Trypanosome Alternative Oxidase Research Articles

Alternative Oxidase: From Molecule and Function to Future Inhibitors.

In the respiratory chain of the majority of aerobic organisms, the enzyme alternative oxidase (AOX) functions as the terminal oxidase and has important roles in maintaining metabolic and signaling homeostasis in mitochondria. AOX endows the respiratory system with flexibility in the coupling among the carbon metabolism pathway, electron transport chain (ETC) activity, and ATP turnover. AOX allows electrons to bypass the main cytochrome pathway to restrict the generation of reactive oxygen species (ROS). The inhibition of AOX leads to oxidative damage and contributes to the loss of adaptability and viability in some pathogenic organisms. Although AOXs have recently been identified in several organisms, crystal structures and major functions still need to be explored. Recent work on the trypanosome alternative oxidase has provided a crystal structure of an AOX protein, which contributes to the structure-activity relationship of the inhibitors of AOX. Here, we review the current knowledge on the development, structure, and properties of AOXs, as well as their roles and mechanisms in plants, animals, algae, protists, fungi, and bacteria, with a special emphasis on the development of AOX inhibitors, which will improve the understanding of respiratory regulation in many organisms and provide references for subsequent studies of AOX-targeted inhibitors.

Imidazoline- and Benzamidine-Based Trypanosome Alternative Oxidase Inhibitors: Synthesis and Structure-Activity Relationship Studies.

The trypanosome alternative oxidase (TAO), a mitochondrial enzyme involved in the respiration of the bloodstream form trypomastigotes of Trypanosoma brucei, is a validated drug target against African trypanosomes. Earlier series of TAO inhibitors having a 2,4-dihydroxy-6-methylbenzoic acid scaffold (“head”) and a triphenylphosphonium or quinolin-1-ium cation as a mitochondrion-targeting group (“tail”) were shown to be nanomolar inhibitors in enzymatic and cellular assays. We investigated here the effect of different mitochondrion-targeting cations and other scaffold modifications on the in vitro activity of this class of inhibitors. Low micromolar range activities were obtained, and the structure–activity relationship studies showed that modulation of the tail region with polar substituents is generally detrimental to the enzymatic and cellular activity of TAO inhibitors.

Antitrypanosomal properties of Anogeissus leiocarpa extracts and their inhibitory effect on trypanosome alternative oxidase

BackgroundAfrican trypanosomiasis is a protozoan disease with huge socio-economic burden to sub-Saharan African exceeding US$4.6 annual loss. To mitigate the incidence of trypanosomal drug resistance, efforts are geared towards discovery of molecules, especially from natural products, with potential to inhibit important molecular target (trypanosome alternative oxidase, TAO) in trypanosomes that are critical to their survival. MethodCrude methanol extract of Anogeissus leiocarpa was subjected to in vitro bioassay-guided antitrypanosomal assay to identify the most active extract with trypanocidal activity. The most active extract was run on a column chromatography yielding five fractions, F1-F5. The fractions were assayed for inhibitory effect on TAO. The most promising TAO inhibitor was subjected to antitrypanosomal evaluation by trypanosome count, drug incubation infectivity test (DIIT) and in vivo studies. Gas chromatography-mass spectrometry (GC-MS) was used to identify and quantify phytochemical constituents of the potential TAO-inhibiting fraction. ResultsEthyl acetate extract (EtOAc) significantly (p<0.05) produced trypanocidal effect and was the most active extract. Of the five fractions, only F4 significantly (p<0.05) inhibited TAO compared to the control. F4 completely immobilised the trypanosomes up to 0.5 µg/µl, yielding an EC50 of 0.024 µg/µl compared to the 0.502 µg/µl of diminazene aceturate positive control group. The DIIT showed that F4 was significantly (p<0.05) potent up to 0.1 µg/µl. F4 significantly (p<0.05) suppressed parasite multiplication in systemic circulation of the treated rats and significantly (p<0.05) maintained high PCV when compared to the 5% DMSO group. Furthermore, F4 significantly (p<0.05) lowered serum concentrations of malondialdehyde. Phytoconstituents identified by the GC-MS include tetradecene; cetene; 3-(benzylthio) acrylic acid, methyl ester; 1-octadecene; 9-heptadecanone; hexadecanoic acid, methyl ester; dibutyl phthalate; eicosene; octadecenoic acid, methyl ester; oleic acid; 2-methyl-Z,Z-3,13-octadecadienol; 1-docosene; 3-phenylthiane, s-oxide; phenol, 3-methyl; phthalic acid, di(2-propylpentyl) ester and 1,4-benzenedicarboxylic acid, bis (2-ethylhexyl) ester. ConclusionF4 from EtOAc contains six carbohydrates (9.58%), two free fatty acids (6.48%), five fatty acid esters (27.73%), two aromatic compounds (50.63%) and one organosulphide (5.61%). It inhibited TAO and demonstrated antitrypanosomal effects.

