Antiamoebic Agents Research Articles

Discovery of Anti-Amoebic Inhibitors from Screening the MMV Pandemic Response Box on Balamuthia mandrillaris, Naegleria fowleri, and Acanthamoeba castellanii.

Pathogenic free-living amoebae, Balamuthia mandrillaris, Naegleria fowleri, and several Acanthamoeba species are the etiological agents of severe brain diseases, with case mortality rates > 90%. A number of constraints including misdiagnosis and partially effective treatments lead to these high fatality rates. The unmet medical need is for rapidly acting, highly potent new drugs to reduce these alarming mortality rates. Herein, we report the discovery of new drugs as potential anti-amoebic agents. We used the CellTiter-Glo 2.0 high-throughput screening methods to screen the Medicines for Malaria Ventures (MMV) Pandemic Response Box in a search for new active chemical scaffolds. Initially, we screened the library as a single-point assay at 10 and 1 µM. From these data, we reconfirmed hits by conducting quantitative dose–response assays and identified 12 hits against B. mandrillaris, 29 against N. fowleri, and 14 against A. castellanii ranging from nanomolar to low micromolar potency. We further describe 11 novel molecules with activity against B. mandrillaris, 22 against N. fowleri, and 9 against A. castellanii. These structures serve as a starting point for medicinal chemistry studies and demonstrate the utility of phenotypic screening for drug discovery to treat diseases caused by free-living amoebae.

Speculative analysis on the electronic structure, IR assignments and molecular docking of N-{4-[(4-amino-3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)sulfonyl]phenyl}acetamide, an anti-amoebic agent

An exhaustive quantum mechanical calculations on a pharmaceutically critical molecule N-{4-[(4-amino-3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)sulfonyl]phenyl}acetamide have been investigated through the B3LYP/6-31G∗∗ Density Functional and HF/6-31G∗∗ Wave Function techniques. Physicochemical parameters along with the advanced electronic structure parameters like; MEP (molecular electrostatic potentials) and highest occupied & lowest unoccupied molecular orbitals (HOMO-LUMO) analysis have additionally been scanned over both methods. The computed HOMO-LUMO energy demonstrates that charge exchange takes place inside the molecule. The estimated small HOMO-LUMO energy gap, through both methods, indicates that the molecule is chemically reactive. Further, the IR vibrational spectra of the molecule have been assigned in the region 400-4000 cm−1 through the DFT technique. The anticipated vibrational assignments have been compared with the experimental values accounted for in the literature. To comprehend the mode of binding, docking investigations of the molecule alongwith the co-crystallized metronidazole (MNZ) molecule were accomplished with O-acetyl-serine-sulfhydrylase (OASS) enzyme using GLIDE-SP and GLIDE-XP modules. Docking simulations and reported biological activities (IC50) demonstrate that the title molecule may act as a lead molecule for constraining the progression of Entamoeba histolytica illness.

Isoniazid Conjugated Magnetic Nanoparticles Loaded with Amphotericin B as a Potent Antiamoebic Agent against Acanthamoeba castellanii.

The pathogenic free-living amoeba, Acanthamoeba castellanii, is responsible for a rare but deadly central nervous system infection, granulomatous amoebic encephalitis and a blinding eye disease called Acanthamoeba keratitis. Currently, a combination of biguanides, amidine, azoles and antibiotics are used to manage these infections; however, the host cell cytotoxicity of these drugs remains a challenge. Furthermore, Acanthamoeba species are capable of transforming to the cyst form to resist chemotherapy. Herein, we have developed a nano drug delivery system based on iron oxide nanoparticles conjugated with isoniazid, which were further loaded with amphotericin B (ISO-NPs-AMP) to cause potent antiamoebic effects against Acanthamoeba castellanii. The IC50 of isoniazid conjugated with magnetic nanoparticles and loaded with amphotericin B was found to be 45 μg/mL against Acanthamoeba castellanii trophozoites and 50 μg/mL against cysts. The results obtained in this study have promising implications in drug discovery as these nanomaterials exhibited high trophicidal and cysticidal effects, as well as limited cytotoxicity against rat and human cells.

Monoclinic cerium(III) picrate tetraethylene glycol complex: design, synthesis and biological evaluation as anti-amoebic activity against Acanthamoeba sp.

