Synthesis, biological, and photophysical studies of molecular rotorbased fluorescent inhibitors of the trypanosome alternative oxidase Cueto Díaz, Eduardo J. González García, María del Carmen Girón González, María Dolores Salto González, Rafael González Vera, Juan Antonio Ruedas Rama, María José Orte Gutiérrez, Ángel Trypanosome alternative oxidase (TAO) Inhibitor Trypanosoma brucei Molecular rotor Fluorescent probe 2,4-dihydroxybenzoic acid derivative Julolidine This work was supported by the Spanish Ministerio de Economia y Competitividad (grant SAF2015-66690-R), the Spanish Ministerio de Ciencia, Innovaci on y Universidades (MCIU/AEI/FEDER, UE; grants RTI2018-093940-B-I00 to CD, and CTQ2017-85658-R to AO) and the Japan Society for the promotion of Science (JSPS grant- 17F17420 to GUE). MAU is funded through a studentship from the Petroleum Technology Development Fund (PTDF), Abuja, Nigeria. IAA was funded through a Ph.D. studentship from the Ministry of Health of Saudi Arabia.We thank Dr. Jos e Cumella for the synthesis of the 1,14-dibromotetradecane precursor and to Prof. Ibon Alkorta for his help with DFT calculations. We also thank Professor Fred Opperdoes and Dr Alena Zíkov a for their insightful discussions on T. brucei energy metabolism. 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. 2021-07-27T11:02:46Z 2021-07-27T11:02:46Z 2021-04-16 info:eu-repo/semantics/article Eduardo J. Cueto-Díaz... [et al.]. Synthesis, biological, and photophysical studies of molecular rotor-based fluorescent inhibitors of the trypanosome alternative oxidase, European Journal of Medicinal Chemistry, Volume 220, 2021, 113470, ISSN 0223-5234, [https://doi.org/10.1016/j.ejmech.2021.113470] http://hdl.handle.net/10481/69941 10.1016/j.ejmech.2021.113470 eng http://creativecommons.org/licenses/by-nc-nd/3.0/es/ info:eu-repo/semantics/openAccess Atribución-NoComercial-SinDerivadas 3.0 España Elsevier