Heterologous production of ascofuranone and ilicicolin A in Aspergillus sojae.

Ascofuranone and its precursor, ilicicolin A, are secondary metabolites with various pharmacological activities that are produced by Acremonium egyptiacum. In particular, ascofuranone strongly inhibits trypanosome alternative oxidase and represents a potential drug candidate against African trypanosomiasis. However, difficulties associated with industrial production of ascofuranone by A. egyptiacum, specifically the co-production of ascochlorin, which inhibits mammalian respiratory chain complex III at low concentrations, has precluded its widespread application. Therefore, in this study, ascofuranone biosynthetic genes (ascA-E and H-J) were heterologously expressed in Aspergillus sojae, which produced very low-levels of endogenous secondary metabolites under conventional culture conditions. As a result, although we obtained transformants producing both ilicicolin A and ascofuranone, they were produced only when an adequate concentration of chloride ions was added to the medium. In addition, we succeeded in increasing the production of ilicicolin A, by enhancing the expression of the rate-determining enzyme AscD, using a multi-copy integration system. The heterologous expression approach described here afforded the production of both ascofuranone and ilicicolin A, allowing for their development as therapeutics.

Identification of megacerotonic acid and a quinazoline derivative from Universal Natural Product Database as potential inhibitors of Trypanosoma brucei brucei alternative oxidase: molecular docking, molecular dynamic simulation and MM/PBSA analysis

African trypanosomiasis is caused by Trypanosoma brucei subspecies and available drugs against it, are unsatisfactory due to poor pharmacokinetic properties. Trypanosomal Alternative Oxidase (TAO) is an attractive target for anti-trypanosome rational drug discovery because it is essential for parasite-specific ATP generation and absent in the mammalian host. In this study, 360 filtered ligands from the Universal Natural Product Database were virtually screened and docked on T. brucei brucei TAO (PDB-ID 3VVA). From the virtual screening, 10 ligands with binding energy from −10.6 to −9.0 kcal/mol were selected as hits and further subjected pharmacokinetic and toxicity analyses where all of them passed Lipinski’s rule of five. Also, the compounds were non-mutagenic, non-tumorigenic and could cross the blood brain barrier. The two topmost hits (UNPD29179; megacerotonic acid and UNPD41551; a quinazoline derivative) interacted with `four glutamates (Glu123, Glu162, Glu213 and Glu266) close to di-iron (2 iron elements) at the catalytic site of the enzyme. Subsequently, 100 ns MD simulations of the two topmost hits were performed using GROMACS where high RMSD values of 0.75 nm (TAO-UNPD29179) and 0.52 nm (TAO- UNPD41551), low residues fluctuations and consistent values of radius of gyration were observed. Moreover, Solvent Accessible Surface Area showed a consistent value of 160 nm2 for both complexes while TAO-UNPD29179 had higher number of hydrogen bonds than the TAO-UNPD41551. Similarly, MM/PBSA calculations indicated that UNPD29179 had higher free binding energy with TAO than UNPD41551. The data suggest that megacerotonic acid and a quinazoline derivative could be potential inhibitors of TAO with improved pharmacokinetic properties. Communicated by Ramaswamy H. Sarma

Partially Purified Leaf Fractions of Azadirachta indica Inhibit Trypanosome Alternative Oxidase and Exert Antitrypanosomal Effects on Trypanosoma congolense.

