Antitrypanosomal Compounds Research Articles

In vitro antitrypanosomal activity of plant terpenes against Trypanosoma brucei

During the course of screening to discover antitrypanosomal compounds, 24 known plant terpenes (6 sesquiterpenes, 14 sesquiterpene lactones and 4 diterpenes) were evaluated for in vitro antitrypanosomal activity against Trypanosoma brucei brucei. Among them, 22 terpenes exhibited antitrypanosomal activity. In particular, α-eudesmol, hinesol, nardosinone and 4-peroxy-1,2,4,5-tetrahydro-α-santonin all exhibited selective and potent antitrypanosomal activities in vitro. Detailed here in an in vitro antitrypanosomal properties and cytotoxicities of the 24 terpenes compared with two therapeutic antitrypanosomal drugs (eflornithine and suramin). This finding represents the first report of promising trypanocidal activity of these terpenes. Present results also provide some valuable insight with regard to structure–activity relationships and the possible mode of action of the compounds.

Synthesis of antitrypanosomal 1,2-dioxane derivatives based on a natural product scaffold.

A short practical synthesis of a new natural product based scaffold (6), based on antitrypanosomal and antimalarial compounds isolated from different Plakortis species is described. The scaffold contains a peroxide unit that is surprisingly stable to chemical manipulation elsewhere in the molecule, enabling it to be elaborated into a small library of derivatives. It is stable to ozonolysis, reductive work-up with dimethylsulfide and the Wittig reaction with stabilized phosphorus ylides. The scaffold along with its Wittig analogues has displayed low to sub-micro molar (0.2-3.3 μM) antitrypanosomal activity.

Ligand-based discovery of novel trypanosomicidal drug-like compounds: In silico identification and experimental support

Two-dimensional bond-based linear indices and linear discriminant analysis are used in this report to perform a quantitative structure–activity relationship study to identify new trypanosomicidal compounds. A database with 143 anti-trypanosomal and 297 compounds having other clinical uses, are utilized to develop the theoretical models. The best discriminant models computed using bond-based linear indices provides accuracies greater than 90 for both training and test sets. Our models identify as anti-trypanosomals five out of nine compounds of a set of already-synthesized substances. The in vitro anti-trypanosomal activity of this set against epimastigote forms of Trypanosoma cruzi is assayed. Both models show a perfect agreement between theoretical predictions and experimental results. The compounds identified as active ones show more than 98% of anti-epimastigote elimination (AE) at a concentration of 100 μg/mL. Besides, three compounds show more than 70% of AE at a concentration of 10 μg/mL. Finally, compounds with the best “activity against epimastigote forms/unspecific cytotoxicity” ratio are evaluated using an amastigote susceptibility assay. It should be noticed that, compound Va7-71 exhibit a 100% of intracellular amastigote elimination and shows similar activity when compared to a standard trypanosomicidal as nifurtimox. Finally, we can emphasize that, the present algorithm constitutes a step forward in the search for efficient ways of discovering new anti-trypanosomal compounds.

The Crystal Structure of the Leishmania major Deoxyuridine Triphosphate Nucleotidohydrolase in Complex with Nucleotide Analogues, dUMP, and Deoxyuridine

Members of the Leishmania genus are the causative agents of the life-threatening disease leishmaniasis. New drugs are being sought due to increasing resistance and adverse side effects with current treatments. The knowledge that dUTPase is an essential enzyme and that the all α-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric β-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding. These findings will aid in the development of dUTPase inhibitors as potential new lead anti-trypanosomal compounds.

