Study On The Antischistosomal Inhibitors Based On The SjTGR Activity

Schistosomiasis(bilharziasis) is a severely parasitic disease for public health caused by schistosome. According to the World Health Organization reports, at least 249 million people worldwide need schistosomiasis treatment in 2012. Schistosomiasis currently relies mainly on praziquantel. However, due to long-term use of praziquantel, Schistosoma has begun to develop potential resistance.Therefore, the development of new drugs against schistosome become an urgent task.The multifunctional enzyme, thioredoxin glutathione reductase from Schistosoma japonicum(SjTGR) containing TrxR and GR activities in vivo is essential for survival of schistosome. In this study, the selenoprotein SjTGR has been expressed and its activities have been measured. The chemical inhibitors have been screened mainly including derivatives of praziquantel and Oxazole-2-oxygen. The toxicity of antischistosomal drug for adult worms and cercariae were also evaluated. The main contents include:(1) Preparation of SjTGR selenoproteins and enzyme kinetic analysis. The soluble target protein has been expressed successfully with a concentration of 0.6 mM IPTG at 24 ℃. Protein was purified to a final concentration of 95% by Ni2+ column and DEAE column. The thioredoxin reductase(TrxR) and glutathione reductase(GR) activities of SjTGR were analysed by UV spectrophotometer at 25 ℃. with the substrates of 5-thio- bis-2-nitrobenzoic acid(DTNB),coenzyme Ⅱ(NADPH), oxidized glutathione(GSSG). Their activities were 7.06±0.23umolmin-1mg-1 and 1.832±0.02 umolmin-1mg-1, respectively. The value of Km were 31.4±3.31μM(NADPH), 454±5.47uM(DTNB),51.2±4.23uM(GSSG)), respectively.(2) Chemial inhibitors screening based on the activities of SjTGR. The 12 chemicals including oxadiazole, praziquantel nitrate, brominated praziquantel and carmustine have been screened. The results indicated that oxadiazole aldehyde?? show a high affinity activity in this type of inhibitors which show a IC50 value of 0.907 n M. Bromo praziquantel and carmustine showed the GR inhibitory activity. For the inhibitory type, carmustine shows a noncompetitive inhibition characterization in TrxR activity and other drugs are competitive inhibition. In GR inhibition experiments, all these drugs are non-competitive inhibition.(3) Toxicity of several compounds against S. japonicum adult worms and cercariae. S.japonicum adult are placed in RPMI 1640 medium and incubated for 30-60 min with different concentrations of the drug(praziquantel and oxadiazole derivatives). Then the parasite viability and death were continuously counted every 6 hours within 72 hours after incubation. The results showed that drugs of praziquantel derivative showed the activity of killing the adult worms. Inaddition, the efficacy is reduced with the length of alkyl chain. Oxadiazole compounds(aldehydes substituted) containing the electron withdrawing group showed stronger insecticidal activity.