title: 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.
authors.affiliation: Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, University of Dundee, Dundee, United Kingdom.
authors.forename: Andrea
authors.initials: A
authors.lastname: Nühs
authors.affiliation: Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, University of Dundee, Dundee, United Kingdom.
authors.forename: Manu
authors.initials: M
authors.lastname: De Rycker
authors.affiliation: Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, University of Dundee, Dundee, United Kingdom.
authors.forename: Sujatha
authors.initials: S
authors.lastname: Manthri
authors.affiliation: Broad Institute, Cambridge, Massachusetts, United States of America.
authors.forename: Eamon
authors.initials: E
authors.lastname: Comer
authors.affiliation: Broad Institute, Cambridge, Massachusetts, United States of America.
authors.forename: Christina A
authors.initials: CA
authors.lastname: Scherer
authors.affiliation: Broad Institute, Cambridge, Massachusetts, United States of America.
authors.forename: Stuart L
authors.initials: SL
authors.lastname: Schreiber
authors.affiliation: Drugs for Neglected Diseases initiative, Geneva, Switzerland.
authors.forename: Jean-Robert
authors.initials: JR
authors.lastname: Ioset
authors.affiliation: Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, University of Dundee, Dundee, United Kingdom.
authors.forename: David W
authors.initials: DW
authors.lastname: Gray
mesh_terms: D000981:Antiprotozoal Agents / Q000494:pharmacology*; D061245:Axenic Culture; D055808:Drug Discovery / Q000379:methods*; D057166:High-Throughput Screening Assays; D006801:Humans; D007893:Leishmania donovani / Q000187:drug effects* / Q000254:growth & development; D007898:Leishmaniasis, Visceral / Q000188:drug therapy / Q000469:parasitology; D011237:Predictive Value of Tests
chemical_list: D000981:Antiprotozoal Agents