Imidazole Carbamates as a Promising Alternative for Treating Trichomoniasis: In Vitro Effects on the Growth and Gene Expression of Trichomonas vaginalis.

Metronidazole (MTZ) is the most common drug used against Trichomonas vaginalis (T. vaginalis) infections; however, treatment failures and high rates of recurrence of trichomoniasis have been reported, suggesting the presence of resistance in T. vaginalis to MTZ. Therefore, research into new therapeutic options against T. vaginalis infections has become increasingly urgent. This study investigated the trichomonacidal activity of a series of five imidazole carbamate compounds (AGR-1, AGR-2, AGR-3, AGR-4, and AGR-5) through in vitro susceptibility assays to determine the IC50 value of each compound. All five compounds demonstrated potent trichomonacidal activity, with IC50 values in the nanomolar range and AGR-2 being the most potent (IC50 400 nM). To gain insight into molecular events related to AGR-induced cell death in T. vaginalis, we analyzed the expression profiles of some metabolic genes in the trophozoites exposed to AGR compounds and MTZ. It was found that both AGR and MTZ compounds reduced the expression of the glycolytic genes (CK, PFK, TPI, and ENOL) and genes involved in metabolism (G6PD, TKT, TALDO, NADHOX, ACT, and TUB), suggesting that disturbing these key metabolic genes alters the survival of the T. vaginalis parasite and that they probably share a similar mechanism of action. Additionally, the compounds showed low cytotoxicity in the Caco-2 and HT29 cell lines, and the results of the ADMET analysis indicated that these compounds have pharmacokinetic properties similar to those of MTZ. The findings offer significant insights that can serve as a basis for future in vivo studies of the compounds as a potential new treatment against T. vaginalis.

Novel anti-Acanthamoeba effects elicited by a repurposed poly (ADP-ribose) polymerase inhibitor AZ9482.

Introduction: Acanthamoeba infection is a serious public health concern, necessitating the development of effective and safe anti-Acanthamoeba chemotherapies. Poly (ADP-ribose) polymerases (PARPs) govern a colossal amount of biological processes, such as DNA damage repair, protein degradation and apoptosis. Multiple PARP-targeted compounds have been approved for cancer treatment. However, repurposing of PARP inhibitors to treat Acanthamoeba is poorly understood. Methods: In the present study, we attempted to fill these knowledge gaps by performing anti-Acanthamoeba efficacy assays, cell biology experiments, bioinformatics, and transcriptomic analyses. Results: Using a homology model of Acanthamoeba poly (ADP-ribose) polymerases (PARPs), molecular docking of approved drugs revealed three potential inhibitory compounds: olaparib, venadaparib and AZ9482. In particular, venadaparib exhibited superior docking scores (-13.71) and favorable predicted binding free energy (-89.28 kcal/mol), followed by AZ9482, which showed a docking score of -13.20 and a binding free energy of -92.13 kcal/mol. Notably, the positively charged cyclopropylamine in venadaparib established a salt bridge (through E535) and a hydrogen bond (via N531) within the binding pocket. For comparison, AZ9482 was well stacked by the surrounding aromatic residues including H625, Y652, Y659 and Y670. In an assessment of trophozoites viability, AZ9482 exhibited a dose-and time-dependent anti-trophozoite effect by suppressing Acanthamoeba PARP activity, unlike olaparib and venadaparib. An Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis assay revealed AZ9482 induced trophozoite necrotic cell death rather than apoptosis. Transcriptomics analyses conducted on Acanthamoeba trophozoites treated with AZ9482 demonstrated an atlas of differentially regulated proteins and genes, and found that AZ9482 rapidly upregulates a multitude of DNA damage repair pathways in trophozoites, and intriguingly downregulates several virulent genes. Analyzing gene expression related to DNA damage repair pathway and the rate of apurinic/apyrimidinic (AP) sites indicated DNA damage efficacy and repair modulation in Acanthamoeba trophozoites following AZ9482 treatment. Discussion: Collectively, these findings highlight AZ9482, as a structurally unique PARP inhibitor, provides a promising prototype for advancing anti-Acanthamoeba drug research.

Combating the causative agent of amoebic keratitis, Acanthamoeba castellanii, using Padina pavonica alcoholic extract: toxicokinetic and molecular docking approaches.

