Antitrypanosomal Drugs Research Articles

In vitro trypanocidal activities and structure-activity relationships of ciprofloxacin analogs.

Tropical diseases, such as African trypanosomiasis, by their nature and prevalence lack the necessary urgency regarding drug development, despite the increasing need for novel, structurally diverse antitrypanosomal drugs, using different mechanisms of action that would improve drug efficacy and safety. Traditionally antibacterial agents, the fluoroquinolones, reportedly possess in vitro trypanocidal activities against Trypanosoma brucei organisms. During our research, the fluroquinolone, ciprofloxacin (1), and its analogs (2-24) were tested against bloodstream forms of T. brucei brucei, T. b. gambiense, T. b. rhodesiense, T. evansi, T. equiperdum, and T. congolense and Madin-Darby bovine kidney cells (cytotoxicity). Ciprofloxacin [CPX (1)] demonstrated selective trypanocidal activity against T. congolense (IC50 7.79µM; SI 39.6), whereas the CPX derivatives (2-10) showed weak selective activity (25 < IC50 < 65µM; 2 < SI < 4). Selectivity and activity of the CPX and 1,2,3-triazole (TZ) hybrids (11-24) were governed by their chemical functionality at C-3 (carboxylic acid, or 4-methylpiperazinyl amide) and their electronic effect (electron-donating or electron-withdrawing para-benzyl substituent), respectively. Trypanocidal hits in the micromolar range were identified against bloodstream forms of T. congolense [CPX (1); CPX amide derivatives 18: IC50 8.95µM; SI 16.84; 22: IC50 5.42µM; SI 25.2] and against T. brucei rhodesiense (CPX acid derivative 13: IC50 4.51µM; SI 10.2), demonstrating more selectivity toward trypanosomes than mammalian cells. Hence, the trypanocidal hit compound 22 may be optimized by retaining the 4-methylpiperazine amide functional group (C-3) and the TZ moiety at position N-15 and introducing other electron-withdrawing ortho-, meta-, and/or para-substituents on the aryl ring in an effort to improve the pharmacokinetic properties and increase the trypanocidal activity.

N-Pyrazolyl- and N-Triazolylamines and -Ureas as Antileishmanial and Antitrypanosomal Drugs.

Three types of modifications of antileishmanial pyrazole lead compounds 7 and 8 were conducted to expand understanding of the relationships between structural features and antileishmanial/antitrypanosomal activity: (1) the pyrazole core was retained or replaced by a 1,2,4-triazole ring; (2) various aryl moieties including 2-fluorophenyl, pyridin-3-yl and pyrazin-2-yl rings were attached at 3-position of the core azole; (3) either arylmethylamino or ureido substituents were introduced at 5-position of the azole core. The synthesis followed established routes starting with esters 9 or 15 and anhydride 21. The synthesized 3-arylpyrazoles and 3-aryl-1,2,4-triazoles had only very low antileishmanial activity. The 2-fluorophenyl-substituted pyrazole 18c revealed the highest antileishmanial activity of this series of compounds, but its IC50 value (20 μM) still indicates low activity. However, low micromolar antitrypanosomal activity was detected for the pyridin-3-yl-substituted pyrazoles 12b (IC50=4.7 μM) and 14a (IC50=2.1 μM). Their IC50 values are comparable with the IC50 values of the reference compounds benznidazole and nifurtimox. Whereas only low unspecific cytotoxicity at the primary peritoneal mouse macrophages (PMM) was detected, considerable cytotoxicity at MRC-5 human fibroblast cells was found for both pyrazoles 12b an 14a. The activity of pyrazole 12b against T. cruzi is 4-fold higher than its unspecific MRC-5 cytotoxicity.

Comparison of Three Widely Employed Extraction Methods for Metabolomic Analysis of Trypanosoma brucei.

