Simultaneous multi-parametric analysis of Leishmania and of its hosting mammal cells: A high content imaging-based method enabling sound drug discovery process

Forestier, Claire-Lise; Späth, Gérald F.; Prina, Éric; Dasari, Sreekanth

doi:10.1016/j.micpath.2014.10.012

Cited by 12 publications

(9 citation statements)

References 67 publications

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“…e . promastigotes or axenic amastigote forms, which are easy to handle and inexpensive to grow, thus allowing for high-throughput (HT), automatized, micro-well plate-based screening 12 . However, the biological relevance of such assays may be called into question given (i) the extensive genetic instability and virulence attenuation observed in cultured parasites 13–15 , and (ii) the biological differences in for example infectivity between axenic and bona fide, tissue-derived amastigotes 16 .…”

Section: Introductionmentioning

confidence: 99%

Discovery of novel hit compounds with broad activity against visceral and cutaneous Leishmania species by comparative phenotypic screening

Lamotte

Aulner

Späth

et al. 2019

Sci Rep

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The limited success of recent phenotypic anti-leishmanial drug screening campaigns calls for new screening strategies for the discovery of clinically relevant hits. Here we present such a novel strategy based on physiologically relevant, ex vivo biology. We established high content phenotypic assays that combine primary murine macrophages and lesion-derived, virulent L. donovani and L. amazonensis amastigotes, which we applied to validate previously identified, anti-leishmanial hit compounds referred to as ‘GSK Leish-Box’. Together with secondary screens using cultured promastigotes, our pipeline distinguished stage- and/or species-specific compounds, including 20 hits with broad activity at 10 µM against intracellular amastigotes of both viscerotropic and dermotropic Leishmania. Even though the GSK Leish-Box hits were identified by phenotypic screening using THP-1 macrophage-like cells hosting culture-derived L. donovani LdBob parasites, our ex vivo assays only validated anti-leishmanial activity at 10 µM on intra-macrophagic L. donovani for 23 out of the 188 GSK Leish-Box hits. In conclusion, our comparative approach allowed the identification of hits with broad anti-leishmanial activity that represent interesting novel candidates to be tested in animal models. Physiologically more relevant screening approaches such as described here may reduce the very high attrition rate observed during pre-clinical and clinical phases of the drug development process.

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Section: Introductionmentioning

confidence: 99%

Discovery of novel hit compounds with broad activity against visceral and cutaneous Leishmania species by comparative phenotypic screening

Lamotte

Aulner

Späth

et al. 2019

Sci Rep

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“…Therefore, a trend towards high-throughput phenotypic screening has been noted in the last years. Also, the introduction of high-content screening platforms emerged as a promising approach, allowing large libraries to be tested against Leishmania intracellular amastigotes (Siqueira-Neto et al 2012; Forestier et al 2015). This approach has several advantages including the differentiation between activity against the parasite or the host cell, and the possibility of identification of singular cell events that can guide the research on the drug's mode of action.…”

Section: Drug Screeningmentioning

confidence: 99%

Chemotherapy of leishmaniasis: present challenges

2017

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Cutaneous and visceral leishmaniasis are amongst the most devastating infectious diseases of our time, affecting millions of people worldwide. The treatment of these serious diseases rely on a few chemotherapeutic agents, most of which are of parenteral use and induce severe side-effects. Furthermore, rates of treatment failure are high and have been linked to drug resistance in some areas. Here, we reviewed data on current chemotherapy practice in leishmaniasis. Drug resistance and mechanisms of resistance are described as well as the prospects for applying drug combinations for leishmaniasis chemotherapy. It is clear that efforts for discovering new drugs applicable to leishmaniasis chemotherapy are essential. The main aspects on the various steps of drug discovery in the field are discussed.

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“…[15][16][17] These screens generally employ transgenic Leishmania lines expressing either fluorescent (e.g., GFP) or bioluminescent (e.g., luciferase) reporter proteins and quantify intracellular growth in infected macrophages from total well fluorescence/luminescence, or the shift in host cell fluorescence by flow cytometry. 15,[18][19][20][21][22][23][24][25] However, per-well measurements cannot distinguish between intracellular and extracellular parasites, and in some studies, the reported IC 50 s for known drugs, using a flow cytometry-based method, are considerably greater than those reported with other assays.…”

mentioning

confidence: 99%

“…21 More recent studies have used high-content screening (HCS) approaches to monitor the intracellular growth of transgenic parasite lines within individual macrophages, providing greater insights into host-parasite interactions and drug toxicity. 10,15,16,26 However, the creation of suitable transgenic parasites for these HTS and HCS assays can be time-consuming and is often associated with a progressive loss of virulence. 27 As an alternative to using transgenic parasite lines, a number of HTS methods have stained the host or parasite with nuclear stains (e.g., 4 0 , 6-diamidino-2-phenylindole [DAPI]) or used synthetic nucleosides (combined with fluorophoreconjugated anti-BrdU antibodies).…”

mentioning

confidence: 99%

High-Content Assay for Measuring Intracellular Growth ofLeishmaniain Human Macrophages

Dagley

Saunders

Simpson

et al. 2015

ASSAY and Drug Development Technologies

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Leishmania species are sandfly-transmitted protozoan parasites that cause a spectrum of diseases, ranging from localized skin lesions to fatal visceral disease, in more than 12 million people worldwide. These parasites primarily target macrophages in their mammalian hosts and proliferate as non-motile amastigotes in the phagolysosomal compartment of these cells. High-throughput screens for measuring Leishmania growth within this intracellular niche are needed to identify host and parasite factors that are required for virulence and to identify new drug candidates. Here we describe the development of a new high-content imaging method for quantifying the intracellular growth of Leishmania mexicana parasites in THP-1 macrophages. Wild-type parasites were pre-stained with the fluorescent dye CellTracker(™) Orange CMRA and used to infect THP-1 macrophages in 384-well plates. Infected and uninfected macrophages were subsequently stained with CellTracker Green CMFDA, allowing accurate quantitation of the number of parasites per macrophage using separate detector channels. We validated this method for use in high-content drug screening by examining the dose dependence of known anti-leishmanial drugs on intracellular growth. Unlike previous protocols, this method does not require the generation of transgenic fluorescent or bioluminescent parasite lines and can be readily adapted for screening different Leishmania species, strains, or mutant lines in a wide range of phagocytic host cell types.

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Simultaneous multi-parametric analysis of Leishmania and of its hosting mammal cells: A high content imaging-based method enabling sound drug discovery process

Cited by 12 publications

References 67 publications

Discovery of novel hit compounds with broad activity against visceral and cutaneous Leishmania species by comparative phenotypic screening

Discovery of novel hit compounds with broad activity against visceral and cutaneous Leishmania species by comparative phenotypic screening

Chemotherapy of leishmaniasis: present challenges

High-Content Assay for Measuring Intracellular Growth ofLeishmaniain Human Macrophages

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