Mechanism of aquaporin-mediated drug resistance of trypanosomes

非洲锥虫病的治疗远不是最佳的，因为化合物集有限且陈旧（20世纪40年代），并且具有显著的，甚至致命的副作用。我们的初步研究表明，大卫霍恩，邓迪，英国，水通道蛋白介导的耐药性（TbAQP 2）的发现，喷他脒/米拉索普可以转化为一个新的原则，穿梭小（药物）分子到寄生虫。霍恩小组假设，TbAQP 2可能促进喷他脒/米拉索普的直接扩散。由于我们对AQPs的经验，我们怀疑像喷他脒这样大的甚至带电荷的分子可以通过TbAQP 2。使用功能测定，我们发现喷他脒对TbAQP 2的完全抑制和非凡的亲和力（IC 50 = 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。我们的工作提示了以TbAQP 2为受体的喷他脒的内吞作用是受体介导的，而不是扩散摄取。我们将通过TbAQP 2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检测TbAQP 2的一般阳离子渗透性/排斥性。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TbAQP 2。我们将引入点突变取代TbAQP 2的一个特殊通道天冬氨酸（D265 N，D265 A），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TbAQP 2相关的TbAQP 3。我们预期，由于所提出的结合区域中的序列差异，情况并非如此。最后，我们将尝试通过点突变将对戊脒不敏感的AQP转化为对戊脒敏感的AQP，以证明我们预测的结合模式。我们将确定受TbAQP 2影响的第二种药物（即美拉胂醇）的结合/渗透。在这里，我们将测试TbAQP 2促进米拉索普摄取到酵母使用原子吸收光谱法利用砷原子米拉索普。此外，我们将使用停流光散射测试美拉胂醇对TbAQP 2的抑制作用。我们将观察喷他脒与TbAQP 2的结合和摄取到活锥虫中。为此，我们将合成荧光喷他脒衍生物，并量化其对TbAQP 2的亲和力。与大卫霍恩合作，将使用具有足够亲和力的化合物标记培养的锥虫。这样，我们将可视化的结合位点，这应该与TbAQP 2在鞭毛口袋共定位。我们将使用高分辨率显微镜和适当的复染进一步分析温度依赖性的内吞摄取荧光pentamidines进入锥虫。荧光标记的摄取将代表一种新的TbAQP 2/pentamidine促进的方式穿梭（药物）分子进入锥虫内部的概念验证。

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.