Simultaneous Determination of A Novel Antitrypanosomal Compound (OSU-36) and its Ester Derivative (OSU-40) in Plasma by HPLC: Application to First Pharmacokinetic Study in Rats

To develop an HPLC-UV method for determination of a novel antitrypanosomal compound (OSU-36) and its ester prodrug (OSU-40) in rat plasma and to apply the method for pharmacokinetic evaluation of both compounds in rats. Since an attempt to assay for OSU-36 and OSU-40 in non-stabilized plasma resulted in highly non-linear calibration curves and poor sensitivity due to instability of the compounds, the plasma was stabilized using paraoxon and ascorbic acid. The sample treatment included protein precipitation by acetonitrile; evaporation; reconstitution with acetonitrile and filtration. The chromatography conditions included Xterra RP18 3.5 µm 4.6X100 mm column and gradient mobile phase system of acetonitrile-water. The limits of quantification (LOQ) were 50 ng/mL and 40 ng/mL for OSU-36 and OSU-40, respectively. The intra- and interday precision and accuracies were below 13% for low, medium and high quality control samples for both compounds. While OSU-40 has been stable in all tested handling conditions, OSU-36 was unstable in plasma after 20 days storage in -80 °C or 4h 28 °C storage. The developed method has been applied for a pharmacokinetic study in rats which revealed that an ester prodrug OSU-40 is rapidly converted to OSU-36 within the plasma compartment by plasma esterases. OSU-36, in turn, relatively quickly undergoes oxidative metabolism, including within the plasma compartment. A supplementation of rat plasma with an esterase inhibitor to prevent degradation of ester prodrug (OSU-40), and with antioxidant to prevent oxidation of OSU-36, is necessary for reliable determination of both compounds. Due to limited stability of OSU-36 in stabilized rat plasma, long-term storage of samples or prolonged handling in room temperature conditions is not recommended.

Erratum: Application of monoclonal antibodies to measure metabolism of an anti-trypanosomal compound in vitro and in vivo

Erratum: Application of monoclonal antibodies to measure metabolism of an anti-trypanosomal compound in vitro and in vivo

Diverse Effects on Mitochondrial and Nuclear Functions Elicited by Drugs and Genetic Knockdowns in Bloodstream Stage Trypanosoma brucei

BackgroundThe options for treating the fatal disease human African trypanosomiasis are limited to a few drugs that are toxic or facing increasing resistance. New drugs that kill the causative agents, subspecies of Trypanosoma brucei, are therefore urgently needed. Little is known about the cellular mechanisms that lead to death of the pathogenic bloodstream stage.Methodology/Principal FindingsWe therefore conducted the first side by side comparison of the cellular effects of multiple death inducers that target different systems in bloodstream form parasites, including six drugs (pentamidine, prostaglandin D2, quercetin, etoposide, camptothecin, and a tetrahydroquinoline) and six RNAi knockdowns that target distinct cellular functions. All compounds tested were static at low concentrations and killed at high concentrations. Dead parasites were rapidly quantified by forward and side scatter during flow cytometry, as confirmed by ethidium homodimer and esterase staining, making these assays convenient for quantitating parasite death. The various treatments yielded different combinations of defects in mitochondrial potential, reactive oxygen species, cell cycle, and genome segregation. No evidence was seen for phosphatidylserine exposure, a marker of apoptosis. Reduction in ATP levels lagged behind decreases in live cell number. Even when the impact on growth was similar at 24 hours, drug-treated cells showed dramatic differences in their ability to further proliferate, demonstrating differences in the reversibility of effects induced by the diverse compounds.Conclusions/SignificanceParasites showed different phenotypes depending on the treatment, but none of them were clear predictors of whether apparently live cells could go on to proliferate after drugs were removed. We therefore suggest that clonal proliferation assays may be a useful step in selecting anti-trypanosomal compounds for further development. Elucidating the genetic or biochemical events initiated by the compounds with the most profound effects on subsequent proliferation may identify new means to activate death pathways.

