## 3 Discussion

Table 4: The GlideScore, Emodel and Lipo/Coulomb/H-bond values resulting from molecular docking study of the compounds Org 43553 and TP4/2 into the transmembrane allosteric sites of rat LHCGR and TSHR. TP derivatives, and a number of other LMW agonists of LHCGR are able to affect, albeit weakly, TSHR activity, which made it possible to create selective allosteric regulators of TSHR [52; 53; 54]. In contrast to LHCGR, the entrance to the transmembrane allosteric site of TSHR is narrower and enriched in hydrophobic amino acids. As a result, TSHR allosteric ligands, including the TP derivatives, should have hydrophobic groups that facilitate their penetration into the transmembrane channel of receptor [54; 55; 56]. The steric orientation of TP4/2 hydrophobic groups allows for reducing the possibility of ligand interaction with the TSHR transmembrane allosteric site. This assumption is supported by our modelling results, which show a low probability of productive protein-ligand TSHR-TP4/2 interaction, described by the GlideScore, Emodel and Lipo parameters (Table 4). This fact is confirmed by our experimental results on the absence of the TP4/2 effect on the basal and thyrotropin-releasing hormone-stimulated production of thyroid hormones (Table S3). These data indicate the specificity of TP4/2 to the LHCGR- but not TSHR-dependent regulations, and largely exclude the negative effect of TP4/2 on the thyroid system. The LHCGR allosteric agonists stimulate the LH/hCG signaling in target cells, including testicular Leydig cells, with less efficiency compared to gonadotropins [16; 21; 22; 36]. This is typical for allosteric agonists, since, due to the cooperative effect, their regulatory effects quickly reach a limit with increasing concentration, and their maximal effects, as a rule, are lower than those for orthosteric agonists [57; 58; 59]. Nevertheless, this is rather their advantage, since it provides moderate, controlled and specific regulation of the LH/hCG pathways and, as a consequence, does not lead to hyperactivation and desensitization of LHCGR and does not induce the tissue resistance to endogenous gonadotropins. In the present study, we showed that TP4/2 is inferior to hCG in its ability to activate AC in the testicular membranes and to stimulate T production when administered to young adult male rats. In this regard, it should be noted that an increase in the intracellular level of cAMP synthesized by AC leads to activation of the StAR protein and stimulation of the steroidogenic genes expression [9; 10; 11; 12; 60; 61]. We have shown for the first time that the dynamics of the steroidogenic effect of LHCGR allosteric agonist significantly differs from that of gonadotropin. With a single administration, 1-3 h after the drugs administration, the steroidogenic effect of hCG exceeded that of TP4/2, but after 5 h there was no significant difference between the steroidogenic effects of TP4/2 and hCG (Figure 2). This may be due to differences in bioavailability and pharmacokinetics of the TP derivatives and gonadotropins, their delivery route and vehicle composition [19], and to differences in the effectiveness and selectivity of their action on cAMP signaling [16; 22; 24]. During the five-day administration, steroidogenic effect of TP4/2 did not decrease, in contrast to that of hCG, and on the third day did not differ significantly from that of gonadotropin, as illustrated by comparable T levels in the Young+TP and Young+hCG groups (Figure 3A). It was also shown that on the fifth day of drugs treatment, there were no significant differences between the groups Young+TP and Young+hCG in the content of intratesticular T and arbostenedione, although the content of 17-hydroxyprogesterone in the Young+hCG group was higher (Table 2). One of the reasons for this may be a decrease in the gene expression of LHCGR in the Young+hCG group and its significant increase in the Young+TP group (Figure 4A).