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A novel Leishmania major amastigote assay in 96-well format for rapid drug screening and its use for discovery and evaluation of a new class of leishmanicidal quinolinium salts.

ABSTRACT: In most laboratories, the screening for leishmanicidal compounds is carried out with Leishmania promastigotes or axenic amastigotes. However, the best approach to identify leishmanicidal compounds is the use of amastigotes residing in macrophages. Reporter gene-based assays are relatively new tools in the search for drugs against eucaryotic protozoa, permitting the development of faster, more automated assays. In this paper, we report on the establishment of a rapid screening assay in a 96-well format. A luciferase-transgenic (Luc-tg) Leishmania major strain was generated and used to infect bone marrow-derived macrophages (BMDM). Amastigote-infected BMDM were treated with different compound concentrations. Cells were lysed with a luciferin-containing buffer, and the resulting luminescence was measured to determine the half-maximal inhibitory concentration (IC50). To validate this new amastigote screening assay, a library of a new class of quinolinium salts was synthesized and tested for leishmanicidal activity. Some of the quinolinium salts showed very promising activities, with IC50s against intracellular amastigotes (IC50 < 1 ?g/ml) and selectivity indices (SI > 20) that match the criteria of World Health Organization (WHO) for hits. Compound 21c (IC50 = 0.03 ?g/ml; SI = 358) could become a new lead structure for the development of improved chemotherapeutic drugs against L. major. In summary, we describe the establishment of a new 96-well format assay with Luc-transgenic L. major for the rapid screening of compounds for leishmanicidal activity against intracellular amastigotes and its application to the identification of a new class of quinolinium salts with most promising leishmanicidal activity.

SUBMITTER: Bringmann G

PROVIDER: S-EPMC3697368 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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A novel Leishmania major amastigote assay in 96-well format for rapid drug screening and its use for discovery and evaluation of a new class of leishmanicidal quinolinium salts.

Bringmann Gerhard G Thomale Katja K Bischof Sebastian S Schneider Christoph C Schultheis Martina M Schwarz Tobias T Moll Heidrun H Schurigt Uta U

Antimicrobial agents and chemotherapy 20130415 7

In most laboratories, the screening for leishmanicidal compounds is carried out with Leishmania promastigotes or axenic amastigotes. However, the best approach to identify leishmanicidal compounds is the use of amastigotes residing in macrophages. Reporter gene-based assays are relatively new tools in the search for drugs against eucaryotic protozoa, permitting the development of faster, more automated assays. In this paper, we report on the establishment of a rapid screening assay in a 96-well ...[more]

PMID: 23587955

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Project description:Phages have demonstrated significant potential as therapeutics in bacterial disease control and as diagnostics due to their targeted bacterial host range. Host range has typically been defined by plaque assays; an important technique for therapeutic development that relies on the ability of a phage to form a plaque upon a lawn of monoculture bacteria. Plaque assays cannot be used to evaluate a phage's ability to recognize and adsorb to a bacterial strain of interest if the infection process is thwarted post-adsorption or is temporally delayed, and it cannot highlight which phages have the strongest adsorption characteristics. Other techniques, such as classic adsorption assays, are required to define a phage's "adsorptive host range." The issue shared amongst all adsorption assays, however, is that they rely on the use of a complete bacteriophage and thus inherently describe when all adsorption-specific machinery is working together to facilitate bacterial surface adsorption. These techniques cannot be used to examine individual interactions between a singular set of a phage's adsorptive machinery (like long tail fibers, short tail fibers, tail spikes, etc.) and that protein's targeted bacterial surface receptor. To address this gap in knowledge we have developed a high-throughput, filtration-based, bacterial binding assay that can evaluate the adsorptive capability of an individual set of a phage's adsorption machinery. In this manuscript, we used a fusion protein comprised of an N-terminal bioluminescent tag translationally fused to T4's long tail fiber binding tip (gp37) to evaluate and quantify gp37's relative adsorptive strength against the Escherichia coli reference collection (ECOR) panel of 72 Escherichia coli isolates. Gp37 could adsorb to 61 of the 72 ECOR strains (85%) but coliphage T4 only formed plaques on 8 of the 72 strains (11%). Overlaying these two datasets, we were able to identify ECOR strains incompatible with T4 due to failed adsorption, and strains T4 can adsorb to but is thwarted in replication at a step post-adsorption. While this manuscript only demonstrates our assay's ability to characterize adsorptive capabilities of phage tail fibers, our assay could feasibly be modified to evaluate other adsorption-specific phage proteins.

Dataset Information

A novel Leishmania major amastigote assay in 96-well format for rapid drug screening and its use for discovery and evaluation of a new class of leishmanicidal quinolinium salts.

Publications

A novel Leishmania major amastigote assay in 96-well format for rapid drug screening and its use for discovery and evaluation of a new class of leishmanicidal quinolinium salts.

Similar Datasets

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