IntroductionTrypanosomiasis is a neglected disease of humans and livestock caused by single-celled flagellated haemo-protozoan parasites belonging to the genus Trypanosoma.PurposeWidespread resistance to trypanocidal drugs creates urgent need for new, more effective drugs with potential to inhibit important trypanosome molecular targets.MethodsNine column chromatographic, partially purified leaf fractions of Azadirachta indica (AIF) were subjected to trypanosome alternative oxidase (TAO) inhibition assay using ubiquinol oxidase assay. The potent TAO inhibitors were evaluated for trypanocidal activities against T. congolense in rat model using in vitro, ex vivo, and in vivo assays. Complete cessation or reduction in parasite motility was scored from 0 (no parasite) to 6 (greater than or equal to 6 × 107 trypanosomes/milliliter of blood), and was used to evaluate the efficacy of in vitro treatments.ResultsOnly AIF1, AIF2, and AIF5 significantly inhibited TAO. AIF1 and AIF5 produced significant, dose-dependent suppression of parasite motility reaching score zero within 1 h with EC50 of 0.005 and 0.004 µg/µL, respectively, while trypanosome-laden blood was still at score six with an EC50 of 44,086 µg/µL. Mice inoculated with the concentrations at scores 0 and 1 (1–2 moribund parasites) at the end of the experiment did not develop parasitaemia. The two fractions significantly (p < 0.05) lowered parasite burden, with the AIF5 exhibiting highest in vivo trypanocidal effects. Packed cell volume was significantly higher in AIF1 (p < 0.05) and AIF5 (p < 0.001) groups compared to DMSO-treated group. Only AIF5 significantly (p < 0.05) lowered malondialdehyde.ConclusionAIF1 and AIF5 offer prospects for the discovery of TAO inhibitor(s).

Synthesis, biological, and photophysical studies of molecular rotor-based fluorescent inhibitors of the trypanosome alternative oxidase.

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.

Weak O2 binding and strong H2O2 binding at the non-heme diiron center of trypanosome alternative oxidase

Alternative oxidase (AOX) catalyzes the four-electron reduction of dioxygen to water as an additional terminal oxidase, and the catalytic reaction is critical for the parasite to survive in its bloodstream form. Recently, the X-ray crystal structure of trypanosome alternative oxidase (TAO) complexed with ferulenol was reported and the molecular structure of the non-heme diiron center was determined. The binding of O2 was a unique side-on type compared to other iron proteins. In order to characterize the O2 binding state of TAO, the O2 binding states were searched at a quantum mechanics/molecular mechanics (QM/MM) theoretical level in the present study. We found that the most stable O2 binding state is the end-on type, and the binding states of the side-on type are higher in energy. Based on the binding energies and electronic structure analyses, O2 binds very weakly to the TAO iron center (ΔE =6.7 kcal mol−1) in the electronic state of Fe(II)…OO, not in the suggested charge transferred state such as the superoxide state (Fe(III)OO· –) as seen in hemerythrin. Coordination of other ligands such as water, Cl−, CN−, CO, N3− and H2O2 was also examined, and H2O2 was found to bind most strongly to the Fe(II) site by ΔE = 14.0 kcal mol−1. This was confirmed experimentally through the measurement of ubiquinol oxidase activity of TAO and Cryptosporidium parvum AOX which was found to be inhibited by H2O2 in a dose-dependent and reversible manner.

Kinetic and structural characterisation of the ubiquinol-binding site and oxygen reduction by the trypanosomal alternative oxidase

