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.

Investigation of pyrimidine nucleoside analogues as chemical probes to assess compound effects on the proliferation of Trypanosoma cruzi intracellular parasites.

Trypanosoma cruzi parasites utilise de novo pyrimidine biosynthesis to produce DNA and survive within mammalian host cells. This pathway can be hijacked to assess the replication of intracellular parasites with the exogenous addition of a DNA specific probe. To identify suitable probe compounds for this application, a collection of pyrimidine nucleoside analogues was assessed for incorporation into T. cruzi intracellular amastigote DNA using image-based technology and script-based analysis. Associated mammalian cell toxicity of these compounds was also determined against both the parasite host cells (3T3 cells) and HEK293 cells. Incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into parasite DNA was the most effective of the probes tested, with minimal growth inhibition observed following either two or four hours EdU exposure. EdU was subsequently utilised as a DNA probe, followed by visualisation with click chemistry to a fluorescent azide, to assess the impact of drugs and compounds with previously demonstrated activity against T. cruzi parasites, on parasite replication. The inhibitory profiles of these molecules highlight the benefit of this approach for identifying surviving parasites post-treatment in vitro and classifying compounds as either fast or slow-acting. F-ara-EdU resulted in <50% activity observed against T. cruzi amastigotes following 48 hours incubation, at 73 µM. Collectively, this supports the further development of pyrimidine nucleosides as chemical probes to investigate replication of the parasite T. cruzi.

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 antitubercular 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 nitroimidazooxazines. Synthetic analogues with a novel nitroimidazopyrazin-one/-e bicyclic nitroimidazole chemotype were designed and synthesized, and structure-activity relationships were generated. Selected derivatives had potent antiparasitic and antitubercular activity while maintaining drug-like properties such as low cytotoxicity, good metabolic stability in liver microsomes and high apparent permeability across Caco-2 cells. 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 that promising subclasses of bicyclic nitroimidazoles containing different core architectures have potential for further development.

Screening a Natural Product-Based Library against Kinetoplastid Parasites.

Kinetoplastid parasites cause vector-borne parasitic diseases including leishmaniasis, human African trypanosomiasis (HAT) and Chagas disease. These Neglected Tropical Diseases (NTDs) impact on some of the world's lowest socioeconomic communities. Current treatments for these diseases cause severe toxicity and have limited efficacy, highlighting the need to identify new treatments. In this study, the Davis open access natural product-based library was screened against kinetoplastids (Leishmania donovani DD8, Trypanosoma brucei brucei and Trypanosoma cruzi) using phenotypic assays. The aim of this study was to identify hit compounds, with a focus on improved efficacy, selectivity and potential to target several kinetoplastid parasites. The IC50 values of the natural products were obtained for L. donovani DD8, T. b. brucei and T. cruzi in addition to cytotoxicity against the mammalian cell lines, HEK-293, 3T3 and THP-1 cell lines were determined to ascertain parasite selectivity. Thirty-one compounds were identified with IC50 values of ≤ 10 µM against the kinetoplastid parasites tested. Lissoclinotoxin E (1) was the only compound identified with activity across all three investigated parasites, exhibiting IC50 values < 5 µM. In this study, natural products with the potential to be new chemical starting points for drug discovery efforts for kinetoplastid diseases were identified.

Identification of compounds with anti-proliferative activity against Trypanosoma brucei brucei strain 427 by a whole cell viability based HTS campaign.

Human African Trypanosomiasis (HAT) is caused by two trypanosome sub-species, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Drugs available for the treatment of HAT have significant issues related to difficult administration regimes and limited efficacy across species and disease stages. Hence, there is considerable need to find new alternative and less toxic drugs. An approach to identify starting points for new drug candidates is high throughput screening (HTS) of large compound library collections. We describe the application of an Alamar Blue based, 384-well HTS assay to screen a library of 87,296 compounds against the related trypanosome subspecies, Trypanosoma brucei brucei bloodstream form lister 427. Primary hits identified against T.b. brucei were retested and the IC(50) value compounds were estimated for T.b. brucei and a mammalian cell line HEK293, to determine a selectivity index for each compound. The screening campaign identified 205 compounds with greater than 10 times selectivity against T.b. brucei. Cluster analysis of these compounds, taking into account chemical and structural properties required for drug-like compounds, afforded a panel of eight compounds for further biological analysis. These compounds had IC(50) values ranging from 0.22 µM to 4 µM with associated selectivity indices ranging from 19 to greater than 345. Further testing against T.b. rhodesiense led to the selection of 6 compounds from 5 new chemical classes with activity against the causative species of HAT, which can be considered potential candidates for HAT early drug discovery. Structure activity relationship (SAR) mining revealed components of those hit compound structures that may be important for biological activity. Four of these compounds have undergone further testing to 1) determine whether they are cidal or static in vitro at the minimum inhibitory concentration (MIC), and 2) estimate the time to kill.

