A strategy to enhance the water solubility of luminescent ß-diketonate-Europium(III) complexes for time-gated luminescence bioassays.

Luminescent ß-diketonate-europium(III) complexes have been found a wide range of applications in time-gated luminescence (TGL) bioassays, but their poor water solubility is a main problem that limits their effective uses. In this work we propose a simple and general strategy to enhance the water solubility of luminescent ß-diketonate-europium(III) complexes that permits facile synthesis and purification. By introducing the fluorinated carboxylic acid group into the structures of ß-diketone ligands, two highly water-soluble and luminescent Eu3+ complexes, PBBHD-Eu3+ and CPBBHD-Eu3+, were designed and synthesized. An excellent solubility exceeding 20 mg/mL for PBBHD-Eu3+ was found in a pure aqueous buffer, while it also displayed strong and long-lived luminescence (quantum yield φ = 26%, lifetime τ = 0.49 ms). After the carboxyl groups of PBBHD-Eu3+ were activated, the PBBHD-Eu3+-labeled streptavidin-bovine serum albumin (SA-BSA) conjugate was prepared, and successfully used for the immunoassay of human α-fetoprotein (AFP) and the imaging of an environmental pathogen Giardia lamblia under TGL mode, which demonstrated the practicability of PBBHD-Eu3+ for highly sensitive TGL bioassays. The carboxyl groups of PBBHD can also be easily derivatized with other reactive chemical groups, which enables PBBHD-Eu3+ to meet diverse requirements of biolabeling technique, to provide new opportunities for developing functional europium(III) complex biolabels serving for TGL bioassays.

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.

Development of New PCR Protocols to Detect Genetic Diversity in the Metronidazole Metabolism Genes in Susceptible and Refractory Clinical Samples of Giardia duodenalis.

PURPOSE: Investigating the genetic variation in thioredoxin reductase (TrxR) and nitroreductase (NR) genes in both treatment-resistant and -sensitive Giardia duodenalis isolates can provide valuable information in identifying potential markers of resistance to metronidazole. The rapid increase in metronidazole treatment failures suggests the presence of genetic resistance mechanisms. By analyzing these genes, researchers can gain insights into the efficacy of metronidazole against G. duodenalis and potentially develop alternative treatment strategies. In this regard, four G. duodenalis isolates (two clinically sensitive and two clinically resistant to metronidazole) were collected from various hospitals of Shiraz, southwestern Iran. METHODS: Parasitological methods including sucrose flotation and microscopy were employed for the primary confirmation of G. duodenalis cysts in stool samples. Microscopy-positive samples were approved by SSU-PCR amplification of the parasite DNA. All four positive G. duodenalis specimens at SSU-PCR were afterward analyzed utilizing designed primers based on important metronidazole metabolism genes including TrxR, NR1, and NR2. RESULTS: Unlike TrxR gene, the results of NR1 and NR2 genes showed that there are non-synonymous variations between sequences of treatment-sensitive and -resistant samples compared to reference sequences. Furthermore, the outcomes of molecular docking revealed that there is an interaction between the protein sequence and spatial shape of treatment-resistant samples and metronidazole in the position of serine amino acid based on the NR1 gene. CONCLUSION: This issue can be one of the possible factors involved in the resistance of Giardia parasites to metronidazole. To reach more accurate results, a large sample size along with simulation and advanced molecular dynamics investigations are needed.

Elimination of Giardia duodenalis BIV in vivo using natural extracts in microbiome and dietary supplements.