The alternative oxidase (AOX) is a monotopic di‑iron carboxylate protein which acts as a terminal respiratory chain oxidase in a variety of plants, fungi and protists. Of particular importance is the finding that both emerging infectious diseases caused by human and plant fungal pathogens, the majority of which are multi-drug resistant, appear to be dependent upon AOX activity for survival. Since AOX is absent in mammalian cells, AOX is considered a viable therapeutic target for the design of specific fungicidal and anti-parasitic drugs.In this work, we have mutated conserved residues within the hydrophobic channel (R96, D100, R118, L122, L212, E215 and T219), which crystallography has indicated leads to the active site. Our data shows that all mutations result in a drastic reduction in Vmax and catalytic efficiency whilst some also affected the Km for quinol and oxygen. The extent to which mutation effects inhibitor sensitivity was also investigated, with mutation of R118 and T219 leading to a complete loss of inhibitor potency. However, only a slight reduction in IC50 values was observed when R96 was mutated, implying that this residue is less important in inhibitor binding. In silico modelling has been used to provide insight into the reason for such changes, which we suggest is due to disruptions in the proton transfer network, resulting in a reduction in overall reaction kinetics. We discuss our results in terms of the structural features of the ubiquinol binding site and consider the implications of such findings on the nature of the catalytic cycle. SignificanceThe alternative oxidase is a ubiquinol oxidoreductase enzyme that catalyses the oxidation of ubiquinol and the reduction of oxygen to water. It is widely distributed amongst the plant, fungal and parasitic kingdoms and plays a central role in metabolism through facilitating the turnover of the TCA cycle whilst reducing ROS production.

Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei.

African trypanosomiasis, sleeping sickness in humans or nagana in animals, is a potentially fatal neglected tropical disease and a threat to 65 million human lives and 100 million small and large livestock animals in sub-Saharan Africa. Available treatments for this devastating disease are few and have limited efficacy, prompting the search for new drug candidates. Simultaneous inhibition of the trypanosomal glycerol kinase (TGK) and trypanosomal alternative oxidase (TAO) is considered a validated strategy toward the development of new drugs. Our goal is to develop a TGK-specific inhibitor for coadministration with ascofuranone (AF), the most potent TAO inhibitor. Here, we report on the identification of novel compounds with inhibitory potency against TGK. Importantly, one of these compounds (compound 17) and its derivatives (17a and 17b) killed trypanosomes even in the absence of AF. Inhibition kinetics revealed that derivative 17b is a mixed-type and competitive inhibitor for TGK and TAO, respectively. Structural data revealed the molecular basis of this dual inhibitory action, which, in our opinion, will aid in the successful development of a promising drug to treat trypanosomiasis. Although the EC50 of compound 17b against trypanosome cells was 1.77 µM, it had no effect on cultured human cells, even at 50 µM.-Balogun, E. O., Inaoka, D. K., Shiba, T., Tsuge, C., May, B., Sato, T., Kido, Y., Nara, T., Aoki, T., Honma, T., Tanaka, A., Inoue, M., Matsuoka, S., Michels, P. A. M., Watanabe, Y.-I., Moore, A. L., Harada, S., Kita, K. Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei.

The Cross Talk between TbTim50 and PIP39, Two Aspartate-Based Protein Phosphatases, Maintains Cellular Homeostasis in Trypanosoma brucei.

Trypanosoma brucei, the infectious agent of a deadly disease known as African trypanosomiasis, undergoes various stresses during its digenetic life cycle. We previously showed that downregulation of T. brucei mitochondrial inner membrane protein translocase 50 (TbTim50), an aspartate-based protein phosphatase and a component of the translocase of the mitochondrial inner membrane (TIM), increased the tolerance of procyclic cells to oxidative stress. Using comparative proteomics analysis and further validating the proteomics results by immunoblotting, here we discovered that TbTim50 downregulation caused an approximately 5-fold increase in the levels of PIP39, which is also an aspartate-based protein phosphatase and is primarily localized in glycosomes. A moderate upregulation of a number of glycosomal enzymes was also noticed due to TbTim50 knockdown. We found that the rate of mitochondrial ATP production by oxidative phosphorylation decreased and that substrate-level phosphorylation increased due to depletion of TbTim50. These results were correlated with relative increases in the levels of trypanosome alternative oxidase and hexokinase and a reduced-growth phenotype in low-glucose medium. The levels and activity of the mitochondrial superoxide dismutase and glutaredoxin levels were increased due to TbTim50 knockdown. Furthermore, we show that TbTim50 downregulation increased the cellular AMP/ATP ratio, and as a consequence, phosphorylation of AMP-activated protein kinase (AMPK) was increased. Knocking down both TbTim50 and TbPIP39 reduced PIP39 levels as well as AMPK phosphorylation and reduced T. brucei tolerance to oxidative stress. These results suggest that TbTim50 and PIP39, two protein phosphatases in mitochondria and glycosomes, respectively, cross talk via the AMPK pathway to maintain cellular homeostasis in the procyclic form of T. bruceiIMPORTANCETrypanosoma brucei, the infectious agent of African trypanosomiasis, must adapt to strikingly different host environments during its digenetic life cycle. Developmental regulation of mitochondrial activities is an essential part of these processes. We have shown previously that mitochondrial inner membrane protein translocase 50 in T. brucei (TbTim50) possesses a dually specific phosphatase activity and plays a role in the cellular stress response pathway. Using proteomics analysis, here we have elucidated a novel connection between TbTim50 and a protein phosphatase of the same family, PIP39, which is also a differentiation-related protein localized in glycosomes. We found that these two protein phosphatases cross talk via the AMPK pathway and modulate cellular metabolic activities under stress. Together, our results indicate the importance of a TbTim50 and PIP39 cascade for communication between mitochondria and other cellular parts in regulation of cell homeostasis in T. brucei.