Alkaloids from the Chinese vine Gnetum montanum.

During a high-throughput screening campaign of a prefractionated natural product library, fractions from the Chinese vine Gnetum montanum showed in vitro activity against Pseudomonas aeruginosa wild-type strain, PAO1. UV-directed isolation of the organic extract from the vine leaves resulted in the purification of the new natural products N-methyllaudanosolinium trifluoroacetate (1), 3'-hydroxy-N,N-dimethylcoclaurinium trifluoroacetate (2), 1,9,10-trihydroxy-2-methoxy-6-methylaporphinium trifluoroacetate (3), and 6a,7-didehydro-1,9,10-trihydroxy-2-methoxy-6-methylaporphinium trifluoroacetate (4). Compound 4 is described here for the first time, and this is the first report of compounds 1-3 as natural products. Compounds 1-3 were found to racemize over time. Starting from commercially available (+)-boldine, through a series of semisynthetic reactions, a mechanism for the racemization of the isolated compounds is proposed. The known natural products (-)-latifolian A (5) and magnocurarine (6) were also isolated during these studies. The antibacterial activity was explained by the presence of 5, which displayed an IC50 value of 9.8 µM (MIC = 35 µM).

New galloylated flavanonols from the Australian plant Glochidion sumatranum.

Bioassay-guided fractionation of the CH(2)Cl(2)/MeOH extract from the Australian plant Glochidion sumatranum resulted in the isolation of four new galloylated flavanonols, (2R,3R)-dihydromyricetin-4'-O-(3''-O-methyl)-gallate (1), (2R,3R)-dihydromyricetin-3'-O-(3''-O-methyl)-gallate (2), (2R,3R)-dihydromyricetin-4'-O-gallate (3), and (2R,3R)-dihydromyricetin-3'-O-gallate (4), along with the known compound, (2R,3R)-dihydromyricetin (5). The structures of 1-5 were determined by NMR and MS analysis and their absolute configuration was elucidated by comparison of the CD data with literature values. Compounds 1/2 and 3/4 are two pairs of structural isomers that were shown to interconvert by transesterification during NMR and LC-MS studies. This process involved the intramolecular migration of the galloyl moieties between C-3' and C-4' of the flavanonol skeleton. Compounds 1 and 3 were identified as the more stable isomers. Compounds 1, 3, and 5 showed weak activity against the gram-negative bacterium Pseudomonas aeruginosa and the gram-positive bacterium Staphylococcus aureus.

Antiparasitic activity of alkaloids from plant species of Papua New Guinea and Australia.

New drugs are needed to help overcome the increasing problem of drug resistance in parasites that cause diseases such as malaria and trypanosomiasis. In this study, alkaloid compounds isolated from extracts of the plants Flindersia amboinensis, Stephania zippeliana and Voacanga papuana from Papua New Guinea and Flindersia acuminata from Australia were examined for their antiparasitic activity against Plasmodium falciparum strains and Trypanosoma brucei brucei as well as their cytotoxicity against the mammalian cell lines HEK 293 and HeLa. The most active compound, dimethylisoborreverine (DMIB), showed submicromolar activity, with 50% inhibitory concentration (IC(50)) values between 20 nM and 810 nM both against drug-sensitive and drug-resistant P. falciparum strains, along with moderate selectivity against T. b. brucei and mammalian cells. Stage specificity studies revealed that P. falciparum trophozoite-stage parasites were more susceptible to DMIB than ring- or schizont-stage parasites. DMIB-treated trophozoites showed changes in food vacuole morphology, with an apparent reduction in haemozoin formation that does not appear to be inhibited via the direct binding of haem. These findings suggest a potential for indole alkaloids from Flindersia spp. as new antiparasitic agents.

Lysianadioic acid, a carboxypeptidase B inhibitor from Lysiana subfalcata.

A new natural product, lysianadioic acid, was isolated from the plant Lysiana subfalcata as a carboxypeptidase B (CPB) inhibitor. It is a potent inhibitor of CPB with an IC(50) of 0.36 microM. This is the first known example of a small molecule CPB inhibitor isolated from plant origin. Its structure was determined by NMR spectroscopy.