In vitro and in vivo anti-Trypanosoma cruzi activity of a novel nitro-derivative

Nitroarylidenemalononitriles and their cyanoacetamide derivatives with remarkable anti-epimastigote properties, were synthesized attempting to obtain new 3,5-diamino-4-(5'-nitroarylidene)-4H-thiadiazine 1,1-dioxide derivatives, which in previous reports had shown anti-Trypanosoma cruzi activity. Tests to evaluate the cytotoxicity of compounds were performed on J774 macrophages. 5-nitro-2-thienyl-malononitrile (5NO2TM), was the only product which maintained a high anti-epimastigote activity at concentrations in which it was no longer cytotoxic, thus it was assayed against intracellular amastigotes. Its anti-amastigote activity was similar to that of nifurtimox. Afterwards in vivo toxicity and anti-chagasic activity were determined. A reduction in parasitemia was observed.     

DNA as molecular target of analogous palladium and platinum anti-Trypanosoma cruzi compounds: a comparative study

In the search for drugs with anti-trypanosome activity, we had previously synthesized two series of platinum and palladium analogous compounds of the formula [M^IICl₂(HL)], where HL were bioactive 5-nitrofuryl or 5-nitroacroleine thiosemicarbazone derivatives. In this work, we thoroughly characterized [M^IICl₂(HL)] complexes interaction with DNA by using different techniques: gel electrophoresis, DNA viscosity measurements, circular dichroism (CD) and atomic force microscopy (AFM). Electrophoresis results showed that all complexes induced a withdrawal of DNA superhelicity demonstrated by a decrease in electrophoretic mobility of supercoiled DNA form. This effect on migration was dependent on dose but also on the nature of both the metal and the ligand. In general, the effect produced by palladium complexes was significantly more intense than that observed for the corresponding platinum analogs. Differences between palladium and platinum complexes were also observed in CD experiments. While palladium complexes induce evident calf thymus (CT)-DNA profile changes compatible with B-DNA to Z-DNA conformational transition, no clear effect was observed for platinum ones. Additionally, AFM studies showed that changes in the shape of plasmid DNA, like supercoiling, kinks and thickness increase resulted more intense for the former. In addition, either Pd or Pt complexes increased the viscosity of CT DNA solutions in a concentration dependent manner. Although the nature of DNA interaction of both series of analogous palladium and platinum complexes seemed to be similar, an explanation for the observed differential intensity of the effect could be related to the known kinetic stability differences between palladium and platinum compounds.     

Optimization of anti-Trypanosoma cruzi oxadiazoles leads to identification of compounds with efficacy in infected mice

We recently showed that oxadiazoles have anti-Trypanosoma cruzi activity at micromolar concentrations. These compounds are easy to synthesize and show a number of clear and interpretable structure–activity relationships (SAR), features that make them attractive to pursue potency enhancement. We present here the structural design, synthesis, and anti-T. cruzi evaluation of new oxadiazoles denoted 5a–h and 6a–h. The design of these compounds was based on a previous model of computational docking of oxadiazoles on the T. cruzi protease cruzain. We tested the ability of these compounds to inhibit catalytic activity of cruzain, but we found no correlation between the enzyme inhibition and the antiparasitic activity of the compounds. However, we found reliable SAR data when we tested these compounds against the whole parasite. While none of these oxadiazoles showed toxicity for mammalian cells, oxadiazoles 6c (fluorine), 6d (chlorine), and 6e (bromine) reduced epimastigote proliferation and were cidal for trypomastigotes of T. cruzi Y strain. Oxadiazoles 6c and 6d have IC₅₀ of 9.5 ± 2.8 and 3.5 ± 1.8 μM for trypomastigotes, while Benznidazole, which is the currently used drug for Chagas disease treatment, showed an IC₅₀ of 11.3 ± 2.8 μM. Compounds 6c and 6d impair trypomastigote development and invasion in macrophages, and also induce ultrastructural alterations in trypomastigotes. Finally, compound 6d given orally at 50 mg/kg substantially reduces the parasitemia in T. cruzi-infected BALB/c mice. Our drug design resulted in potency enhancement of oxadiazoles as anti-Chagas disease agents, and culminated with the identification of oxadiazole 6d, a trypanosomicidal compound in an animal model of infection.     

Thiosemicarbazones derived from 1-indanones as new anti-Trypanosoma cruzi agents

In the present work, we synthesized a series of thiosemicarbazones derived from 1-indanones with good anti-Trypanosoma cruzi activity. Most of them displayed remarkable trypanosomicidal activity. All the compounds showed nonspecific cytotoxicity on human erythrocytes. The ability of the new compounds to inhibit cruzipain, the major cysteine protease of T. cruzi, was also explored. Thiosemicarbazones 12 and 24 inhibited this enzyme at the dose assayed. This interaction was also studied in terms of molecular docking.     