In an effort to develop effective anti-amoebic agent, a salt type complex, namely [Ce(Pic)2(H2O)(EO4)](Pic)·H2O complex, was synthesized and screened for in vitro anti-amoebic activity against Acanthamoeba sp. This Ce(III) complex has been characterized by microelemental analysis, and IR and NMR spectroscopy, and the data suggested the structural formula. Single X-ray crystallography structure showed that the Ce(III) ion is bicapped square antiprismatic with the O2 and O5 atoms occupying the top of the capping position forming a 10-coordination number. The cytotoxicity indicated that Ce salt and Ce(III) complex have an excellent anti-amoebic activity with IC50 values as low as 3.75 and 3 µg/mL, respectively, with a significant decrease (p < 0.05) in Acanthamoeba sp. viability. The mode of cell death in Acanthamoeba cells upon treated with the Ce(III) complex was in the early apoptosis and necrosis. This is in contrast to the Ce(NO3)3·6H2O salt which exhibited necrosis. Both Ce(III) complex and Ce salt on the Acanthamoeba cell exhibited the genotoxic effect on the amoeba cells by looking at scoring DNA damage in types 3 and 4. The complexation form of Ce salt, picric acid (HPic) and tetraethylene glycol (EO4) to produce Ce(III) complex is expected to exhibit synergistic effects to show anti-amoebic activity. The molecular formula of Ce(III) complex and crystal packing hydrogen bonding of this compound is responsible for its cytotoxic and genotoxic effects. Overall results suggested the promising cytotoxic and genotoxic activities of monoclinic Ce(III) complex against Acanthamoeba sp., the preparation method using slow evaporation as a synthetic strategy to design lanthanide complex and the advancement in the synthesis of novel lanthanide complexes with comprehensive evaluation as anti-amoebic agents.

Combined 3D-QSAR Modeling and Molecular Docking Study on metronidazole-triazole-styryl hybrids as antiamoebic activity

A series of twenty-two metronidazole-triazole-styryl hybrids as antiamoebic agents were studied based on the combination of 3D-QSAR and surflex-docking. The CoMFA and CoMSIA models were carried out using eighteen compounds in the training set and four compounds in the test set gives Q 2 values of 0.684 and 0.664 respectively, and R 2 values of 0.882 and 0.894 respectively. The adapted alignment method with the suitable parameters resulted in reliable models. Based on contour maps produced by the CoMFA and CoMSIA, we suggested new compounds with high predicted activities, Surflex-docking revealed the important interactions between the ligand and receptor. Therefore, it confirmed the stability of predicted molecules in the receptor with PDB: 4CCQ.

The effect of antiamoebic agents and Ce6‐PDT on acanthamoeba castellani trophozoites and cysts, in vitro

AbstractPurposeTo analyze the concentration dependent effect of diamidines (hexamidine‐diisethionat (HD), propamidin‐isethionate (PD), dibromopropamidine‐diisethionat (DD)); biguanides (polyhexamethylen biguanid (PHMB), chlorhexidine (CH)); natamycin (NM); miltefosine (MF); povidone iodine (PVPI) and chlorin e6 photodynamic therapy (PDT) on Acanthamoeba castellani trophozoites and cysts, in vitro.MethodsAcanthamoeba castellani strain was cultured in 712 peptone‐yeast extract‐glucose (PYG) medium. Thereafter, trophozoites or cysts were cultured in 0.25–0.1% HD, PD or DD, or 0.005–0.02% PHMB, CH or NM or 0.001625–0.0065 MF or 0.25–1% PVPI containing PYG medium for 2 h or underwent Chlorin e6‐PDT. Then, the percentage of dead trophozoites was determined by CytoTox 96® Non‐Radioactive Cytotoxicity assay and trypan blue staining and those of cysts using trypan blue staining. Treated trophozoites and cysts were also inoculated to non‐nutrient agar Eshericia coli plates and were observed for 5 weeks.ResultsAll concentrations of different antiamoebic agents had a significant cytotoxic effect on AK trophozoites and cysts (p &lt; 0.05), except 0.02% PHMB and CH, using trypan blue assay for trophozoites, and Ce6‐PDT for cysts. Observing the agar plates, HD, PD, PHMB, CH, NM and PVPI led to morphological changes of acanthamoeba trophozoites, which could not form cysts within 5 weeks. DD and MF treated cysts could excyst and later encyst again.ConclusionsConservative treatment using different antiamoebic agents or Ce6‐PDT can not eradicate all Acanthamoeba castellani trophozoites and cysts. In vitro analyzis of treatment efficacy of different antiamoebic agents may alow a more specific treatment in acanthamoeba keratitis.

Synergy Testing of Antiamoebic Agents for Acanthamoeba: Antagonistic Effect of Voriconazole.