Natural products play a significant role in providing the current demand as antiparasitic agents, which offer an attractive approach for the discovery of novel drugs. The present study aimed to evaluate in vitro the potential impact of seaweed Padina pavonica (P. pavonica) extract in combating Acanthamoeba castellanii (A. castellanii). The phytochemical constituents of the extract were characterized by Gas chromatography-mass spectrometry. Six concentrations of the algal extract were used to evaluate its antiprotozoal activity at various incubation periods. Our results showed that the extract has significant inhibition against trophozoites and cysts viability, with complete inhibition at the high concentrations. The IC50 of P. pavonica extract was 4.56 and 4.89 µg/mL for trophozoites and cysts, respectively, at 24 h. Morphological alterations of A. castellanii trophozoites/cysts treated with the extract were assessed using inverted and scanning electron microscopes and showed severe damage features upon treatment with the extract at different concentrations. Molecular Docking of extracted compounds against Acanthamoeba cytochrome P450 monooxygenase (AcCYP51) was performed using Autodock vina1.5.6. A pharmacokinetic study using SwissADME was also conducted to investigate the potentiality of the identified bioactive compounds from Padina extract to be orally active drug candidates. In conclusion, this study highlights the in vitro amoebicidal activity of P. pavonica extract against A. castellanii adults and cysts and suggests potential AcCYP51 inhibition.

The anti-amoebic activity of Pinus densiflora leaf extract against the brain-eating amoeba Naegleria fowleri.

Naegleria fowleri invades the brain and causes a fatal primary amoebic meningoencephalitis (PAM). Despite its high mortality rate of approximately 97%, an effective therapeutic drug for PAM has not been developed. Approaches with miltefosine, amphotericin B, and other antimicrobials have been clinically attempted to treat PAM, but their therapeutic efficacy remains unclear. The development of an effective and safe therapeutic drug for PAM is urgently needed. In this study, we investigated the anti-amoebic activity of Pinus densiflora leaf extract (PLE) against N. fowleri. PLE induced significant morphological changes in N. fowleri trophozoites, resulting in the death of the amoeba. The IC50 of PLE on N. fowleri was 62.3±0.95 µg/ml. Alternatively, PLE did not significantly affect the viability of the rat glial cell line C6. Transcriptome analysis revealed differentially expressed genes (DEGs) between PLE-treated and non-treated amoebae. A total of 5,846 DEGs were identified, of which 2,189 were upregulated, and 3,657 were downregulated in the PLE-treated amoebae. The DEGs were categorized into biological process (1,742 genes), cellular component (1,237 genes), and molecular function (846 genes) based on the gene ontology analysis, indicating that PLE may have dramatically altered the biological and cellular functions of the amoeba and contributed to their death. These results suggest that PLE has anti-N. fowleri activity and may be considered as a potential candidate for the development of therapeutic drugs for PAM. It may also be used as a supplement compound to enhance the therapeutic efficacy of drugs currently used to treat PAM.

Andrographolide induced cytotoxicity and cell cycle arrest in Giardia trophozoites.

Giardiasis is a prevalent parasitic diarrheal disease caused by Giardia lamblia, affecting people worldwide. Recently, the availability of several drugs for its treatment has highlighted issues such as multidrug resistance, limited effectiveness and undesirable side effects. Therefore, it is necessary to develop alternative new drugs and treatment strategies that can enhance therapeutic outcomes and effectively treat giardiasis. Natural compounds show promise in the search for more potent anti-giardial agents. Our investigation focused on the effect of Andrographolide (ADG), an active compound of the Andrographis paniculata plant, on Giardia lamblia, assessing trophozoite growth, morphological changes, cell cycle arrest, DNA damage and inhibition of gene expression associated with pathogenic factors. ADG demonstrated anti-Giardia activity almost equivalent to the reference drug metronidazole, with an IC50 value of 4.99 µM after 24 h of incubation. In cytotoxicity assessments and morphological examinations, it showed significant alterations in trophozoite shape and size and effectively hindered the adhesion of trophozoites. It also caused excessive ROS generation, DNA damage, cell cycle arrest and inhibited the gene expression related to pathogenesis. Our findings have revealed the anti-giardial efficacy of ADG, suggesting its potential as an agent against Giardia infections. This could offer a natural and low-risk treatment option for giardiasis, reducing the risk of side effects and drug resistance.

Flucofuron as a Promising Therapeutic Agent against Brain-Eating Amoeba.

