An Image-Based High-Content Screening Assay for Compounds Targeting Intracellular Leishmania donovani Amastigotes in Human Macrophages

Abstract

Leishmaniasis is a tropical disease threatening 350 million people from endemic regions. The available drugs for treatment are inadequate, with limitations such as serious side effects, parasite resistance or high cost. Driven by this need for new drugs, we developed a high-content, high-throughput image-based screening assay targeting the intracellular amastigote stage of different species of Leishmania in infected human macrophages. The in vitro infection protocol was adapted to a 384-well-plate format, enabling acquisition of a large amount of readouts by automated confocal microscopy. The reading method was based on DNA staining and required the development of a customized algorithm to analyze the images, which enabled the use of non-modified parasites. The automated analysis generated parameters used to quantify compound activity, including infection ratio as well as the number of intracellular amastigote parasites and yielded cytotoxicity information based on the number of host cells. Comparison of this assay with one that used the promastigote form to screen 26,500 compounds showed that 50% of the hits selected against the intracellular amastigote were not selected in the promastigote screening. These data corroborate the idea that the intracellular amastigote form of the parasite is the most appropriate to be used in primary screening assay for Leishmania.

(+)-Spectaline, a Piperidine Alkaloid from <i>Senna spectabilis</i> DC. Effective in Reducing the <i>In Vitro</i> Infection of <i>Leishmania major</i>

“…Compound 1 was identified as (+)-spectaline, a known piperidine alkaloid (Figure 1) that agrees in every respect (IR, NMR and MS) with those in literature [32] which the comparison of NMR data are summarised in Table 2 and 3. With an EC 50 value of 20.58 ± 1.65 µg/mL, compound 1 decreased the parasite burden or, it has the activity in macrophage clearance. This value seems to be more potent than activity of Amp-B against L.donovani infection on THP-1 which is around 231.02 µg/mL (0.25 µM) [33]. However, this value is less potent compared to berberine (a quaternary alkaloid found in several plant families) with an EC 50 value of 9.08 µg/mL (0.027 µM) [29].…”

MicroRNA expression profiling ofLeishmania donovani-infected host cells uncovers the regulatory role of MIR30A-3p in host autophagy

“…54 Briefly, cells were fixed using 4% PFA, washed and were subsequently incubated with DNA fluorescent probe DRAQ5 (5 mM) (Thermo Fisher Scientific, 62251) for 5 min at RT. Cells were washed with 1 £ PBS to remove excess dye and images were acquired at 63x using a Leica TCS SP5 II (Leica Microsystems, Wetzlar, Germany) confocal microscope.…”

Yeast-based systems for tropical disease drug discovery

“…[3] Furthermore, phenotypic screenings can be performed only against pathogens that can be cultured. [4] This requirement is particularly problematic when designing screenings for antiparasitic compounds, since the culture of parasites outside the animal host may be extremely difficult or even impossible. For example, there is no assay for the hypnozoite stage of Plasmodium vivax.…”