Podophyllotoxin analogues active versus Trypanosoma brucei

In an effort to discover novel anti-trypanosomal compounds, a series of podophyllotoxin analogues coupled to non-steroidal anti-inflammatory drugs (NSAIDs) has been synthesized and evaluated for activity versus Trypanosoma brucei and a panel of human cell lines, revealing compounds with low nano-molar potencies. It was discovered that coupling of NSAIDs to podophyllotoxin increased the potencies of both compounds over 1300-fold. The compounds were shown to be cytostatic in nature and seem to act via de-polymerization of tubulin in a manner consistent with the known activities of podophyllotoxin. The potencies against T. brucei correlated directly with Log P values of the compounds, suggesting that the conjugates are acting as hydrophobic tags allowing podophyllotoxin to enter the cell.

Antitrypanosomal Activity of 1,2-Dihydroquinolin-6-ols and Their Ester Derivatives

The current chemotherapy for second stage human African trypanosomiasis is unsatisfactory. A synthetic optimization study based on the lead antitrypanosomal compound 1,2-dihydro-2,2,4-trimethylquinolin-6-yl 3,5-dimethoxybenzoate (TDR20364, 1a) was undertaken in an attempt to discover new trypanocides with potent in vivo activity. While 6-ether derivatives were less active than the lead compound, several N1-substituted derivatives displayed nanomolar IC(50) values against T. b. rhodesiense STIB900 in vitro, with selectivity indexes up to >18000. 1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate (10a) displayed an IC(50) value of 0.014 microM against these parasites and a selectivity index of 1700. Intraperitoneal administration of 10a at 50 (mg/kg)/day for 4 days caused a promising prolongation of lifespan in T. b. brucei STIB795-infected mice (>14 days vs 7.75 days for untreated controls). Reactive oxygen species were produced when T. b. brucei were exposed to 10a in vitro, implicating oxidative stress in the trypanocidal mode of action of these 1,2-dihydroquinoline derivatives.

Application of monoclonal antibodies to measure metabolism of an anti-trypanosomal compound in vitro and in vivo.

Human African trypanosomiasis (HAT), also called African sleeping sickness, is a neglected tropical parasitic disease indigenous to sub-Saharan Africa. Diamidine compounds, including pentamidine and CPD-0801, are potent anti-trypanosomal molecules. The latter is a potential drug in the development at the UNC based Consortium for Parasitic Drug Development. An orally bioavailable prodrug of CPD-0801, DB868, is metabolized primarily in the liver to the active form. A monoclonal antibody developed against a pentamidine derivative has shown significant reactivity with CPD-0801 (EC(50) 65.1 nM), but not with the prodrug (EC(50)>18,000 nM). An inhibitory enzyme-linked immunosorbent assay (IELISA) has been used to quantitatively monitor prodrug metabolism by detecting the production of the active compound over time in a sandwich culture rat hepatocyte system and in rats. These results were compared with the results of the standard LC/MS/MS assay. Spearman coefficients of 0.96 and 0.933 (in vitro and in vivo, respectively) indicate a high correlation between these two measurement methods. This novel IELISA provides a facile, inexpensive, and accurate method for drug detection that may aide in elucidating the mechanisms of action and toxicity of existing and future diamidine compounds.

Computational discovery of novel trypanosomicidal drug-like chemicals by using bond-based non-stochastic and stochastic quadratic maps and linear discriminant analysis

