in type 2 diabetes mellitus are comparable, despite different mechanisms of action.

Stimulation of Ovulation in Immature Female Rats Using Orthosteric and Allosteric Luteinizing Hormone Receptor Agonists.

Human chorionic gonadotropin (hCG) and luteinizing hormone (LH) are widely used for the treatment of reproductive disorders and for controlled ovulation induction, but their use is limited by side effects. Allosteric agonists of the LH/hCG receptor, including thieno[2,3-d]thienopyrimidine TP03 developed by us, can become an alternative. TP03 (50 mg/rat, i.p.) when administered to immature female rats treated 48 h before with Follimag has been shown to increase progesterone levels (maximum 8 h post-treatment) and induce ovulation, as indicated by the appearance at 24 h corpus luteum (8.6 ± 0.5 per ovary). In terms of its activity, TP03 is comparable to hCG, although it acts more moderately. In the ovaries, unlike hCG, TP03 does not lead to an increase in the expression of vascular endothelial growth factor, which can cause ovarian hyperstimulation syndrome. Thus, TP03 is a promising drug as an ovulation inducer and ovarian steroidogenesis stimulator.

The Effect of Low-Molecular-Weight Allosteric Agonist of Luteinizing Hormone Receptor on Functional State of the Testes in Aging and Diabetic Rats.

Human chorionic gonadotropin that is widely used for improving spermatogenesis. The effect of chorionic gonadotropin is mediated through luteinizing hormone receptor. Treatment with gonadotropin is associated with undesirable effects due to hyperactivation of testosterone production and luteinizing hormone receptor desensitization. A promising alternative could be low-molecular-weight agonists of luteinizing hormone receptors, but their effects on spermatogenesis have not been investigated. Here we analyzed the effect of a thieno[2,3-d]pyrimidines (TP), 4-((3-(5-amino-6-(tert-butylcarbamoyl)-2-(methylthio)thieno [2,3-d]pyrimidine-4-yl) phenyl)carbamoyl)pyridine 1-oxide (TP22), an allosteric agonist of luteinizing hormone receptors, on the seminiferous tubules and spermatogenic cells in 4- and 18-month-old male rats and in animals with diabetes mellitus. TP22 and gonadotropin were administered in daily doses of 15 mg/kg and 20 U/rat for 5 days. Blood testosterone level, morphology of the seminiferous tubules, and the number of germ cells in them were estimated. Being comparable by the efficiency to gonadotropin, TP22 increased the testosterone level in all the studied groups of rats and restored epithelium thickness in the seminiferous tubules and the number of spermatogonia and pachytenic spermatocytes that are reduced in aging and diabetes, but, unlike gonadotropin, did not suppress the expression of luteinizing hormone receptor. The efficacy of TP22 as a stimulator of testicular spermatogenesis has been demonstrated both under normal conditions and in age-related and diabetes-associated reproductive dysfunctions.

Normalization of Testicular Steroidogenesis and Spermatogenesis in Male Rats with Type 2 Diabetes Mellitus under the Conditions of Metformin Therapy.

One of the complications of type 2 diabetes mellitus in men is steroidogenic and spermatogenic dysfunctions. There is evidence of a restoring effect of the antidiabetic drug metformin on them. We studied the effect of MF therapy (4 weeks, 200 mg/kg/day) on the hormonal parameters of the gonad axis and on the morphological characteristics of epididymal spermatozoa in male rats with a severe form of T2DM caused by a high-fat diet and a low-dose streptozotocin. It has been shown that MF therapy, along with the restoration of the metabolic parameters, normalizes the plasma levels of testosterone and leptin and the content of testosterone, its precursors, leptin and its receptors in the testes, and also increases sperm motility, which is reduced in T2DM. This is the result of both the systemic action of MF and its direct effect on testicular cells.

New Thieno-[2,3-d]pyrimidine-Based Functional Antagonist for the Receptor of Thyroid Stimulating Hormone.

Thyroid stimulating hormone (TSH) receptor antagonists are required for the treatment of TSH-dependent tumors and Graves disease. We developed the compound 5-amino-N-(tert-butyl)-4-(4-iodophenyl)-2-(methylthio)thieno[2,3-d]pyrimidine-6-carboxamide (TP48) and showed that it reduces the TSH-stimulated adenylate cyclase activity in rat thyroid membranes. Pretreatment of rats with compound TP48 (ip, 40 mg/kg) reduced the increase in the levels of total and free thyroxin in blood and the increase in the expression of thyroglobulin and D2 deiodinase genes in the thyroid gland, which are responsible for the synthesis of thyroid hormones, which were caused by intranasal administration of thyroliberin to animals (300 µg/kg). These data indicate that compound TP48 is a functional antagonist of the TSH receptor and can be used to correct the thyroid status in hyperthyroidism.