Primary amoebic meningoencephalitis (PAM) is a rare and fulminant neurodegenerative disease caused by the free-living amoeba Naegleria fowleri. Currently, there is a lack of standardized protocols for therapeutic action. In response to the critical need for effective therapeutic agents, we explored the Global Health Priority Box, a collection of 240 compounds provided by the Medicines for Malaria Venture (MMV). From this pool, flucofuron emerged as a promising candidate, exhibiting high efficacy against trophozoites of both N. fowleri strains (ATCC 30808 IC50 : 2.58 ± 0.64 µM and ATCC 30215 IC50: 2.47 ± 0.38 µM), being even active against the resistant cyst stage (IC50: 0.88 ± 0.07 µM). Moreover, flucofuron induced diverse metabolic events that suggest the triggering of apoptotic cell death. This study highlights the potential of repurposing medications for treating challenging diseases, such as PAM.

Promising effects of 1,8 Cineole to control Giardia lamblia infection: Targeting the inflammation, oxidative stress, and infectivity.

Reportedly, synthetic drugs such as metronidazole, furazolidone, tinidazole, and quinacrine are used for the treatment of giardiasis but are associated with adverse effects. In this study, we aimed to investigate the in vitro and in vivo effects of eucalyptol (ECT, 1,8 cineole) alone and in combination with metronidazole (MNZ) on Giardia lamblia. The effects of ECT on cell viability, plasma membrane permeability, and gene expression levels of adenylate cyclase (AK) and extracellular signal kinases 1 and 2 (ERK1 and ERK2) in trophozoites of G. lamblia were assessed. In vivo, the effects of ECT alone and in combination with MNZ were assessed on mice infected with G. lamblia. In addition, the gene expression of inflammatory genes (e.g., TNF-α, IL-1ß, and IL-10) and antioxidant genes (catalase (CAT), superoxide dismutase 1 (SOD1), glutathione peroxidase 2 (GPX2)) was determined by real-time PCR. The IC50 values of ECT, MNZ, and ECT+MNZ on trophozoites were 30.2 µg/mL, 21.6 µg/mL, and 8.5 µg/mL, respectively. The estimated Fractional inhibitory concentration index (FICI) values for ECT and MNZ were 0.28 and 0.39, respectively. The application of ECT on G. lamblia trophozoites resulted in a dose-dependent increase in plasma membrane permeability, particularly at concentrations of ½ IC50 and IC50 (P < 0.05). The treatment of infected mice with various doses of ECT, mainly in combination with MNZ for 7 days, resulted in a significant decrease (P < 0.001) in the average number and viability of cysts. ECT, especially when combined with MNZ, caused a significant (P < 0.001) reduction in the expression of TNF-α and IL-6 genes, and an increase (P < 0.05) in the expression of IL-10 genes. ECT alone and mainly in combination with MNZ leads to a significant (P < 0.001) increase in the gene expression of CAT, SOD, and GPX genes. These findings demonstrate that the use of ECT in these doses, even for 14 days, does not have any toxic effects on the function of vital liver and kidney tissues. The study findings confirmed the promising effects of ECT against G. lamblia infection both in vitro and in vivo. Considering the possible mechanisms, ECT increases plasma membrane permeability and reduces the expression levels of infectivity-related genes. In addition, ECT suppresses inflammation and oxidative stress, controlling giardiasis in mice. More studies are needed to clarify these findings.

Alpha-terthienyl increases filamentous actin of Entamoeba histolytica.

This study aimed to know if alpha terthienyl (α-T) affects E. histolytica viability and to analyze its effect on the actin cytoskeleton. Trophozoites of E. histolytica HM1-IMSS were treated with α-T, then, cell viability and morphology were evaluated using tetrazolium salts and scanning electron microscopy, respectively; while actin filaments (F-actin) were stained with rhodamine-phalloidin, observed by confocal microscopy and quantified by fluorometry. Data showed that α-T inhibited cell viability of trophozoites (IC50, 19.43 µg / mL), affected the cell morphology, and increased the F-actin in a dose-dependent manner. Production of reactive oxygen species and RhoA-GTP levels remained normal in α-T-treated amebas. Two inhibitors that affect the organization of the trophozoites cytoskeleton, one that interacts directly with actin, Cytochalasin D (CD), and one that affects the Rho signaling pathway by inhibiting the downstream effector Rock, Y27632, were tested. Y27632 did not affect the increase of polymerized actin observed with α-T, this compound partially ameliorates the potent disrupting effects of CD on actin filaments. Docking results suggest that α-T could be an antagonist of CD for the same interaction zone in actin, however, more studies are needed to define the action mechanism of this compound.

