Comparative Study of the Steroidogenic Effects of Human Chorionic Gonadotropin and Thieno[2,3-D]pyrimidine-Based Allosteric Agonist of Luteinizing Hormone Receptor in Young Adult, Aging and Diabetic Male Rats

Abstract

Low-molecular-weight agonists of luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor (LHCGR), which interact with LHCGR transmembrane allosteric site and, in comparison with gonadotropins, more selectively activate intracellular effectors, are currently being developed. Meanwhile, their effects on testicular steroidogenesis have not been studied. The purpose of this work is to perform a comparative study of the effects of 5-amino-N-tert-butyl-4-(3-(1-methylpyrazole-4-carboxamido)phenyl)-2-(methylthio)thieno[2,3-d] pyrimidine-6-carboxamide (TP4/2), a LHCGR allosteric agonist developed by us, and hCG on adenylyl cyclase activity in rat testicular membranes, testosterone levels, testicular steroidogenesis and spermatogenesis in young (four-month-old), aging (18-month-old) and diabetic male Wistar rats. Type 1 diabetes was caused by a single streptozotocin (50 mg/kg) injection. TP4/2 (20 mg/kg/day) and hCG (20 IU/rat/day) were administered for 5 days. TP4/2 was less effective in adenylyl cyclase stimulation and ability to activate steroidogenesis when administered once into rats. On the 3rd–5th day, TP4/2 and hCG steroidogenic effects in young adult, aging and diabetic rats were comparable. Unlike hCG, TP4/2 did not inhibit LHCGR gene expression and did not hyperstimulate the testicular steroidogenesis system, moderately increasing steroidogenic proteins gene expression and testosterone production. In aging and diabetic testes, TP4/2 improved spermatogenesis. Thus, during five-day administration, TP4/2 steadily stimulates testicular steroidogenesis, and can be used to prevent androgen deficiency in aging and diabetes.

The Effects of Separate and Combined Treatment of Male Rats with Type 2 Diabetes with Metformin and Orthosteric and Allosteric Agonists of Luteinizing Hormone Receptor on Steroidogenesis and Spermatogenesis

“…With a single injection of LH/hCG-R-agonists, in both control and diabetic rats the stimulating effect of hCG on testosterone production was higher than that of TP3, especially 1 and 3 h after administration ( Table 2 , Figure 1 ), which is due to the higher affinity of hCG to the receptor and the high efficiency of hCG-induced activation of cAMP-dependent signaling responsible for triggering testicular steroidogenesis. This is not surprising since an orthosteric agonist is generally more effective in activating the hormonal receptors than an allosteric agonist [ 38 , 71 ]. However, starting from the second day of LH/hCG-R-agonists treatment, in both control and diabetic rats the steroidogenic effect of hCG weakened and did not significantly differ from the corresponding effect of TP3, which changed to a small extent ( Table 3 and Table 5 , Figure 2 ).…”

“…In this regard, low-molecular-weight allosteric LH/hCG-R-agonists, which interact with an allosteric site located in the transmembrane domain of LH/hCG-R, are of considerable interest [ 34 ]. Among allosteric agonists, thieno[2,3- d ]-pyrimidine derivatives, developed in 2002 by Dutch scientists [ 35 ], demonstrated a high efficiency [ 36 , 37 , 38 ]. We further developed thieno[2,3- d ]-pyrimidine derivatives 5-amino- N-tert -butyl-2-(methylsulfanyl)-4-(3-(nicotinamido)phenyl)thieno[2,3- d ] pyrimidine-6-carboxamide (TP3) ( Figure S1 ) and its analogues TP1 and TP4/2, which showed significant steroidogenic activity in the in vitro and in vivo conditions [ 38 , 39 , 40 ].…”

“…Among allosteric agonists, thieno[2,3- d ]-pyrimidine derivatives, developed in 2002 by Dutch scientists [ 35 ], demonstrated a high efficiency [ 36 , 37 , 38 ]. We further developed thieno[2,3- d ]-pyrimidine derivatives 5-amino- N-tert -butyl-2-(methylsulfanyl)-4-(3-(nicotinamido)phenyl)thieno[2,3- d ] pyrimidine-6-carboxamide (TP3) ( Figure S1 ) and its analogues TP1 and TP4/2, which showed significant steroidogenic activity in the in vitro and in vivo conditions [ 38 , 39 , 40 ]. They regulated the testosterone production in aging rats and animals with streptozotocin (STZ) diabetes [ 38 , 41 , 42 , 43 ].…”

“…We further developed thieno[2,3- d ]-pyrimidine derivatives 5-amino- N-tert -butyl-2-(methylsulfanyl)-4-(3-(nicotinamido)phenyl)thieno[2,3- d ] pyrimidine-6-carboxamide (TP3) ( Figure S1 ) and its analogues TP1 and TP4/2, which showed significant steroidogenic activity in the in vitro and in vivo conditions [ 38 , 39 , 40 ]. They regulated the testosterone production in aging rats and animals with streptozotocin (STZ) diabetes [ 38 , 41 , 42 , 43 ]. Thieno[2,3- d ]-pyrimidine derivatives selectively stimulated the enzyme adenylyl cyclase, increasing the level of intracellular cAMP and activating protein kinase A [ 38 , 40 , 44 ].…”

“…They regulated the testosterone production in aging rats and animals with streptozotocin (STZ) diabetes [ 38 , 41 , 42 , 43 ]. Thieno[2,3- d ]-pyrimidine derivatives selectively stimulated the enzyme adenylyl cyclase, increasing the level of intracellular cAMP and activating protein kinase A [ 38 , 40 , 44 ]. This resulted in the activation of steroidogenic acute regulatory protein (StAR), a mitochondrial cholesterol-transporting protein, which catalyzes the first, rate-limiting stage of steroidogenesis [ 45 ].…”