Development ofenzyme inlulkar far infectious diseace including malaria and clarification ofthe inlubitor activity

人类疟疾、利什曼病、恰加斯病、人类非洲锥虫病、登革热和血吸虫病等热带疾病继续造成严重的发病率和死亡率。在本项目中，我们对寄生虫中与这三种传染病有关的重要酶进行了X射线结晶学研究，为利用硅胶方法开发药物奠定了结构基础。这些重要的酶是：(1)两种与人类疟疾的嘧啶核苷酸从头合成有关的酶；(2)查格斯病和利什曼原虫的前列腺素(PG)F合成酶。从大肠杆菌中纯化并结晶了重组酶：轮状磷酸核糖转移酶(PYR5)和单磷酸脱羧酶(PYR6)。结果表明，PYR6的天然和产物UMP络合物均得到结晶，并进行了X-射线结构分析。脱辅酶PYR6和复合体PYR6的2.7和2.6A拆分结构提供了脱羧基机制的细节(J.Bioch.，2008)。克氏锥虫和梅约利什曼原虫的PGF合成酶也进行了结晶，测定了1.7A分辨率(Acta Cryst.F，2007)。测定了前列腺素F合成酶与吲哚美辛的络合物结构，为新型抑制剂的开发提供了结构信息。

Tropical diseases such as human malaria, leishmaniasis, Chagas disease, human African trypanosomiasis, dengue fever and schistosomiasis continue to cause significant morbidity and mortality. In this project, we performed X-ray crystallographic studies of important enzymes from parasite concerning three these infectious disease to construct the structural basis for the drug development by the in-silica method. The important enzymes are (1) two kinds of enzymes concerning the de novo synthesis of pyrimidine nucleotide from human malaria, (2) prostaglandin (PG) F synthases from parasites of Chagas' disease and Leishmania.Recombinant enzymes of orotate phosphoribosyltransferase (pyr5) and Orotidine 5'-monophosphate decarboxylase (pyr6) were purified from E.coli and crystallized. As the result, the native and product UMP complex form of pyr6 were crystallized and carried out with the X-ray structural analyses. The 2.7 and 2.6 A resolutional structures of apo- and complex forms of pyr6 provided the details of the decarboxylation mechanism (J. Biochem., 2008). As for the PGF synthases from Trypanosoma cruzi and Leishmania mejor were also performed crystallization and the structure of PGF synthase from T. cruzi were determined at 1.7 A resolution (Acta Cryst. F, 2007). The complex structure of PGF sytnthase with indomethacin were also determined, providing the structural information for new inhibitor development.