| Schistosomiasis remains one of the most endemic parasitic diseases threatening to human health. In China, Schistosoma japonicum is the major pathogen of the disease. No effective vaccine has been developed to date and the treatment exclusively depends on praziquantel. However, praziquantel has been used for nearly 40 years and long periods of utilizing a single drug inevitably evolved drug-resistant parasites. Therefore it is urgent to develop new vaccines or drugs to prevent and treat schistosomiasis. Drug or vaccine development depends on the in-depth study of the metabolic process of schistosomes as well as their interaction with host. Recently, more attention is paid to the redox metabolism of S. japonicum or redox-associated proteins, since the redox metabolic is not only important for energy supply, gene regulation, cell growth and differentiation, but also helpful for protecting the schistosomes from reactive oxygen species (ROS) attack. In the present study, we selected three redox-related proteins and explored their functions, structures and applications.In the study about S. japonicum aldose reductase (SjAR), we successfully cloned, expressed, purified recombinant Sj AR protein and analyzed its enzyme activity in vitro. In the regard of considering SjAR as drug target, we first resolved SjAR tertiary structure through X-ray diffrattion method and then identified 10 compounds from Maybridge HitFinder library through molecular docking based on the structure. Further antischistosomal activity analysis showed that one compound renamed AR9 has good activity with the LD50 value (5.48 ± 2.38) μg/ml. Subsequently, AR9 was found to effectively inhibit the recombinant SjAR activity with the IC50 value 11.75 μg/ml, which directly supported that SjAR was indeed the target of AR9. Cytotoxicity analysis showed AR9 almost exhibited no toxic against host cells even at twice of antischistosomal concentration, suggesting its potential value as a lead compound for guiding new drugs development. Additionally, we also evaluated the protective efficiency of the recombinant SjAR against schistosome infection in murine model. The results indicated that the worm burden in Sj AR immunized group reduced by 32.9%(P<0.01) and the egg number in liver and fecal were also reduced by 28.3% (P>0.05) and 42.8% (P<0.05) respectively. The data suggested that SjAR might be also a potential vaccine candidate which was worthy for further investigation. RT-PCR showed that the transcriptional levels of SjAR were significantly higher in male adult worms than female worms and further immunolocalization found that SjAR was abundant in the gynecophoral canal. These results implied that SjAR was likely to be a male-preference protein.In the study of 3-oxoacyl-ACP reductase (SjOAR) of S. japonicum. we successfully cloned, expressed, purified recombinant SjOAR protein and analyzed its enzyme activity in vitro. Moreover, we also determined the kinetic parameters of SjOAR and demonstrated the enzymatic reaction followed a sequential mechanism. In the study of regarding SjOAR as a drug target, firstly, we predicted SjOAR structure by homology modeling and then identified 30 small molecules from Maybridge HitFinder library (3 compounds were not obtained because of shortage). BIAcore analysis demonstrated that all the compounds were able to bind SjOAR protein. Further kinetic assays indicated that the dissociation constants (KD) of partial compounds were up to 10 μM. The best binding was found between OAR27 and SjOAR with the KD reached as low as 1.85×10-8 M. Antischistosomal activity analysis showed 3 compounds renamed OAR19, OAR22 and OAR27 had significant antischistosomal effects, and their LD50 values were (19.7±8.7) μg/ml, (18.0±7.6) 0μg/ml and (18.5±6.1) μg/ml, respectively. To investigate whether SjOAR was indeed the target of the three active compounds, we detected their inhibition effects on SjOAR activity. The results revealed that OAR22 and OAR27 could inhibit SjOAR activity in varying degrees [IC50 values were>80 μg/ml and (21.79±1.33) μg/ml] while OAR19 could not. These results suggested SjOAR was indeed the target of compounds OAR22 and OAR27, but not the target of OAR19. Cytotoxicity assay showed that OAR 19 had the strongest toxic against host cells; by contrast, the cytotoxicity of OAR22 and OAR27 was mild. Additionally, we also evaluated the protective efficiency against schistosome infection in murine model. The results indicated that SjOAR immunized mouse induced partial reduction in worm and egg number; however, these data were not statistically significant. Immunolocalization showed SjOAR was abundant in the inner of adult worms.In this study, another redox-associated protein, S.japonicum thioredoxin-1(SjTrx-1), was also cloned, expressed, purified and demonstrated to own activity in vitro. In the study of regarding SjTrx-1 as drug target, we demonstrated auranofin, a known inhibitor of thioredoxin glutathione reductase (TGR), could also inhibited SjTrx-1 activity, which suggested that the known TGR was not the only target of auranofin. Auranofin exhibited significant antischistosomal activity in vitro, moreover, its effect on adult worms were more effective than schistosomula. Cytotoxicity test indicated that auranofin displayed strong toxic at 5 μg/ml, which challenged the previous research that a mammalian cell lines could tolerate 100 μM (68 μg/ml) for 5 days. Immunolocalization confirmed that SjOAR was widely spread in various tissues with high levels.In conclusion, this study integrated various methods such as bioinformatics, molecular biology, enzymology, structural biology and immunology to study the functions, structures and applications of 3 redox-associated proteins of S. japonicum. The results might provide a valuable reference to prevent and treat schistosomiasis. |
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