To determine whether combinations of commonly used antiamoebic agents display synergy in their ability to kill Acanthamoeba cysts in vitro. Synergy testing was performed with a microdilution checkerboard assay on 10 clinical Acanthamoeba keratitis isolates collected at the Proctor Foundation from 2008 to 2012. Each isolate was exposed to pairwise combinations of chlorhexidine, propamidine, and voriconazole. The minimum cysticidal concentration (MCC) for each drug pair was estimated for each isolate, and the summed fractional cysticidal concentration (ΣFCC) was calculated for each drug combination in the checkerboard, with synergy defined as a lack of growth at a ΣFCC ≤ 0.5 and antagonism as growth at a ΣFCC > 4. Chlorhexidine and propamidine were cysticidal, with median MCCs of 12.5 (range 1.5-50) and 11.7 (range 0.2-250), respectively. Voriconazole was not cysticidal, with a median MCC of >10,000 μg/mL. The combination of chlorhexidine and propamidine did not markedly change the cysticidal activity compared with either drug alone. By contrast, voriconazole antagonized the cysticidal activity of both chlorhexidine and propamidine, with Acanthamoeba growth observed at antagonistic ΣFCCs in 27 of 49 (55.1%, 95% confidence interval 35.7%-78.6%) checkerboard combinations of voriconazole and chlorhexidine and in 58 of 147 (39.5%, 95% confidence interval 14.3%-50.3%) combinations of voriconazole and propamidine. In an in vitro assay, voriconazole reduced the cysticidal activity of 2 commonly used antiamoebic drugs. Although the in vivo drug interactions could be different, these observations may be useful in cases of nonhealing Acanthamoeba keratitis being treated with combination therapies that include voriconazole.

Oleic acid-conjugated silver nanoparticles as efficient antiamoebic agent against Acanthamoeba castellanii.

Acanthamoeba castellanii belonging to the T4 genotype is an opportunistic pathogen which is associated with blinding eye keratitis and rare but fatal central nervous system infection. A. castellanii pose serious challenges in antimicrobial chemotherapy due to its ability to convert into resistant, hardy shell-protected cyst form that leads to infection recurrence. The fatty acid composition of A. castellanii trophozoites is known to be most abundant in oleic acid which chemically is an unsaturated cis-9-Octadecanoic acid and naturally found in animal and vegetable fats and oils. This study was designed to evaluate antiacanthamoebic effects of oleic acid against trophozoites, cysts as well as parasite-mediated host cell cytotoxicity. Moreover, oleic acid-conjugated silver nanoparticles (AgNPs) were also synthesized and tested against A. castellanii. Oleic acid-AgNPs were synthesized by chemical reduction method and characterized by ultraviolet-visible spectrophotometry, atomic force microscopy, dynamic light scattering analysis, and Fourier transform infrared spectroscopy. Viability, growth inhibition, encystation, and excystation assays were performed with 10 and 5μM concentration of oleic acid alone and oleic acid-conjugated AgNPs. Bioassays revealed that oleic acid alone and oleic acid-conjugated AgNPs exhibited significant antiamoebic properties, whereas nanoparticle conjugation further enhanced the efficacy of oleic acid. Phenotype differentiation assays also showed significant inhibition of encystation and excystation at 5μM. Furthermore, oleic acid and oleic acid-conjugated AgNPs also inhibited amoebae-mediated host cell cytotoxicity as determined by lactate dehydrogenase release. These findings for the first time suggest that oleic acid-conjugated AgNPs exhibit antiacanthamoebic activity that hold potential for therapeutic applications against A. castellanii.

Tannic acid-modified silver nanoparticles as a novel therapeutic agent against Acanthamoeba

Free-living amoebae belonging to Acanthamoeba genus are widely distributed protozoans which are able to cause infection in humans and other animals such as keratitis and encephalitis. Acanthamoeba keratitis is a vision-threatening corneal infection with currently no available fully effective treatment. Moreover, the available therapeutic options are insufficient and are very toxic to the eye. Therefore, there is an urgent need for the development of more effective anti-amoebic agents. Nanotechnology approaches have been recently reported to be useful for the elucidation antimicrobial, antiviral, antifungal and antiprotozoal activities and thus, they could be a good approach for the development of anti-Acanthamoeba agents. Therefore, this study was aimed to explore the activity and cytotoxicity of tannic acid-modified silver nanoparticles, pure silver nanoparticles and pure gold nanoparticles against clinical strains of Acanthamoeba spp. The obtained results showed a significant anti-amoebic effect of the tannic acid-modified silver nanoparticles which also presented low cytotoxicity. Moreover, tannic acid-modified silver nanoparticles were well absorbed by the trophozoites and did not induce encystation. On the other hand, pure silver nanoparticles were only slightly active against the trophozoite stage and pure gold nanoparticles did not show any activity. In conclusion and based on the observed results, silver nanoparticle conjugation with tannic acid may be considered as potential agent against Acanthamoeba spp.