Alternative oxidase inhibitors: Mitochondrion-targeting as a strategy for new drugs against pathogenic parasites and fungi.

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.

SAR of 4-Alkoxybenzoic Acid Inhibitors of the Trypanosome Alternative Oxidase.

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 T. brucei. Remarkably, two analogues containing the 2-hydroxy-6-methyl scaffold (9e and 16e) displayed single digit nanomolar TAO inhibition, which constitute the most potent 4-alkoxybenzoic acid derivatives described to date. 9e was 50-times more potent against TAO and 10-times more active against T. brucei compared to its benzaldehyde analogue 1. The farnesyl derivative 16e was as potent a TAO inhibitor as ascofuranone with IC50 = 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.

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. brucei and T. congolense

African trypanosomiasis is a neglected parasitic disease that is still of great public health relevance, and a severe impediment to agriculture in endemic areas. The pathogens possess certain unique metabolic features that can be exploited for the development of new drugs. Notably, they rely on an essential, mitochondrially-localized enzyme, Trypanosome Alternative Oxidase (TAO) for their energy metabolism, which is absent in the mammalian hosts and therefore an attractive target for the design of safe drugs. In this study, we cloned, expressed and purified the physiologically relevant form of TAO, which lacks the N-terminal 25 amino acid mitochondrial targeting sequence (ΔMTS-TAO). A new class of 32 cationic and non-cationic 4-hydroxybenzoate and 4-alkoxybenzaldehyde inhibitors was designed and synthesized, enabling the first structure-activity relationship studies on ΔMTS-TAO. Remarkably, we obtained compounds with enzyme inhibition values (IC50) as low as 2 nM, which were efficacious against wild type and multidrug-resistant strains of T. brucei and T. congolense. The inhibitors 13, 15, 16, 19, and 30, designed with a mitochondrion-targeting lipophilic cation tail, displayed trypanocidal potencies comparable to the reference drugs pentamidine and diminazene, and showed no cross-resistance with the critical diamidine and melaminophenyl arsenical classes of trypanocides. The cationic inhibitors 15, 16, 19, 20, and 30 were also much more selective (900 - 344,000) over human cells than the non-targeted neutral derivatives (selectivity >8-fold). A preliminary in vivo study showed that modest doses of 15 and 16 reduced parasitaemia of mice infected with T. b. rhodesiense (STIB900). These compounds represent a promising new class of potent and selective hits against African trypanosomes.

Toward More Drug Like Inhibitors of Trypanosome Alternative Oxidase.

New tools are required to ensure the adequate control of the neglected tropical disease human African trypanosomiasis. Annual reports of infection have recently fallen to fewer than 5000 cases per year; however, current therapies are hard to administer and have safety concerns and, hence, are far from ideal. Trypanosome alternative oxidase is an exciting target for controlling the infection; it is unique to the parasite, and inhibition of this enzyme with the natural product ascofuranone has shown to clear in vivo infections. We report the synthesis and associated structure activity relationships of inhibitors based upon this natural product with correlation to T.b.brucei growth inhibition in an attempt to generate molecules that possess improved physicochemical properties and potential for use as new treatments for human African trypanosomiasis.