Trypanosoma brucei (Tb) is the causative agent of human African trypanosomiasis (HAT), also known as sleeping sickness, which can be fatal if left untreated. An understanding of the parasite's cellular metabolism is vital for the discovery of new antitrypanosomal drugs and for disease eradication. Metabolomics can be used to analyze numerous metabolic pathways described as essential to Tb. brucei but has some limitations linked to the metabolites' physicochemical properties and the extraction process. To develop an optimized method for extracting and analyzing Tb. brucei metabolites, we tested the three most commonly used extraction methods, analyzed the extracts by hydrophilic interaction liquid chromatography high-resolution mass spectrometry (HILIC LC-HRMS), and further evaluated the results using quantitative criteria including the number, intensity, reproducibility, and variability of features, as well as qualitative criteria such as the specific coverage of relevant metabolites. Here, we present the resulting protocols for untargeted metabolomic analysis of Tb. brucei using (HILIC LC-HRMS). © 2024 Wiley Periodicals LLC. Basic Protocol 1: Culture of Trypanosoma brucei brucei parasites Basic Protocol 2: Preparation of samples for metabolomic analysis of Trypanosoma brucei brucei Basic Protocol 3: LC-HRMS-based metabolomic data analysis of Trypanosoma brucei brucei.

Synthesis of (-) - 6,6'-dinitrohinokinin from hinokinin natural product and in silico and in vitro trypanocidal activity assessment

Chagas disease is a public health problem affecting approximately seven million people worldwide. Thus, there is a need to discover drugs for the adequate treatment of this disease because currently available drugs have serious side effects. Therefore, this study aimed to evaluate the in vitro trypanocidal activity of (-)-6,6'-dinitrohinokinin, obtained from the partial synthesis of (-)-hinokinin, on the trypomastigotes and amastigotes forms. For the trypomastigote assay, blood was collected from mice infected with Trypanosoma cruzi through cardiac puncture at the parasitemic peak. The results show that (-)-6,6'-dinitrohinokinin was effective against the trypomastigote forms, presenting an IC50 of 19.83 µM and lysis percentage values ​​of 78.4% and 69.4% at concentrations of 200 and 100 µM, respectively. Molecular docking calculations indicate that (-)-6,6'-dinitrohinokinin favorably interacts with the amino acids present in the active site of the protein trypanothione reductase, a typical target for anti-trypanosomal drug development. According to the results, the (-)-6,6'-dinitrohinokinin showed more significant trypanocidal activity with IC50 of 1.83 µM than benzonidazole positive control with IC50 of 53.2 µM, showing to be a prototype molecule promising for the development of a new antiparasitic drug.

Parasitological, Serological and Molecular Prevalence of Trypanosoma evansi among Arabian Camels (Camelus dromedaries) with Evaluation of Antitrypanosomal Drugs.

This study was carried out to assess the prevalence of Trypanosoma evansi infection in naturally diseased Dromedary camels in Dammam, Eastern region of Saudi Arabia. The detection of Trypanosoma evansi was performed using the parasitological, serological, and molecular diagnosis and a comparison between such methods were analyzed. In addition, evaluation of therapeutic efficacy of selected antitrypanosomal drugs, cymelarsan and quinapyrmine (aquin-1.5), was trialed for treatment of diagnosed infected cases. A total 350 randomly selected camels were evaluated using thin blood smear (TBS), RoTat1.2 PCR and CATT/T. evansi techniques. The total prevalence was 6.9%, 7.7%, and 32.8% by TBS, RoTat1.2 PCR and CATT/T. evansi techniques, respectively. Although PCR detect T. evansi in more samples than TBS, the agreement was good (K = 0.9). Among the CATT/T. evansi results, PCR detect T. evansi in 12 and 15 CATT positive and negative camels, respectively, with low agreement (Kappa = 0.1). The use of cymelarsan and quinapyramine sulfate in the treatment of naturally infected cases demonstrated a very efficient therapeutic response. It was found that 1. Comparing the CATT/T. evansi and PCR results, the positivity of CATT was higher than PCR detection, while the agreement was poor (K = 0.1). 2. Cymelarsan and aquin-1.5 proved to be effective in the treatment of naturally infected camels, but cymelarsan presented with higher effectiveness (100%) than aquin-treated camels (83.3%). a 3. The use of cymelarsan and CATT is recommended for disease treatment and control.

