Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity

Abstract

Visceral leishmaniasis is an important parasitic disease of the developing world with a limited arsenal of drugs available for treatment. The existing drugs have significant deficiencies so there is an urgent need for new and improved drugs. In the human host, Leishmania are obligate intracellular parasites which poses particular challenges in terms of drug discovery. To achieve sufficient throughput and robustness, free-living parasites are often used in primary screening assays as a surrogate for the more complex intracellular assays. We and others have found that such axenic assays have a high false positive rate relative to the intracellular assays, and that this limits their usefulness as a primary platform for screening of large compound collections. While many different reasons could lie behind the poor translation from axenic parasite to intracellular parasite, we show here that a key factor is the identification of growth slowing and cytostatic compounds by axenic assays in addition to the more desirable cytocidal compounds. We present a screening cascade based on a novel cytocidal-only axenic amastigote assay, developed by increasing starting density of cells and lowering the limit of detection, and show that it has a much improved translation to the intracellular assay. We propose that this assay is an improved primary platform in a new Leishmania screening cascade designed for the screening of large compound collections. This cascade was employed to screen a diversity-oriented-synthesis library, and yielded two novel antileishmanial chemotypes. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery.

Targeting the Cytochrome bc 1 Complex of Leishmania Parasites for Discovery of Novel Drugs

“…5A). For infected macrophages treated with DMSO, the number of intracellular amastigotes decreased by ϳ20% over 96 h, while treatment with 10 M buparvaquone resulted in complete death of the amastigotes by 96 h. This result indicates that buparvaquone is cytocidal, because application of this compound leads to parasite death rather than simply growth arrest (40).…”

Identification of Trypanocidal Activity for Known Clinical Compounds Using a New Trypanosoma cruzi Hit-Discovery Screening Cascade

“…Our previous experience in finding startpoints for kinetoplastid drug discovery has highlighted the issue of separating growth-slowing from cytocidal compounds [ 44 , 45 ]. We have observed significant replication of the T .…”

Challenges in drug discovery targeting TriTryp diseases with an emphasis on leishmaniasis

“…The development of new therapeutics focuses on screening potentially effective compounds in parasite growth/multiplication assays. There are several screening assays available against Leishmania based on different stages of the parasites: promastigotes ( Siqueira-Neto et al, 2010 ), axenic amastigotes ( Nühs et al, 2015 ) or intracellular amastigotes ( Siqueira-Neto et al, 2012 ). Screenings for axenic forms of Leishmania present several advantages; (i) limited number of parasites is sufficient to test many compounds; (ii) faster read-outs; (iii) higher throughput; and (iv) reproducibility.…”