High-Throughput Chemical Screening Facility, Structural Biology Division, Walter+Eliza Hall Institute of Medical Research, Melbourne, Australia.
Antimicrob Agents Chemother. 2010 May;54(5):1712-9. doi: 10.1128/AAC.01634-09. Epub 2010 Feb 16.
The current treatment for leishmaniasis is based on chemotherapy, which relies on a handful of drugs with serious limitations, such as high cost, toxicity, and a lack of efficacy in regions of endemicity. Therefore, the development of new, effective, and affordable antileishmanial drugs is a global health priority. Leishmania synthesizes a range of mannose-rich glycoconjugates that are essential for parasite virulence and survival. A prerequisite for glycoconjugate biosynthesis is the conversion of monosaccharides to the activated mannose donor, GDP-mannose, the product of a reaction catalyzed by GDP-mannose pyrophosphorylase (GDP-MP). The deletion of the gene encoding GDP-MP in Leishmania led to a total loss of virulence, indicating that the enzyme is an ideal drug target. We developed a phosphate sensor-based high-throughput screening assay to quantify the activity of GDP-MP and screened a library containing approximately 80,000 lead-like compounds for GDP-MP inhibitors. On the basis of their GDP-MP inhibitory properties and chemical structures, the activities of 20 compounds which were not toxic to mammalian cells were tested against ex vivo amastigotes and in macrophage amastigote assays. The most potent compound identified in the primary screen (compound 3), a quinoline derivative, demonstrated dose-dependent activity in both assays (50% inhibitory concentration = 21.9 microM in the macrophage assay) and was shown to be nontoxic to human fibroblasts. In order to elucidate signs of an early structure-activity relationship (SAR) for this class of compounds, we obtained and tested analogues of compound 3 and undertook limited medicinal chemistry optimization, which included the use of a number of SAR probes of the piperazinyl aryl substituent of compound 3. We have identified novel candidate compounds for the design and synthesis of antileishmanial therapeutics.
当前治疗利什曼病的方法基于化疗，该方法依赖于少数几种具有严重局限性的药物，如成本高、毒性大以及在流行地区疗效不佳。因此，开发新的、有效且负担得起的抗利什曼病药物是全球卫生的重点。利什曼原虫合成了一系列富含甘露糖的糖缀合物，这些糖缀合物对寄生虫的毒力和存活至关重要。糖缀合物生物合成的前提是将单糖转化为激活的甘露糖供体 GDP-甘露糖，该产物是 GDP-甘露糖焦磷酸化酶（GDP-MP）催化反应的产物。利什曼原虫中编码 GDP-MP 的基因缺失导致毒力完全丧失，表明该酶是一个理想的药物靶点。我们开发了一种基于磷酸盐传感器的高通量筛选测定法来定量 GDP-MP 的活性，并筛选了一个包含大约 80,000 个类似先导化合物的文库，以寻找 GDP-MP 抑制剂。基于其 GDP-MP 抑制特性和化学结构，测试了对哺乳动物细胞无毒性的 20 种化合物对体外无鞭毛体和巨噬细胞无鞭毛体试验的活性。在初步筛选中鉴定出的最有效化合物（化合物 3，一种喹啉衍生物），在两种试验中均表现出剂量依赖性活性（在巨噬细胞试验中 50%抑制浓度为 21.9 μM），并且对人成纤维细胞无毒性。为了阐明此类化合物的早期结构-活性关系（SAR）的迹象，我们获得并测试了化合物 3 的类似物，并进行了有限的药物化学优化，其中包括使用化合物 3 的哌嗪基芳基取代基的一些 SAR 探针。我们已经确定了用于设计和合成抗利什曼病治疗药物的新型候选化合物。
