Mechanism of aquaporin-mediated drug resistance of trypanosomes

非洲锥虫病的治疗还远未达到最佳效果，因为复方药物有限且陈旧（20世纪40年代），而且有严重甚至致命的副作用。我们的初步研究表明，David Horn, Dundee， UK发现的水通道蛋白介导的对喷他脒/美拉唑醇的耐药性（TbAQP2）可以转化为将小（药物）分子输送到寄生虫中的新原理。Horn小组认为TbAQP2可能促进喷他脒/美拉胂醇的直接扩散。根据我们对aqp的经验，我们怀疑像喷他脒这样大且甚至带电的分子是否能通过TbAQP2。通过功能分析，我们发现喷他脒对TbAQP2具有完全的抑制作用和极强的亲和力（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，以建立其结合模式。我们的研究表明，受体介导的喷他脒内吞作用以TbAQP2为受体，而不是扩散摄取。我们将努力实现三个目标：1。我们将通过TbAQP2分析喷他脒的结合与通透性问题。在这方面，我们将使用已建立的铵和甲基铵测定法测试TbAQP2的一般阳离子渗透性/排他性。如果这些小阳离子被排斥，可以合理地假设较大的喷他脒也不会渗透到TbAQP2中。我们将引入点突变取代TbAQP2 （D265N, D265A）的特殊通道天冬氨酸，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。我们预计，由于所提出的结合区域的序列差异，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转化为对戊脒抑制的AQP，以证明我们预测的结合模式。我们将测定受TbAQP2影响的第二种药物的结合/渗透，即美拉胂醇。在这里，我们将测试tbaqp2促进三聚氰胂醇进入酵母利用原子吸收光谱利用砷原子的三聚氰胂醇。此外，我们将使用停流光散射测试三聚氰胺对TbAQP2的抑制作用。我们将可视化喷他脒与TbAQP2结合并被活锥虫摄取。为此，我们将合成荧光喷他脒衍生物，并定量其与TbAQP2的亲和力。具有足够亲和力的化合物将与David Horn合作用于标记培养的锥虫。通过这种方式，我们可以看到与TbAQP2在鞭毛囊内共定位的结合位点。我们将使用高分辨率显微镜和适当的反染色进一步分析荧光喷他脒在锥虫体内的温度依赖性内吞摄取。荧光标记的吸收将代表一种新的TbAQP2/喷他脒促进方式将（药物）分子穿梭到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.