The Effect of Antiamoebic Agents on Viability, Proliferation and Migration of Human Epithelial Cells, Keratocytes and Endothelial Cells, In Vitro

ABSTRACTPurpose: To analyze the effect of diamidines (hexamidine-diisethionat (HD), propamidin-isethionate (PD), dibromopropamidine-diisethionat (DD)), and biguanides (polyhexamethylen biguanid (PHMB), chlorhexidine (CH)) on human corneal epithelial cell, keratocyte and endothelial cell viability, proliferation, and migration, in vitro.Methods: For epithelial and endothelial cells a human cell line and for keratocytes primary cultures were used (n = 6 each). We used 3.9x10−4–0.1% HD, PD or DD, 3.9x10−4–0.0125% PD, 7.8x10−5–0.02% PHMB or CH concentration for 24 h to determine viability (Cell Proliferation Kit XTT), proliferation (Cell Proliferation ELISA BrdU kit), and migration using wound healing assay. Viability/proliferation/migration values of each drug were summarized as “area under curve” (AUC) together with a Mann–Whitney test.Results: HCEC, keratocyte, and HCEC-12 viability AUC, comparing PD and PHMB (p ≤ 0.014 for all; PD better) or PD and HD (p ≤ 0.011 for all; PD better) differed significantly. Keratocyte and HCEC-12 viability AUC comparing CH and HD (p ≤ 0.027; CH better), HCEC-12 viability AUC comparing PD and HD (p = 0.005; PD better) and HCEC viability AUC comparing CH and PHMB (p = 0.014; CH better) differed significantly. HCEC proliferation AUC, comparing PD with PHMB, CH, DD, HD (p ≤ 0.016; PD worse for all) and keratocyte proliferation AUC, comparing PHMB with HD, PD (p = 0.004; p = 0.002; PHMB better for both), CH with HD, PD (p ≤ 0.001; CH better for both) and DD with PD (p = 0.043; DD better) differed significantly. Keratocyte migration AUC comparing PD with control, PHMB, CH, DD and HD differed significantly (p ≤ 0.012; PD worse for all).Conclusions: Propamidin-isethionate as diamidine and chlorhexidin as biguanide may be used clinically to reduce cytotoxicity of antiamoebic treatment on human corneal cells. Diamidines reduce proliferation of human epithelial cells and keratocytes more than biguanides and propamidin-isethionate reduces migration of keratocytes. Therefore, in spite of lower cytotoxicity, the inhibitory effect on proliferation and migration indicates that extended use of propamidin-isethionate should be avoided in patients.

Novel Pyrimidine‐based Ferrocenyl substituted Organometallic Compounds: Synthesis, Characterization and Biological Evaluation

Some novel pyrimidine‐based ferrocenyl substituted organometallic compounds were synthesized via multistep reactions, well characterized by different spectroscopic techniques and elemental analyses and evaluated for in vitro antiprotozoal susceptibility against HM1: IMSS strain of Entamoeba histolytica. The results of antiprotozoal susceptibility unveiled these compounds, as new leads in protozoal chemotherapy as most of the organometallics displayed an exceptionally higher antiamoebic activity (IC50 = 0.055 μM ‐ 0.815 μM) than the reference drug metronidazole which gave IC50 (50% inhibitory concentration) value 1.781 μM in our experiments, concluding that newly synthesized organometallic compounds have potential to be employed as effective antiamoebic agents and these organometallics can be very useful for further optimization work on amoebic chemotherapy.

Silver nanoparticle conjugation affects antiacanthamoebic activities of amphotericin B, nystatin, and fluconazole.

Infectious diseases are the leading cause of morbidity and mortality, killing more than 15 million people worldwide. This is despite our advances in antimicrobial chemotherapy and supportive care. Nanoparticles offer a promising technology to enhance drug efficacy and formation of effective vehicles for drug delivery. Here, we conjugated amphotericin B, nystatin (macrocyclic polyenes), and fluconazole (azole) with silver nanoparticles. Silver-conjugated drugs were synthesized successfully and characterized by ultraviolet-visible spectrophotometry, Fourier transform infrared spectroscopy, and atomic force microscopy. Conjugated and unconjugated drugs were tested against Acanthamoeba castellanii belonging to the T4 genotype using amoebicidal assay and host cell cytotoxicity assay. Viability assays revealed that silver nanoparticles conjugated with amphotericin B (Amp-AgNPs) and nystatin (Nys-AgNPs) exhibited significant antiamoebic properties compared with drugs alone or AgNPs alone (P < 0.05) as determined by Trypan blue exclusion assay. In contrast, conjugation of fluconazole with AgNPs had limited effect on its antiamoebic properties. Notably, AgNP-coated drugs inhibited amoebae-mediated host cell cytotoxicity as determined by measuring lactate dehydrogenase release. Overall, here we present the development of a new formulation of more effective antiamoebic agents based on AgNPs coated with drugs that hold promise for future applications.