African trypanosomiasis: Synthesis & SAR enabling novel drug discovery of ubiquinol mimics for trypanosome alternative oxidase

African trypanosomiasis is a parasitic disease affecting 5000 humans and millions of livestock animals in sub-Saharan Africa every year. Current treatments are limited, difficult to administer and often toxic causing long term injury or death in many patients. Trypanosome alternative oxidase is a parasite specific enzyme whose inhibition by the natural product ascofuranone (AF) has been shown to be curative in murine models. Until now synthetic methods to AF analogues have been limited, this has restricted both understanding of the key structural features required for binding and also how this chemotype could be developed to an effective therapeutic agent. The development of 3 amenable novel synthetic routes to ascofuranone-like compounds is described. The SAR generated around the AF chemotype is reported with correlation to the inhibition of T. b. brucei growth and corresponding selectivity in cytotoxic assessment in mammalian HepG2 cell lines. These methods allow access to greater synthetic diversification and have enabled the synthesis of compounds that have and will continue to facilitate further optimisation of the AF chemotype into a drug-like lead.

Aquaglyceroporin-null trypanosomes display glycerol transport defects and respiratory-inhibitor sensitivity.

Aquaglyceroporins (AQPs) transport water and glycerol and play important roles in drug-uptake in pathogenic trypanosomatids. For example, AQP2 in the human-infectious African trypanosome, Trypanosoma brucei gambiense, is responsible for melarsoprol and pentamidine-uptake, and melarsoprol treatment-failure has been found to be due to AQP2-defects in these parasites. To further probe the roles of these transporters, we assembled a T. b. brucei strain lacking all three AQP-genes. Triple-null aqp1-2-3 T. b. brucei displayed only a very moderate growth defect in vitro, established infections in mice and recovered effectively from hypotonic-shock. The aqp1-2-3 trypanosomes did, however, display glycerol uptake and efflux defects. They failed to accumulate glycerol or to utilise glycerol as a carbon-source and displayed increased sensitivity to salicylhydroxamic acid (SHAM), octyl gallate or propyl gallate; these inhibitors of trypanosome alternative oxidase (TAO) can increase intracellular glycerol to toxic levels. Notably, disruption of AQP2 alone generated cells with glycerol transport defects. Consistent with these findings, AQP2-defective, melarsoprol-resistant clinical isolates were sensitive to the TAO inhibitors, SHAM, propyl gallate and ascofuranone, relative to melarsoprol-sensitive reference strains. We conclude that African trypanosome AQPs are dispensable for viability and osmoregulation but they make important contributions to drug-uptake, glycerol-transport and respiratory-inhibitor sensitivity. We also discuss how the AQP-dependent inverse sensitivity to melarsoprol and respiratory inhibitors described here might be exploited.

Conjugates of 2,4-Dihydroxybenzoate and Salicylhydroxamate and Lipocations Display Potent Antiparasite Effects by Efficiently Targeting the Trypanosoma brucei and Trypanosoma congolense Mitochondrion.

We investigated a chemical strategy to boost the trypanocidal activity of 2,4-dihydroxybenzoic 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 multidrug resistant strains of African trypanosomes (Trypanosoma brucei brucei and T. congolense). This represented an improvement in trypanocidal potency of at least 200-fold, and up to >10 000-fold, compared with that of 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 trypanosome alternative oxidase and the mitochondrial membrane potential was also studied for selected compounds. We conclude that effective mitochondrial targeting greatly potentiated the activity of these series of compounds.

The trypanosome alternative oxidase: a potential drug target?

New drugs against Trypanosoma brucei, the causative agent of Human African Trypanosomiasis, are urgently needed to replace the highly toxic and largely ineffective therapies currently used. The trypanosome alternative oxidase (TAO) is an essential and unique mitochondrial protein in these parasites and is absent from mammalian mitochondria, making it an attractive drug target. The structure and function of the protein are now well characterized, with several inhibitors reported in the literature, which show potential as clinical drug candidates. In this review, we provide an update on the functional activity and structural aspects of TAO. We then discuss TAO inhibitors reported to date, problems encountered with in vivo testing of these compounds, and discuss the future of TAO as a therapeutic target.

Investigation into the ubiquinol-binding site of the trypanosomal alternative oxidase

Investigation into the ubiquinol-binding site of the trypanosomal alternative oxidase