Inhibition of trypanosome alternative oxidase without its N-terminal mitochondrial targeting signal (ΔMTS-TAO) by cationic and non-cationic 4-hydroxybenzoate and 4-alkoxybenzaldehyde derivatives active against T.

Introduction A previous study showed the unique character of Nigerian red propolis from Rivers State, Nigeria (RSN), with regards to chemical composition and activity against Trypanosoma brucei in comparison with other African propolis. Objective To carry out fractionation and biological testing of Nigerian propolis in order to isolate compounds with anti‐trypanosomal activity. To compare the composition of the RSN propolis with the composition of Brazilian red propolis. Methodology Profiling was carried out using HPLC‐UV‐ELSD and HPLC‐Orbitrap‐FTMS on extracts of two samples collected from RSN with data extraction using MZmine software. Isolation was carried out by normal phase and reversed phase MPLC. Elucidation of the compounds with a purity > 95% was performed by 1D/2D NMR HRMS and HRLC‐MSn. Results Ten phenolic compounds were isolated or in the case of liquiritigenin partially purified. Data for nine of these correlated with literature reports of known compounds i.e. one isoflavanone, calycosin (1); two flavanones, liquiritigenin (2) and pinocembrin (5); an isoflavan, vestitol (3); a pterocarpan, medicarpin (4); two prenylflavanones, 8‐prenylnaringenin (7) and 6‐prenylnaringenin (8); and two geranyl flavonoids, propolin D (9) and macarangin (10). The tenth was elucidated as a previously undescribed dihydrobenzofuran (6). The isolated compounds were tested against Trypanosoma brucei and displayed moderate to high activity. Some of the compounds tested had similar activity against wild type T. brucei and two strains displaying pentamidine resistance. Conclusion Nigerian propolis from RSN has some similarities with Brazilian red propolis. The propolis displayed anti‐trypanosomal activity at a potentially useful level. Copyright © 2015 John Wiley & Sons, Ltd.

Profiling of extracts from twelve propolis samples collected from eight regions in Nigeria was carried out using high performance liquid chromatography (LC) coupled with evaporative light scattering (ELSD), ultraviolet detection (UV) and mass spectrometry (MS), gas chromatography mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR). Principal component analysis (PCA) of the processed LC-MS data demonstrated the varying chemical composition of the samples. Most of the samples were active against Trypanosoma b. brucei with the highest activity being in the samples from Southern Nigeria. The more active samples were fractionated in order to isolate the component(s) responsible for their activity using medium pressure liquid chromatography (MPLC). Three xanthones, 1,3,7-trihydroxy-2,8-di-(3-methylbut-2-enyl)xanthone, 1,3,7-trihydroxy-4,8-di-(3-methylbut-2-enyl)xanthone a previously undescribed xanthone and three triterpenes: ambonic acid, mangiferonic acid and a mixture of α-amyrin with mangiferonic acid (1:3) were isolated and characterised by NMR and LC-MS. These compounds all displayed strong inhibitory activity against T.b. brucei but none of them had higher activity than the crude extracts. Partial least squares (PLS) modelling of the anti-trypanosomal activity of the sample extracts using the LC-MS data indicated that high activity in the extracts, as judged from LCMS2 data, could be correlated to denticulatain isomers in the extracts.

We investigated a chemical strategy to boost the trypanocidal activity of 2,4dihydroxybenzoic acid (2,4-DHBA)-and salicylhydroxamic acid (SHAM)-based trypanocides with triphenylphosphonium and quinolinium lipophilic cations (LC). Three series of LC conjugates were synthesized that were active in the submicromolar (5a-d and 10d-f) to low nanomolar (6a-f) range against wild-type and multi-drug resistant strains of African trypanosomes (Trypanosoma brucei brucei and T. congolense). This represented an improvement in trypanocidal potency of at least 200fold, and up to >10,000-fold, compared with the non-LC coupled parent compounds 2,4-DHBA and SHAM. Selectivity over human cells was >500 and reached >23,000 for 6e. Mechanistic studies showed that 6e did not inhibit the cell cycle but affected parasite respiration in a dose-dependent manner. Inhibition of the trypanosome alternative oxidase (TAO) and the mitochondrial membrane potential was also studied for selected compounds. We conclude that effective mitochondrial targeting greatly potentiated the activity of these compound series.