Mutagenicity of CL 64855, a potent anti-Trypanosoma cruzi drug

The nitroimidazole-thiadiazole derivative CL 64855 (2-amino-5 (1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole), a potent antimicrobial agent with curative action against Trypanosoma cruzi, was assayed in the Salmonella/microsome test. CL 64855 proved to be a potent mutagen to the frameshift indicator tester strains TA98 and TA102. No activity was observed with the base-pair substitution mutagen indicator strain TA100 in spot tests. No significant increase in the number of induced mutants could be detected in the presence of rat-liver microsome fraction. The excision-repair-deficient strain TA98 was much more sensitive to the killing action of CL 64855 than TA102, a repair-proficient strain. Possible differences among the mutagenic effects of CL 64855 and those observed with other anti-trypanosomal drugs are discussed.     

Synthesis and anti-Trypanosoma cruzi activity of derivatives from nor-lapachones and lapachones

New naphthoquinone derivatives were synthesized and assayed against bloodstream trypomastigote forms of Trypanosoma cruzi, the etiological agent of Chagas’ disease. The compounds were rationalized based on hybrid drugs and appear as important compounds against this parasite. From nor-lapachol were prepared five substituted ortho-naphthofuranquinones, a non-substituted para-naphthofuranquinone, a new oxyrane and an azide and from -lapachone a new non-substituted para-naphthofuranquinone. Other five substituted ortho-naphthofuranquinones recently designed as cytotoxic, were also evaluated. The most active compounds were the ortho naphthofuranquinones 3-(4-methoxyphenylamino)-2,3-dihydro-2,2-dimethylnaphtho[1,2-b]furan-4,5-dione and 3-(3-nitrophenylamino)-2,3-dihydro-2,2-dimethylnaphtho[1,2-b]furan-4,5-dione with trypanocidal activity higher than that of benznidazole, the standard drug. The compounds were rationalized based on hybrid drugs and appear as important compounds against T. cruzi. The trypanocidal activity of these substances endowed with redox properties representing a good starting point for a medicinal chemistry program aiming the chemotherapy of Chagas’ disease.     

Predictive model of rat reproductive toxicity from ToxCast high throughput screening

The U.S. Environmental Protection Agency's ToxCast research program uses high throughput screening (HTS) for profiling bioactivity and predicting the toxicity of large numbers of chemicals. ToxCast Phase I tested 309 well-characterized chemicals in more than 500 assays for a wide range of molecular targets and cellular responses. Of the 309 environmental chemicals in Phase I, 256 were linked to high-quality rat multigeneration reproductive toxicity studies in the relational Toxicity Reference Database. Reproductive toxicants were defined here as having achieved a reproductive lowest-observed-adverse-effect level of less than 500 mg kg(-1) day(-1). Eight-six chemicals were identified as reproductive toxicants in the rat, and 68 of those had sufficient in vitro bioactivity to model. Each assay was assessed for univariate association with the identified reproductive toxicants. Significantly associated assays were linked to gene sets and used for the subsequent predictive modeling. Using linear discriminant analysis and fivefold cross-validation, a robust and stable predictive model was produced capable of identifying rodent reproductive toxicants with 77% ± 2% and 74% ± 5% (mean ± SEM) training and test cross-validation balanced accuracies, respectively. With a 21-chemical external validation set, the model was 76% accurate, further indicating the model's potential for prioritizing the many thousands of environmental chemicals with little to no hazard information. The biological features of the model include steroidal and nonsteroidal nuclear receptors, cytochrome P450 enzyme inhibition, G protein-coupled receptors, and cell signaling pathway readouts-mechanistic information suggesting additional targeted, integrated testing strategies and potential applications of in vitro HTS to risk assessment.     

Quantitative microtiter fibronectin fibrillogenesis assay: use in high throughput screening for identification of inhibitor compounds