Herein we present results of a quantitative structure–activity relationship (QSAR) studies to classify and design, in a rational way, new antitrypanosomal compounds by using non-stochastic and stochastic bond-based quadratic indices. A data set of 440 organic chemicals, 143 with antitrypanosomal activity and 297 having other clinical uses, is used to develop QSAR models based on linear discriminant analysis (LDA). Non-stochastic model correctly classifies more than 93% and 95% of chemicals in both training and external prediction groups, respectively. On the other hand, the stochastic model shows an accuracy of about the 87% for both series. As an experiment of virtual lead generation, the present approach is finally satisfactorily applied to the virtual evaluation of 9 already synthesized in house compounds. The in vitro antitrypanosomal activity of this series against epimastigote forms of Trypanosoma cruzi is assayed. The model is able to predict correctly the behaviour for the majority of these compounds. Four compounds (FER16, FER32, FER33 and FER 132) showed more than 70% of epimastigote inhibition at a concentration of 100 μg/mL (86.74%, 78.12%, 88.85% and 72.10%, respectively) and two of these chemicals, FER16 (78.22% of AE) and FER33 (81.31% of AE), also showed good activity at a concentration of 10 μg/mL. At the same concentration, compound FER16 showed lower value of cytotoxicity (15.44%), and compound FER33 showed very low value of 1.37%. Taking into account all these results, we can say that these three compounds can be optimized in forthcoming works, but we consider that compound FER33 is the best candidate. Even though none of them resulted more active than Nifurtimox, the current results constitute a step forward in the search for efficient ways to discover new lead antitrypanosomals.

Designing novel antitrypanosomal agents from a mixed graph‐theoretical substructural approach

Chagas disease is nowadays the most serious parasitic health problem. This disease is caused by Trypanosoma cruzi. The great number of deaths and the insufficient effectiveness of drugs against this parasite have alarmed the scientific community worldwide. In an attempt to overcome this problem, a model for the design and prediction of new antitrypanosomal agents was obtained. This used a mixed approach, containing simple descriptors based on fragments and topological substructural molecular design descriptors. A data set was made up of 188 compounds, 99 of them characterized an antitrypanosomal activity and 88 compounds that belong to other pharmaceutical categories. The model showed sensitivity, specificity and accuracy values above 85%. Quantitative fragmental contributions were also calculated. Then, and to confirm the quality of the model, 15 structures of molecules tested as antitrypanosomal compounds (that we did not include in this study) were predicted, taking into account the information on the abovementioned calculated fragmental contributions. The model showed an accuracy of 100% which means that the "in silico" methodology developed by our team is promising for the rational design of new antitrypanosomal drugs.

In vitro and in vivo antitrypanosomal activities of three peptide antibiotics: leucinostatin A and B, alamethicin I and tsushimycin

In the course of our screening for antitrypanosomal compounds from soil microorganisms, as well as from the antibiotics library of the Kitasato Institute for Life Sciences, we found three peptide antibiotics, leucinostatin (A and B), alamethicin I and tsushimycin, which exhibited potent or moderate antitrypanosomal activity. We report here the in vitro and in vivo antitrypanosomal properties and cytotoxicities of leucinostatin A and B, alamethicin I and tsushimycin compared with suramin. We also discuss their possible mode of action. This is the first report of in vitro and in vivo trypanocidal activity of leucinostatin A and B, alamethicin I and tsushimycin.

Novel linear triaryl guanidines, N-substituted guanidines and potential prodrugs as antiprotozoal agents

A series of triaryl guanidines and N-substituted guanidines designed to target the minor groove of DNA were synthesized and evaluated as antiprotozoal agents. Selected carbamate prodrugs of these guanidines were assayed for their oral efficacy. The linear triaryl bis-guanidines 6a, b were prepared from their corresponding diamines 4a, b through the intermediate BOC protected bis-guanidines 5a, b followed by acid catalyzed deprotection. The N-substituted guanidino analogues 9c– f were obtained in three steps starting by reacting the diamines 4a, b with ethyl isothiocyanatoformate to give the carbamoyl thioureas 7a, b. Subsequent condensation of 7a, b with various amines in the presence of EDCI provided the carbamoyl N-substituted guanidine intermediates 8a– f which can also be regarded as potential prodrugs for the guanidino derivatives. Compounds 9c– f were obtained via the base catalyzed decarbamoylation of 8a– f. The DNA binding affinities for the target dicationic bis-guanidines were assessed by Δ T m values. In vitro antiprotozoal screening of the new compounds showed that derivatives 6a, 9c and 9e possess high to moderate activity against Trypanosoma brucei rhodesiense ( T.b.r.) and Plasmodium falciparum ( P.f.). While the prodrugs did not yield cures upon oral administration in the antitrypanosomal STIB900 mouse model, compounds 8a and 8c prolonged the survival of the treated mice.