Amoebicidal Activity of Poly-Epsilon-Lysine Functionalized Hydrogels.

Purpose: To determine the amoebicidal activity of functionalized poly-epsilon-lysine hydrogels (pɛK+) against Acanthamoeba castellanii. Methods: A. castellanii trophozoites and cysts were grown in the presence of pɛK solution (0-2.17 mM), pɛK or pɛK+ hydrogels, or commercial hydrogel contact lens (CL) for 24 hours or 7 days in PBS or Peptone-Yeast-Glucose (PYG) media (nutrient-deplete or nutrient-replete cultures, respectively). Toxicity was determined using propidium iodide and imaged using fluorescence microscopy. Ex vivo porcine corneas were inoculated with A. castellanii trophozoites ± pɛK, pɛK+ hydrogels or commercial hydrogel CL for 7 days. Corneal infection was assessed by periodic acid-Schiff staining and histologic analysis. Regrowth of A. castellanii from hydrogel lenses and corneal discs at 7 days was assessed using microscopy and enumeration. Results: The toxicity of pɛK+ hydrogels resulted in the death of 98.52% or 83.31% of the trophozoites at 24 hours or 7 days, respectively. The toxicity of pɛK+ hydrogels resulted in the death of 70.59% or 82.32% of the cysts in PBS at 24 hours or 7 days, respectively. Cysts exposed to pɛK+ hydrogels in PYG medium resulted in 75.37% and 87.14% death at 24 hours and 7 days. Ex vivo corneas infected with trophozoites and incubated with pɛK+ hydrogels showed the absence of A. castellanii in the stroma, with no regrowth from corneas or pɛK+ hydrogel, compared with infected-only corneas and those incubated in presence of commercial hydrogel CL. Conclusions: pɛK+ hydrogels demonstrated pronounced amoebicidal and cysticidal activity against A. castellanii. pɛK+ hydrogels have the potential for use as CLs that could minimize the risk of CL-associated Acanthamoeba keratitis.

Giardia duodenalis and Its Secreted PPIB Trigger Inflammasome Activation and Pyroptosis in Macrophages through TLR4-Induced ROS Signaling and A20-Mediated NLRP3 Deubiquitination.

The extracellular protozoan parasite Giardia duodenalis is a well-known and important causative agent of diarrhea on a global scale. Macrophage pyroptosis has been recognized as an important innate immune effector mechanism against intracellular pathogens. Yet, the effects of noninvasive Giardia infection on macrophage pyroptosis and the associated molecular triggers and regulators remain poorly defined. Here we initially observed that NLRP3 inflammasome-mediated pyroptosis was activated in Giardia-treated macrophages, and inhibition of ROS, NLRP3, or caspase-1 could block GSDMD cleavage, IL-1ß, IL-18 and LDH release, and the cell viability reduction. We also confirmed that Giardia-induced NLRP3 inflammasome activation was involved in its K63 deubiquitination. Thus, six candidate deubiquitinases were screened, among which A20 was identified as an effective regulator. We then screened TLRs on macrophage membranes and found that upon stimulation TLR4 was tightly correlated to ROS enhancement, A20-mediated NLRP3 deubiquitination, and pyroptotic signaling. In addition, several Giardia-secreted proteins were predicted as trigger factors via secretome analysis, of which peptidyl-prolyl cis-trans isomerase B (PPIB) independently induced macrophage pyroptosis. This was similar to the findings from the trophozoite treatment, and also led to the TLR4-mediated activation of NLRP3 through K63 deubiquitination by A20. Collectively, the results of this study have significant implications for expanding our understanding of host defense mechanisms after infection with G. duodenalis.

The transcriptome of Balamuthia mandrillaris trophozoites for structure-guided drug design.

