Authors

Discussion

African trypanosomiasis is regarded as one of the neglected tropical diseases of public health concern, and a severe challenge to agriculture within endemic regions. However, the parasites possess certain unique metabolic features that can be exploited for the purpose of effective drug development.  In earlier studies, we reported the development and trypanocidal efficacies of two classes of inhibitors based on 4-hydroxybenzoate and 4-alkoxybenzaldehyde scaffolds, designed to target the distinctive and critical enzyme of trypanosome respiration, the Trypanosome Alternative Oxidase (TAO), which is situated in the mitochondrion of the parasite. These inhibitors were targeted to the mitochondrial matrix by coupling to lipophilic cations (LC; triphenylphosphonium or quinolinium salts). To enable optimal interaction with the enzyme’s active site, the coupling used a long and flexible C14 linker. In addition, the structure of the inhibitor was optimised by systematically exploring substitutions on the central phenyl ring.         

The result of this strategy was a much-enhanced anti-parasite effect, which we attributed to an increased accumulation of the inhibitor at the target, i.e., the mitochondrion, compared to previous inhibitors that were not linked to LCs.  In order to concretely establish that the compounds reached the ascribed localization, we synthesized a series of fluorescent analogues, to be used for visualizing the distribution and accumulation of the inhibitor within the cell. Remarkable, the fluorophore used, a julolidine moiety, has the interesting feature of also being able to sense and respond to changes in viscosity within its environment, by acting as a molecular rotor, which changes the fluorescent emission. This is of interest consequent of the cellular glycerol known to be produced during TAO inhibition. The probes would display a red emission under such condition. In the present work, we report the biological activities of two series of molecular rotor-based fluorescent inhibitors of the trypanosome alternative oxidase. Using live-cell imaging, the fluorescent inhibitors 1a and 2a were shown to be selectively taken up into the mitochondrion of T. brucei or mammalian cells upon incubation.  The compounds were active against wild-type (Lister 427) and multidrug-resistant Trypanosoma brucei strains (B48, AQP1-3 KO) in the sub micromolar range. They were also potent inhibitors of purified recombinant TAO protein in the low nanomolar (2a) to sub micromolar (1a,2c,2d) range. We also observed good selectivity (SI>29) over the mammalian (Human Embryonic Kidney, HEK) cells. However, no substantial viscosity-related shift in emission wavelength was detected within trypanosomes; apparently because the probe located, essentially exclusively, in the parasite’s mitochondrion was not in contact with sufficient amounts of glycerol, which was cytosolically produced, and believed to be exported out of the cell through aquaglyceroporins.