Ethyl Cellulose Coated Chitosan Microspheres of Metronidazole as Potential Anti-Amoebic Agent

Ethyl Cellulose Coated Chitosan Microspheres of Metronidazole as Potential Anti-Amoebic Agent

In Vitro Testing of Potential Entamoeba histolytica Pyruvate Phosphate Dikinase Inhibitors.

Adverse effects and resistance to metronidazole have motivated the search for new antiamoebic agents against Entamoeba histolytica. Control of amoeba growth may be achieved by inhibiting the function of the glycolytic enzyme and pyruvate phosphate dikinase (PPDK). In this study, we screened 10 compounds using an in vitro PPDK enzyme assay. These compounds were selected from a virtual screening of compounds in the National Cancer Institute database. The antiamoebic activity of the selected compounds was also evaluated by determining minimal inhibitory concentrations (MICs) and IC50 values using the nitro-blue tetrazolium reduction assay. Seven of the 10 compounds showed inhibitory activities against the adenosine triphosphate (ATP)/inorganic phosphate binding site of the ATP-grasp domain. Two compounds, NSC349156 (pancratistatin) and NSC228137 (7-ethoxy-4-[4-methylphenyl] sulfonyl-3-oxido-2, 1, 3-benzoxadiazol-3-ium), exhibited inhibitory effects on the growth of E. histolytica trophozoites with MIC values of 25 and 50 μM, and IC50 values of 14 and 20.7 μM, respectively.

ChemInform Abstract: Recent Progress on the Discovery of Antiamoebic Agents

AbstractReview: 92 refs.

Synthesis, antiamoebic and molecular docking studies of furan-thiazolidinone hybrids

In continuation of our previous work, a series of furan-thiazolidinone hybrids was prepared by Knoevenagel condensation of 3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-thiazolidin-4-one with different aryl aldehydes in presence of strong base. Some members of the series exhibited remarkable antiamoebic activity and cell viability. Three compounds (3, 6 and 11) showed excellent binding energy for Entamoeba histolytica O-acetyle-l-serine sulfohydrolase and Entamoeba histolytica thioredoxin reductase. These compounds demonstrated significant inhibition of O-acetyle-l-serine sulfohydrolase. The promising antiamoebic activity and enzymatic assay of 3, 6 and 11 make them promising molecules for further lead optimization in the development of novel antiamoebic agents.

Synthesis and biological evaluation of 4-(2-(dimethylamino)ethoxy)benzohydrazide derivatives as inhibitors of Entamoeba histolyica

In the quest for potent antiamoebic agents, a series of hydrazone hybrids (H1H30) have been designed and sequentially synthesized. The dimethylaminoethoxy and hydrazone entities incorporated into one molecule proved to be more persuasive and selective approach towards designing of antiamoebic agent. The synthesized compounds exhibited promising results against E. histolytica. The compound N′-(2-chlorobenzylidene)-4-(2-(dimethylamino) ethoxy)benzohydrazide was most impending among the series. Cytotoxicity profile showed better cell viability on lung cancer cell line (A549 cells) by MTT assay.

Design, synthesis and biological evaluation of quinazolin-4(3H)-one Schiff base conjugates as potential antiamoebic agents

In an effort to develop novel antiamoebic scaffolds having better efficacy than the standard drug metronidazole (IC50=1.80μM) used against Entamoeba histolytica, quinazolin-4(3H)-one Schiff base conjugates were synthesized and evaluated against HM1: IMSS strain of E. histolytica. Out of the thirteen compounds (S2-S14), six compounds (S2, S3, S4, S5, S6 and S11) were found to be better inhibitors than metronidazole and showed low cytotoxicity on HeLa cells, a cervical cancer cell line. The structure of intermediate compound S1 was confirmed by crystal structure studies.

ChemInform Abstract: Azole‐Based Compounds as Antiamoebic Agents: A Perspective Using Theoretical Calculations

AbstractReview: 98 refs.

Azole-based compounds as antiamoebic agents: a perspective using theoretical calculations

Diseases caused by protozoal organisms are responsible for significant mortality and morbidity worldwide. Amoebiasis caused byEntamoeba histolyticais an example of such diseases.