A luciferase based viability assay for ATP detection in 384-well format for high throughput whole cell screening of Trypanosoma brucei brucei bloodstream form strain 427

<p>Abstract</p> <p>Background</p> <p>Human African Trypanosomiasis (HAT) is caused by two trypanosome species, <it>Trypanosoma brucei rhodesiense </it>and <it>Trypanosoma brucei gambiense</it>. Current drugs available for the treatment of HAT have significant issues related to toxicity, administration regimes with limited effectiveness across species and disease stages, thus there is a considerable need to find alternative drugs. A well recognised approach to identify new drug candidates is high throughput screening (HTS) of large compound library collections.</p> <p>Results</p> <p>We describe here the development of a luciferase based viability assay in 384-well plate format suitable for HTS of <it>T.b.brucei</it>. The parameters that were explored to determine the final HTS assay conditions are described in detail and include DMSO tolerability, Z', diluents and cell inoculum density. Reference compound activities were determined for diminazene, staurosporine and pentamidine and compared to previously published IC₅₀data obtained. The assay has a comparable sensitivity to reference drugs and is more cost effective than the 96-well format currently reported for <it>T.b.brucei</it>.</p> <p>Conclusion</p> <p>Due to the reproducibility and sensitivity of this assay it is recommended for potential HTS application. As it is commercially available this assay can also be utilised in many laboratories for both large and small scale screening.</p

Tripanocidno i citotoksično djelovanje etanolskoga iscrpka lišća Psidium guajava određivano bojanjem alamarskim plavilom

Ethanolic extracts prepared from the leaves of Psidium guajava were evaluated for anti-trypanosoma and cytotoxicity activity in the bloodstream species of Trypanosoma brucei brucei (BS427) and HEK293 in 384-well Alamar Blue assays respectively. Cytotoxicity activity in HEK293 cells was subsequently used to estimate the selectivity index of the extracts. The activities of the plant extracts were determined to evaluate if further chemical and biological profi ling may be warranted for potential development in early drug discovery for African Sleeping Sickness. Two trypanocides, pentamidine and diminazene, were employed as reference drugs, while puromycin was also included as control for general cell growth inhibition. The results show that the extracts inhibited growth of T. b. brucei with an IC50 of 6.3 μg/mL and 48.9 μg/mL for 80% and 20% ethanolic preparations respectively, with corresponding activity of less than 50% against HEK293 at the highest screening dose of 238.10 μg/mL. The estimated selectivity index of the extracts compares favourably with pentamidine and diminazene. Meanwhile the reference compounds were found to have activities in agreement with published sensitivities at the doses screened. The lack of cytotoxicity at the doses screened and direct activity against T. b. brucei whole cells, make these extracts suitable candidates for further chemical elucidation and biological profiling.Istraženo je tripanocidno djelovanje etanolskog iscrpka lišća Psidium guajava na vrstu Trypanosoma brucei brucei (BS427) i njegova citotoksičnost na stanice HEK293 bojanjem alamarskim plavilom u 384 jažice. Citotoksični učinak na stanice HEK293 rabljen je za procjenu indeksa selektivnosti. Učinkovitost biljnih iscrpaka određivana je da bi se procijenila svrhovitost budućih kemijskih i bioloških istraživanja potencijalnoga lijeka za afričku bolest spavanja. U istraživanju su rabljena dva tripanocida, pentamidin i diminazen, te puromicin kao sredstvo koje usporava rast stanica. Rezultati su pokazali da 80% etanolskih pripravaka s IC50 od 6,3 μg/mL koči rast i razvoj tripanosoma, a samo 20% onih s IC50 od 48,9 μg/mL, s odgovarajućom aktivnosti manjom od 50% na stanice HEK293 u najvećoj dozi od 238,10 μg/mL. Indeks selektivnosti iscrpaka bio je sukladan s aktivnošću pentamidina i diminazena. Aktivnost istraživanih sastojaka bila je sukladna s razinom prije objavljene osjetljivosti. Izostanak citotoksičnosti na razini rabljenih koncentracija i izravna djelotvornost na stanice T. b. brucei daju osnovu za daljnja kemijska i biološka istraživanja predmetnih pripravaka

Re-evaluating pretomanid analogues for Chagas disease:Hit-to-lead studies reveal both in vitro and in vivo trypanocidal efficacy