In vitro activity of para-guanidinoethylcalix[4]arene against susceptible and antibiotic-resistant Gram-negative and Gram-positive bacteria

Emergence of multidrug-resistant bacteria has encouraged vigorous efforts to develop antimicrobial agents with new mechanisms of action. In this study, the in vitro antibacterial activity of para-guanidinoethylcalix[4]arene was evaluated and compared with that of its constitutive monomer, para-guanidinoethylphenol. Hexamidine, a widely used antiseptic, and synthalin A, an old antidiabetic and anti-trypanosomal compound, were chosen as references. MIC and MBC were determined for five reference strains (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and ATCC 29213, Enterococcus faecalis ATCC 29212 and Pseudomonas aeruginosa ATCC 27853), as well as five antibiotic-resistant clinical isolates. Toxicity on MRC-5 and HaCaT eukaryotic cell lines was also evaluated by MTT and Neutral Red assays. No antibacterial activity was observed for para-guanidinoethylphenol (MIC >or= 512 mg/L) and synthalin A (MIC >or= 64 mg/L). Conversely, para-guanidinoethylcalix[4]arene and hexamidine: (i) showed a broad antibacterial spectrum, both on Gram-positive and on Gram-negative bacteria (MIC = 4 mg/L against E. coli and 8 mg/L against S. aureus for para-guanidinoethylcalix[4]arene), to a lesser degree against E. faecalis and P. aeruginosa (MIC = 32 mg/L); (ii) were bacteriostatic (MBC >or= 256 mg/L); and (iii) MICs and MBCs obtained for clinical isolates were similar to those obtained with reference strains. Both compounds, the monomer and the calixarene, showed no apparent cytotoxicity, whereas hexamidine and synthalin A had significant toxic effects that increased with time and concentration and in a range of 100-1000 times that for calixarene. In conclusion, results confirm para-guanidinoethylcalix[4]arene as a broad-spectrum new agent or an auxiliary in antimicrobial chemotherapy.

Natural Products to Anti-trypanosomal Drugs: An Overview of New Drug Prototypes for American Trypanosomiasis

Protozoan tropical diseases cause great suffering throughout developing countries, with high rates of morbidity and mortality. American Trypanosomiasis affects 16-18 million people in Latin America, representing a dramatic disease among symptomatic patients. Old, toxic and ineffective chemotherapeutic agents continue to be used for the treatment of Chagas' disease. Since the early days of medicine, chemical substances derived from plants and animals have been used to treat human diseases. In the marine ecosystem ecological pressures, such as competition for space and predation, may have favored several invertebrate organisms to select unique metabolites with an assortment of astonishing biological activities. In terrestrial ecosystems, amphibians present a unique efficient skin secretion system with a variety of glands which produce a myriad of potent bioactive compounds such as peptides, alkaloids, biogenic amines and lipids. Plants contribute with several antitrypanosomal compounds derived mainly from their secondary metabolism. Proteins and peptides from snake venoms have also been considered as novel drug candidates, showing effective activities. In this review, we broadly discuss the epidemiology, pathology, and current treatment of Chagas' disease as well as the contributions of pharmacologically tested marine invertebrate, amphibian, snake and plant compounds which have shown promising antitrypanosomal activities. We also explore these possibilities for developing new chemotherapeutics against Chagas' disease.