Extracts from twelve samples of propolis collected from different regions of Libya were tested for their activity against Trypanosoma brucei, Leishmania donovani, Plasmodium falciparum, Crithidia fasciculata and Mycobacterium marinum and the cytotoxicity of the extracts was tested against mammalian cells. All the extracts were active to some degree against all of the protozoa and the mycobacterium, exhibiting a range of EC50 values between 1.65 and 53.6 μg/ml. The toxicity against mammalian cell lines was only moderate; the most active extract against the protozoan species, P2, displayed an IC50 value of 53.2 μg/ml. The extracts were profiled by using liquid chromatography coupled to high resolution mass spectrometry. The data sets were extracted using m/z Mine and the accurate masses of the features extracted were searched against the Dictionary of Natural Products (DNP). A principal component analysis (PCA) model was constructed which, in combination with hierarchical cluster analysis (HCA), divided the samples into five groups. The outlying groups had different sets of dominant compounds in the extracts, which could be characterised by their elemental composition. Orthogonal partial least squares (OPLS) analysis was used to link the activity of each extract against the different micro-organisms to particular components in the extracts.

The SAR of 4-hydroxybenzaldehyde inhibitors of the trypanosome alternative oxidase (TAO), a critical enzyme for the respiration of bloodstream forms of trypanosomes, was investigated. Replacing the aldehyde group with a methyl ester resulted in a 10-fold increase in TAO inhibition and activity against . Remarkably, two analogues containing the 2-hydroxy-6-methyl scaffold ( and ) displayed single digit nanomolar TAO inhibition, which constitute the most potent 4-alkoxybenzoic acid derivatives described to date. was 50-times more potent against TAO and 10-times more active against compared to its benzaldehyde analogue. The farnesyl derivative was as potent a TAO inhibitor as ascofuranone with IC = 3.1 nM. Similar to ascofuranone derivatives, the 2-hydroxy and 6-methyl groups seemed essential for low nanomolar TAO inhibition of acid derivatives, suggesting analogous binding interactions with the TAO active site.

The alternative oxidase (AOX) is a ubiquitous terminal oxidase of plants and many fungi, catalyzing the four‐electron reduction of oxygen to water alongside the cytochrome‐based electron transfer chain. Unlike the classical electron transfer chain, however, the activity of AOX does not generate adenosine triphosphate but has functions such as thermogenesis and stress response. As it lacks a mammalian counterpart, it has been investigated intensely in pathogenic fungi. However, it is in African trypanosomes, which lack cytochrome‐based respiration in their infective stages, that trypanosome alternative oxidase (TAO) plays the central and essential role in their energy metabolism. TAO was validated as a drug target decades ago and among the first inhibitors to be identified was salicylhydroxamic acid (SHAM), which produced the expected trypanocidal effects, especially when potentiated by coadministration with glycerol to inhibit anaerobic energy metabolism as well. However, the efficacy of this combination was too low to be of practical clinical use. The antibiotic ascofuranone (AF) proved a much stronger TAO inhibitor and was able to cure Trypanosoma vivax infections in mice without glycerol and at much lower doses, providing an important proof of concept milestone. Systematic efforts to improve the SHAM and AF scaffolds, aided with the elucidation of the TAO crystal structure, provided detailed structure‐activity relationship information and reinvigorated the drug discovery effort. Recently, the coupling of mitochondrion‐targeting lipophilic cations to TAO inhibitors has dramatically improved drug targeting and trypanocidal activity while retaining target protein potency. These developments appear to have finally signposted the way to preclinical development of TAO inhibitors.

Trypanosoma brucei, the causative agent of sleeping sickness (Human African Trypanosomiasis, HAT), contains a kinetoplast with the mitochondrial DNA (kDNA), comprising of >70% AT base pairs. This has prompted studies of drugs interacting with AT-rich DNA, such as the N-phenylbenzamide bis(2-aminoimidazoline) derivatives 1 [4-((4,5-dihydro-1H-imidazol-2-yl)amino)-N-(4-((4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)benzamide dihydrochloride] and 2 [N-(3-chloro-4-((4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)-4-((4,5-dihydro-1H-imidazol-2-yl)amino)benzamide] as potential drugs for HAT. Both compounds show in vitro effects against T. brucei and in vivo curative activity in a mouse model of HAT. The main objective was to identify their cellular target inside the parasite. We were able to demonstrate that the compounds have a clear effect on the S-phase of T. brucei cell cycle by inflicting specific damage on the kinetoplast. Surface plasmon resonance (SPR)–biosensor experiments show that the drug can displace HMG box-containing proteins essential for kDNA function from their kDNA binding sites. The crystal structure of the complex of the oligonucleotide d[AAATTT]2 with compound 1 solved at 1.25 Å (PDB-ID: 5LIT) shows that the drug covers the minor groove of DNA, displaces bound water and interacts with neighbouring DNA molecules as a cross-linking agent. We conclude that 1 and 2 are powerful trypanocides that act directly on the kinetoplast, a structure unique to the order Kinetoplastida.