In Vitro Trypanocidal Effect of the Extract and Isolated Compound of Corymbia torelliana Stem Bark against Trypanosoma brucei brucei (Federe Strain)

African animal trypanosomosis (AAT) is a severe debilitating protozoan disease of livestock and some wild animal species. It is caused by the pathogenic trypanosome species, and transmitted by Tsetse flies. It affects about 60 million cattle in Africa, with about US$1.5 billion in annual losses. Eradication of the disease through chemotherapy and vaccine development has been hindered by parasite antigenic variation and its resistance to drugs. In this study, the crude ethanol stem bark extract of Corymbia torelliana was prepared by cold maceration in 70% Ethanol. It was further purified using the modified Kupchan partition method into n-hexane, Ethyl acetate, N-butanol and Aqueous pure fractions. A pure compound was isolated from the ethyl acetate purified fraction using the column and thin layer chromatographic techniques. The isolated compound was named Makoshi_Mak1. The in vitro antitrypanosomal effects of the crude extract, the purified fractions and the isolated compound were then tested against the blood stream forms of Trypanosoma brucei brucei (Federe strain). 0.2 ml of prepared trypanosome suspension containing about 2x104 parasites was dispensed into each of seven clean test tubes using micropipettes. 0.2ml of prepared Ringer’s solution was also added to stabilize the parasites. Five varying concentrations of the crude extract, the extract fractions and the isolated compound (Makoshi_Mak1) were added to test tubes 1-5. Test tube 6 and 7 served as the negative and positive controls respectively. Aliquots of the contents of each test tube were examined under the microscope at x400 magnification for parasites motility at different time interval for 2 hours. The crude extract was effective at the concentrations of 12 mg/ml, 1.2 mg/ml,0.12 mg/ml and 0.012 mg/ml. the n-hexane fraction had efficacy at 0.12mg/ml and 0.096mg/ml. The Eethyl acetate fractions were effective at 0.12mg/ml, 0.096 mg/ml, 0.048mg/ml and 0.024mg/ml, the N-butanol fraction had efficacy at 0.12mg/ml and 0.096mg/ml, while the Aqueous fraction was effective at 0.2mg/ml, 0.16mg/ml and 0.08mg/ml. Makoshi_Mak1 was effective in vitro against T. brucei beucei at all the tested concentrations of 0.219mg/ml, 0.109mg/ml, 0.055mg/ml and 0.027mg/ml. Efficacy of the extracts and isolated compound was determined by parasite mortality, and was seen to be concentration dependent. Dead parasites appeared to float freely on the fluid environment, while the lysed parasites were seen as floating debris. The results of the study showed that both the crude extract, the purified extract and the pure isolated compound of Corymbia torelliana had in vitro activity against Trypanosoma brucei brucei in a concentration dependent manner. Corymbia torelliana can then be considered as a potential herbal antitrypanosomal drug candidate.

Tackling Sleeping Sickness: Current and Promising Therapeutics and Treatment Strategies.

Human African trypanosomiasis is a neglected tropical disease caused by the extracellular protozoan parasite Trypanosoma brucei, and targeted for eradication by 2030. The COVID-19 pandemic contributed to the lengthening of the proposed time frame for eliminating human African trypanosomiasis as control programs were interrupted. Armed with extensive antigenic variation and the depletion of the B cell population during an infectious cycle, attempts to develop a vaccine have remained unachievable. With the absence of a vaccine, control of the disease has relied heavily on intensive screening measures and the use of drugs. The chemotherapeutics previously available for disease management were plagued by issues such as toxicity, resistance, and difficulty in administration. The approval of the latest and first oral drug, fexinidazole, is a major chemotherapeutic achievement for the treatment of human African trypanosomiasis in the past few decades. Timely and accurate diagnosis is essential for effective treatment, while poor compliance and resistance remain outstanding challenges. Drug discovery is on-going, and herein we review the recent advances in anti-trypanosomal drug discovery, including novel potential drug targets. The numerous challenges associated with disease eradication will also be addressed.

