Mechanism of aquaporin-mediated drug resistance of trypanosomes

非洲锥虫病的治疗远远不是最佳的，因为复方药物有限且陈旧(20世纪40年代)，并有戏剧性的、甚至是致命的副作用。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耐药(TbAQP2)可以转变为将小(药物)分子穿梭到寄生虫中的新原理。Horn小组认为TbAQP2可能促进了五烷双胺/美拉索的直接扩散。由于我们在AQP方面的经验，我们怀疑像五烷双胺这样的大分子甚至带电分子能否通过TbAQP2。在功能分析中，我们发现了对TbAQP2的完全抑制和极强的亲和力(IC50=130 nM)。我们滴定了五胺结合部位的pKa，并合成了五胺的衍生物，以建立结合模式。我们的工作是提出以TbAQP2为受体而不是扩散摄取的受体介导的五烷双胺的内吞作用。我们将致力于三个目标：1.我们将通过TbAQP2分析五烷双胺的结合与渗透问题。在这方面，我们将使用已建立的铵和甲基铵检测方法来测试TbAQP2的一般阳离子渗透性/排斥性。如果这些小阳离子被排斥，可以合理地假设较大的五烷胺也不会渗透到TbAQP2中。我们将引入点突变来取代TbAQP2(D265N，D265A)的一个特殊通道天冬氨酸，以确认我们提出的五胺结合位点。我们将确定五烷双胺是否也抑制TbAQP2相关的TbAQP3。我们预计，由于所提议的结合区的序列差异，情况并非如此。最终，我们将尝试通过点突变将不敏感的五烷胺AQP转变为可抑制五烷双胺的AQP，以证明我们预测的结合模式。我们将确定TbAQP2影响的第二种药物，即美拉索洛尔的结合/渗透。在这里，我们将利用美拉索中的砷原子，利用原子吸收光谱来测试TbAQP2促进的美拉索在酵母中的吸收。此外，我们还将利用停流光散射检测美拉索对TbAQP2的抑制作用。我们将可视化五烷双胺与TbAQP2的结合并摄取到活的锥虫体内。为此，我们将合成荧光五烷胺类化合物，并对它们与TbAQP2的亲和力进行定量。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。这样，我们就可以可视化结合部位，它应该与TbAQP2在鞭毛口袋中共同定位。我们将使用高分辨率显微镜和适当的反染色进一步分析锥体对荧光五胺类物质的内吞与温度的关系。荧光标记的吸收将代表一种新的TbAQP2/五烷胺促进的将(药物)分子穿梭到锥体内部的方法的概念验证。

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.