In this study, a combination therapy of several natural products was evaluated in vivo in the Giardia duodenalis infection model. G. duodenalis infected mice were treated as follows: distilled water (infected control C+), BIOintestil® (BIO; natural products of Cymbopogon martinii and Zingiber officinale), MicrobiomeX® (MBX; extract of Citrus sinensis and Citrus paradisi), MBX + BIO, Camellia sinensis tea (CPR; black tea). These natural compounds were administered in a dose of 100 mg/day and were compared to G. duodenalis-infected mice treated with albendazole (ALB; 50 mg/Kg/day) and metronidazole (MET; 500 mg/Kg/day), the conventional therapies used to this day. One group remained un-infected and untreated as our control group (C-). Treatment started 8 days after infection, and after 5 days of treatment (7 days for MET), all animals were followed for 15 days. We continuously checked for the presence of G. duodenalis by Faust method, in association with detection of the parasite by PCR from feces, as well for the presence of trophozoites in the intestinal mucosa after sacrifice. Animals treated with MBX, BIO and MBX + BIO presented an undetectable parasitic load until the 15th day of monitoring, while animals treated with CPR, MET and ALB continued to release cysts. Animals in the MBX, MBX + BIO, ALB groups consumed lower feed, MBX, CPR, MET had greater weight and MBX, MBX + BIO, BIO, CPR, C- consumed more water when compared to infected-group control. MBX and BIO alone or associated eliminated G. duodenalis without apparent adverse effects and animals of these groups showed better clinical performance in relation to those with high parasitic load. MET, ALB and CPR only decreased the number of cysts, indicating limitations and therapeutic failure.

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.

Effect of dexamethasone on experimental enteritis produced by Giardia lamblia in a Meriones unguiculatus model.

Our aim was to evaluate the impact of immunosuppression on the development of giardiasis. Thirty-six gerbils (4-6 weeks old) were distributed in four groups containing nine animals each: Control (CT); Control-Infected by Giardia lamblia (CTIn), Immunosuppressed (IS), and Immunosuppressed-Infected by G. lamblia (ISIn). Animals in the IS and ISIn groups received intramuscular dexamethasone solution for 25 days. On the 11th day, the animals in the CTIn and ISIn groups were inoculated with G. lamblia. After 14 days of infection, the 25th day of the experiment, all groups were euthanized. Four hours after euthanasia, the intestinal permeability was evaluated and sections of the duodenum and spleen were harvested for morphometric and histopathological analyses. Immunosuppressed groups showed a significant increase in intestinal permeability compared to control and infected groups. Considering that the infection can become chronic in immunosuppressed groups, we should be alert to the possibilities of chronic inflammatory changes, both locally and systemically, due to the loss of the intestinal barrier. Lesions were observed in the duodenal mucosa of the gerbils of the CTIn group, with reduced villi size, crypt hyperplasia, edema, and the presence of inflammatory infiltrate in the lamina propria. In the ISIn group, we observed no inflammation, long and intact villi, and a significant increase in the area of intestinal mucins, despite the large number of trophozoites identified. Our results suggest that exacerbation of the immune response has a direct relationship with the appearance of lesions during enteritis produced by G. lamblia in the assessed model.

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.

A novel yeast-based high-throughput method for the identification of protein palmitoylation inhibitors.

Protein S-acylation or palmitoylation is a widespread post-translational modification that consists of the addition of a lipid molecule to cysteine residues of proteins through a thioester bond. Palmitoylation and palmitoyltransferases (PATs) have been linked to several types of cancers, diseases of the central nervous system and many infectious diseases where pathogens use the host cell machinery to palmitoylate their effectors. Despite the central importance of palmitoylation in cell physiology and disease, progress in the field has been hampered by the lack of potent-specific inhibitors of palmitoylation in general, and of individual PATs in particular. Herein, we present a yeast-based method for the high-throughput identification of small molecules that inhibit protein palmitoylation. The system is based on a reporter gene that responds to the acylation status of a palmitoylation substrate fused to a transcription factor. The method can be applied to heterologous PATs such as human DHHC20, mouse DHHC21 and also a PAT from the parasite Giardia lamblia. As a proof-of-principle, we screened for molecules that inhibit the palmitoylation of Yck2, a substrate of the yeast PAT Akr1. We tested 3200 compounds and were able to identify a candidate molecule, supporting the validity of our method.

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).

