Open-access drug discovery provides a substantial resource for diseases primarily affecting the poor and disadvantaged. The open-access Pathogen Box collection is comprised of compounds with demonstrated biological activity against specific pathogenic organisms. The supply of this resource by the Medicines for Malaria Venture has the potential to provide new chemical starting points for a number of tropical and neglected diseases, through repurposing of these compounds for use in drug discovery campaigns for these additional pathogens. We tested the Pathogen Box against kinetoplastid parasites and malaria life cycle stages in vitro. Consequently, chemical starting points for malaria, human African trypanosomiasis, Chagas disease, and leishmaniasis drug discovery efforts have been identified. Inclusive of this in vitro biological evaluation, outcomes from extensive literature reviews and database searches are provided. This information encompasses commercial availability, literature reference citations, other aliases and ChEMBL number with associated biological activity, where available. The release of this new data for the Pathogen Box collection into the public domain will aid the open-source model of drug discovery. Importantly, this will provide novel chemical starting points for drug discovery and target identification in tropical disease research.

Abstract. There is an urgent need for new compounds for the drug development pipeline for treatment of patients with African sleeping sickness. One approach for identifying such compounds is by high throughput screening (HTS) of compound collections. For time and cost considerations, there is a need for the development of an assay that uses at least 384-well formats. To our knowledge, there are currently no viability assays for whole cell screening of trypanosomes in the 384-well plate format. We have developed and optimized an Alamar Blue viability assay in a 384-well format for Trypanosoma brucei brucei bloodstream form strain 427 (BS427). The assay had a Z′ > 0.5 and tolerated a final dimethylsulfoxide concentration of 0.42%. Drug sensitivity was compared with those reported from previously developed 96-well methods and was found to be comparable. The sensitivity and cost benefit of the Alamar Blue assay make it an excellent candidate for HTS application.

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 IC50 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 IC50 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.

We have developed a high content 384-well, image-based assay to estimate the effect of compound treatment on Trypanosoma cruzi amastigotes in 3T3 fibroblasts. In the same well, the effect of compound activity on host cells can also be determined, as an initial indicator of cytotoxicity. This assay has been used to identify active compounds from an in-house library of compounds with either known biological activity or that are FDA approved, and separately, from the Medicines for Malaria Venture Malaria Box collection. Active compounds were screened against T. cruzi trypomastigotes, utilising an assay developed with the viability dye resazurin. Twelve compounds with reconfirmed solid sample activity, with IC50 values of less than 10 μM and selectivity indices to T. cruzi amastigotes over 3T3 host cells of between >22 and 319 times were identified from these libraries. As 3T3 cells are contact inhibited, with limited proliferation in the assay, selective compounds of interest were profiled in a separate assay to estimate the viability of compound treated, replicating HEK293 cells. Selective compounds that were not previously reported in the literature were further profiled by extending the incubation time against amastigote infected 3T3 cells to determine if there were residual amastigotes post-treatment, important for the consideration of the exposure time required for further biological characterisation. The assay development process and the suitability of identified compounds as hit molecules for Chagas disease research are discussed.

Determining the activity of a compound and the potential impact on a diseased state is frequently undertaken using phenotypic or target-based approaches. Phenotypic screens have the advantage of the whole organism being exposed to the compound and thus all the targets and biological pathways associated with it. Cell penetration and access to targets in their "natural" environment are taken into account. Unless utilizing a genetically modified organism with an additional target associated indicator, elucidation of specific target(s) of active compounds is necessary. Target discovery is desirable to allow development of chemical entities based upon knowledge of the target structure. Phenotypic drug discovery has successfully identified new molecular entities for neglected protozoan disease research. In this perspective, the phenotypic approaches used to identify chemical entities for drug discovery and for use as tools against the parasites Plasmodium falciparum, Trypanosoma brucei brucei, and Trypanosoma cruzi will be outlined.

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.

Using high throughput, high-content imaging, a proprietary library was screened against intracellular Trypanosoma cruzi amastigotes to identify compounds with novel activity against the parasite. Five inhibitors were discovered, which did not clear all of the parasites from 3T3 host cells following 48 hours exposure, and were identified as putative T. cruzi cytochrome P450 (TcCYP51) inhibitors. TcCYP51 inhibitors are not favourable for the drug discovery pipeline for treatment of Chagas Disease infection due to clinical and pre-clinical failures. To determine if there were in vitro inhibitory characteristics of these compounds that could aid the prediction of TcCYP51 inhibition further profiling using imaging and fluorescence based assays was undertaken. It was determined that in vitro profiles, coupled with analysis of chemical structure, could support the early prediction of putative TcCYP51 activity and thus enable early de-prioritisation of these compounds from progression through the drug discovery pipeline.

A high-throughput screening campaign using a prefractionated natural product library and an in vitro Pseudomonas aeruginosa (PAO200 strain) assay identified two antibacterial fractions derived from the marine sponge Suberea ianthelliformis. Mass-directed isolation of the CH(2)Cl(2)/CH(3)OH extract from S. ianthelliformis resulted in the purification of three new bromotyrosine-derived metabolites, ianthelliformisamines A-C (1-3), together with the known natural products aplysamine 1 (4) and araplysillin I (5). The structures of 1-3 were determined following analysis of 1D and 2D NMR and MS spectroscopic data. This is the first report of chemistry from the marine sponge S. ianthelliformis. Ianthelliformisamine A (1) showed inhibitory activity against the Gram-negative bacterium P. aeruginosa with an IC(50) value of 6.8 μM (MIC = 35 μM).