Automated genome-wide visual profiling of cellular proteins involved in HIV infection.

Genovesio, Auguste,Kwon, Yong-Jun,Windisch, Marc P,Kim, Nam Youl,Choi, Seo Yeon,Kim, Hi Chul,Jung, Sungyong,Mammano, Fabrizio,Perrin, Virginie,Boese, Annette S,Casartelli, Nicoletta,Schwartz, Olivier Mary Ann Liebert, Inc 2011 Journal of biomolecular screening Vol.16 No.9

<P>Recent genome-wide RNAi screens have identified >842 human genes that affect the human immunodeficiency virus (HIV) cycle. The list of genes implicated in infection differs between screens, and there is minimal overlap. A reason for this variance is the interdependence of HIV infection and host cell function, producing a multitude of indirect or pleiotropic cellular effects affecting the viral infection during RNAi screening. To overcome this, the authors devised a 15-dimensional phenotypic profile to define the viral infection block induced by CD4 silencing in HeLa cells. They demonstrate that this phenotypic profile excludes nonspecific, RNAi-based side effects and viral replication defects mediated by silencing of housekeeping genes. To achieve statistical robustness, the authors used automatically annotated RNAi arrays for seven independent genome-wide RNAi screens. This identified 56 host genes, which reliably reproduced CD4-like phenotypes upon HIV infection. The factors include 11 known HIV interactors and 45 factors previously not associated with HIV infection. As proof of concept, the authors confirmed that silencing of PAK1, Ku70, and RNAseH2A impaired HIV replication in Jurkat cells. In summary, multidimensional, visual profiling can identify genes required for HIV infection.</P>

Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes

Arhel, Nathalie,Genovesio, Auguste,Kim, Kyeong-Ae,Miko, Sarah,Perret, Emmanuelle,Olivo-Marin, Jean-Christophe,Shorte, Spencer,Charneau, Pierre Nature Publishing Group 2006 Nature methods Vol.3 No.10

Emerging real-time techniques for imaging viral infections provide powerful tools for understanding the dynamics of virus-host cell interactions. Here we labeled human immunodeficiency virus-1 (HIV-1) integrase with a small tetracysteine tag, which preserved the virus' infectivity while allowing it to be labeled with the bis-arsenical fluorescein derivative FlAsH. This labeling allowed us to image both intracytoplasmic and intranuclear HIV-1 complexes in three dimensions over time (4D) in human cells and enabled us to analyze HIV-1 kinetics by automated 4D quantitative particle tracking. In the cytoplasm, HIV-1 complexes underwent directed movements toward the nuclear compartment, kinetically characteristic of both microtubule- and actin-dependent transport. The complexes then adopted smaller movements in a very confined volume once associated with the nuclear membrane and more diffuse movements once inside the nucleus. This work contributes new insight into the various movements of HIV-1 complexes within infected cells and provides a useful tool for the study of virus-host cell interactions during infection.

3D Detection of Mutant Hairs in Drosophila Wing Images

Jihoon Kwak,Chun-Taek Oh,Sung-Jun Han,Michael Adsetts Edberg Hansen,Auguste Genovesio,Myungjoo Kang 한국산업응용수학회 2011 한국산업응용수학회 학술대회 논문집 Vol.6 No.1

We developed an automated mutant hair counting system in Drosophila wing images. In our previous work [2], we developed the image acquisition method using multi-focused image stack, hair separation method into upper and lower hair, and hair detection method using template matching. In this work, the hair detection and mutant classification algorithms are enhanced and extended to 3D, and the wing area segmentation is newly developed.

Quantification of protein interaction in living cells by two‐photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed‐through

Kim, Jiho,Li, Xiaolan,Kang, Moon‐,Sik,Im, Kang‐,Bin,Genovesio, Auguste,Grailhe, Regis Wiley Subscription Services, Inc., A Wiley Company 2012 Cytometry. the journal of the International Societ Vol.a81 No.2

<P><B>Abstract</B></P><P>Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful method to visualize and quantify protein–protein interaction in living cells. Unfortunately, the emission bleed‐through of FPs limits the usage of this complex technique. To circumvent undesirable excitation of the acceptor fluorophore, using two‐photon excitation, we searched for FRET pairs that show selective excitation of the donor but not of the acceptor fluorescent molecule. We found this property in the fluorescent cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) and YFP/mCherry FRET pairs and performed two‐photon excited FRET spectral imaging to quantify protein interactions on the later pair that shows better spectral discrimination. Applying non‐negative matrix factorization to unmix two‐photon excited spectral imaging data, we were able to eliminate the donor bleed‐through as well as the autofluorescence. As a result, we achieved FRET quantification by means of a single spectral acquisition, making the FRET approach not only easy and straightforward but also less prone to calculation artifacts. As an application of our approach, the intermolecular interaction of amyloid precursor protein and the adaptor protein Fe65 associated with Alzheimer's disease was quantified. We believe that the FRET approach using two‐photon and fluorescent YFP/mCherry pair is a promising method to monitor protein interaction in living cells. © 2011 International Society for Advancement of Cytometry</P>