Regulatory role of the intestinal microbiota in the immune response against Giardia.

Giardia duodenalis is one of the most commonly found intestinal parasites in mammalian hosts. Infections can generally be cleared by mounting an adequate protective immune response that is orchestrated through IL-17A. This study was aimed to investigate if and how the intestinal microbiome affects the protective Th17 response against Giardia by analysing and comparing the immune response following a G. muris and G. duodenalis infection in antibiotic treated and untreated mice. Depletion of the intestinal flora by antibiotic treatment had a severe effect on the infection dynamics of both Giardia species. Not only duration of infection was affected, but also the parasite burden increased significantly. Markers associated with a protective immune response, such as IL-17A and mannose binding lectin 2 were still significantly upregulated following infection in the antibiotic-treated mice, despite the lack of protection. On the other hand, the antibiotic treatment significantly decreased the level of IgA in the intestinal lumen by affecting its transporter and by reducing the number of IgA+ B-cells at the Peyer's patches. Furthermore, the depletion of the gut microbiota by antibiotics also significantly lowered the intestinal motility. The combination of these factors likely results in a decreased clearance of the parasite from the intestinal tract.

Synthesis, Antiprotozoal Activity, and Cheminformatic Analysis of 2-Phenyl-2H-Indazole Derivatives.

Indazole is an important scaffold in medicinal chemistry. At present, the progress on synthetic methodologies has allowed the preparation of several new indazole derivatives with interesting pharmacological properties. Particularly, the antiprotozoal activity of indazole derivatives have been recently reported. Herein, a series of 22 indazole derivatives was synthesized and studied as antiprotozoals. The 2-phenyl-2H-indazole scaffold was accessed by a one-pot procedure, which includes a combination of ultrasound synthesis under neat conditions as well as Cadogan's cyclization. Moreover, some compounds were derivatized to have an appropriate set to provide structure-activity relationships (SAR) information. Whereas the antiprotozoal activity of six of these compounds against E. histolytica, G. intestinalis, and T. vaginalis had been previously reported, the activity of the additional 16 compounds was evaluated against these same protozoa. The biological assays revealed structural features that favor the antiprotozoal activity against the three protozoans tested, e.g., electron withdrawing groups at the 2-phenyl ring. It is important to mention that the indazole derivatives possess strong antiprotozoal activity and are also characterized by a continuous SAR.

Saccharomyces boulardii as therapeutic alternative in experimental giardiasis.

AIMS: The objective of this study was to evaluate the effect of treatment with the probiotic Saccharomyces boulardii with or without metronidazole in experimental giardiasis. METHODS AND RESULTS: The effect of treatment with S. boulardii with or without metronidazole on the intestinal mucosa, the antioxidant defence system and the parasitic load was determined in experimental giardiasis. Eight groups of animals with infection and/or treatment with the probiotic and/or drugs for 1 week after infection with Giardia lamblia were used. A reduction of approximately 90% in the parasitic load was observed in all the treated groups. Saccharomyces boulardii attenuated the damage caused by infection in the intestinal mucosa preserving its architecture and inhibiting the oxidative stress induced by parasite and metronidazole. CONCLUSIONS: Saccharomyces boulardii was effective alone or in combination with metronidazole in resolving already established G. lamblia infection. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest the use of S. boulardii as an alternative treatment for giardiasis mainly in cases of resistance or intolerance to conventional treatment.

Intestinal protozoa and helminths in ulcerative colitis and the influence of anti-parasitic therapy on the course of the disease.