Phenotypic screening of a 900 compound library of antitubercular nitroimidazole derivatives related to pretomanid against the protozoan parasite Trypanosoma cruzi (the causative agent for Chagas disease) identified several structurally diverse hits with an unknown mode of action. Following initial profiling, a first proof-of-concept in vivo study was undertaken, in which once daily oral dosing of a 7-substituted 2-nitroimidazooxazine analogue suppressed blood parasitemia to low or undetectable levels, although sterile cure was not achieved. Limited hit expansion studies alongside counter-screening of new compounds targeted at visceral leishmaniasis laid the foundation for a more in-depth assessment of the best leads, focusing on both drug-like attributes (solubility, metabolic stability and safety) and maximal killing of the parasite in a shorter timeframe. Comparative appraisal of one preferred lead (58) in a chronic infection mouse model, monitored by highly sensitive bioluminescence imaging, provided the first definitive evidence of (partial) curative efficacy with this promising nitroimidazooxazine class

Design, synthesis and biological evaluation of 2-nitroimidazopyrazin-one/-es with antitubercular and antiparasitic activity

Tuberculosis and parasitic diseases, such as giardiasis, amebiasis, leishmaniasis and trypanosomiasis, all urgently require improved treatment options. Recently, it has been shown that anti-tubercular bicyclic nitroimidazoles such as pretomanid and delamanid have potential as repurposed therapeutics for the treatment of visceral leishmaniasis. Here we show that pretomanid also possesses potent activity against Giardia lamblia and Entamoeba histolytica, thus expanding the therapeutic potential of nitroimidazo-oxazines. Synthetic analogs with the novel nitroimidazopyrazin-one/-e bicyclic nitroimidazole chemotype were designed, synthesized and structure activity relationships generated. Selected derivatives had potent antiparasitic and antitubercular activity whilst maintaining drug-like properties such as low cytotoxicity against mammalian cell lines (CC50 >100 μM), good metabolic stability in human and mouse liver microsomes and high apparent permeability in a Caco-2 model of intestinal absorption. The kinetic solubility of the new bicyclic derivatives varied, and was found to be a key parameter for future optimization. Taken together, these results suggest promising subclasses of bicyclic nitroimidazoles containing different core architectures have potential for further development

Preclinical data do not support the use of amiodarone or dronedarone as antiparasitic drugs for Chagas disease at the approved human dosing regimen

The repurposing of approved drugs is an appealing method to fast-track the development of novel therapies for neglected diseases. Amiodarone and dronedarone, two approved antiarrhythmic agents, have been reported to have potential for the management of Chagas disease patients displaying symptomatic heart pathology. More recently, it has been suggested that both molecules not only have an antiarrhythmic effect, but also have trypanocidal activity against Trypanosoma cruzi, the causative agent of Chagas disease. In this work, we assessed the in vitro activity of these compounds against T. cruzi, the in vivo pharmacokinetics, and pharmacodynamics, to determine the potential for repurposing these drugs as therapies for Chagas disease. Based on these results, we were unable to reproduce the in vitro potencies of amiodarone and dronedarone described in the literature, and both drugs were found to be inactive or cytotoxic against a variety of different mammalian cell lines. The evaluation of in vivo efficacy in a bioluminescent murine model of T. cruzi did not show antiparasitic activity at the highest tolerated dose tested. While the potential of amiodarone and dronedarone as antiarrhythmic agents in Chagas cardiomyopathic patients cannot be completely excluded, a trypanocidal effect in patients treated with these two drugs appears unlikely

Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019

Background: In an era of shifting global agendas and expanded emphasis on non-communicable diseases and injuries along with communicable diseases, sound evidence on trends by cause at the national level is essential. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) provides a systematic scientific assessment of published, publicly available, and contributed data on incidence, prevalence, and mortality for a mutually exclusive and collectively exhaustive list of diseases and injuries. Methods: GBD estimates incidence, prevalence, mortality, years of life lost (YLLs), years lived with disability (YLDs), and disability-adjusted life-years (DALYs) due to 369 diseases and injuries, for two sexes, and for 204 countries and territories. Input data were extracted from censuses, household surveys, civil registration and vital statistics, disease registries, health service use, air pollution monitors, satellite imaging, disease notifications, and other sources. Cause-specific death rates and cause fractions were calculated using the Cause of Death Ensemble model and spatiotemporal Gaussian process regression. Cause-specific deaths were adjusted to match the total all-cause deaths calculated as part of the GBD population, fertility, and mortality estimates. Deaths were multiplied by standard life expectancy at each age to calculate YLLs. A Bayesian meta-regression modelling tool, DisMod-MR 2.1, was used to ensure consistency between incidence, prevalence, remission, excess mortality, and cause-specific mortality for most causes. Prevalence estimates were multiplied by disability weights for mutually exclusive sequelae of diseases and injuries to calculate YLDs. We considered results in the context of the Socio-demographic Index (SDI), a composite indicator of income per capita, years of schooling, and fertility rate in females younger than 25 years. Uncertainty intervals (UIs) were generated for every metric using the 25th and 975th ordered 1000 draw values of the posterior distribution. Findings: Global health has steadily improved over the past 30 years as measured by age-standardised DALY rates. After taking into account population growth and ageing, the absolute number of DALYs has remained stable. Since 2010, the pace of decline in global age-standardised DALY rates has accelerated in age groups younger than 50 years compared with the 1990–2010 time period, with the greatest annualised rate of decline occurring in the 0–9-year age group. Six infectious diseases were among the top ten causes of DALYs in children younger than 10 years in 2019: lower respiratory infections (ranked second), diarrhoeal diseases (third), malaria (fifth), meningitis (sixth), whooping cough (ninth), and sexually transmitted infections (which, in this age group, is fully accounted for by congenital syphilis; ranked tenth). In adolescents aged 10–24 years, three injury causes were among the top causes of DALYs: road injuries (ranked first), self-harm (third), and interpersonal violence (fifth). Five of the causes that were in the top ten for ages 10–24 years were also in the top ten in the 25–49-year age group: road injuries (ranked first), HIV/AIDS (second), low back pain (fourth), headache disorders (fifth), and depressive disorders (sixth). In 2019, ischaemic heart disease and stroke were the top-ranked causes of DALYs in both the 50–74-year and 75-years-and-older age groups. Since 1990, there has been a marked shift towards a greater proportion of burden due to YLDs from non-communicable diseases and injuries. In 2019, there were 11 countries where non-communicable disease and injury YLDs constituted more than half of all disease burden. Decreases in age-standardised DALY rates have accelerated over the past decade in countries at the lower end of the SDI range, while improvements have started to stagnate or even reverse in countries with higher SDI. Interpretation: As disability becomes an increasingly large component of disease burden and a larger component of health expenditure, greater research and developm nt investment is needed to identify new, more effective intervention strategies. With a rapidly ageing global population, the demands on health services to deal with disabling outcomes, which increase with age, will require policy makers to anticipate these changes. The mix of universal and more geographically specific influences on health reinforces the need for regular reporting on population health in detail and by underlying cause to help decision makers to identify success stories of disease control to emulate, as well as opportunities to improve. Funding: Bill & Melinda Gates Foundation. © 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licens

Temporal and Wash-Out Studies Identify Medicines for Malaria Venture Pathogen Box Compounds with Fast-Acting Activity against Both Trypanosoma cruzi and Trypanosoma brucei

Chagas disease caused by the protozoan Trypanosoma cruzi is endemic to 21 countries in the Americas, effects approximately 6 million people and on average results in 12,000 deaths annually. Human African Trypanosomiasis (HAT) is caused by the Trypanosoma brucei sub-species, endemic to 36 countries within sub-Saharan Africa. Treatment regimens for these parasitic diseases are complicated and not effective against all disease stages; thus, there is a need to find improved treatments. To identify new molecules for the drug discovery pipelines for these diseases, we have utilised in vitro assays to identify compounds with selective activity against both T. cruzi and T.b. brucei from the Medicines for Malaria Venture (MMV) Pathogen Box compound collection. To prioritise these molecules for further investigation, temporal and wash off assays were utilised to identify the speed of action and cidality of compounds. For translational relevance, compounds were tested against clinically relevant T.b. brucei subspecies. Compounds with activity against T. cruzi cytochrome P450 (TcCYP51) have not previously been successful in clinical trials for chronic Chagas disease; thus, to deprioritise compounds with this activity, they were tested against recombinant TcCYP51. Compounds with biological profiles warranting progression offer important tools for drug and target development against kinetoplastids