Association of the receptor for activated C-kinase 1 with ribosomes in Plasmodium falciparum.

The receptor for activated C-kinase 1 (RACK1), a highly conserved eukaryotic protein, is known to have many varying biological roles and functions. Previous work has established RACK1 as a ribosomal protein, with defined regions important for ribosome binding in eukaryotic cells. In Plasmodium falciparum, RACK1 has been shown to be required for parasite growth, however, conflicting evidence has been presented about RACK1 ribosome binding and its role in mRNA translation. Given the importance of RACK1 as a regulatory component of mRNA translation and ribosome quality control, the case could be made in parasites that RACK1 either binds or does not bind the ribosome. Here, we used bioinformatics and transcription analyses to further characterize the P. falciparum RACK1 protein. Based on homology modeling and structural analyses, we generated a model of P. falciparum RACK1. We then explored mutant and chimeric human and P. falciparum RACK1 protein binding properties to the human and P. falciparum ribosome. We found that WT, chimeric, and mutant RACK1 exhibit distinct ribosome interactions suggesting different binding characteristics for P. falciparum and human RACK1 proteins. The ribosomal binding of RACK1 variants in human and parasite cells shown here demonstrates that although RACK1 proteins have highly conserved sequences and structures across species, ribosomal binding is affected by species-specific alterations to this protein. In conclusion, we show that in the case of P. falciparum, contrary to the structural data, RACK1 is found to bind ribosomes and actively translating polysomes in parasite cells.

Plasmodium falciparum translational machinery condones polyadenosine repeats.

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.

Discovery and Optimization of a Compound Series Active against Trypanosoma cruzi, the Causative Agent of Chagas Disease.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. It is endemic in South and Central America and recently has been found in other parts of the world, due to migration of chronically infected patients. The current treatment for Chagas disease is not satisfactory, and there is a need for new treatments. In this work, we describe the optimization of a hit compound resulting from the phenotypic screen of a library of compounds against T. cruzi. The compound series was optimized to the level where it had satisfactory pharmacokinetics to allow an efficacy study in a mouse model of Chagas disease. We were able to demonstrate efficacy in this model, although further work is required to improve the potency and selectivity of this series.

Scaffold and Parasite Hopping: Discovery of New Protozoal Proliferation Inhibitors.

Utilizing a target repurposing and parasite-hopping approach, we tested a previously reported library of compounds that were active against Trypanosoma brucei, plus 31 new compounds, against a variety of protozoan parasites including Trypanosoma cruzi, Leishmania major, Leishmania donovani, and Plasmodium falciparum. This led to the discovery of several compounds with submicromolar activities and improved physicochemical properties that are early leads toward the development of chemotherapeutic agents against kinetoplastid diseases and malaria.

Adaptation of Translational Machinery in Malaria Parasites to Accommodate Translation of Poly-Adenosine Stretches Throughout Its Life Cycle.

Malaria is caused by unicellular apicomplexan parasites of the genus Plasmodium, which includes the major human parasite Plasmodium falciparum. The complex cycle of the malaria parasite in both mosquito and human hosts has been studied extensively. There is tight control of gene expression in each developmental stage, and at every level of gene synthesis: from RNA transcription, to its subsequent translation, and finally post-translational modifications of the resulting protein. Whole-genome sequencing of P. falciparum has laid the foundation for significant biological advances by revealing surprising genomic information. The P. falciparum genome is extremely AT-rich (∼80%), with a substantial portion of genes encoding intragenic polyadenosine (polyA) tracks being expressed throughout the entire parasite life cycle. In most eukaryotes, intragenic polyA runs act as negative regulators of gene expression. Recent studies have shown that translation of mRNAs containing 12 or more consecutive adenosines results in ribosomal stalling and frameshifting; activating mRNA surveillance mechanisms. In contrast, P. falciparum translational machinery can efficiently and accurately translate polyA tracks without activating mRNA surveillance pathways. This unique feature of P. falciparum raises interesting questions: (1) How is P. falciparum able to efficiently and correctly translate polyA track transcripts, and (2) What are the specifics of the translational machinery and mRNA surveillance mechanisms that separate P. falciparum from other organisms? In this review, we analyze possible evolutionary shifts in P. falciparum protein synthesis machinery that allow efficient translation of an AU rich-transcriptome. We focus on physiological and structural differences of P. falciparum stage specific ribosomes, ribosome-associated proteins, and changes in mRNA surveillance mechanisms throughout the complete parasite life cycle, with an emphasis on the mosquito and liver stages.