Dimerization, Oligomerization, and Aggregation of Human Amyotrophic Lateral Sclerosis Copper/Zinc Superoxide Dismutase 1 Protein Mutant Forms in Live Cells

Kim, Jiho,Lee, Honggun,Lee, Joo Hyun,Kwon, Do-yoon,Genovesio, Auguste,Fenistein, Denis,Ogier, Arnaud,Brondani, Vincent,Grailhe, Regis American Society for Biochemistry and Molecular Bi 2014 The Journal of biological chemistry Vol.289 No.21

<P>More than 100 copper/zinc superoxide dismutase 1 (SOD1) genetic mutations have been characterized. These mutations lead to the death of motor neurons in ALS. In its native form, the SOD1 protein is expressed as a homodimer in the cytosol. <I>In vitro</I> studies have shown that SOD1 mutations impair the dimerization kinetics of the protein, and <I>in vivo</I> studies have shown that SOD1 forms aggregates in patients with familial forms of ALS. In this study, we analyzed WT SOD1 and 9 mutant (mt) forms of the protein by non-invasive fluorescence techniques. Using microscopic techniques such as fluorescence resonance energy transfer, fluorescence complementation, image-based quantification, and fluorescence correlation spectroscopy, we studied SOD1 dimerization, oligomerization, and aggregation. Our results indicate that SOD1 mutations lead to an impairment in SOD1 dimerization and, subsequently, affect protein aggregation. We also show that SOD1 WT and mt proteins can dimerize. However, aggregates are predominantly composed of SOD1 mt proteins.</P>

High Content Screening Pipe Line for Leishmaniasis

Seunghyun Moon,Jair L. Siqueira-Neto,Lucio H. Freitas-Junoir,Auguste Genovesio,Myungjoo Kang 한국산업응용수학회 2009 한국산업응용수학회 학술대회 논문집 Vol.4 No.2

An Image-Based High-Content Screening Assay for Compounds Targeting Intracellular <i>Leishmania donovani</i> Amastigotes in Human Macrophages

Siqueira-Neto, Jair L.,Moon, Seunghyun,Jang, Jiyeon,Yang, Gyongseon,Lee, Changbok,Moon, Hong Kee,Chatelain, Eric,Genovesio, Auguste,Cechetto, Jonathan,Freitas-Junior, Lucio H. Public Library of Science 2012 PLoS neglected tropical diseases Vol.6 No.6

<▼1><P>Leishmaniasis is a tropical disease threatening 350 million people from endemic regions. The available drugs for treatment are inadequate, with limitations such as serious side effects, parasite resistance or high cost. Driven by this need for new drugs, we developed a high-content, high-throughput image-based screening assay targeting the intracellular amastigote stage of different species of <I>Leishmania</I> in infected human macrophages. The <I>in vitro</I> infection protocol was adapted to a 384-well-plate format, enabling acquisition of a large amount of readouts by automated confocal microscopy. The reading method was based on DNA staining and required the development of a customized algorithm to analyze the images, which enabled the use of non-modified parasites. The automated analysis generated parameters used to quantify compound activity, including infection ratio as well as the number of intracellular amastigote parasites and yielded cytotoxicity information based on the number of host cells. Comparison of this assay with one that used the promastigote form to screen 26,500 compounds showed that 50% of the hits selected against the intracellular amastigote were not selected in the promastigote screening. These data corroborate the idea that the intracellular amastigote form of the parasite is the most appropriate to be used in primary screening assay for <I>Leishmania</I>.</P></▼1><▼2><P><B>Author Summary</B></P><P>Leishmaniasis, one of the most neglected tropical diseases, affects over 2 million people each year. Visceral leishmaniasis (VL), also known as Kala-azar, is caused by the protozoan parasites <I>Leishmania donovani</I> and <I>Leishmania infantum</I> and is fatal if left untreated. Because existing treatments are often ineffective due to parasite resistance and/or toxicity new drugs are urgently needed. Leishmaniasis is transmitted to humans by the bite of an infected sandfly. In the insect vector, parasites exist as flagellated forms—promastigotes, which infect macrophage cells of the human host, where they differentiate to round forms known as amastigotes. Amastigotes and promastigotes are substantially different from a molecular perspective. Drug discovery for leishmaniasis has traditionally been complicated by the unavailability of validated drug targets and of relevant drug assays. Whole cell-based assays have been widely used, as they bypass the need for a validated target. However, they use the insect form of the parasite; indeed, the human form, the intracellular amastigote, is difficult to obtain in the laboratory in quantities compatible with drug screening. We describe here the technical advances that made it possible to adapt the intracellular amastigote form of <I>L. donovani</I> to a drug assay compatible with high-throughput screening.</P></▼2>

