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>

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>

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

Pectin and SDS as auxiliary flocculants for complementary treatment of textile wastewater by electrocoagulation

Mauro Cosme de Carvalho Góes,Marcos Paulo Ribeiro Garcez,Andréa Roberta Ferreira Siqueira,Thiago Palhares Farias,Claudemir Gomes de Santana,Jonas de Jesus Gomes da Costa Neto,Cicero Wellington Brito B 한국화학공학회 2021 Korean Journal of Chemical Engineering Vol.38 No.8

Textile wastewaters currently remain as one of the major sources of environmental pollution. In addition to the presence of several recalcitrant species, the volume of the effluent to be treated is usually quite high, due to the excessive consumption of water in this kind of processing. To avoid all the negative impacts associated with the discharge of these untreated effluents, effective remediation techniques should be applied. Although there are currently several methods available, due to complexity and volume of the wastewater, combined technologies can provide better efficiency, lower cost and less time consumption. In this work, electrocoagulation (EC) combined with the conventional flocculation process was studied for the removal of methylene blue dye (MB) in aqueous medium. Under specific EC conditions (4 pairs of electrodes, 2.5 cm spacing, electrolysis time of 203min, pHinitial 4, 32 V, 1.5 A) it was possible to remove 63% of the dye. Combining EC with conventional flocculation, a notable reduction in electrolysisrequired time (203 to 60 min), dye (99.8%), color (100%) and turbidity (99.2%) was observed.

Synthesis and biological evaluation of 2-acetamidothiophene-3-carboxamide derivatives against Leishmania donovani

Oh, S.,Kwon, B.,Kong, S.,Yang, G.,Lee, N.,Han, D.,Goo, J.,Siqueira-Neto, J.,Freitas-Junior, L.,Liuzzi, M. TEH ROYAL SOCIETY OF CHEMISTRY 2014 MedChemComm Vol.5 No.2

A high-throughput (HTS) and high-content screening (HCS) campaign of a commercial library identified 2-acetamidothophen-3-carboxamide as a novel scaffold for developing new anti-leishmanial agents. A series of chemical modifications were performed to study the structure-activity relationship (SAR) and in vitro anti-leishmanial activities were evaluated using biological assays of not only extracellular promastigotes but also intracellular amastigotes. Compound 6a showed promising anti-amastigote activity (EC50 = 6.41 mu M) against L. donovani without any cytotoxicity (CC50 > 50 mu M) towards human macrophages.