Anilinoquinoline based inhibitors of trypanosomatid proliferation.

We recently reported the medicinal chemistry re-optimization of a series of compounds derived from the human tyrosine kinase inhibitor, lapatinib, for activity against Plasmodium falciparum. From this same library of compounds, we now report potent compounds against Trypanosoma brucei brucei (which causes human African trypanosomiasis), T. cruzi (the pathogen that causes Chagas disease), and Leishmania spp. (which cause leishmaniasis). In addition, sub-micromolar compounds were identified that inhibit proliferation of the parasites that cause African animal trypanosomiasis, T. congolense and T. vivax. We have found that this set of compounds display acceptable physicochemical properties and represent progress towards identification of lead compounds to combat several neglected tropical diseases.

Series of Alkynyl-Substituted Thienopyrimidines as Inhibitors of Protozoan Parasite Proliferation.

Discovery of new chemotherapeutic lead agents can be accelerated by optimizing chemotypes proven to be effective in other diseases to act against parasites. One such medicinal chemistry campaign has focused on optimizing the anilinoquinazoline drug lapatinib (1) and the alkynyl thieno[3,2-d]pyrimidine hit GW837016X (NEU-391, 3) into leads for antitrypanosome drugs. We now report the structure-activity relationship studies of 3 and its analogs against Trypanosoma brucei, which causes human African trypanosomiasis (HAT). The series was also tested against Trypanosoma cruzi, Leishmania major, and Plasmodium falciparum. In each case, potent antiparasitic hits with acceptable toxicity margins over mammalian HepG2 and NIH3T3 cell lines were identified. In a mouse model of HAT, 3 extended life of treated mice by 50%, compared to untreated controls. At the cellular level, 3 inhibited mitosis and cytokinesis in T. brucei. Thus, the alkynylthieno[3,2-d]pyrimidine chemotype is an advanced hit worthy of further optimization as a potential chemotherapeutic agent for HAT.

Optimization of physicochemical properties for 4-anilinoquinazoline inhibitors of trypanosome proliferation.

Human African trypanosomiasis (HAT) is a deadly disease in need of new chemotherapeutics that can cross into the central nervous system. We previously reported the discovery of 2 (NEU-617), a small molecule with activity against T. brucei bloodstream proliferation. Further optimization of 2 to improve the physicochemical properties (LogP, LLE, [1], and MPO score) [2] have led us to twelve sub-micromolar compounds, most importantly the headgroup variants 9i and 9j, and the linker variant 18. Although these 3 compounds had reduced potency compared to 2, they all had improved LogP, LLE and MPO scores. Cross-screening these analogs against other protozoan parasites uncovered 9o with potent activity towards T. brucei, T. cruzi and L. major, while four others compounds (17, 18, 21, 26) showed activity towards P. falciparum D6. This reinforces the effectiveness of lead repurposing for the discovery of new protozoan disease therapeutics.

Antiparasitic Lead Discovery: Toward Optimization of a Chemotype with Activity Against Multiple Protozoan Parasites.

Human African trypanosomiasis (HAT), Chagas disease, and leishmaniasis present a significant burden across the developing world. Existing therapeutics for these protozoal neglected tropical diseases suffer from severe side effects and toxicity. Previously, NEU-1045 (3) was identified as a promising lead with cross-pathogen activity, though it possessed poor physicochemical properties. We have designed a library of analogues with improved calculated physicochemical properties built on the quinoline scaffold of 3 incorporating small, polar aminoheterocycles in place of the 4-(3-fluorobenzyloxy)aniline substituent. We report the biological activity of these inhibitors against Trypanosoma brucei (HAT), T. cruzi (Chagas disease), and Leishmania major (cutaneous leishmaniasis) and describe the identification of N-(5-chloropyrimidin-2-yl)-6-(4-(morpholinosulfonyl)phenyl)quinolin-4-amine (13t) as a promising inhibitor of L. major proliferation and 6-(4-(morpholinosulfonyl)phenyl)-N-(pyrimidin-4-yl)quinolin-4-amine (13j), a potent inhibitor of T. brucei proliferation with improved drug-like properties.

