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.

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 STIMULATING EFFECT OF THIENOPYRIMIDINES, THE STRUCTURAL ANALOGS OF ORG 43553, ON THE ACTIVITY OF ADENYLYL CYCLASE IN THE TESTES AND ON THE TESTOSTERONE PRODUCTION IN MALE RATS

The regulation of the functional activity of luteinizing hormone (LH) receptor can be carried out by using gonadotropins or low-molecular weight agonists of this receptor, which, unlike gonadotropins, bind to an allosteric site located in the transmembrane channel of the receptor. Thienopyrimidine derivatives, the analogs of the compound Org 43553, the greatest interest among the low-molecular weight agonists. The aim of the work was synthesis of the novel thienopyrimidine derivatives, such as 5-amino-N-(tert-butyl)-4-(3-(2-methoxynicotinamido) phenyl)-2-(methylthio)thieno [2,3-d]pyrimidine-6-carboxamide (TP-21), 4-(3-(5-amino-6-(tert-butylcarbamoyl)- 2-) methylthio)thieno[2,3-d] pyrimidine-4-yl)phenyl)carbamoyl)pyridine 1-ocide (TP-22) and 5-amino-N-(tert-butyl)-4-(3-(2-chloronicotinamido)-2-(methylthio)thieno[2,3-d] pyrimidine-6-carboxamide (TP-23), and the study of their effects in vitro on adenylyl cyclase (AC) activity in testicular membranes of rats as well in vivo on the testosterone level in the case of their intratesticular and intraperitoneally administration into male rats. The compounds TP-21, TP-22 and TP-23 stimulated the basal AS activity in rat testicular membranes with the EC50 values, such as 1556, 358 and 372 nM, and ranked according to their efficiency in the following order: TP-23 > TP-21 TP-22. In the case of combined action of thienopyrimidines (10­4 M) and human chorionic gonadotropin (hCG, 10­8 M), the AC stimulating effect of gonadotropin was preserved, but at a concentration of 10­4 M, the additivity of AC effects of thienopyrimidines and hCG was observed. The TP-21, TP-22 and TP-23, when i. t. administered into male rats at a dose of 10 mg/kg, increased the testosterone levels, and, 5 h after treatment, the increase of concentration of testosterone over its value in the control group was 32.8, 36.4 and 76.9 nM respectively. When administered intraparenterally, TP-21 and TP-22 had a little effect on the testosterone level, while the compound TP-23 showed significant increase in the testosterone level at 1 and 3 h (the increase over control amounted 34.8 and 18.9 nM). The data obtained indicate a high activity of TP-23, as a stimulator of the synthesis and secretion of testosterone, as well as the prospect of development on its basis of highly effective agonists of LH receptor.

[Activation of adenylyl cyclase in rats testes and ovaries using thienopyrimidine derivatives].

Signaling systems regulated by luteinizing hormone (LH) and human chorionic gonadotropin (hCG) and having LH receptor as a sensor component play an important role in the functioning of the reproductive tissues. The use of LH and hCG in medicine for the treatment of diseases of the reproductive system and in auxiliary reproductive technologies is limited by their high cost, the need to use parenteral administration, and side effects. In recent years there has been the development of low molecular weight agonists of LH receptor that are devoid of these disadvantages and can be administered orally. The most effective among them are thienopyrimidine derivatives, in particular compound Org 43553. The purpose of this study was to investigate the influence of newly synthesized analogs of Org 43553, 5-amino-N-(tert-butyl)-4-(3-(isonicotinamide)phenyl)-2-(methylthio)thieno[2,3-d]pyrimidine-6-carboxamide (compound 1) and 5-amino-N-(tert-butyl)-2-(methylthio)-4--(3-(thiophene-3-carboxamido)phenyl)thieno[2,3-d]pyrimidine-6-carboxamide (compound 2), on the basal and LH-stimulated adenylyl cyclase (AC) activity in plasma membranes fractions isolated from rat testes and ovaries. Compounds 1 and 2 have been shown to stimulated the basal AC activity in membranes isolated from the testes and ovaries in a dose dependent manner, and compound 2 was more effective in comparison with compound 1. EC50 values for the effects of the compounds 1 and 2 on the AC activity were 1.05-1.12 and 0.28-0.37 µM, respectively. Stimulating effect of hCG on the AC activity retained in the presence of the thienopyrimidine derivatives, and at low, non-saturating, concentrations of the hormone the additivity of the effects ofhCG and compounds 1 and 2 on AC activity was observed. This indicates that the thienopyrimidine derivatives interact with allosteric site localized in the transmembrane channel of LH receptor, and do not overlap with the binding site of gonadotropins, located in the N-terminal ectodomain. Effect of compounds 1 and 2 was tissue specific, and was not found in tissues where no LH receptors. Our data indicate that compounds 1 and 2 may be a prototype for drugs that regulate the function of male and female reproductive systems.