[The effect of high-dose metformin on the metabolic parameters and functional state of the liver of agouti-mice with the melanocortin obesity.]

In recent years, the effectiveness of high-dose metformin (MF) to treat the endocrine and oncological diseases has been shown. However, the use of high-dose MF may be associated with the lactic acidosis and the liver dysfunctions. The aim of the work was to study the effect of long-term (10 days) oral administration of a relatively high dose of MF (600 mg/kg per day) into yellow C57Bl/6J (Ay/a) Agouti line mice with the melanocortin type obesity on the liver function, which was evaluated by the morphology of hepatocytes and the severity of steatosis, the expression of the inflammatory and apoptotic factors of and the activity of aminotransferases, as well as on the plasma lactate level in the animals. In Agouti line mice, MF (600 mg/kg per day) caused a decrease in the body and fat weight, led to the reduced hyperglycemia, hyperinsulinemia and hyperleptinemia, and restored the sensitivity to glucose and insulin. At the same time, in the liver of Agouti line mice treated with MF, the small-drop and large-drop fatty degeneration and the hydropic degeneration were attenuated, and the expression of pro-inflammatory IL-1ß and pro-apoptotic Bax protein and the Bax/Bcl-2 ratio did not differ from the control C57Bl/6J (a/a) mice. In the blood of Agouti line mice treated with MF, the activity of alanine aminotransferase was normalized, and the lactate levels was increased, but to a moderate degree. It was concluded that the high-dose MF did not induce the lactic acidosis in Agouti line mice, and at the same time it restored the liver functions impaired in the melanocortin obesity. This allows us to consider the use of the high doses of MF as one of the possible ways to treat obesity and metabolic disorders that are associated with the hepatic steatosis.

[The effect of different types of bariatric surgery on the metabolic and hormonal parameters in rats with decompensed form of type 2 diabetes mellitus.]

Currently, one of the approaches to correct metabolic disorders in the type 2 diabetes mellitus (DM2) with obesity are bariatric surgery (BS), including sleeve gastrectomy (SG), gastric bypass (GB) and ileal transposition (IT). However, their effectiveness and impact on the hypothalamic signaling and hormonal status in severe forms of DM2 without obesity remain little studied. The aim of the work was to study the effect of IT, SG and GB on the insulin, leptin, ghrelin and glucagon-like peptide-1 (GLP-1) levels in the blood and on the expression of the genes encoding the main components of the hypothalamic signaling systems in rats with decompensated form of DM2, which was induced by a high-fat diet (3 months) and a single low dose of streptozotocin (25 mg/kg, 2 months after the start of the diet). In diabetic rats, a significantly expressed hyperglycemia, an impaired glucose tolerance, a decrease in glucose-stimulated GLP-1 level, a slight decrease in the insulin and leptin levels and an slight increase in ghrelin level were detected. In the hypothalamus, the expression of the genes encoding GLP-1 receptor, orexigenic agouti-related peptide (AgRP), as well as phosphotyrosine phosphatase 1B and SOCS3, the negative regulators of the leptin and insulin pathways was increased. In diabetic rats, the IT reduced the glucose levels 120 minutes after glucose load, increased the basal and glucose-stimulated GLP-1 levels, normalized the gene expression for phosphotyrosine phosphatase 1B, SOCS3, AgRP and GLP-1 receptor, which indicates the restoration of the hypothalamic signaling responsible for the control of energy metabolism and insulin sensitivity. In the case of SG and GB, an improvement in the glucose tolerance was found, and in the case of SG, an increase in the basal and glucose-stimulated GLP-1 levels was shown. However, no significant effect on the expression of the hypothalamic genes in SG and GB was found. Thus, IT is the most effective of all studied BS in the treatment of severe forms of DM2 without obesity.

Conservation of Steroidogenic Effect of the Low-Molecular-Weight Agonist of Luteinizing Hormone Receptor in the Course of Its Long-Term Administration to Male Rats.