Identification of "Preferred" Human Kinase Inhibitors for Sleeping Sickness Lead Discovery. Are Some Kinases Better than Others for Inhibitor Repurposing?

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".

Protozoan Parasite Growth Inhibitors Discovered by Cross-Screening Yield Potent Scaffolds for Lead Discovery.

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.

EVALUATION OF AROMATIC 6-SUBSTITUTED THIENOPYRIMIDINES AS SCAFFOLDS AGAINST PARASITES THAT CAUSE TRYPANOSOMIASIS, LEISHMANIASIS, AND MALARIA.

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.

Small-molecule xenomycins inhibit all stages of the Plasmodium life cycle.

Widespread resistance to most antimalaria drugs in use has prompted the search for novel candidate compounds with activity against Plasmodium asexual blood stages to be developed for treatment. In addition, the current malaria eradication programs require the development of drugs that are effective against all stages of the parasite life cycle. We have analyzed the antimalarial properties of xenomycins, a novel subclass of small molecule compounds initially isolated for anticancer activity and similarity to quinacrine in biological effects on mammalian cells. In vitro studies show potent activity of Xenomycins against Plasmodium falciparum. Oral administration of xenomycins in mouse models result in effective clearance of liver and blood asexual and sexual stages, as well as effective inhibition of transmission to mosquitoes. These characteristics position xenomycins as antimalarial candidates with potential activity in prevention, treatment and elimination of this disease.

Replication attempt: "Effect of BMAP-28 antimicrobial peptides on Leishmania major promastigote and amastigote growth: role of leishmanolysin in parasite survival".

This study describes an attempt to replicate experiments from the paper "Effect of BMAP-28 Antimicrobial Peptides on Leishmania major Promastigote and Amastigote Growth: Role of Leishmanolysin in Parasite Survival," which was submitted to the Reproducibility Initiative for independent validation. The cathelicidin bovine myeloid antimicrobial peptide 28 (BMAP-28) and its isomers were previously shown to have potent antiparasitic activity against Leishmania major. We tested the effectiveness of L-BMAP-28 and two of its isomers, the D-amino acid form (D-BMAP-28) and the retro-inverso form (RI-BMAP-28), in both unamidated and amidated forms, as anti-leishmanial agents against Leishmania major promastigotes in vitro. We observed that L-BMAP-28, as well as its D and RI isomers, demonstrate anti-leishmanial activity against L. major promastigotes in vitro. The inhibitory effect was lower than what was seen in the original study. At 2 µM of amidated peptides, the viability was 94%, 36%, and 66% with L-, D- and RI-peptides, versus 57%, 6%, and 18% in the original study.

Repurposing human Aurora kinase inhibitors as leads for anti-protozoan drug discovery.

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.

A high-efficiency microfluidic device for size-selective trapping and sorting.

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 introduced 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.

Imaging and identification of waterborne parasites using a chip-scale microscope.

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.

Wide field-of-view Talbot grid-based microscopy for multicolor fluorescence imaging.

The capability to perform multicolor, wide field-of-view (FOV) fluorescence microscopy imaging is important in screening and pathology applications. We developed a microscopic slide-imaging system that can achieve multicolor, wide FOV, fluorescence imaging based on the Talbot effect. In this system, a light-spot grid generated by the Talbot effect illuminates the sample. By tilting the excitation beam, the Talbot-focused spot scans across the sample. The images are reconstructed by collecting the fluorescence emissions that correspond to each focused spot with a relay optics arrangement. The prototype system achieved an FOV of 12 × 10 mm(2) at an acquisition time as fast as 23 s for one fluorescence channel. The resolution is fundamentally limited by spot size, with a demonstrated full-width at half-maximum spot diameter of 1.2 µm. The prototype was used to nimage green fluorescent beads, double-stained human breast cancer SK-BR-3 cells, Giardia lamblia cysts, and the Cryptosporidium parvum oocysts. This imaging method is scalable and simple for implementation of high-speed wide FOV fluorescence microscopy.

Antitrypanosomal lead discovery: identification of a ligand-efficient inhibitor of Trypanosoma cruzi CYP51 and parasite growth.

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.