Development of a semi-automated image-based high-throughput drug screening system.

We previously reported that the innate sensing of the endosymbiont <i>Leishmania</i> RNA virus 1 (LRV1) within <i>Leishmania (Viannia) guyanensis</i> through Toll-like receptor 3, worsens the pathogenesis of parasite infection in mice. The presence of LRV1 has been associated with the failure of first-line treatment in patients infected with LRV1 containing - <i>L. guyanensis</i> and - <i>L. braziliensis</i> parasites. Here, we established a semi-automated image-based high-throughput drug screening (HTDS) protocol to measure parasiticidal activity of the Prestwick chemical library in primary murine macrophages infected with LRV1-containing <i>L. guyanensis</i> . The two-independent screens generated 14 hit compounds with over sixty-nine percent reduction in parasite growth compared to control, at a single dose in both screens. Our screening strategy offers great potential in the search for new drugs and accelerates the discovery rate in the field of drug repurposing against <i>Leishmania</i> . Moreover, this technique allows the concomitant assessment of the effect of drug toxicity on host cell number

High-throughput, nonperturbing quantification of lipid droplets with digital holographic microscopy.

In vitro differentiating adipocytes are sensitive to liquid manipulations and have the tendency to float. Assessing adipocyte differentiation using current microscopy techniques involves cell staining and washing, while using flow cytometry involves cell retrieval in suspension. These methods induce biases, are difficult to reproduce, and involve tedious optimizations. In this study, we present digital holographic microscopy (DHM) as a label-free, nonperturbing means to quantify lipid droplets in differentiating adipocytes in a robust medium- to high-throughput manner. Taking advantage of the high refractive index of lipid droplets, DHM can assess the production of intracellular lipid droplets by differences in phase shift in a quantitative manner. Adipocytic differentiation, combined with other morphological features including cell confluence and cell death, was tracked over 6 days in live OP9 mesenchymal stromal cells. We compared DHM with other currently available methods of lipid droplet quantification and demonstrated its robustness with modulators of adipocytic differentiation in a dose-responsive manner. This study suggests DHM as a novel marker-free nonperturbing method to study lipid droplet accumulation and may be envisioned for drug screens and mechanistic studies on adipocytic differentiation

Novel high-throughput fluorescence-based assay for the identification of nematocidal compounds that target the blood-feeding pathway

Hookworm infections cause a neglected tropical disease (NTD) affecting ~740 million people worldwide, principally those living in disadvantaged communities. Infections can cause high morbidity due to their impact on nutrient uptake and their need to feed on host blood, resulting in a loss of iron and protein, which can lead to severe anaemia and impaired cognitive development in children. Currently, only one drug, albendazole is efficient to treat hookworm infection and the scientific community fears the rise of resistant strains. As part of on-going efforts to control hookworm infections and its associated morbidities, new drugs are urgently needed. We focused on targeting the blood-feeding pathway, which is essential to the parasite survival and reproduction, using the laboratory hookworm model Nippostrongylus brasiliensis (a nematode of rodents with a similar life cycle to hookworms). We established an in vitro-drug screening assay based on a fluorescent-based measurement of parasite viability during blood-feeding to identify novel therapeutic targets. A first screen of a library of 2654 natural compounds identified four that caused decreased worm viability in a blood-feeding-dependent manner. This new screening assay has significant potential to accelerate the discovery of new drugs against hookworms

Homology modelling and spectroscopy, a never-ending love story

Homology modelling is normally the technique of choice when experimental structure data are not available but three-dimensional coordinates are needed, for example, to aid with detailed interpretation of results of spectroscopic studies. Herein, the state of the art of homology modelling will be described in the light of a series of recent developments, and an overview will be given of the problems and opportunities encountered in this field. The major topic, the accuracy and precision of homology models, will be discussed extensively due to its influence on the reliability of conclusions drawn from the combination of homology models and spectroscopic data. Three real-world examples will illustrate how both homology modelling and spectroscopy can be beneficial for (bio)medical research

Screening out irrelevant cell-based models of disease

The common and persistent failures to translate promising preclinical drug candidates into clinical success highlight the limited effectiveness of disease models currently used in drug discovery. An apparent reluctance to explore and adopt alternative cell-and tissue-based model systems, coupled with a detachment from clinical practice during assay validation, contributes to ineffective translational research. To help address these issues and stimulate debate, here we propose a set of principles to facilitate the definition and development of disease-relevant assays, and we discuss new opportunities for exploiting the latest advances in cell-based assay technologies in drug discovery, including induced pluripotent stem cells, three-dimensional (3D) co-culture and organ-on-a-chip systems, complemented by advances in single-cell imaging and gene editing technologies. Funding to support precompetitive, multidisciplinary collaborations to develop novel preclinical models and cell-based screening technologies could have a key role in improving their clinical relevance, and ultimately increase clinical success rates