In vivo Metabolism of Nifurtimox and the Drug-Drug Interaction Potential Including its Major Metabolites.

Nifurtimox is an effective treatment for patients with Chagas disease, but knowledge of its biotransformation and excretion is limited. This study aimed to better understand the fate of oral nifurtimox in vivo. We investigated the exposure and excretion pathways of [14C]-labeled nifurtimox and its metabolites in rats. We then quantified the prominent metabolites and nifurtimox in the urine and plasma of patients receiving nifurtimox using LC-HRMS with reference standards and quantified these compounds in rat plasma after a single, high dose of nifurtimox. We also investigated potential drug-drug interactions (DDIs) of these compounds in vitro. In rats, orally administered nifurtimox was rapidly absorbed (tmax 0.5 h) and eliminated (t½ 1.4 h). Metabolism of nifurtimox yielded six predominant metabolites (M-1 to M-6) in urine and plasma, and the dose was excreted equally via the renal and fecal routes with only traces of unchanged nifurtimox detectable due to its instability in excreta. In patients with Chagas disease, only M-6 and M-4 achieved relevant exposure levels, and the total amount of excreted metabolites in urine was higher in fed versus fasted patients, consistent with the higher systemic exposure. For nifurtimox, M-6, and M-4, no potential perpetrator pharmacokinetic DDIs with the main cytochrome P- 450 enzymes and drug transporters were identified in vitro. This contemporary analysis of the complex metabolite profile and associated exposures emerging after oral dosing of nifurtimox in rats and humans, together with the expected low risk for clinically relevant DDIs, expands the understanding of this important anti-trypanosomal drug.

Intracellular ROS production and apoptotic effect of quinoline and isoquinoline alkaloids on the growth of Trypanosoma evansi

Trypanosoma evansi, a hemoflagellate poses huge economic threat to the livestock industry of several countries of Asia, Africa, South America and Europe continents of the world. Limited number of available chemical drugs, incidents of growing drug resistance, and related side effects encouraged the use of herbal substitutes. In the present investigation, the impact of six alkaloids of quinoline and isoquinoline group was evaluated on the growth and multiplication of Trypanosoma evansi and their cytotoxic effect was examined on horse peripheral blood mononuclear cells in an in vitro system. Quinine, quinindine, cinchonine, cinchonidine, berbamine and emetine showed potent trypanocidal activities with IC50/24 h values 6.631 ± 0.244, 8.718 ± 0.081, 16.96 ± 0.816, 33.38 ± 0.653, 2.85 ± 0.065, and 3.12 ± 0.367 µM, respectively, which was comparable to the standard anti-trypanosomal drug, quinapyramine sulfate (20 µM). However, in the cytotoxicity assay, all the drugs showed dose dependent cytotoxic effect and quinine, berbamine and emetine showed selectivity index more than 5, based of ration of CC50 to IC50. Among the selected alkaloids, quinidine, berbamine and emetine exhibited higher apoptotic effects in T. evansi. Likewise, drug treated parasites showed a dose-dependent and time-dependent increase in reactive oxygen species (ROS) production. Therefore, increased apoptosis in combination with ROS generation could be responsible for the observed trypanocidal effect which could be further evaluated in T. evansi-infected mice model.

In-silico analysis of bufadienolides as potential inhibitors of Trypanosoma nucleoside hydrolase

Trypanosomiasis is one of the neglected tropical diseases which curtails the productivity of the victim and causes mortality in extreme cases. Non-availability of vaccines, low therapeutic index of trypanocidal drugs and development of resistance lead to the need for research focused on developing alternative treatment options against this disease. The aim of the current work is to use computer-assisted drug discovery techniques to examine the anti-trypanosomal action of bufadienolides, which are commonly detected in the skin secretions of toads. A library of 16 compounds from the bufadienolides family was tested for toxicity and docked against the Trypanosoma brucei hydrolase. The oral bioavailability and good suitability of the compounds for transdermal delivery was also indicated by the ADMET study. The findings based on molecular docking and dynamics simulation along with toxicity assessment showed that three compounds, marinobufagin, resibufogenin and resibufaginol, have the lowest toxicity and maximum binding energy against Trypanosoma brucei hydrolase. According to the results obtained by molecular docking interactions between drugs and target proteins are observed and contain a number of hydrogen bonds near the residues in the active pocket of the enzyme. The results of this study contribute to the understanding of the SAR of bufadienolides and provide a foundation for further research and drug development efforts. The computational analysis, including the density functional theory study, the knowledge of inhibitory mechanisms and aids in the design and optimization of novel anti-trypanosomal drugs.

