| The difference between parasites and their host in the respiratory chains is very huge. In order to adapt to the hypoxic environment in the host, the parasities use the anaerobic respiratory chain (NFRD-fumarate reductase, NFRD) for electron transfer, but the NFRD does not exit in the host.So the anaerobic respiratory chain can be used as a new target for drug development against parasites. At the same time, the anaerobic respiratory chain inhibitors can selectively kill tumor cells DLD-1 and Panc-1 by inhibiting the activity of NFRD. Therefore, the anaerobic respiratory chain inhibitors are also potential antitumor drugs.Wortmannilactones are a class of compounds isolated from Talaromyces wortmannii, which have a similar structure with the reported anaerobic respiratory chain inhibitor Ukulactones. In this study,12 strains of marine fungi were selected for Wortmannilactone F biotransformation firstly. All the fungi can transform Wortmannilactone F into its structural analogues, but the abilities were different.The conversion rate of DL1103 was 10.7%, which was the highest. The DL1103 was identified in molecular level, a fragment of 562 bp was amplified by PCR. BLAST results indicated that DL1103 had a rather high sequence identity (100%) with Aspergillus sp. nov. Fl. Scale up transformation experiment was performed and the conversion product was isolated and purified.The evaluation system of antiheminth activity was developed. NFRD and its component of Complex I, the aerobic respiratory chain (NADH oxidase)and its components of Complex I, Complex II models in total of 5 were built. Optimized reaction conditions of various models and the results were as following. NFRD model:37℃, pH 7.2,350 μM NADH, the concentration of mitochondrial protein is 120 μg/ml (under anaerobic environment). The Complex I of NFRD model:pH 7.2,0.32 mM RQ,0.35 mM NADH, the protein concentration of mitochondrial fragments (SMP) is 160 μg/ml. NADH oxidase model:37 ℃, pH 6.8,350 μM NADH, mitochondrial protein concentration was 20 μg/ml. The Complex I of NADH oxidase model:pH 6.8,0.74 mM DCIP,0.35 mM NADH,0.135 mM UQ and 1 mM Antimycin A, the protein concentration of SMP is 45 μg/ml. The Complex II of NADH oxidase model:pH 6.8,0.74 mM DCIP,0.35 mM sodium succinate,0.135 mM UQ and 1 mM Antimycin A, the protein concentration of mitochondrial fragments is 45 μg/ml. The NFRD model in this study revised inaccuracy for activity evaluation in orignial method by creating anaerobic environment and optimizing reaction conditions.The activities of Wortmannilactone E, F and transformation products were evaluated based on the works above.The results showed that Wortmannilactone E and F inhibited NFRD with an IC50 of 0.57 and 0.63 μM respectively, Complex I from NFRD with an IC50 of 0.56 and 0.51 μM seperately; NADH oxidase with an IC50 > 100 μM; Complex I and Complex II from NADH oxidase with an IC50 > 100 μM, which showed that Wortmannilactones had a strong selective activity to inhibit the anaerobic respiratory chain. The transformation products inhibit anaerobic respiratory chain (NFRD) with an ICso=13.5 μM. It inhibited less than 20% activity of NADH oxidase in the concerntration of 135μM, suggesting that they were selective inhibitor against helminth.The study lay a foundation for obtaining the derivatives of Wortmanilactone F, finding the anaerobic repiration chain inhibitor with better activity and revealing the relationship between the Wortmannilactones and the activity of inhibiting anaerobic respiratory chain, also provided a theoretical basis for the development of the Wortmannilactones as a new kind of anti-worm and anti-tumor drugs. |
PDF Full Text Request