Chagas disease is caused by the intracellular protozoan parasite Trypanosomal cruzi, and current drugs are lacking in terms of desired safety and efficacy profiles. Following on a recently reported high-throughput screening campaign, we have explored initial structure-activity relationships around a class of imidazole-based compounds. This profiling has uncovered compounds 4c (NEU321) and 4j (NEU704), which are potent against in vitro cultures of T. cruzi and are greater than 160-fold selective over host cells. We report in vitro drug metabolism and properties profiling of 4c and show that this chemotype inhibits the T cruzi CYP51 enzyme, an observation confirmed by X-ray crystallographic analysis. We compare the binding orientation of 4c to that of other, previously reported inhibitors. We show that 4c displays a significantly better ligand efficiency and a shorter synthetic route over previously disclosed CYP51 inhibitors, and should therefore be considered a promising lead compound for further optimization.

Tropical protozoal infections are a significant cause of morbidity and mortality worldwide; four in particular (human African trypanosomiasis (HAT), Chagas disease, cutaneous leishmaniasis, and malaria) have an estimated combined burden of over 87 million disability-adjusted life years. New drugs are needed for each of these diseases. Building on the previous identification of NEU-617 (1) as a potent and nontoxic inhibitor of proliferation for the HAT pathogen (Trypanosoma brucei), we have now tested this class of analogs against other protozoal species: T. cruzi (Chagas disease), Leishmania major (cutaneous leishmaniasis), and Plasmodium falciparum (malaria). Based on hits identified in this screening campaign, we describe the preparation of several replacements for the quinazoline scaffold and report these inhibitors’ biological activities against these parasites. In doing this, we have identified several potent proliferation inhibitors for each pathogen, such as 4-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-6-(4-((4-methyl-1,4-diazepan-1-yl)sulfonyl)phenyl)quinoline-3-carbonitrile (NEU-924, 83) for T. cruzi and N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-7-(4-((4-methyl-1,4-diazepan-1-yl)sulfonyl)phenyl)cinnolin-4-amine (NEU-1017, 68) for L. major and P. falciparum.

Hesperadin, an established human Aurora B inhibitor, was tested against cultures of Trypanosoma brucei, Leishmania major, and Plasmodium falciparum, and was identified to be a potent proliferation inhibitor. A series of analogs was designed and tested to establish the initial structure-activity relationships for each parasite. In this study, we identified multiple non-toxic compounds with high potency against T. brucei and P. falciparum with good selectivity. These compounds may represent an opportunity for continued optimization.

We report the development of a simple poly(dimethylsiloxane) microfluidic device for high-efficiency trapping and sorting of micron-size particles. In this device, hydrodynamic fluid flow through the sieve-like microfluidic channel sequentially fills the trap positions with particles of the trap size, and particles smaller than the trap size pass through the sieve and are trapped by smaller traps downstream. By incorporating side channels alongside the main channel, we were able to decouple the fluidic flow in one stage from the flows in the other stages. This decoupling allows us to modularize each stage of the device regardless of the size of the entire device. In our demonstration experiment with the prototype, we showed that more than 85% of the polystyrene microspheres (of sizes 15 μm, 6 μm and 4 μm) were sorted in the correct segment of the device that targets their respective sizes. Moreover, this high-efficiency device was able to trap all microspheres which were indtroduced into the device. Finally, we tested the device's ability to trap and sort three different species of waterborne parasites (Entamoeba, Giardia, and Cryptosporidium) and obtained excellent sorting performance.

A kinase-targeting cell-based high-throughput screen (HTS) against Trypanosoma brucei was recently reported, and this screening set included the Published Kinase Inhibitor Set (PKIS). From the PKIS was identified 53 compounds with pEC50 ≥ 6. Utilizing the published data available for the PKIS, a statistical analysis of these active antiparasitic compounds was performed, allowing identification of a set of human kinases having inhibitors that show a high likelihood for blocking T. brucei cellular proliferation in vitro. This observation was confirmed by testing other established inhibitors of these human kinases and by mining past screening campaigns at GlaxoSmithKline. Overall, although the parasite targets of action are not known, inhibitors of this set of human kinases displayed an enhanced hit rate relative to a random kinase-targeting HTS campaign, suggesting that repurposing efforts should focus primarily on inhibitors of these specific human kinases. We therefore term this statistical analysis-driven approach “preferred lead repurposing”.

We demonstrate a compact portable imaging system for the detection of waterborne parasites in resource-limited settings. The previously demonstrated sub-pixel sweeping microscopy (SPSM) technique is a lens-less imaging scheme that can achieve high-resolution (<1 µm) bright-field imaging over a large field-of-view (5.7 mm×4.3 mm). A chip-scale microscope system, based on the SPSM technique, can be used for automated and high-throughput imaging of protozoan parasite cysts for the effective diagnosis of waterborne enteric parasite infection. We successfully imaged and identified three major types of enteric parasite cysts, Giardia, Cryptosporidium, and Entamoeba, which can be found in fecal samples from infected patients. We believe that this compact imaging system can serve well as a diagnostic device in challenging environments, such as rural settings or emergency outbreaks.

Target repurposing is a proven method for finding new lead compounds that target Trypanosoma brucei, the causative agent of human African trypanosomiasis. Due to the recent discovery of a lapatinib-derived analog 2 with excellent potency against T. brucei (EC50 = 42 nM) and selectivity over human host cells, we have explored other classes of human tyrosine kinase inhibitor scaffolds in order to expand the range of chemotypes for pursuit. Following library expansion, we found compound 11e to have an EC50 of 84 nM against T. brucei cells while maintaining selectivity over human hepatocytes. In addition, the library was tested against causative agents of Chagas’ disease, leishmaniasis, and malaria. Two analogs with sub-micromolar potencies for T. cruzi (4j) and Plasmodium falciparum (11j) were discovered, along with an analog with considerable potency against Leishmania major amastigotes (4e). Besides identifying new and potent protozoan growth inhibitors, these data highlight the value of concurrent screening of a chemical library against different protozoan parasites.

Plasmodium falciparum is a causative agent of human malaria. Sixty percent of mRNAs from its extremely AT-rich (81%) genome harbor long polyadenosine (polyA) runs within their ORFs, distinguishing the parasite from its hosts and other sequenced organisms. Recent studies indicate polyA runs cause ribosome stalling and frameshifting, triggering mRNA surveillance pathways and attenuating protein synthesis. Here, we show that P. falciparum is an exception to this rule. We demonstrate that both endogenous genes and reporter sequences containing long polyA runs are efficiently and accurately translated in P. falciparum cells. We show that polyA runs do not elicit any response from No Go Decay (NGD) or result in the production of frameshifted proteins. This is in stark contrast to what we observe in human cells or T. thermophila, an organism with similar AT-content. Finally, using stalling reporters we show that Plasmodium cells evolved not to have a fully functional NGD pathway.