PURPOSE: The aim of this study is to determine the prevalence of intestinal helminths and protozoa in patients with ulcerative colitis (UC) and to estimate the influence of the anti-parasitic therapy on the course of the disease. METHODS: The study was conducted at the Research Institute of Epidemiology, Microbiology and Infectious Diseases and Coloproctology Department of the Republic Clinical Hospital №1 of the Ministry of Health of the Republic of Uzbekistan. One hundred UC patients and 200 healthy individuals were examined by triple coproscopy. Additionally, 20, 25 and 22 UC patients with Blastocystis infection were treated with nitazoxanide (1.0 g/day), mesalazine (1.5-2 g/day) or a combination of nitazoxanide (1.0 g/day) and mesalazine (≥1.5-2 g/day) for 14 consecutive days, respectively. Parasitological, clinical and endoscopic examinations were conducted before therapy, immediately after and 6 and 12 weeks after therapy completion. RESULTS: The overall prevalence of helminths in UC patients and control individuals was not significantly different: 14±3.4% and 8.5±1.9%, respectively (OR: 1.7524; 95% CI: 0.8258 to 3.7186; P=0.1). Giardia lamblia was the most prevalent parasite in both groups, but the difference compared to the control was insignificant (OR: 0.4565; 95% CI: 0.2020 to 1.0318; P=0.05). A significantly higher prevalence of Blastocystis sp., Chilomastix mesnili and Iodamoeba butschlii in UC patients compared to control individuals was found (P<0.0005): 65.0%, 14.0% and 22.0%, respectively. During all follow-up periods, the clinical response and clinical remission were not statistically different between the groups (P>0.05). Mucosal healing immediately and 6 weeks after therapy with a combination of nitazoxanide with mesalazine was significantly better than with a monotherapy of nitazoxanide, respectively (P<0.05). UC patients treated with a combination of nitazoxanide with mesalazine showed better mucosal healing than in patients treated with a monotherapy of mesalazine (P>0.05). CONCLUSIONS: Diagnosis of Blastocystis sp. should be introduced in the complex examination of UC patients. Further clinical studies are necessary for assessment of the efficiency of anti-Blastocystis therapy in UC patients.

In vitro-induction of metronidazole-resistant Giardia duodenalis is not associated with nucleotide alterations in the genes involved in pro-drug activation.

Giardiasis is an infectious disease caused by Giardia duodenalis. The pro-drug metronidazole (MTZ) is the first-line treatment for giardiasis. Parasite's proteins as pyruvate:ferredoxin oxidoreductase (PFOR), ferredoxin (Fd), nitroreductase-1 (NR-1) and thioredoxin reductase (TrxR) participate in MTZ activation. Here, we showed Giardia trophozoites long-term exposed to MTZ presented higher IC50 than controls, showing the drug influenced the parasite survival. That reduction in MTZ's susceptibility does not seem to be related to mutations in the genes pfor, fd, nr-1 or trxr. It points that different mechanism as alterations in other metabolic pathways can account for Giardia resistance to MTZ therapy.

Ultrastructural and proapoptotic-like effects of kaempferol in Giardia duodenalis trophozoites and bioinformatics prediction of its potential protein target.

BACKGROUND: Kaempferol (KPF) is a flavonoid with antiparasitic activity including experimental giardiasis which mechanism of action is unknown. OBJECTIVE: To analyse the cytotoxic effects of KPF on Giardia duodenalis trophozoites and to identify a likely parasite target of this compound. METHODS: We used inhibitory concentrations of KPF (IC25, IC50 and IC100) and albendazole (ABZ) as reference drug. The ultrastructure of the trophozoites was analysed by transmission electron microscopy (TEM) whilst apoptosis/necrosis, production of reactive oxygen species (ROS) and cell cycle progression were assessed by flow cytometry (FCM) and confocal laser microscopy (CLM). Ligand-protein docking analyses were carried out using KPF structure from a drug library and crystal structure of a G. duodenalis aldose reductase (GdAldRed) homolog. RESULTS: KPF provoked appearance of perinuclear and periplasmic spaces devoid of cytosolic content and multilamellar structures. KPF induced proapoptotic death associated with partial arrest in the S phase without ROS production. Bioinformatics approaches predicted that GdAldRed is a viable KPF target (ΔG = -7.09 kCal/mol), exhibiting 92% structural identity and a similar coupling pattern as its human homolog. CONCLUSIONS: KPF exerted a proapoptotic effect on G. duodenalis trophozoites involving partial interruption of DNA synthesis without oxidative stress or structure damage to chromatin and cytoskeletal structures. GdAldRed is a likely target underlying its antigiardial activity.