Balamuthia mandrillaris, a pathogenic free-living amoeba, causes cutaneous skin lesions as well as granulomatous amoebic encephalitis, a 'brain-eating' disease. As with the other known pathogenic free-living amoebas (Naegleria fowleri and Acanthamoeba species), drug discovery efforts to combat Balamuthia infections of the central nervous system are sparse; few targets have been validated or characterized at the molecular level, and little is known about the biochemical pathways necessary for parasite survival. Current treatments of encephalitis due to B. mandrillaris lack efficacy, leading to case fatality rates above 90%. Using our recently published methodology to discover potential drugs against pathogenic amoebas, we screened a collection of 85 compounds with known antiparasitic activity and identified 59 compounds that impacted the growth of Balamuthia trophozoites at concentrations below 220 µM. Since there is no fully annotated genome or proteome of B. mandrillaris, we sequenced and assembled its transcriptome from a high-throughput RNA-sequencing (RNA-Seq) experiment and located the coding sequences of the genes potentially targeted by the growth inhibitors from our compound screens. We determined the sequence of 17 of these target genes and obtained expression clones for 15 that we validated by direct sequencing. These will be used in the future in combination with the identified hits in structure guided drug discovery campaigns to develop new approaches for the treatment of Balamuthia infections.

Multilevel Approach for the Treatment of Giardiasis by Targeting Arginine Deiminase.

Giardiasis represents a latent problem in public health due to the exceptionally pathogenic strategies of the parasite Giardia lamblia for evading the human immune system. Strains resistant to first-line drugs are also a challenge. Therefore, new antigiardial therapies are urgently needed. Here, we tested giardial arginine deiminase (GlADI) as a target against giardiasis. GlADI belongs to an essential pathway in Giardia for the synthesis of ATP, which is absent in humans. In silico docking with six thiol-reactive compounds was performed; four of which are approved drugs for humans. Recombinant GlADI was used in enzyme inhibition assays, and computational in silico predictions and spectroscopic studies were applied to follow the enzyme's structural disturbance and identify possible effective drugs. Inhibition by modification of cysteines was corroborated using Ellman's method. The efficacy of these drugs on parasite viability was assayed on Giardia trophozoites, along with the inhibition of the endogenous GlADI. The most potent drug against GlADI was assayed on Giardia encystment. The tested drugs inhibited the recombinant GlADI by modifying its cysteines and, potentially, by altering its 3D structure. Only rabeprazole and omeprazole decreased trophozoite survival by inhibiting endogenous GlADI, while rabeprazole also decreased the Giardia encystment rate. These findings demonstrate the potential of GlADI as a target against giardiasis.

Virtual Screening of FDA-Approved Drugs against Triose Phosphate Isomerase from Entamoeba histolytica and Giardia lamblia Identifies Inhibitors of Their Trophozoite Growth Phase.

Infectious diseases caused by intestinal protozoan, such as Entamoeba histolytica (E. histolytica) and Giardia lamblia (G. lamblia) are a worldwide public health issue. They affect more than 70 million people every year. They colonize intestines causing primarily diarrhea; nevertheless, these infections can lead to more serious complications. The treatment of choice, metronidazole, is in doubt due to adverse effects and resistance. Therefore, there is a need for new compounds against these parasites. In this work, a structure-based virtual screening of FDA-approved drugs was performed to identify compounds with antiprotozoal activity. The glycolytic enzyme triosephosphate isomerase, present in both E. histolytica and G. lamblia, was used as the drug target. The compounds with the best average docking score on both structures were selected for the in vitro evaluation. Three compounds, chlorhexidine, tolcapone, and imatinib, were capable of inhibit growth on G. lamblia trophozoites (0.05-4.935 µg/mL), while folic acid showed activity against E. histolytica (0.186 µg/mL) and G. lamblia (5.342 µg/mL).

In vitro anti-Acanthamoeba activity of flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and Aconitum napellus.

Acanthamoeba spp. are widely distributed in the environment and cause serious infections in humans. Treatment of Acanthamoeba infections is very challenging and not always effective which requires the development of more efficient drugs against Acanthamoeba spp. The purpose of the present study was to test medicinal plants that may be useful in the treatment of Acanthamoeba spp. Here we evaluated the trophozoital and cysticidal activity of 13 flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and from Aconitum napellus subsp. Lusitanicum against the amoeba Acanthamoeba castellanii. AlamarBlue Assay Reagent® was used to determine the activity against trophozoites of A. castellanii, and cytotoxic using Vero cells. Cysticidal activity was assessed on treated cysts by light microscopy using a Neubauer chamber to quantify cysts and trophozoites. Flavonoids 1, 2, 3 and 4 showed higher trophozoital activity and selectivity indexes than the reference drug chlorhexidine digluconate. In addition, flavonoid 2 showed 100% cysticidal activity at a concentration of 50 µm, lower than those of the reference drug and flavonoid 3 (100 µm). These results suggest that flavonoids 2 and 3 might be used for the development of novel therapeutic approaches against Acanthamoeba infections after satisfactory in vivo evaluations.