In Vitro Effects of Aminopyridyl Ligands Complexed to Copper(II) on the Physiology and Interaction Process of Trypanosoma cruzi.

Chagas disease is derived from the infection by the protozoan Trypanosoma cruzi. In many countries, benznidazole is the only drug approved for clinical use despite several side effects and the emergence of resistant parasite strains. In this context, our group has previously pointed out that two novel aminopyridine derivatives complexed with Cu2+, namely, cis-aquadichloro(N-[4-(hydroxyphenyl)methyl]-2-pyridinemethamino)copper (3a) and its glycosylated ligand cis-dichloro (N-{[4-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyloxy)pheny]lmethyl}-2-pyridinemethamino)copper (3b), are effective against T. cruzi trypomastigote forms. With this result in mind, the present work aimed to investigate the effects of both compounds on trypomastigotes physiology and on the interaction process with host cells. Apart from loss of plasma membrane integrity, an increased generation of reactive oxygen species (ROS) and decreased mitochondrial metabolism were observed. Pretreatment of trypomastigotes with these metallodrugs inhibited the association index with LLC-MK2 cells in a typical dose-dependent manner. Both compounds showed low toxicity on mammalian cells (CC50 > 100 µM), and the IC50 values calculated for intracellular amastigotes were determined as 14.4 µM for 3a and 27.1 µM for 3b. This set of results demonstrates the potential of these aminopyridines complexed with Cu2+ as promising candidates for further antitrypanosomal drug development.

Comparative Analysis of the Interaction of Silver Nanoparticles with Hexokinase from Trypanosoma brucei and Humans.

Regardless of the efforts to ease cases of human African trypanosomiasis (HAT), an increased number of cases get reported annually. This is because of drug resistant Trypanosoma brucei (Tb), the causative agent of the illness. This has renewed the need for creative methods to find new anti-trypanosomal drugs. The blood stream form (BSF) of the parasite depends exclusively on the glycolytic pathway for energy production while it is in the human host. Interruptions in this pathway efficiently kills the parasite. Trypanosoma brucei hexokinase (TbHK) is the first enzyme in glycolysis, and any effectors or inhibitors of TbHK would have potential as anti-trypanosomal agents. TbHK and human glucokinase (hGCK) were over-expressed with a 6 histidine-tag in E. coli BL21(DE3) cells having the pRARE2 plasmid. TbHK had thermal and pH stability between 30°C and 55°C and 7.5 and 8.5, respectively, while hGCK exhibited thermal and pH stability between 30°C and 40°C and 7.0 and 8.0, respectively. Kinetically, TbHK had a Km of 39.3 µM, Vmax of 0.066 µmol.min-1.mL-1, kcat of 2.05 min-1 and kcat/Km of 0.0526 min-1.µmol-1. hGCK exhibited a Km of 4.5 µM, Vmax of 0.032 µnmol.min-1.mL-1, kcat of 11.25 min-1, and kcat/Km of 2.5 min-1.µmol-1. Interaction kinetic studies of silver nanoparticles (AgNPs) (0.1 µM) of average size of 6 nm with TbHK and hGCK were conducted. AgNPs selectively inhibited TbHK over hGCK. TbHK showed a non-competitive inhibition with a 50% and 28% decrease in Vmax, and kcat/km, respectively. HsGCK showed a 33% increase in affinity, 9% decrease in Vmax, and a 50% increase in enzyme efficiency. The observed pattern of hGCK and AgNPs falls under the uncompetitive inhibition. The observed highly selective inhibitory effects of AgNPs between TbHK and hGCK may be used in development of new anti-trypanosomal drugs.