Enhanced Antigiardial Effect of Omeprazole Analog Benzimidazole Compounds.

Giardiasis is a diarrheal disease that is highly prevalent in developing countries. Several drugs are available for the treatment of this parasitosis; however, failures in drug therapy are common, and have adverse effects and increased resistance of the parasite to the drug, generating the need to find new alternative treatments. In this study, we synthesized a series of 2-mercaptobenzimidazoles that are derivatives of omeprazole, and the chemical structures were confirmed through mass, 1H NMR, and 13C NMR techniques. The in vitro efficacy compounds against Giardia, as well as its effect on the inhibition of triosephosphate isomerase (TPI) recombinant, were investigated, the inactivation assays were performed with 0.2 mg/mL of the enzyme incubating for 2 h at 37 °C in TE buffer, pH 7.4 with increasing concentrations of the compounds. Among the target compounds, H-BZM2, O2N-BZM7, and O2N-BZM9 had greater antigiardial activity (IC50: 36, 14, and 17 µM on trophozoites), and inhibited the TPI enzyme (K2: 2.3, 3.2, and 2.8 M-1 s-1) respectively, loading alterations on the secondary structure, global stability, and tertiary structure of the TPI protein. Finally, we demonstrated that it had low toxicity on Caco-2 and HT29 cells. This finding makes it an attractive potential starting point for new antigiardial drugs.

Triazoxins: Novel nucleosides with anti-Giardia activity.

Novel nucleoside analogues named "triazoxins" were synthesized. Of these, two analogues were found to be highly effective against Giardia lamblia, an intestinal parasite and a major cause of waterborne infection, worldwide. While compound 7 reduced the growth of trophozoites in culture (IC50, ~5 µM), compound 21 blocked the in vitro cyst production (IC50 ~5 µM). Compound 21 was also effective against trophozoites (IC50, ~36 µM). A third analogue (compound 8) was effective against both trophozoites (IC50, ~36 µM) and cysts (IC50, ~20 µM) although at higher concentration. Thus triazoxin analogues are unique and exhibit morphology (i.e., trohozoites or cysts) -specific effects against Giardia.

Click chemistry-facilitated comprehensive identification of proteins adducted by antimicrobial 5-nitroimidazoles for discovery of alternative drug targets against giardiasis.

Giardiasis and other protozoan infections are major worldwide causes of morbidity and mortality, yet development of new antimicrobial agents with improved efficacy and ability to override increasingly common drug resistance remains a major challenge. Antimicrobial drug development typically proceeds by broad functional screens of large chemical libraries or hypothesis-driven exploration of single microbial targets, but both strategies have challenges that have limited the introduction of new antimicrobials. Here, we describe an alternative drug development strategy that identifies a sufficient but manageable number of promising targets, while reducing the risk of pursuing targets of unproven value. The strategy is based on defining and exploiting the incompletely understood adduction targets of 5-nitroimidazoles, which are proven antimicrobials against a wide range of anaerobic protozoan and bacterial pathogens. Comprehensive adductome analysis by modified click chemistry and multi-dimensional proteomics were applied to the model pathogen Giardia lamblia to identify dozens of adducted protein targets common to both 5'-nitroimidazole-sensitive and -resistant cells. The list was highly enriched for known targets in G. lamblia, including arginine deiminase, α-tubulin, carbamate kinase, and heat shock protein 90, demonstrating the utility of the approach. Importantly, over twenty potential novel drug targets were identified. Inhibitors of two representative new targets, NADP-specific glutamate dehydrogenase and peroxiredoxin, were found to have significant antigiardial activity. Furthermore, all the identified targets remained available in resistant cells, since giardicidal activity of the respective inhibitors was not impacted by resistance to 5'-nitroimidazoles. These results demonstrate that the combined use of click chemistry and proteomics has the potential to reveal alternative drug targets for overcoming antimicrobial drug resistance in protozoan parasites.

