Efficacy Evaluation Of Amino Alcohols On Echinococcus Granulosus And Preliminary Identification Of The Drug Targets

Echinococcosis, also called hydatid diseases, is an important zoonotic disease with worldwide distribution, caused by larval stages of Echinococcus spp.. Humans acquire the infection by oral ingestion of parasite eggs. The eggs hatch in the gastrointestinal tract, penetrate through the intestinal walls, enter into the bloodstream, and eventually locate mostly in the liver and lungs. The growth of hydatid cysts in patients is slow and always asymptomatic either for many years or permanently. Without effective treatments, the development of cysts eventually results in organ malfunction and even death. Nowadays, chemotherapy based on benzimidazoles is the most important choice for the patients. However, albendazole and mebendazole are the only two drugs licensed for the treatment of echinococcosis. According to the results of clinical trials organized by WHO and other subsequent clinical practices, the cure rate of mebendazole and albendazole in treatment of echinococcosis was only about 30%. Therefore, it is essential to find novel alternatives for chemotherapy against these diseases.The amino alcohols discussed in the present study are the alkane compounds with hydroxyl (-OH) and amino (-NH,-NHR and -NR2). The amino alcohols were reported with effects on malaria, schistosoma, filarial, tumor, bacterial and so on. In this study, the effects of amino alcohols on the protoscoleces and germinal cell of Echinococcus granulosus were evaluated based on the high throughput drug screening method. In addition, the drug targets were also identified and validated.1. The establishment of the high throughput drug screening method for anti-Echinococcus granulosusThe culture methods for the protoscoleces from infected sheep and the germinal cell from infected KM mouse were modified. Then, The viability of protoscoleces and germinal cell were assessed by methylene blue staining and CCK-8 (MTT) method separately. After the establishment of the screening method, the in vivo and in vitro effects of mebendazole, mefloquine, nitazoxanide and tizoxanide against Echinococcus granulosus were tested. The results indicated that the high throughput drug screening method was effective and accurate in the study of anti-echinococcus compounds.2. The efficacy, druggability and toxicity of amino alcoholsA total of 131 amino alcohol compounds were evaluated based on the high throughput screening method established above. More then 20 compounds showed the activity against protoscoleces and germinal cell. According to the LC50 and IC50 values of these active compounds, thirteen kinds of amino alcohols with the both values less than 10 μg/ml were selected for subsequent analysis. Then the druggability and toxicity of these active compounds were predicted, and their toxicity on normal host cell were also tested. Finally, the compound 16,54 and 124 were identified as the candidate lead compounds against Echinococcus granulosus.3. The pharmacophore model of active amino alcohol compounds and virtual screeningThe simulation of pharmacophore was achieved by of the software Discovery studio. Firstly, the active amino alcohols were prepared as training set, and 10 pharmacophore models were outputted. Then the test set was prepared and the best pharmacophore was selected. This model included a positive center, a hydrophobic aromatic, two hydrophobic centers and a hydrogen bond donor. The virtual screening of 1000 compounds deposited in the ZINC database based on the pharmacophore showed that there were 101 compounds with the FitValue value greater than 3.0.4. The identification and validation of drug target for amino alcoholsFirstly, the drug target of amino alcohols were predicted by reverse docking method (idTarget server). After a series of drug target screening procedures, such as sequence alignment, homology modeling, molecular docking (Autodock and Autocock vina) and so on, the glycogen phosphorylase was identified as the candidate target of amino alcohols. However, mefloquine only inhibited the activity of glycogen phosphorylase from Echinococcus granulosus protoscoleces at 482.2 μM in vitro, with the inhibition rate of 57.8%.Based on this study, the high throughput drug screening method for anti-Echinococcus granulosuses is established by using the protoscoleces and germinal cell cultured in vitro. Then amino alcohols was tested the efficacy aginst E. granulosus and some of them were identified as the candidate active compounds. In addition, the pharmacophore model of active amino alcohol compounds and the virtual screening were achieved. After a series of drug target screening procedures, one E. granulosus enzyme was identified as the drug target.