Fibronectin (FN) is a plasma glycoprotein that circulates in the near micromolar concentration range and is deposited along with locally produced FN in the extracellular matrices of many tissues. The control of FN deposition is tightly controlled by cells. Agents that modulate FN assembly may be useful therapeutically in conditions characterized by excessive FN deposition, such as fibrosis, inflammatory diseases, and malignancies. To identify such agents by high throughput screening (HTS), we developed a microtiter assay of FN deposition by human fibroblasts. The assay provides a robust read-out of FN assembly. Alexa 488-FN (A488-FN) was added to cell monolayers, and the total fluorescence intensity of deposited A488-FN was quantified. The fluorescence intensity of deposited A488-FN correlated with the presence of FN fibrils visualized by fluorescence microscopy. The assay Z′ values were 0.67 or 0.54, respectively, when using background values of fluorescence either with no added A488-FN or with A488-FN added together with a known inhibitor of FN deposition. The assay was used to screen libraries comprising 4160 known bioactive compounds. Nine compounds were identified as non- or low-cytotoxic inhibitors of FN assembly. Four (ML-9, HA-100, tyrphostin and imatinib mesylate) are kinase inhibitors, a category of compounds known to inhibit FN assembly; two (piperlongumine and cantharidin) are promoters of cancer cell apoptosis; and three (maprotiline, CGS12066B, and aposcopolamine) are modulators of biogenic amine signaling. The latter six compounds have not been recognized heretofore as affecting FN assembly. The assay is straight-forward, adapts to 96- and 384-well formats, and should be useful for routine measurement of FN deposition and HTS. Screening of more diverse chemical libraries and identification of specific and efficient modulators of FN fibrillogenesis may result in therapeutics to control excessive connective tissue deposition.     

Statistical considerations for high throughput screening data

High throughput screening (HTS) is a widely used effective approach in genome-wide association and large scale protein expression studies, drug discovery, and biomedical imaging research. How to accurately identify candidate ‘targets’ or biologically meaningful features with a high degree of confidence has led to extensive statistical research in an effort to minimize both false-positive and false-negative rates. A large body of literature on this topic with in-depth statistical contents is available. We examine currently available statistical methods on HTS and aim to summarize some selected methods into a concise, easy-tofollow introduction for experimental biologists.     

Imaging polarimetry for high throughput chiral screening

Imaging polarimetry was demonstrated as a highly parallel method of determining optical rotation of biochemical samples. The imaging polarimeter utilized a bright, uniform light source wavelength-filtered to near the sodium D line, a sample array flanked by inlet and analyzing polarizers, and a CCD camera fitted with an equal-perspective telecentric lens. The prototype apparatus was demonstrated to have an optical resolution better than 0.08 degrees. The potential for high throughput screening was demonstrated by imaging chiral solutions in 1536-well microtiter plates and by real-time monitoring of 30 simultaneous chiral enzymatic reactions. Improvements in polarizer and CCD technology may broadly expand the technique's applicability to fields such as directed evolution and combinatorial chemistry, where screening throughput is currently limiting for chiral applications.     

A non-radiolabelled ferriprotoporphyrin IX biomineralisation inhibition test for the high throughput screening of antimalarial compounds

Intraerythrocytic malaria parasites produce large amounts of toxic ferriprotoporphyrin IX (FP) during their digestion of host cell haemoglobin. The inhibition of biomineralisation of FP to haemozoin (or beta-haematin) by antimalarial drugs underlies their mode of action. We have developed an in vitro microassay for testing the inhibition of biomineralisation by drugs. It is based on the detection by optical density measurement of solubilised beta-haematin remaining after contact with drugs. The assay uses a 192-microM haemin chloride solution in dimethyl sulfoxide, 96-well filtration microplates as well as normal microplates; it lasts 18-24h and requires a spectrophotometer. We determined by this assay the IC(50) of chloroquine phosphate (28microM) and quinine base (324microM) and showed that unlike previous methods it is insensitive to inorganic anions. We also determined the activity of synthetic dyes and plant extract to determinate the interference of coloured compounds on the accuracy of the test. We found that methylene blue, thionine (IC(50) 38 and 87microM, respectively), and an extract of plants that contains quinoline derivatives, inhibited the biomineralisation of FP regardless of their intrinsic colour.     

GFP-reporter for a high throughput assay to monitor estrogenic compounds

In vitro test systems using yeast cells are a useful tool for the determination of the estrogenic activity of estrogens, phyto- and xeno-estrogens and can be used for monitoring large sample numbers in a routine analysis procedure. Our conventional transactivation assay functions with an expression plasmid expressing estrogen receptor α (ERα) under the control of a copper-inducible CUP1 promoter and a reporter plasmid expressing β-galactosidase under the control of the vitellogenin estrogen response element (ERE). In the novel yeast screen system the lacZ gene in the reporter plasmid was substituted by a gene for green fluorescent protein (GFP). Incubation of yeast with various concentrations of estrogenically active substances led to expression of the reporter gene product GFP in a dose dependent manner. The yeast transactivation assay was further down-scaled to be performed in a microplate scale, which is an important step to facilitate handling of large sample numbers. The sensitivity and reproducibility of the novel test system could be confirmed by analysis of the potencies of various estrogenically active substances. Thus, the newly developed yeast estrogen screen using GFP as a reporter can substitute the assay that has been used for a period of several years.     

Isotype determination of anti-Trypanosoma cruzi antibody in murine Chagas' disease.

