A Novel Leishmania major Amastigote Assay in 96-Well Format for Rapid Drug Screening and Its Use for Discovery and Evaluation of a New Class of Leishmanicidal Quinolinium Salts
A Novel Leishmania major Amastigote Assay in 96-Well Format for Rapid Drug Screening and Its Use for Discovery and Evaluation of a New Class of Leishmanicidal Quinolinium Salts
复制标题
DOI：
10.1128/aac.02201-12
复制
发表时间：
2013-07-01
影响因子：
4.9
通讯作者：
Schurigt, Uta
中科院分区：
文献类型：
--
作者：
Bringmann, Gerhard;Thomale, Katja;Schurigt, Uta
在大多数实验室中，用利什曼原虫前鞭毛体或无鞭毛体进行杀利什曼原虫化合物的筛选。然而，鉴定杀利什曼化合物的最佳方法是使用巨噬细胞中的无鞭毛体。报告基因为基础的分析是相对较新的工具，在寻找药物对真核原生动物，允许开发更快，更自动化的检测。在本文中，我们报告建立一个快速筛选测定在96孔格式。产生了一种转β-内酰胺酶（Luc-tg）的硕大利什曼原虫株，并用于感染骨髓源性巨噬细胞（BMDM）。用不同浓度的化合物处理无鞭毛体感染的BMDM。用含胡萝卜素的缓冲液裂解细胞，并测量所得发光以确定半数最大抑制浓度（IC 50）。为了验证这种新的无鞭毛体筛选测定，合成了一类新的喹啉鎓盐的文库并测试了杀利什曼活性。一些喹啉盐显示出非常有前景的活性，其对细胞内无鞭毛体的IC（50）（IC 50 < 1 μ g/ml）和选择性指数（SI > 20）符合世界卫生组织（WHO）的命中标准。化合物21 c（IC_（50）= 0.03 μ g/ml; SI = 358）可能成为开发抗L.少校总之，我们描述了一个新的96孔板检测法的建立与吕克转基因L。主要用于快速筛选具有抗细胞内无鞭毛体的杀利什曼活性的化合物，及其在鉴定具有最有希望的杀利什曼活性的一类新喹啉盐中的应用。
In most laboratories, the screening for leishmanicidal compounds is carried out with Leishmania promastigotes or axenic amastigotes. However, the best approach to identify leishmanicidal compounds is the use of amastigotes residing in macrophages. Reporter gene-based assays are relatively new tools in the search for drugs against eucaryotic protozoa, permitting the development of faster, more automated assays. In this paper, we report on the establishment of a rapid screening assay in a 96-well format. A luciferase-transgenic (Luc-tg) Leishmania major strain was generated and used to infect bone marrow-derived macrophages (BMDM). Amastigote-infected BMDM were treated with different compound concentrations. Cells were lysed with a luciferin-containing buffer, and the resulting luminescence was measured to determine the half-maximal inhibitory concentration (IC50). To validate this new amastigote screening assay, a library of a new class of quinolinium salts was synthesized and tested for leishmanicidal activity. Some of the quinolinium salts showed very promising activities, with IC(50)s against intracellular amastigotes (IC50 < 1 mu g/ml) and selectivity indices (SI > 20) that match the criteria of World Health Organization (WHO) for hits. Compound 21c (IC50 = 0.03 mu g/ml; SI = 358) could become a new lead structure for the development of improved chemotherapeutic drugs against L. major. In summary, we describe the establishment of a new 96-well format assay with Luc-transgenic L. major for the rapid screening of compounds for leishmanicidal activity against intracellular amastigotes and its application to the identification of a new class of quinolinium salts with most promising leishmanicidal activity.