'de Novo' repurposing of Daflon as anti-intestinal parasitic drug in experimental giardiasis.

BACKGROUND: There is a necessity to develop or discover an alternative drug to combat the drug resistance by Giardia duodenalis and minimize the multiple doses and frequency of the conventional drug administration. Progressive repositioning or 'repurposing' of drugs has become widespread due to economic circumstances and medical emergency needs. Daflon 500 mg (DFL) is a natural product used safely as a nutrient supplement and an antidiabetic drug in many European countries and the US. OBJECTIVE: This study aimed at investigating the efficiency of DFL, in vivo, in a murine model as a safe alternative or co-drug for giardiasis. MATERIALS AND METHODS: Swiss Albino mice (n = 32) were inoculated with 1X104Giardia cysts and assigned to four groups: One group was the infected non-treated control mice and three experimental groups that were treated differently, either with Metronidazole (MTZ), DFL, or combined therapy of DFL/MTZ. Also, eight normal mice served as a control group. All mice were sacrificed 13 days post-infection for the parasitic, histopathological, and oxidative stress analysis. RESULTS: MTZ, DFL, and the combined therapy significantly reduced the number of trophozoites and cysts compared to their counterparts of the infected mice. The histopathological analysis of the small intestines of the mice treated with the combined therapy retained typical intestinal architecture and normal levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione. CONCLUSION: This study indicated promising actions of Daflon 500 as an anti-giardial drug, and the results demonstrated its potential effect in improving the intestinal epithelial tissue and disturbing the Giardia stages when it was taken collectively with Metronidazole.

The amoebicidal activity of three substances derived from benzothiazole on Acanthamoeba castellanii cysts and trophozoites and its cytotoxic potentials.

Acanthamoeba species are free-living amoebae isolated from many ecological areas such as swimming pools, dams, lakes, soil, and air filters. These amoebae are usually causing granulomatous amebic encephalitis and amebic keratitis in immunosuppressive individuals. In this study, the reproductive potential and morphological changes determined of Acanthamoeba castellanii trophozoite and cyst forms exposed to three different active substances derived from benzothiazole. Furthermore, the cytotoxic potential of these active substances determined by XTT analysis. In the study, axenic cultures prepared for Acanthamoeba castellanii cyst and trophozoite forms and parasite exposed to different concentrations of active substances. Cell counts of parasite cultures were performed at the 30 minutes, 1st, 6th, 12th, 24th, and 48th hour periods. As a result of the study, the reproductive potential suppressive effects of all three substances on Acanthamoeba castellanii trophozoites and cysts were determined. The most effective of these substances was 2-Amino-6(trifluoromethoxy)-benzothiazole. In the first three concentrations of this substance (0.1%, 0.05%, 0.025%), no determined trophozoite and cysts at the end of twenty four. Due to its strong ameobicidal effect, it is thought that 2-Amino-6(trifluoromethoxy)-benzothiazole may be a new therapeutic agent in diseases caused by acanthamoeba parasites by supporting this study with animal experiments.

Gold(I) Phosphine Derivatives with Improved Selectivity as Topically Active Drug Leads to Overcome 5-Nitroheterocyclic Drug Resistance in Trichomonas vaginalis.

Trichomonas vaginalis causes the most common, nonviral sexually transmitted infection. Only metronidazole (Mz) and tinidazole are approved for treating trichomoniasis, yet resistance is a clinical problem. The gold(I) complex, auranofin, is active against T. vaginalis and other protozoa but has significant human toxicity. In a systematic structure-activity exploration, we show here that diversification of gold(I) complexes, particularly as halides with simple C1-C3 trialkyl phosphines or as bistrialkyl phosphine complexes, can markedly improve potency against T. vaginalis and selectivity over human cells compared to that of the existing antirheumatic gold(I) drugs. All gold(I) complexes inhibited the two most abundant isoforms of the presumed target enzyme, thioredoxin reductase, but a subset of compounds were markedly more active against live T. vaginalis than the enzyme, suggesting that alternative targets exist. Furthermore, all tested gold(I) complexes acted independently of Mz and were able to overcome Mz resistance, making them candidates for the treatment of Mz-refractory trichomoniasis.

Antineoplastic kinase inhibitors: A new class of potent anti-amoebic compounds.

