New compound sets identified from high throughput phenotypic screening against three kinetoplastid parasites:an open resource

Using whole-cell phenotypic assays, the GlaxoSmithKline high-throughput screening (HTS) diversity set of 1.8 million compounds was screened against the three kinetoplastids most relevant to human disease, i.e. Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei. Secondary confirmatory and orthogonal intracellular anti-parasiticidal assays were conducted, and the potential for non-specific cytotoxicity determined. Hit compounds were chemically clustered and triaged for desirable physicochemical properties. The hypothetical biological target space covered by these diversity sets was investigated through bioinformatics methodologies. Consequently, three anti-kinetoplastid chemical boxes of ~200 compounds each were assembled. Functional analyses of these compounds suggest a wide array of potential modes of action against kinetoplastid kinases, proteases and cytochromes as well as potential host–pathogen targets. This is the first published parallel high throughput screening of a pharma compound collection against kinetoplastids. The compound sets are provided as an open resource for future lead discovery programs, and to address important research questions.The support and funding of Tres Cantos Open Lab Foundation is gratefully acknowledgedPeer reviewe

Hundreds of dual-stage antimalarial molecules discovered by a functional gametocyte screen.

Plasmodium falciparum stage V gametocytes are responsible for parasite transmission, and drugs targeting this stage are needed to support malaria elimination. We here screen the Tres Cantos Antimalarial Set (TCAMS) using the previously developed P. falciparum female gametocyte activation assay (Pf FGAA), which assesses stage V female gametocyte viability and functionality using Pfs25 expression. We identify over 400 compounds with activities <2 μM, chemically classified into 57 clusters and 33 singletons. Up to 68% of the hits are chemotypes described for the first time as late-stage gametocyte-targeting molecules. In addition, the biological profile of 90 compounds representing the chemical diversity is assessed. We confirm in vitro transmission-blocking activity of four of the six selected molecules belonging to three distinct scaffold clusters. Overall, this TCAMS gametocyte screen provides 276 promising antimalarial molecules with dual asexual/sexual activity, representing starting points for target identification and candidate selection

Automated high-content assay for compounds selectively toxic to Trypanosoma cruzi in a myoblastic cell line.

BACKGROUND:Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, represents a very important public health problem in Latin America where it is endemic. Although mostly asymptomatic at its initial stage, after the disease becomes chronic, about a third of the infected patients progress to a potentially fatal outcome due to severe damage of heart and gut tissues. There is an urgent need for new drugs against Chagas disease since there are only two drugs available, benznidazole and nifurtimox, and both show toxic side effects and variable efficacy against the chronic stage of the disease. METHODOLOGY/PRINCIPAL FINDINGS:Genetically engineered parasitic strains are used for high throughput screening (HTS) of large chemical collections in the search for new anti-parasitic compounds. These assays, although successful, are limited to reporter transgenic parasites and do not cover the wide T. cruzi genetic background. With the aim to contribute to the early drug discovery process against Chagas disease we have developed an automated image-based 384-well plate HTS assay for T. cruzi amastigote replication in a rat myoblast host cell line. An image analysis script was designed to inform on three outputs: total number of host cells, ratio of T. cruzi amastigotes per cell and percentage of infected cells, which respectively provides one host cell toxicity and two T. cruzi toxicity readouts. The assay was statistically robust (Z´ values >0.6) and was validated against a series of known anti-trypanosomatid drugs. CONCLUSIONS/SIGNIFICANCE:We have established a highly reproducible, high content HTS assay for screening of chemical compounds against T. cruzi infection of myoblasts that is amenable for use with any T. cruzi strain capable of in vitro infection. Our visual assay informs on both anti-parasitic and host cell toxicity readouts in a single experiment, allowing the direct identification of compounds selectively targeted to the parasite

High resolution image of controls.

<p>Representative <i>T. cruzi</i> infected and uninfected H9c2 pictures at 40X. Kinetoplastid kDNA (circle) and nDNA (bean-like) can be distinguished at this magnification. Bar is 30 μm.</p

Discrimination of spots using ‘Corrected intensity’ as parameter.