High Content Screening Identifies Decaprenyl-Phosphoribose 2′ Epimerase as a Target for Intracellular Antimycobacterial Inhibitors

Christophe, Thierry,Jackson, Mary,Jeon, Hee Kyoung,Fenistein, Denis,Contreras-Dominguez, Monica,Kim, Jaeseung,Genovesio, Auguste,Carralot, Jean-Philippe,Ewann, Fanny,Kim, Eun Hye,Lee, Sae Yeon,Kang, S Public Library of Science 2009 PLoS pathogens Vol.5 No.10

<▼1><P>A critical feature of <I>Mycobacterium tuberculosis</I>, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of <I>M. tuberculosis</I> within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against <I>M. tuberculosis</I>, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of <I>M. tuberculosis</I> with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2′ epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials.</P></▼1><▼2><P><B>Author Summary</B></P><P>Tuberculosis is still a major threat to global health. The disease in humans is caused by a bacterium, <I>Mycobacterium tuberculosis</I>, and treatment of an infected individual requires more than six months of chemotherapy. Because such a long course of treatment is required, compliance is low, which can result in the development of multidrug resistant strains (MDR-TB) and even extremely resistant strains (XDR-TB). Identifying new drug targets and potential lead therapeutic compounds are needed to combat MDR-XDR-TB. We developed a new type of assay based on the visualization of mycobacterium replication within host cells and applied it for the search of compounds that are able to chase the pathogen from its hideout. As a result, we found 20 new series of drug candidates that are effective against the bacilli in its hiding place, potentially addressing a crucial aspect in the resilience of the disease. We also showed that one series of compounds acts by inhibiting a key enzyme required for the synthesis of an essential component from the mycobacterial cell wall that is not targeted by any of the commercially available antituberculosis drugs. Altogether, our results pave the way for development of the next generation of antibacterial agents.</P></▼2>

Antileishmanial High-Throughput Drug Screening Reveals Drug Candidates with New Scaffolds

Siqueira-Neto, Jair L.,Song, Ok-Ryul,Oh, Hyunrim,Sohn, Jeong-Hun,Yang, Gyongseon,Nam, Jiyoun,Jang, Jiyeon,Cechetto, Jonathan,Lee, Chang Bok,Moon, Seunghyun,Genovesio, Auguste,Chatelain, Eric,Christoph Public Library of Science 2010 PLoS neglected tropical diseases Vol.4 No.5

<▼1><P>Drugs currently available for leishmaniasis treatment often show parasite resistance, highly toxic side effects and prohibitive costs commonly incompatible with patients from the tropical endemic countries. In this sense, there is an urgent need for new drugs as a treatment solution for this neglected disease. Here we show the development and implementation of an automated high-throughput viability screening assay for the discovery of new drugs against <I>Leishmania</I>. Assay validation was done with <I>Leishmania</I> promastigote forms, including the screening of 4,000 compounds with known pharmacological properties. In an attempt to find new compounds with leishmanicidal properties, 26,500 structurally diverse chemical compounds were screened. A cut-off of 70% growth inhibition in the primary screening led to the identification of 567 active compounds. Cellular toxicity and selectivity were responsible for the exclusion of 78% of the pre-selected compounds. The activity of the remaining 124 compounds was confirmed against the intramacrophagic amastigote form of the parasite. <I>In vitro</I> microsomal stability and cytochrome P450 (CYP) inhibition of the two most active compounds from this screening effort were assessed to obtain preliminary information on their metabolism in the host. The HTS approach employed here resulted in the discovery of two new antileishmanial compounds, bringing promising candidates to the leishmaniasis drug discovery pipeline.</P></▼1><▼2><P><B>Author Summary</B></P><P>Every year, more than 2 million people worldwide suffer from leishmaniasis, a neglected tropical disease present in 88 countries. The disease is caused by the single-celled protozoan parasite species of the genus <I>Leishmania</I>, which is transmitted to humans by the bite of the sandfly. The disease manifests itself in a broad range of symptoms, and its most virulent form, named visceral leishmaniasis, is lethal if not treated. Most of the few available treatments for leishmaniasis were developed decades ago and are often toxic, sometimes even leading to the patient's death. Furthermore, the parasite is developing resistance to available drugs, making the discovery and development of new antileishmanials an urgent need. To tackle this problem, the authors of this study employed the use of high-throughput technologies to screen a large library of small, synthetic molecules for their ability to interfere with the viability of <I>Leishmania</I> parasites. This study resulted in the discovery of two novel compounds with leishmanicidal properties and promising drug-like properties, bringing new candidates to the leishmaniasis drug discovery pipeline.</P></▼2>