Diphenyl Furans and Aza Analogs: Effects of Structural Modification on In Vitro Activity, DNA Binding, and Accumulation and Distribution in Trypanosomes

Human African trypanosomiasis is a devastating disease with only a few treatment options, including pentamidine. Diamidine compounds such as pentamidine, DB75, and DB820 are potent antitrypanosomal compounds. Previous investigations have shown that diamidines accumulate to high concentrations in trypanosomes. However, the mechanism of action of this class of compounds remains unknown. A long-hypothesized mechanism of action has been binding to DNA and interference with DNA-associated enzymes. The fluorescent diamidines, DB75 and DB820, have been shown to localize not only in the DNA-containing nucleus and kinetoplast of trypanosomes but also to the acidocalcisomes. Here we investigate two series of analogs of DB75 and DB820 with various levels of in vitro antitrypanosomal activity to determine whether any correlation exists between trypanosome accumulation, distribution, and in vitro activity. Despite wide ranges of in vitro antitrypanosomal activity, all of the compounds investigated accumulated to millimolar concentrations in trypanosomes over a period of 8 h. Interestingly, some of the less potent compounds accumulated to concentrations much higher than those of more potent compounds. All of the compounds were localized to the DNA-containing nucleus and/or kinetoplast, and many were also found in the acidocalcisomes. Accumulation in the nucleus and kinetoplast should be important to the mechanism of action of these compounds. The acidocalcisomes may also play a role in the mechanism of action of these compounds. This investigation suggests that the extent of accumulation alone is not responsible for killing trypanosomes and that organelle-specific accumulation may not predict in vitro activity.

Novel Potential Drug Against T. cruzi and Its Interaction with Surfactant Micelles

The interaction of 5-nitro-2-furfurilylidene benzhydrazide (5NFB), potential anti-trypanosomal compound, with micellar solutions was studied. The results indicated that 50 μg of 5NFB completely kills 20 million T. cruzi epimastigote cells within 3 days, whereas the same amount of benznidazole kills 30% of the cells after 4 days. 5NFB solubility in surfactants solutions (SDS, DTAB, C12EO8) increased linearly with surfactant concentration. According to small angle X-ray scattering (SAXS), 5NFB does not affect micellar structural features. A comparison between C12EO8 effects on T. cruzi epimastigote cells and on erythrocytes showed that surfactant lytic effect is stronger in parasite cells, enlightening the potential of 5NFB micellar formulations.

DNA Binding Affinity of Bisguanidine and Bis(2-aminoimidazoline) Derivatives with in Vivo Antitrypanosomal Activity

A new antitrypanosomal hit compound that cures an acute (STIB 900) mouse model of Trypanosoma brucei rhodesiense trypanosomiasis is described. This bis(2-aminoimidazolinium) dicationic compound proved to be an excellent DNA minor groove binder, suggesting a possible mechanism for its trypanocidal activity. From these studies, the 4,4'-diaminodiphenylamine skeleton emerged as a good scaffold for antitrypanosomal drugs.

Cytoskeleton reassembly in cardiomyocytes infected by Trypanosoma cruzi is triggered by treatment with ergosterol biosynthesis inhibitors

We have previously shown that Trypanosoma cruzi-infected cardiomyocytes present alterations in cytoskeletal organisation in vitro. The remarkable change in the host cell cytoskeleton opened up the question of whether treatment of infected cells with antitrypanosomal compounds, such as ergosterol biosynthesis inhibitors (EBIs), allows the reconstruction of myofibrils and the microtubule network, restoring the cell biological function. Therefore, 48-h-old T. cruzi-infected cardiomyocyte cultures were treated with 10 nM ketoconazole or posaconazole and cytoskeletal remodelling of the host cells was analysed by indirect immunofluorescence assay. Both compounds displayed a potent antiparasitic effect and dramatically reduced the infection ratio. After 120 h of treatment, actin polygonal configuration was frequently visualised in the host cell cytoplasm, suggesting the initial stage of actin framework restoration. Rearrangement of myofibrils and the microtubule network was achieved 168 h after the start of drug treatment. Our data demonstrate that the trypanocidal effect of EBIs lead to reconstruction of the cytoskeleton of T. cruzi-infected cardiomyocytes in vitro.