## 3 Discussion  
The orthosteric site of LHCGR is localized in its large extracellular domain, and LH and hCG bind to it with high affinity, activating various types of heterotrimic G-proteins and \(\beta\)-arrestins [5; 6; 7]. LHCGR also contains the allosteric sites that are located in both the intracellular and extracellular regions and within the transmembrane domain formed by seven hydrophobic helices [16; 17; 45]. It should be noted that, in a large number of GPCRs, a high-affinity orthosteric site is located within the transmembrane domain. It can be assumed that in evolutionary precursors of the LHCGR and other receptors of pituitary glycoprotein hormones, the transmembrane orthosteric site, due to structural changes in the transmembrane domain, including the replacement of the proline residue in the fifth transmembrane region, which is highly conserved in Class A GPCRs [46; 47; 48], has lost the ability to bind hormonal regulators and, as a result, transformed into an allosteric site. It can be assumed that in the course of evolution, in LHCGR and structurally related TSHR and follicle-stimulating hormone receptor, the transmembrane orthosteric site has lost the ability to bind hormones and converted to an allosteric site. Despite the fact that during activation by gonadotropins the LHCGR allosteric site remains free, it ensures the transfer of conformational rearrangements from the extracellular ligand-bound domain to the intracellular loops, and provides an active conformation or a set of active conformations of gonadotropin-activated LHCGR, which is required for effective interaction with heterotrimic G-proteins and \(\beta\)-arrestins [6; 7]. The low specificity of LH and hCG preparations to the LH-regulated signaling cascades and the possibility of their hyperactivation under conditions of using high-dose gonadotropins are the main reasons for the undesirable effects of LH and hCG when used in the clinic [6; 7; 13; 14]. In this regard, over the past two decades, the search and development of allosteric regulators of the LHCGR, which would be devoid of these disadvantages, have been carried out. As a result, heterocyclic compounds with the activity of allosteric full and inverse agonists and allosteric neutral antagonists of LHCGR have been developed and studied, and they specifically bind to the transmembrane allosteric site of the receptor [49; 50; 51]. Based on the structure-activity relationship of LMW agonists of LHCGR, a suitable three-dimensional model for the LHCGR allosteric site was built [52]. The most active among LHCGR allosteric agonists is Org 43553, which belongs to the class of TPs [16; 18; 19; 20]. Based on this, we earlier developed a series of the TP derivatives, among which the compounds TP1 and TP3 were more active in the in vitro and in vivo conditions [21; 22; 23; 36]. In the present work, with the use of molecular modelling, we designed the compound TP4/2, interacting with the transmembrane allosteric site of LHCGR, with suitable parameters (Table 4, Figure 8). Our experimental results confirm the ability of TP4/2 to stimulate LHCGR-dependent testicular steroidogenesis (Figures 2 and 3, Table 2). Despite the functional differences, the structural organization of the transmembrane allosteric sites LHCGR and TSHR have features of similarity [53]. As a result, it is important to exclude the effect of LHCGR allosteric agonists on TSHR activity. Earlier, it was shown that Org 41841, which belongs to the \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Compound** & **GlideScore** & **Emodel** & **Lipo** & **Coulomb** & **H-Bond** \\ \hline \multicolumn{6}{c}{Rat LHCGR} \\ \hline Org 43553 & \(-\)5.53 & \(-\)51.36 & \(-\)3.23 & \(-\)4.87 & \(-\)0.28 \\ \hline TP4/2 & \(-\)5.07 & \(-\)52.84 & \(-\)2.47 & \(-\)1.12 & 0.00 \\ \hline \multicolumn{6}{c}{Rat TSHR} \\ \hline Org 43553 & \(-\)4.06 & \(-\)57.83 & \(-\)1.33 & \(-\)6.44 & 0.00 \\ \hline TP4/2 & \(-\)3.82 & \(-\)38.71 & \(-\)0.84 & \(-\)6.66 & \(-\)0.26 \\ \hline \hline \end{tabular}
\end{table}