[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.

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.

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 RELATIONSHIP BETWEEN PHYSICAL-CHEMICAL CHARACTERISTICS AND BIOLOGICAL ACTIVITY OF PEPTIDE 562­572 OF LUTEINIZING HORMONE RECEPTOR MODIFIED BY DECANOYL RADICALS AT THE N- AND C-TERMINI.

Lipophilic derivatives of peptides corresponding to the cytoplasmic regions associated with the G-protein coupled receptors (GPCRs) are capable of functioning as an intracellular agonist. Previously, we have shown that peptides corresponding to region 562­572 of luteinizing hormone receptor (LHR) and modified by decanoate and palmitate at the C-terminus activate adenylyl cyclase (AC) in the testes of rats. The stimulating effect of peptide 562­572 modified by decanoates at the N- and C-termini (IV) peaked at a concentration of 10(­5) M and then subsequently decreased with increasing concentration. We hypothesized that this may be due to ability of the peptide IV to micelle formation. To test this suggestion, we examined the relationship between biological activity, hydrophobicity and ability to micelle formation for peptide IV and other acylated derivatives of peptide 562­572 including the derivatives containing C-terminal decanoate (III) and palmitate (VI). It has been shown that the stimulating effect of peptide IV at a concentration of 10(­5) M on AC activity in the plasma membranes of rat testes and ovaries is only slightly inferior to that of peptide VI and superior to the corresponding effect of peptide III. The effect of peptide IV at a concentration of 10­3 M was reduced by 20­27 % and amounted to 50­51 and 87­88 % of that of peptides VI and III, respectively. Despite the high hydrophobicity, the peptide IV had abnormally low retention time in reverse-phase HPLC when it was eluted from the Nucleosil C8 column, even lower than that of unmodified peptide 562­572. However, with increasing concentration of trifluoroacetic acid in the eluent from 0.1 to 0.5 % causing the destruction of micelle-like structures, the retention time of the peptide IV was significantly increased, whereas it remained unchanged in the case of the other peptides. Surface tension of aqueous solution of peptide IV insignificantly decreased with the increase of its concentration, but then, at peptide concentration of 710(­6) M, the sharp decline and the plateau were found, which indicates the beginning of the formation of micelles. Thus, at concentration of 10(­5) M and higher the peptide IV forms micelles which prevents its interaction with the receptor. The ability of GPCR-peptides to self-aggregation and micelle formation should be taken into account when developing their membrane-active analogues.