Discovery of Sustainable Drugs for Neglected Tropical Diseases: Cashew Nut Shell Liquid (CNSL)‐Based Hybrids Target Mitochondrial Function and ATP Production in Trypanosoma brucei

Abstract

In the search for effective and sustainable drugs for human African trypanosomiasis (HAT), we developed hybrid compounds by merging the structural features of quinone 4 (2‐phenoxynaphthalene‐1,4‐dione) with those of phenolic constituents from cashew nut shell liquid (CNSL). CNSL is a waste product from cashew nut processing factories, with great potential as a source of drug precursors. The synthesized compounds were tested against Trypanosoma brucei brucei , including three multidrug‐resistant strains, T. congolense , and a human cell line. The most potent activity was found against T. b. brucei , the causative agent of HAT. Shorter‐chain derivatives 20 (2‐(3‐(8‐hydroxyoctyl)phenoxy)‐5‐methoxynaphthalene‐1,4‐dione) and 22 (5‐hydroxy‐2‐(3‐(8‐hydroxyoctyl)phenoxy)naphthalene‐1,4‐dione) were more active than 4 , displaying rapid micromolar trypanocidal activity, and no human cytotoxicity. Preliminary studies probing their mode of action on trypanosomes showed ATP depletion, followed by mitochondrial membrane depolarization and mitochondrion ultrastructural damage. This was accompanied by reactive oxygen species production. We envisage that such compounds, obtained from a renewable and inexpensive material, might be promising bio‐based sustainable hits for anti‐trypanosomatid drug discovery.

European propolis is highly active against trypanosomatids including Crithidia fasciculata

“…The standard laboratory strain Lister 427WT 36 was used as drug sensitive standard and the multi-drug resistant clone B48 37 was used to assess the potential for cross-resistance with the diamidine and melaminophenyl arsenical classes of trypanocides. T. congolense strain IL3000 (Savannah-type) was cultured as described previously in Minimal Essential Medium (MEM) base with 10% goat serum, supplemented with 14 µL/L β-mercapto-ethanol, glutamine and antibiotics as described 38 .…”

Novel flavanones with anti-trypanosomal activity isolated from Zambian and Tanzanian propolis samples

“…Culturing of T. brucei bloodstream forms was performed in complete HMI-9 medium with 10% fetal bovine serum at 37 °C/5% CO 2 , as described ( Gudin et al, 2006 ). The T. congolense bloodstream forms of strain IL3000 (Savannah-type), and the derived diminazene-resistant clone 6C3, were cultured in Minimal Essential Medium (MEM) base with 10% goat serum, supplemented with 14 μL/L β-mercapto-ethanol, glutamine and antibiotics at 34 °C/5% CO 2 as described ( Cerone et al, 2019 ). The extract and the purified compounds were tested against T. brucei and T. congolense using the resazurin viability indicator exactly as described previously ( Alotaibi et al, 2019 ; Omar et al, 2017 ).…”

Diminazene resistance inTrypanosoma congolenseis linked to reduced mitochondrial membrane potential and not to reduced transport capacity

“…This drug susceptibility assay was performed essentially as described [63] and is based on only live cells reducing the blue and non-fluorescent viability indicator dye resazurin sodium salt (‘Alamar blue’; Sigma) to pink, fluorescent metabolite resorufin [36]. The fluorescence was quantified using a FLUOstar Optima (BMG Labtech, Durham, NC, USA) at wavelength of 540 nm (excitation), 590 nm (emission) and the data plotted to a sigmoidal curve with variable slope using Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA).…”

“…Changes in Ψm were determined by flow cytometry using the indicator dye tetramethylrhodamine ethyl ester (TMRE) as the probe, exactly as previously described [47,63] Briefly, bloodstream forms of 1×10 7 T. congolense IL3000 and several IL3000-adapted DA-Res clonal lines in mid-log growth phase were centrifuged at 1600 × g for 10 min at 4 °C and washed in 1 mL pf PBS (pH 7.4) and finally resuspended in 1 mL PBS containing 200 nM TMRE. These cells were incubated for 30 min at room temperature and then placed on ice for a further 30 min prior to analysis on a Becton Dickinson FACSCalibur™ system (BD Biosciences) using a FL2-height detector, and CellQuest and FlowJo 10 software.…”