Isotypic analysis of anti-parasite humoral responses of C57B1/6 and C3H (He) mice surviving acute Trypanosoma cruzi infection showed that both mouse strains demonstrate IgG1, IgG2a, IgG2b, and IgM enzyme-linked immunosorbent assay titers from days 21 to 300 of infection. Using the western blot technique to determine the antigen specificity of the isotypic responses, 100-day infected C3H mice showed strong IgG1, IgG2a, and IgG2b responses to many antigens, whereas C57B1/6 mice showed weak responses to fewer antigens. Isotype western blots showed that reactivity to the T. cruzi antigen of 75-77 kDa is present in the humoral response of day 21-infected mice that will survive and missing in those that will not survive. In general, surviving immunized C3H mice respond with IgG1, IgG2a, and IgG2b reactions to the 75-77-kDa and other antigens, whereas resistant B6 mice concentrate their anti-T. cruzi response in the IgG2b isotype to the 75-77-kDa antigen. Perhaps induction of ineffective antibody responses to nonprotective antigens is beneficial to the parasite and detrimental to the host.     

Novel fluorescence sensing methods for high throughput screening

We describe two new methods of fluorescence sensing for use in high throughput screening (HTS). Modulation sensing transforms analyte-dependent intensity changes into a change in the low-frequency modulation signal. Polarization sensing transforms an intensity change into a change in polarization. Both methods are internally calibrated by using a reference film immediately adjacent to the sample, which can be readily located on the HTS plate or on a nearby optical component and provides an intensity or polarization reference. Modulation sensing and polarization sensing were both shown useful for measurements of fluorophore concentrations, pH, or calcium concentrations in the wells of HTS plates. Sensing with a reference film provides the opportunity to internally reference HTS measurements without the need for additions to the sample. This approach can provide standardization for assays performed at different times.     

Evaluation of thiosemicarbazones and semicarbazones as potential agents anti-Trypanosoma cruzi

Synthetic thiosemicarbazones and semicarbazones were evaluated for their Trypanosoma cruzi trypomastigotes obtained from LLC-MK2 cell cultures. In general, thiosemicarbazone derivatives were most effective and among them the 4-N-(2′-methoxy styryl)-thiosemicarbazone was chosen, to compare the in vitro effect against amastigotes of T. cruzi lodged in mouse peritoneal and human macrophages. A potent trypanocidal effect was observed that was more pronounced against parasites internalized in human macrophages. A potential target for this compound was also evaluated by measuring the nitric oxide synthase activity through NADPH consumption. A significant decrease in enzyme activity was observed. In contrast to the cytotoxic effect observed with benznidazole, no macrophage toxicity was observed for any of the compounds, indicating that their activity was specific for the parasite forms investigated.     

Heteroallyl-containing 5-nitrofuranes as new anti-Trypanosoma cruzi agents with a dual mechanism of action

New heteroallyl-containing 5-nitrofuranes were synthesized as potential anti-Trypanosoma cruzi agents with a dual mechanism of action, oxidative stress and inhibition of membrane sterol biosynthesis. Some of the derivatives were found to have high and selective activity against the proliferative stages of the parasite, with IC(50) values against the clinically relevant intracellular amastigote forms in the low micromolar to sub-micromolar range. Oxidative stress was verified measuring cyanide dependent respiration. Inhibition of the de novo sterol biosynthesis at the level of squalene epoxidase was confirmed, using high-resolution gas-liquid chromatography coupled to mass spectrometry, by the disappearance of the parasite's mature sterols and the concomitant accumulation of squalene. The in vitro activities of these novel compounds were superior to that of nifurtimox, a nitrofuran currently used in the treatment of human Chagas' disease, and terbinafine, a commercially available allylamine-based squalene epoxidase inhibitor. The results support further in vivo studies of some of these nitrofuran derivatives.     

Adaptation of aequorin functional assay to high throughput screening

AequoScreen, a cellular aequorin-based functional assay, has been optimized for luminescent high-throughput screening (HTS) of G protein-coupled receptor (GPCRs). AequoScreen is a homogeneous assay in which the cells are loaded with the apoaequorin cofactor coelenterazine, diluted in assay buffer, and injected into plates containing the samples to be tested. A flash of light is emitted following the calcium increase resulting from the activation of the GPCR by the sample. Here we have validated a new plate reader, the Hamamatsu Photonics FDSS6000, for HTS in 96- and 384-well plates with CHO-K1 cells stably coexpressing mitochondrial apoaequorin and different GPCRs (AequoScreen cell lines). The acquisition time, plate type, and cell number per well have been optimized to obtain concentration-response curves with 4000 cells/well in 384-well plates and a high signal:background ratio. The FDSS6000 and AequoScreen cell lines allow reading of twenty 96- or 384-well plates in 1 h with Z' values of 0.71 and 0.78, respectively. These results bring new insights to functional assays, and therefore reinforce the interest in aequorin-based assays in a HTS environment.