Alchornea laxiflora as therapeutic agent in the treatment of Trypanosoma brucei brucei infection using experimental wistar rats; invivo and gene expression studies

Aims: African trypanosomosis is a zoonotic tropical disease of Sub-Saharan Africa. Its etiology is a group of extracellular protozoans of the genera Trypanosoma which has numerous species including Trypanosoma brucei brucei. Inavailability of vaccines and numerous side effects arising from few available antitrypanosomal drugs gives the indication for search for local alternatives. This experimental study investigates the antitrypanosomal efficacy of chloroform extract of A. laxiflora leaves in wistar albino rats infected with Trypanosoma brucei brucei. Place and Duration of Study: Animal Parasitology and Microbiology Research Unit, Department of Animal Production and Health, Federal University of Technology, Akure, Nigeria, between July and November, 2022. Methodology: Thirty-six rats of both sexes were infected with Trypanosoma brucei brucei and treated with Alchornea laxiflora extracts intraperitoneally at dosages of 100, 200 and 400mg/kg for three consecutive days. Diminacene aceturate at 3.5mg/kg as single dose was used as positive control. Daily parasiteamia, rectal temperature and body weight were monitored for 26 days. Antioxidant (SOD, CAT, Nrf2) and pro-inflammatory (TNF-α, IL-6) gene expressions were determined. Results: Treatment with Alchornea laxiflora caused significant (p &lt; 0.05) decrease in parasitemia of infected rats compared to negative control. Increase in weight gain was recorded after the initial weight loss post infection. There was prolongation of survival days to 22days at 400mg/kg treatment dose compared to 7days in the untreated control. Significant (p &lt; 0.05) dose dependent upward regulation of SOD, CAT and Nrf2 antioxidant genes and down-regulation of TNF-α and IL-6 inflammatory genes were observed in all treatment groups compared to the untreated control in the spleen and liver. Conclusion: These observations suggest that A. laxiflora extract possess excellent anti-trypanosomal properties with the downregulation of pro inflammatory genes and up-regulation of antioxidant genes

Hit-to-lead optimization of a 2-aminobenzimidazole series as new candidates for chagas disease

Chagas disease is a neglected tropical disease caused by Trypanosoma cruzi. Because current treatments present several limitations, including long duration, variable efficacy and serious side effects, there is an urgent need to explore new antitrypanosomal drugs. The present study describes the hit-to-lead optimization of a 2-aminobenzimidazole hit 1 identified through in vitro phenotypic screening of a chemical library against intracellular Trypanosoma cruzi amastigotes, which focused on optimizing potency, selectivity, microsomal stability and lipophilicity. Multiparametric Structure−Activity Relationships were investigated using a set of 277 derivatives. Although the physicochemical and biological properties of the initial hits were improved, a combination of low kinetic solubility and in vitro cytotoxicity against mammalian cells prevented progression of the best compounds to an efficacy study using a mouse model of Chagas disease.

Trypanocidal and cell swelling activity of 20-deoxysalinomycin

The naturally occurring polyether ionophore salinomycin was previously found to display promising anti-proliferative activity against bloodstream forms of Trypanosoma brucei. Here, we report the evaluation of 20-deoxysalinomycin, a naturally occurring homolog to salinomycin, for trypanocidal and cell swelling activity. The concentration of 20-deoxysalinomycin required to reduce the growth rate of bloodstream-form trypanosomes by 50% was determined to be 0.12 μM and found to be 8 times more trypanocidal than that of salinomycin. Moreover, 20-deoxysalinomycin and salinomycin displayed similar cytotoxic activity against human HL-60 cells. Measured as the ratio of cytotoxic to trypanocidal activity, 20-deoxysalinomycin thus exhibits a four-fold higher selectivity compared to salinomycin. The stronger trypanocidal activity of 20-deoxysalinomycin is attributed to an enhanced ability to induce cell swelling in trypanosomes. The findings support 20-deoxysalinomycin as a useful lead in the rational development of new and improved anti-trypanosomal drugs.

