Alpha-fetoprotein (AFP) is a major embryo- and tumor-associated protein capable of binding and transporting a variety of hydrophobic ligands, including estrogens. AFP has been shown to inhibit estrogen receptor (ER)-positive tumor growth, which can be attributed to its estrogen-binding ability. Despite AFP having long been investigated, its three-dimensional (3D) structure has not been experimentally resolved and molecular mechanisms underlying AFP–ligand interaction remains obscure. In our study, we constructed a homology-based 3D model of human AFP (HAFP) with the purpose of molecular docking of ERα ligands, three agonists (17β-estradiol, estrone and diethylstilbestrol), and three antagonists (tamoxifen, afimoxifene and endoxifen) into the obtained structure. Based on the ligand-docked scoring functions, we identified three putative estrogen- and antiestrogen-binding sites with different ligand binding affinities. Two high-affinity binding sites were located (i) in a tunnel formed within HAFP subdomains IB and IIA and (ii) on the opposite side of the molecule in a groove originating from a cavity formed between domains I and III, while (iii) the third low-affinity binding site was found at the bottom of the cavity. Here, 100 ns molecular dynamics (MD) simulation allowed us to study their geometries and showed that HAFP–estrogen interactions were caused by van der Waals forces, while both hydrophobic and electrostatic interactions were almost equally involved in HAFP–antiestrogen binding. Molecular mechanics/Generalized Born surface area (MM/GBSA) rescoring method exploited for estimation of binding free energies (ΔGbind) showed that antiestrogens have higher affinities to HAFP as compared to estrogens. We performed in silico point substitutions of amino acid residues to confirm their roles in HAFP–ligand interactions and showed that Thr132, Leu138, His170, Phe172, Ser217, Gln221, His266, His316, Lys453, and Asp478 residues, along with two disulfide bonds (Cys224–Cys270 and Cys269–Cys277), have key roles in both HAFP–estrogen and HAFP–antiestrogen binding. Data obtained in our study contribute to understanding mechanisms underlying protein–ligand interactions and anticancer therapy strategies based on ERα-binding ligands.

Разработаны методы получения новых гетероциклических соединений, производных 1,3,4-тиадиазолов. Показано, что данные соединения обладают выраженной антифунгальной активностью и могут быть рекомендованы для дальнейшего изучения в качестве потенциальных антимикробных средств c фунгицидным действием.

Alpha-fetoprotein (AFP) is a major embryo-and tumor-associated protein capable of binding and transporting variety of hydrophobic ligands including estrogens. AFP has been shown to inhibit estrogen receptor (ER)-positive tumor growth and this can be attributed to its estrogen-binding ability. Despite AFP has long been investigated, its three-dimensional (3D) structure has not been experimentally resolved and molecular mechanisms underlying AFPligand interaction remain obscure. In our study we constructed homology-based 3D model of human AFP (HAFP) with the purpose to perform docking of ERα ligands, three agonists (17βestradiol, estrone and diethylstilbestrol) and three antagonists (tamoxifen, afimoxifene and endoxifen) into the obtained structure. Based on ligand docked scoring function, we identified three putative estrogen-and antiestrogen-binding sites with different ligand binding affinities.Two high-affinity sites were located in (i) a tunnel formed within HAFP subdomains IB and IIA and (ii) opposite side of the molecule in a groove originating from cavity formed between domains I and III, while (iii) the third low-affinity site was found at the bottom of the cavity. 100 ns MD simulation allowed studying their geometries and showed that HAFP-estrogen interactions occur due to van der Waals forces, while both hydrophobic and electrostatic interactions were almost equally involved in HAFP-antiestrogen binding. MM/GBSA rescoring method estimated binding free energies (ΔGbind) and showed that antiestrogens have higher affinities to HAFP as compared to estrogens. We performed in silico point substitutions of amino acid residues to confirm their roles in HAFP-ligand interactions and showed that Thr132, Leu138, His170, Phe172, Ser217, Gln221, His266, His316, Lys453, and Asp478 residues along two disulfide bonds, Cys224-Cys270 and Cys269-Cys277 have key roles in both HAFP-estrogen and HAFP-antiestrogen binding. Data obtained in our study contribute to understanding mechanisms underlying protein-ligand interactions and anti-cancer therapy strategies based on ER-binding ligands. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted:Investigation of protein surface topology and elucidating channels, cavities, grooves and pockets in its three-dimensional (3D) structure play important roles in predicting protein-ligand interactions and explaining molecular mechanisms underlying protein functioning. Current computational approaches are becoming powerful tools to create 3D model of a protein structure if it has not been obtained by experimental methods and to assess at atomic level mechanisms of protein-ligand binding capabilities. Mostly, this is based on ligand docking procedure followed by calculation of scoring functions with subsequent molecular dynamics (MD) simulations and binding free energy (ΔG) estimations. In this study we used the in silico approaches to predict three major binding sites in human alpha-fetoprotein (HAFP) and to compare binding affinities of natural and synthetic estrogens and antiestrogens...