Serrão Vitor Hugo Balasco, Romanello Larissa, Cassago Alexandre, de Souza Juliana Roberta Torini, Cheleski Juliana, DeMarco Ricardo, Brandão-Neto José, Pereira Humberto D'Muniz
Physics Institute of São Carlos, University of São Paulo, São Carlos-SP, CEP13566-590, Brazil.
National Laboratory of Nanotechnology - LNNano/CNPEM, Campinas-SP, CEP 3083-970, Brazil.
Acta Trop. 2017 Jun;170:190-196. doi: 10.1016/j.actatropica.2017.03.007. Epub 2017 Mar 11.
The parasite Schistosoma mansoni possesses all pathways for pyrimidine biosynthesis, in which dihydrofolate reductase (DHFR), thymidylate cycle participants, is essential for nucleotide metabolism to obtain energy and structural nucleic acids. Thus, DHFRs have been widely suggested as therapeutic targets for the treatment of infectious diseases. In this study, we expressed recombinant SmDHFR in a heterologous manner to obtain structural, biochemical and kinetic information. X-ray diffraction of recombinant SmDHFR at 1.95Å resolution showed that the structure exhibited the canonical DHFR fold. Isothermal titration calorimetry was used to determine the kinetic constants for NADP and dihydrofolate. Moreover, inhibition assays were performed using the commercial folate analogs methotrexate and aminopterin; these analogs are recognized as folate competitors and are used as chemotherapeutic agents in cancer and autoimmune diseases. This study provides information that may prove useful for the future discovery of novel drugs and for understanding these metabolic steps from this pathway of S. mansoni, thus aiding in our understanding of the function of these essential pathways for parasite metabolism.
曼氏血吸虫这种寄生虫拥有嘧啶生物合成的所有途径，其中二氢叶酸还原酶（DHFR）作为胸苷酸循环参与者，对于获取能量和构建核酸的核苷酸代谢至关重要。因此，二氢叶酸还原酶被广泛认为是治疗传染病的靶点。在本研究中，我们以异源方式表达重组曼氏血吸虫二氢叶酸还原酶（SmDHFR），以获取其结构、生化和动力学信息。重组SmDHFR的X射线衍射在1.95Å分辨率下显示，该结构呈现出典型的二氢叶酸还原酶折叠。采用等温滴定量热法测定了烟酰胺腺嘌呤二核苷酸磷酸（NADP）和二氢叶酸的动力学常数。此外，使用市售叶酸类似物甲氨蝶呤和氨基蝶呤进行抑制试验；这些类似物被认为是叶酸竞争者，并在癌症和自身免疫性疾病中用作化疗药物。本研究提供的信息可能对未来新型药物的发现以及理解曼氏血吸虫这一途径的这些代谢步骤有用，从而有助于我们了解这些对寄生虫代谢至关重要的途径的功能。