In Vitro Mechanism of Action of Acanthospermum hispidum in Trypanosoma brucei

African trypanosomiasis is a major neglected tropical disease with significant health and economic concerns in sub-Saharan Africa. In the absence of vaccines for African trypanosomiasis, there is a consideration for alternative sources of chemotherapy. Acanthospermum hispidum DC (A. hispidum) is a herbal species of the Asteraceae family that is endowed with rich phytochemicals with unknown mechanisms of antitrypanosomal effects. This study aimed to investigate the cellular mechanisms of antitrypanosomal and antioxidant activities of A. hispidum against Trypanosoma brucei (T. brucei), a causative protozoan species of African trypanosomiasis. Fractions were prepared from the whole plant of A. hispidum through solvent partitioning by employing solvents of varying polarities (hexane, HEX; dichloromethane, DCM; ethyl acetate, EA; aqueous, AQ). The in vitro efficacies and mechanisms of antitrypanosomal activities of A. hispidum were investigated using a panel of cell biological approaches. GC-MS analysis was used to identify the major compounds with a possible contribution to the trypanocidal effects of A. hispidum. A. hispidum fractions displayed significant antitrypanosomal activities in terms of half-maximal effective concentrations (EC50) and selectivity indices (SI) (AH-HEX, EC50 = 2.4 μg/mL, SI = 35.1; AH-DCM, EC50 = 2.2 μg/mL, SI = 38.3; AH-EA, EC50 = 1.0 μg/mL, SI = 92.8; AH-AQ, EC50 = 2.0 μg/mL, SI = 43.8). Fluorescence microscopic analysis showed that at their EC50 values, the fractions of A. hispidum altered the cell morphology as well as the organization of the mitochondria, nucleus, and kinetoplast in T. brucei. At their maximum tested concentrations, the prepared fractions exhibited antioxidant absorbance intensities comparable to the reference antioxidant, Trolox, in contrast to the oxidant intensity of an animal antitrypanosomal drug, diminazene (Trolox, 0.11 A; diminazene, 0.65 A; AH-HEX, 0.20 A, AH-DCM, 0.20 A, AH-EA, 0.13 A, AH-AQ, 0.22 A). GC-MS analysis of the various fractions identified major compounds assignable to the group of alkaloids and esters or amides of aliphatic acids. The results provide useful pharmacological insights into the chemotherapeutic potential of A. hispidum toward drug discovery for African trypanosomiasis.

Anti-viral activity of suramin against influenza A virus in A549 cells

Influenza A virus (IAV) is highly contagious global factor of causing pandemics and epidemics occasionally. Suramin, an anti-trypanosomal drug, also under evaluation as anti-cancer and anti-viral agent. However, inhibitory effect of suramin against influenza A virus have not yet been explored. The purpose of this study was to investigate the anti-viral activity of suramin against IAV. Cytotoxicity assay (IC) of suramin and oseltamivir was determined using MTT assay in A549 cells. The anti-viral effect of suramin was determined by RT-qPCR, western blot and CPE (Cytopathic effect). Oseltamivir, an antiviral drug that has established anti influenza activity was used as positive control drug. Half-maximal toxic concentration of suramin was found to be at 269.2 µg/ml (Log IC=2.43 µg/ml) and IC for oseltamivir was 145.4 µg/ml (Log IC=2.16 µg/ml) in A549 cells at 24hr. Suramin exhibited anti-viral activity at all the concentration, however at 250 µg/ml and 125 µg/ml demonstrated significant down regulation of viral RNA and protein in A549 cells. Oseltamivir, a positive control drug also showed significant viral inhibition at 200 and 100 µg/ml at 24hr. : Suramin is effective in inhibiting influenza A virus replication in A549 cells at lower concentration.

Gallic Acid Alkyl Esters: Trypanocidal and Leishmanicidal Activity, and Target Identification via Modeling Studies.