Entamoeba histolytica is a protozoan parasite which infects approximately 50 million people worldwide, resulting in an estimated 70,000 deaths every year. Since the 1960s E. histolytica infection has been successfully treated with metronidazole. However, drawbacks to metronidazole therapy exist, including adverse effects, a long treatment course, and the need for an additional drug to prevent cyst-mediated transmission. E. histolytica possesses a kinome with approximately 300-400 members, some of which have been previously studied as potential targets for the development of amoebicidal drug candidates. However, while these efforts have uncovered novel potent inhibitors of E. histolytica kinases, none have resulted in approved drugs. In this study we took the alternative approach of testing a set of twelve previously FDA-approved antineoplastic kinase inhibitors against E. histolytica trophozoites in vitro. This resulted in the identification of dasatinib, bosutinib, and ibrutinib as amoebicidal agents at low-micromolar concentrations. Next, we utilized a recently developed computational tool to identify twelve additional drugs with human protein target profiles similar to the three initial hits. Testing of these additional twelve drugs led to the identification of ponatinib, neratinib, and olmutinib were identified as highly potent, with EC50 values in the sub-micromolar range. All of these six drugs were found to kill E. histolytica trophozoites as rapidly as metronidazole. Furthermore, ibrutinib was found to kill the transmissible cyst stage of the model organism E. invadens. Ibrutinib thus possesses both amoebicidal and cysticidal properties, in contrast to all drugs used in the current therapeutic strategy. These findings together reveal antineoplastic kinase inhibitors as a highly promising class of potent drugs against this widespread and devastating disease.

Derivatisation of metronidazole enhances cytotoxic effect against Acanthamoeba genotype T4 isolates and leads to cytomorphological changes in trophozoites.

Amoebae of the genus Acanthamoeba are worldwide distributed causative agents of serious human infections such as granulomatous amoebic encephalitis (GAE) and Acanthamoeba keratitis (AK). To date, treatment of these infections is non-uniform and frequently unsuccessful. Recently, the phosphonium salts were studied for their high levels of antimicrobial activity. This work was aimed to investigate the cytotoxic effect of metronidazole and two phosphonium salts (PS1, PS2) on two clinical Acanthamoeba isolates. The isolates showed distinctly higher susceptibility to both phosphonium salts than to metronidazole. The highest susceptibility was noted to PS1 after 48 h of incubation. Metronidazole derivate PS2 showed higher susceptibility than metronidazole. The values of EC50 of PS2 were approximately twenty times lower than EC50 of metronidazole for Acanthamoeba lugdunensis strain and sixteen times lower for Acanthamoeba quina strain after 48 h. Although the therapeutic effect of metronidazole in Acanthamoeba infections is usually insufficient, its derivatisation can result in a significantly higher amoebicidal effect. Cytomorphological changes of trophozoites after exposure to tested compounds included rounding up of the cells, damage of membrane integrity, presence of pathological protrusions, elongation of the cells or pseudocyst-like stages. Obtained results indicate possible therapeutic potential of studied phosphonium salts.

'Targeting the feast of a sleeping beast': Nutrient and mineral dependencies of encysted Acanthamoeba castellanii.

Acanthamoeba spp. cause a corneal infection, Acanthamoeba keratitis (AK), and a cerebral infection, granulomatous amoebic encephalitis (GAE). Though aggressive chemotherapy has been able to kill the active trophozoite form of Acanthamoeba, the encysted form of this parasite has remained problematic to resist physiological concentrations of drugs. The emergence of encysted amoeba into active trophozoite form poses a challenge to eradicate this parasite. Acanthamoeba trophozoites have active metabolic machinery that furnishes energy in the form of ATPs by subjecting carbohydrates and lipids to undergo pathways including glycolysis and beta-oxidation of free fatty acids, respectively. However, very little is known about the metabolic preferences and dependencies of an encysted trophozoite on minerals or potential nutrients that it consumes to live in an encysted state. Here, we investigate the metabolic and nutrient preferences of the encysted trophozoite of Acanthamoeba castellanii and the possibility to target them by drugs that act on calcium ion dependencies of the encysted amoeba. The experimental assays, immunostaining coupled with bioinformatics tools show that the encysted Acanthamoeba uses diverse nutrient pathways to obtain energy in the quiescent encysted state. These findings highlight potential pathways that can be targeted in eradicating amoebae cysts successfully.