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Mechanism of aquaporin-mediated drug resistance of trypanosomes

水通道蛋白介导的锥虫耐药机制

基本信息

批准号：
263523902
负责人：
Professor Dr. Eric Beitz
金额：
--
依托单位：
Pharmazeutisches Institut
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2014
资助国家：
德国
起止时间：
2013-12-31 至 2017-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/263523902?language=en
关键词：
Mechanism aquaporin mediated drug resistance

项目摘要

The treatment of African trypanosomiasis is far from being optimal because the compound set is limited and old (1940s), and has dramatic, even fatal side effects. Our preliminary studies suggest that the discovery by David Horn, Dundee, UK, of aquaporin-mediated resistance (TbAQP2) to pentamidine/melarsoprol can be turned into a new principle to shuttle small (drug-) molecules into the parasite.The Horn group assumed that TbAQP2 may facilitate direct diffusion of pentamidine/ melarsoprol. Due to our experience with AQPs we doubt that such large and even charged molecules as pentamidine could pass TbAQP2. Using functional assays we found full inhibition and extraordinary affinity of pentamidine to TbAQP2 (IC50 = 130 nM). We titrated the pKa of the pentamidine binding site and synthesized derivatives of pentamidine to establish a binding mode. Our work is suggestive of receptor-mediated endocytosis of pentamidine with TbAQP2 as receptor rather than diffusional uptake.We will work on three objectives:1. We will analyze the binding vs. permeability issue of pentamidine via TbAQP2. In this regard, we will test for general cation permeability/exclusion of TbAQP2 using established assays for ammonium and methylammonium. If these small cations are repelled, it is fair to assume that the larger pentamidine will also not permeate TbAQP2. We will introduce point mutations replacing a peculiar channel aspartate of TbAQP2 (D265N, D265A) to confirm our proposed pentamidine binding site. We will determine whether pentamidine also inhibits the TbAQP2-related TbAQP3. We expect that this is not the case due to sequence differences in the proposed binding region. Eventually, we will try to convert a pentamidine-insensitive AQP into a pentamidine-inhibitable AQP by point mutation to proof our predicted binding mode.2. We will determine binding/permeation of the second drug affected by TbAQP2, i.e. melarsoprol. Here, we will test for TbAQP2-facilitated melarsoprol uptake into yeast using atomic absorption spectroscopy making use of the arsenic atom in melarsoprol. Further, we will test for inhibition of TbAQP2 by melarsoprol using stopped-flow light scattering.3. We will visualize binding of pentamidine to TbAQP2 and uptake into live trypanosomes. To this end, we will synthesize fluorescent pentamidine derivatives, and quantify their affinity to TbAQP2. Compounds of sufficient affinity will be used for labeling of cultured trypanosomes in collaboration with David Horn. This way we will visualize the binding site, which should co-localize with TbAQP2 in the flagellar pocket. We will further analyze temperature-dependent endocytic uptake of fluorescent pentamidines into trypanosomes using high-resolution microscopy and appropriate counterstaining. Uptake of the fluorescent label would represent a proof-of-concept for a novel TbAQP2/pentamidine-facilitated way to shuttle (drug-)molecules into the trypanosome interior.

非洲锥虫病的治疗远非最佳，因为药物组合有限且陈旧（20 世纪 40 年代），并且具有严重甚至致命的副作用。我们的初步研究表明，英国邓迪的 David Horn 发现的水通道蛋白介导的喷他脒/melarsoprol 耐药性 (TbAQP2) 可以转化为将小（药物）分子运送到寄生虫中的新原理。Horn 小组假设 TbAQP2 可能促进喷他脒/melarsoprol 的直接扩散。根据我们在 AQP 方面的经验，我们怀疑像喷他脒这样大的甚至带电的分子能否通过 TbAQP2。通过功能测定，我们发现喷他脒对 TbAQP2 具有完全抑制作用和非凡的亲和力 (IC50 = 130 nM)。我们滴定了喷他脒结合位点的pKa并合成了喷他脒的衍生物以建立结合模式。我们的工作表明以TbAQP2为受体的受体介导的喷他脒的内吞作用而不是扩散摄取。我们将致力于三个目标：1．我们将通过 TbAQP2 分析喷他脒的结合与渗透性问题。在这方面，我们将使用已建立的铵和甲基铵测定法来测试 TbAQP2 的一般阳离子渗透性/排除性。如果这些小阳离子被排斥，则可以合理地假设较大的喷他脒也不会渗透 TbAQP2。我们将引入点突变来替换 TbAQP2 的特殊天冬氨酸通道（D265N、D265A），以确认我们提出的喷他脒结合位点。我们将确定喷他脒是否也抑制 TbAQP2 相关的 TbAQP3。由于所提议的结合区域的序列差异，我们预计情况并非如此。最终，我们将尝试通过点突变将喷他脒不敏感的AQP转化为喷他脒抑制的AQP，以证明我们预测的结合模式。2．我们将确定受 TbAQP2 影响的第二种药物（即 melarsoprol）的结合/渗透。在这里，我们将利用 melarsoprol 中的砷原子，使用原子吸收光谱法测试 TbAQP2 促进的 melarsoprol 被酵母吸收。此外，我们将使用停流光散射测试 melarsoprol 对 TbAQP2 的抑制作用。 3．我们将可视化喷他脒与 TbAQP2 的结合以及活锥虫的摄取。为此，我们将合成荧光喷他脒衍生物，并量化它们与TbAQP2的亲和力。与 David Horn 合作，具有足够亲和力的化合物将用于标记培养的锥虫。通过这种方式，我们将可视化结合位点，该位点应与鞭毛袋中的 TbAQP2 共定位。我们将使用高分辨率显微镜和适当的复染进一步分析荧光喷他脒进入锥虫的温度依赖性内吞摄取。荧光标记的摄取将代表一种新的 TbAQP2/喷他脒促进的将（药物）分子穿梭到锥虫内部的方式的概念验证。