Abstract-It was shown that the thienopyrimidine derivative TP03, a low-molecular-weight agonist of the luteinizing hormone receptor (LHR), during the treatment of male rats for 7 days steadily increased the production of testosterone (T), whose elevated level was retained for 7 days, and increased the expression of the gene for LHR, which indicates the maintenance of the sensitivity of Leydig cells to gonadotropins. At the same time, the steroidogenic effect of human chorionic gonadotropin (hCG), which significantly increased the T level on the first day of administration, was further weakened, which was accompanied by a decrease in the expression of the gene for LHR in the testes, indicating the development of resistance of Leydig cells to hCG. Along with this, in the case of hCG administration, a compensatory increase in the expression of genes of the steroidogenic enzymes, such as cytochrome P450scc and dehydrogenase 3ß-HSD, was shown in the testes, while in the case of TP03 administration this effect was absent.

[The decrease in the basal and luteinizing hormone receptor agonist-stimulated production of testosterone in aging male rats.]

The aging leads to a weakening of the steroid function of the testes and a decrease in their sensitivity to gonadotropins. However, the mechanisms of this are poorly understood. The aim of this work was to study the stimulating effects of human chorionic gonadotropin (hCG) and TP03, a low-molecular-weight agonist of luteinizing hormone (LH)/hCG receptor, on testosterone (T) production and the expression of steroidogenic proteins in young (3 months) and aging (15 months) male rats, and to investigate the activity of the adenylyl cyclase system in the membranes isolated from the testes of rats. The treatment with hCG (100 IU/rat/day) and TP03 (15 mg/rat/day) was carried out for 3 days. In the testes of aging rats the stimulation of adenylyl cyclase (AC) by gonadotropin and guanine nucleotide was decreased, indicating a weakening of the coupling of LH/hCG receptor and Gs protein, the main components of the adenylyl cyclase system regulating the steroidogenesis. In elderly rats, the T level in the blood and the expression of the Star, Cyp11a1 and Cyp17a1 genes encoding the StAR protein and the steroidogenic enzymes cytochromes P450scc and P450-17α in the testes were decreased. With increasing age, the stimulating effect of hCG and TP03 on the T production was weakened, despite the different mechanisms of their action on LH/hCG receptor. The treatment of both young and aging rats with hCG led to an increase in the expression of the genes encoding StAR, P450scc and dehydrogenase 3ß-HSD, while in aging rats, in addition, the expression of the Hsd17B gene was increased and in young rats the expression of the genes encoding Р450-17α and 17ß-HSD was reduced. The treatment of in young rats with TP03 led to an increase in the Star and Cyp17a1 expression, and the TP03 treatment of aging rats increased the Star and Hsd17B expression. Thus, in the testes of aging rats, the coupling between LH/hCG receptor and Gs-protein and the sensitivity of LH/hCG receptor to agonists were weakened, which leads to a decrease in the hCG- and TP03-induced production of T, and the basal and LH/hCG receptor agonists-stimulated levels of gene expression for some steroidogenic proteins were changed.

THE MOLECULAR MECHANISMS OF STEROIDOGENESIS REGULATION IN LEYDIG CELLS.

The synthesis of testosterone (T) in the male organism is carried out by Leydig cells located in the testes and controlled by luteinizing hormone (LH) produced by the anterior pituitary. The LH specifically binds to LH receptors located in the plasma membrane of Leydig cells and stimulates the activity of intracellular signaling pathways coupled with the receptor, which regulate the steroidogenesis. The main role in this regulation belongs to the adenylyl cyclase signaling system which, along with LH receptor, includes Gs-protein, the enzyme adenylyl cyclase catalyzing the cAMP synthesis, and cAMP-dependent effector proteins. In mammals, the process of steroidogenesis is carried out with the participation of StAR protein which transports cholesterol into the mitochondria, where the initial steps of T synthesis occur. The process also involves four steroidogenic enzymes, such as cytochrome P450scc, 3b-hydroxysteroid dehydrogenase (3b-HSD), cytochrome P450-17a possessing 17a-hydroxylase and C17,20-lyase activities, and 17b-hydroxysteroid dehydrogenase (17b-HSD). In different species of mammals and at the different stages of ontogenesis, the order of the reactions of T synthesis and their regulation are characterized by a number of differences. There are D4 and D5 ways of T synthesis, which differ in the stage of isomerization including the displacement of the double bond from the C5­C6 position to the C4­C5. The regulation of T synthesis can take place both by direct interaction of LH-dependent signaling pathways with StAR protein, and indirectly, through the regulation of transcription factors controlling the expression of genes encoding the steroidogenesis enzymes. In the present review the data on the molecular mechanisms of regulation and the ways of T synthesis in mammalian Leydig cells, the species specificity of these ways, as well as on the factors influencing the steroidogenesis are summarized and analyzed.