Eight gallic acid alkyl esters (1–8) were synthesized via Fischer esterification and evaluated for their trypanocidal and leishmanicidal activity using bloodstream forms of Trypanosoma brucei and promastigotes of Leishmania major. The general cytotoxicity of the esters was evaluated with human HL-60 cells. The compounds displayed moderate to good trypanocidal but zero to low leishmanicidal activity. Gallic acid esters with alkyl chains of three or four carbon atoms in linear arrangement (propyl (4), butyl (5), and isopentyl (6)) were found to be the most trypanocidal compounds with 50% growth inhibition values of ~3 μM. On the other hand, HL-60 cells were less susceptible to the compounds, thus, resulting in moderate selectivity indices (ratio of cytotoxic to trypanocidal activity) of >20 for the esters 4–6. Modeling studies combining molecular docking and molecular dynamics simulations suggest that the trypanocidal mechanism of action of gallic acid alkyl esters could be related to the inhibition of the T. brucei alternative oxidase. This suggestion is supported by the observation that trypanosomes became immobile within minutes when incubated with the esters in the presence of glycerol as the sole substrate. These results indicate that gallic acid alkyl esters are interesting compounds to be considered for further antitrypanosomal drug development.

Two Tags in One Probe: Combining Fluorescence- and Biotin-based Detection of the Trypanosomal Cysteine Protease Rhodesain.

Rhodesain is the major cysteine protease of the protozoan parasite Trypanosoma brucei and a therapeutic target for sleeping sickness, a fatal neglected tropical disease. We designed, synthesized and characterized a bimodal activity-based probe that binds to and inactivates rhodesain. This probe exhibited an irreversible mode of action and extraordinary potency for the target protease with a kinac /Ki value of 37,000 M-1 s-1 . Two reporter tags, a fluorescent coumarin moiety and a biotin affinity label, were incorporated into the probe and enabled highly sensitive detection of rhodesain in a complex proteome by in-gel fluorescence and on-blot chemiluminescence. Furthermore, the probe was employed for microseparation and quantification of rhodesain and for inhibitor screening using a competition assay. The developed bimodal rhodesain probe represents a new proteomic tool for studying Trypanosoma pathobiochemistry and antitrypanosomal drug discovery.

Activation of angiotensin-converting enzyme 2 ameliorates metabolic syndrome-induced renal damage in rats by renal TLR4 and nuclear transcription factor κB downregulation.

BackgroundMetabolic syndrome (MetS) is an independent risk factor for chronic kidney disease (CKD) through many mechanisms, including activation of the renin–angiotensin system. The deleterious effects of angiotensin II (Ang II) can be counterbalanced by angiotensin-converting enzyme 2 (ACE2). Diminazene aceturate (DIZE), an anti-trypanosomal drug, can activate ACE2.ObjectiveThis study aimed to investigate the possible reno-protective effects of DIZE in MetS rats with elucidation of related mechanisms.Materials and methodsThirty adult male Wistar albino rats were divided equally into control, MetS, and MetS + DIZE groups. Body weight, systolic blood pressure (SBP), and urinary albumin levels were measured. Serum levels of fasting blood glucose (FBG), insulin, uric acid, lipid profile, urea, and creatinine were measured. Homeostasis Model Assessment Index (HOMA-IR) was estimated. Subsequently, renal levels of ACE2, Ang II, malondialdehyde (MDA), reduced glutathione (GSH), and tumor necrosis factor-α (TNF-α) were measured with histopathological and immunohistochemical assessment of TLR4 and NF-κB in renal tissues.ResultsMetS caused dyslipidemia with significant increases in body weight, SBP, FBG, serum insulin, HOMA-IR, uric acid, urea, creatinine, urinary albumin, and renal levels of Ang II, MDA, and TNF-α, whereas renal ACE2 and GSH were significantly decreased. Renal TLR4 and NF-κB immunoreactivity in MetS rats was upregulated. DIZE supplementation of MetS rats induced significant improvements in renal function parameters; this could be explained by the ability of DIZE to activate renal ACE2 and decrease renal Ang II levels with downregulation of renal TLR4 and NF-κB expression.ConclusionDIZE exerts a reno-protective effect in MetS, mainly by downregulating renal TLR4 and NF-κB levels.