Instituto de Química Médica, IQM-CSIC, Juan de la Cierva 3, E-28006, Madrid, Spain.
Graduate School of Science and Technology, Department of Applied Biology, Kyoto Institute of Technology, Kyoto, 606-8585, Japan; Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom; School of Health and Life Sciences, Teesside University, Middlesbrough, United Kingdom.
Eur J Med Chem. 2021 Aug 5;220:113470. doi: 10.1016/j.ejmech.2021.113470. Epub 2021 Apr 16.
We have recently reported on the development and trypanocidal activity of a class of inhibitors of Trypanosome Alternative Oxidase (TAO) that are targeted to the mitochondrial matrix by coupling to lipophilic cations via C14 linkers to enable optimal interaction with the enzyme's active site. This strategy resulted in a much-enhanced anti-parasite effect, which we ascribed to the greater accumulation of the compound at the location of the target protein, i.e. the mitochondrion, but to date this localization has not been formally established. We therefore synthesized a series of fluorescent analogues to visualize accumulation and distribution within the cell. The fluorophore chosen, julolidine, has the remarkable extra feature of being able to function as a viscosity sensor and might thus additionally act as a probe of the cellular glycerol that is expected to be produced when TAO is inhibited. Two series of fluorescent inhibitor conjugates incorporating a cationic julolidine-based viscosity sensor were synthesized and their photophysical and biological properties were studied. These probes display a red emission, with a high signal-to-noise ratio (SNR), using both single- and two-photon excitation. Upon incubation with T. brucei and mammalian cells, the fluorescent inhibitors 1a and 2a were taken up selectively in the mitochondria as shown by live-cell imaging. Efficient partition of 1a in functional isolated (rat liver) mitochondria was estimated to 66 ± 20% of the total. The compounds inhibited recombinant TAO enzyme in the submicromolar (1a, 2c, 2d) to low nanomolar range (2a) and were effective against WT and multidrug-resistant trypanosome strains (B48, AQP1-3 KO) in the submicromolar range. Good selectivity (SI > 29) over mammalian HEK cells was observed. However, no viscosity-related shift could be detected, presumably because the glycerol was produced cytosolically, and released through aquaglyceroporins, whereas the probe was located, virtually exclusively, in the trypanosome's mitochondrion.
我们最近报道了一类通过 C14 键连接脂溶性阳离子靶向线粒体基质的新型锥虫 Alternative Oxidase（TAO）抑制剂的开发和杀锥虫活性，这种策略可以使化合物与酶的活性位点最佳相互作用。这一策略显著增强了抗寄生虫效果，我们将其归因于化合物在靶蛋白（即线粒体）位置的积累增加，但到目前为止，这种定位尚未得到正式证实。因此，我们合成了一系列荧光类似物来可视化化合物在细胞内的积累和分布。选择的荧光团 julolidine 具有一个显著的额外特点，即能够作为粘度传感器，因此还可以作为 TAO 被抑制时预期产生的细胞甘油的探针。我们合成了两个系列包含阳离子 julolidine 基粘度传感器的荧光抑制剂缀合物，并研究了它们的光物理和生物学性质。这些探针在使用单光子和双光子激发时均显示出红色发射，具有高信噪比（SNR）。在用 T. brucei 和哺乳动物细胞孵育后，荧光抑制剂 1a 和 2a 被选择性地摄取到线粒体中，这一点通过活细胞成像得到证实。在功能分离的（大鼠肝脏）线粒体中，1a 的有效分配估计为总分配的 66 ± 20%。这些化合物以亚微摩尔（1a、2c、2d）至低纳摩尔范围（2a）抑制重组 TAO 酶，对亚微摩尔范围内的 WT 和多药耐药锥虫株（B48、AQP1-3 KO）有效。在哺乳动物 HEK 细胞中观察到良好的选择性（SI > 29）。然而，由于甘油在细胞质中产生，并通过水通道蛋白释放，而探针几乎只位于锥虫的线粒体中，因此没有检测到与粘度相关的位移。