<p>A total of 1.18×10⁶ spots were acquired from infected and non-infected control wells (five fields per well from 96 wells for each control) from 6 independent plates each. Spots were distributed in 9 groups and classified accordingly to their ‘Corrected intensity’ parameter based on the Hartigan-Wong algorithm [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003493#pntd.0003493.ref034" target="_blank">34</a>]. The three parametric regions (low, intermediate and high) are indicated by red threshold lines in the box plot. Percentage of spots from infected and non-infected controls in each of the nine classes is shown below</p

Image analysis development.

<p>Representative images of the <i>T. cruzi</i> infected control, non-infected control, and 30 μM benznidazole treated infected cells (BNZ) for: (a) raw image acquisition (at 635 nm); (b) nuclei selection (selected in green and discarded in red; as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003493#sec002" target="_blank">Methods</a>) and their cytoplasm boundaries identification (green lines); (c) discrimination of true amastigotes (green) from other non-parasitic cytoplasmic spots (red) amongst all cytoplasmic spots detected; (d) identification of infected cells (those with >1 amastigote inside, green) and non-infected cells (red); (e) scatter plots of all detected spots used to discriminate true (green dots) from false amastigote spots (red dots) based on their area and intensity (‘Spot area’ and ‘Corrected intensity’ thresholds lines are shown; ‘Corrected Intensity’ is expressed in arbitrary units). Bar is 30 μm.</p

List of compounds used to validate the assay.

<p>Their host cell toxicity (TC₅₀) and anti-<i>T. cruzi</i> (IC₅₀ for the ‘Am/Cell’ and ‘%Infectivity’ assay outputs) mean values of three independent experiments plus their sd are shown (mean ± sd).</p><p>^aIC₅₀ values for the ‘Am/Cell’ output;</p><p>^bIC₅₀ values for the ‘%Infected’ output;</p><p>^cratio between the mean TC₅₀ values and their corresponding ‘Am/Cell’ IC₅₀ measurement.</p><p>^dliterature reference;</p><p>^end, not determined;</p><p>^#values obtained at the anti-Chagas HTS campaign recently performed at GSK (Peña et al., manuscript in preparation);</p><p>*not actually an IC₅₀ value but the minimal compound dose to eradicate T. cruzi amastigotes in Vero cells [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003493#pntd.0003493.ref026" target="_blank">26</a>].</p><p>List of compounds used to validate the assay.</p

Determination of thresholds.

<p>‘True amastigote’ discrimination thresholds based on their (a) ‘Corrected intensity’, and (b) ‘Spot area’ parameters were defined from the spots populations within both, infected and non-infected control wells (lognormal adjustments, blue lines; lower and upper ‘Corrected intensity’ thresholds (respectively at 48 and 274 a.u.), and the upper ‘Spot area’ cut off (51 px²), green lines). Inset in panel (a) zooms in at the spots distribution in the intermediate ‘Corrected intensity’ region (true amastigotes).</p

Imaging-based high-throughput screening assay to identify new molecules with transmission-blocking potential against Plasmodium falciparum female gamete formation.

In response to a call for the global eradication of malaria, drug discovery has recently been extended to identify compounds that prevent the onward transmission of the parasite, which is mediated by Plasmodium falciparum stage V gametocytes. Lately, metabolic activity has been used in vitro as a surrogate for gametocyte viability; however, as gametocytes remain relatively quiescent at this stage, their ability to undergo onward development (gamete formation) may be a better measure of their functional viability. During gamete formation, female gametocytes undergo profound morphological changes and express translationally repressed mRNA. By assessing female gamete cell surface expression of one such repressed protein, Pfs25, as the readout for female gametocyte functional viability, we developed an imaging-based high-throughput screening (HTS) assay to identify transmission-blocking compounds. This assay, designated the P. falciparum female gametocyte activation assay (FGAA), was scaled up to a high-throughput format (Z' factor, 0.7 ± 0.1) and subsequently validated using a selection of 50 known antimalarials from diverse chemical families. Only a few of these agents showed submicromolar 50% inhibitory concentrations in the assay: thiostrepton, methylene blue, and some endoperoxides. To determine the best conditions for HTS, a robustness test was performed with a selection of the GlaxoSmithKline Tres Cantos Antimalarial Set (TCAMS) and the final screening conditions for this library were determined to be a 2 μM concentration and 48 h of incubation with gametocytes. The P. falciparum FGAA has been proven to be a robust HTS assay faithful to Plasmodium transmission-stage cell biology, and it is an innovative useful tool for antimalarial drug discovery which aims to identify new molecules with transmission-blocking potential