Synthesis, characterization and antiparasitic activity of organometallic derivatives of the anthelmintic drug albendazole.

Helminthiases, a group of neglected tropical diseases, affect more than one billion people mainly in tropical and subtropical regions. Moreover, major intestinal protozoa have a significant impact on global public health. Albendazole (ABZ) is a broad-spectrum anthelmintic recommended by the World Health Organisation (WHO). However, drug resistance is emerging due to its widespread use. In order to tackle this problem, taking into account the spectacular results obtained with ferroquine, an organometallic derivatization of the antimalarial drug chloroquine, we have prepared, in this study, a series of new ferrocenyl and ruthenocenyl derivatives of the organic drug ABZ and assessed their activity against different helminths and protozoans, namely Trichuris muris, Heligmosomoides polygygrus, Schistosoma mansoni, Giardia lamblia, Haemonchus contortus and Toxoplasma gondii. The ferrocene-containing ABZ analogue 2d exhibited over 70% activity against T. muris adults in vitro at 200 µM and no toxicity to mammalian cells (IC50 >100 µM). H. polygyrus adults were not affected by any of the derivatives tested. Against T. gondii, the ferrocene-containing ABZ analogues 1a and 2d showed better in vitro activity than ABZ and low toxicity to the host cells. The activity of the analogous ruthenocenyl compound 2b against S. mansoni and T. gondii in vitro might be attributed to its toxicity towards the host cells rather than a specific antiparasitic activity. These results demonstrate that the derivatives show a species specific in vitro activity and the choice of the organometallic moieties attached to the organic drug is playing a very important role. Two of our organometallic compounds, namely 1b and 2d, were tested in T. muris infected mice. At a 400 mg kg-1 dose, the compounds showed moderate worm burden reductions but low worm expulsion rates. Overall, this work, which is one of the first studies reporting the potential of organometallic compounds on a very broad range of parasitic helminths and protozoan, is a clear confirmation of the potential of organometallic complexes against parasites of medical and veterinary importance.

An image-based Pathogen Box screen identifies new compounds with anti-Giardia activity and highlights the importance of assay choice in phenotypic drug discovery.

Giardia duodenalis, the most prevalent human intestinal parasite causes the disease, giardiasis. On an annual basis G. duodenalis infects ~1 billion people, of which ~280 million develop symptomatic disease. Giardiasis can be severe and chronic, causing malnutrition, stunted growth and poor cognitive development in children. Current treatment options rely on drugs with declining efficacy and side-effects. To improve the health and well-being of millions of people world-wide, new anti-Giardia drugs with different modes of action to currently used drugs are required. The Medicines for Malaria Venture's Pathogen Box, a collection of bio-active compounds specifically chosen to stimulate infectious disease drug discovery, represents an opportunity for the discovery of new anti-Giardia agents. While the anti-Giardia activity of Pathogen Box compounds has been reported, this work failed to identify known anti-Giardia controls within the compound set. It also reported the activity of compounds previously screened and shown to be inactive by others, suggesting data may be inaccurate. Given these concerns the anti-Giardia activity of Pathogen Box compounds was re-assessed in the current study. Data from this work identified thirteen compounds with anti-Giardia IC50 values ≤2 µM. Five of these compounds were reference compounds (marketed drugs with known anti-microbial activity), or analogues of compounds with previously described anti-Giardia activity. However, eight, including MMV676358 and MMV028694, which demonstrated potent sub-µM IC50s against assemblage A, B and metronidazole resistant parasites (0.3 µM and 0.9 µM respectively), may represent new leads for future drug development. Interestingly, only four of these compounds were identified in the previously reported Pathogen Box screen highlighting the importance of assay selection and design when assessing compounds for activity against infectious agents.