Mechanism of aquaporin-mediated drug resistance of trypanosomes

• Applicant: Professor Dr. Eric Beitz
• Subject Area: Pharmacy ; Biochemistry
• Term: from 2014 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 263523902

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.

• DFG Programme: Research Grants
• International Connection: United Kingdom
• Participating Person: Professor Dr. David Horn