Reporter genes facilitating discovery of drugs targeting protozoan parasites

Dube, Anuradha; Gupta, Reema; Singh, Nasib

doi:10.1016/j.pt.2009.06.006

Cited by 72 publications

(77 citation statements)

References 78 publications

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“…As part of the drive to find new treatments, there has been a refocus on the assays and models used to identify and develop new molecules as antileishmanial drugs. For in vitro screens and assays, this has ranged from the need to develop methods that (i) are adaptable to and enable high-throughput screens against the replicative intracellular-macrophage amastigote stage of Leishmania donovani, one of the causative species of VL (7); and (ii) include high-throughput technologies that enable the collection of more information compared to the traditionally used assays based on manual counting and reporter genes (8,9). For example, highcontent screening (HCS) systems that permit the screening of large sets of compounds using imaging techniques that also capture information about compound toxicity against host cells and mode of action (10,11) have been applied to antileishmanial drug discovery (12)(13)(14)(15)(16)(17).…”

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confidence: 99%

A Replicative In Vitro Assay for Drug Discovery against Leishmania donovani

Tegazzini

Díaz

Aguilar

et al. 2016

Antimicrob Agents Chemother

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The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis, a disease potentially fatal if not treated. Current available treatments have major limitations, and new and safer drugs are urgently needed. In recent years, advances in high-throughput screening technologies have enabled the screening of millions of compounds to identify new antileishmanial agents. However, most of the compounds identified in vitro did not translate their activities when tested in in vivo models, highlighting the need to develop more predictive in vitro assays. In the present work, we describe the development of a robust replicative, high-content, in vitro intracellular L. donovani assay. Horse serum was included in the assay media to replace standard fetal bovine serum, to completely eliminate the extracellular parasites derived from the infection process. A novel phenotypic in vitro infection model has been developed, complemented with the identification of the proliferation of intracellular amastigotes measured by EdU incorporation. In vitro and in vivo results for miltefosine, amphotericin B, and the selected compound 1 have been included to validate the assay.T he leishmaniases are a complex of diseases, with visceral and cutaneous manifestations caused by protozoan parasites of the genus Leishmania. Visceral leishmaniasis (VL) has been the main focus for drug research and development over the past 2 decades, due to the large disease burden in East Africa and South Asia (1) and potential patient death if not treated. For VL, there has been progress in treatment over the past decade, with clinical evidence for efficacy of and registration for use of oral miltefosine, paromomycin, and the liposomal formulation of amphotericin B (AmBisome, Gilead, USA) in South Asia (2), as well as combinations of these standard drugs (3). The need for new drugs to treat VL remains, as (i) miltefosine is the only approved oral treatment but requires 28 days of treatment and potential teratogenicity limits its use (4), (ii) paromomycin requires 21 days of treatment and intramuscular administration (http://www.dndi.org/diseases-projects/diseases /vl/current-treatment/current-treatment-vl.html), and (iii) liposomal amphotericin B formulations, which have successful cure rates with a single dose (5), require intravenous (i.v.) infusion, have a high cost if not donated, and have a requirement for cold storage, limiting use in countries where the disease is endemic (6). As part of the drive to find new treatments, there has been a refocus on the assays and models used to identify and develop new molecules as antileishmanial drugs. For in vitro screens and assays, this has ranged from the need to develop methods that (i) are adaptable to and enable high-throughput screens against the replicative intracellular-macrophage amastigote stage of Leishmania donovani, one of the causative species of VL (7); and (ii) include high-throughput technologies that enable the collection of more information compared to the traditionally use...

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mentioning

confidence: 99%

A Replicative In Vitro Assay for Drug Discovery against Leishmania donovani

Tegazzini

Díaz

Aguilar

et al. 2016

Antimicrob Agents Chemother

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“…Transgenic rodent and human malaria parasites that express fluorescent and luminescent reporter proteins have been used in screening assays to efficiently and rapidly measure inhibition of parasite growth at different points of the parasite life cycle [6,17,22,51]. These assays have been used to identify and characterize anti-Plasmodium drugs and small-molecule inhibitors, as well as vaccines targeting parasite development at different points of the life cycle.…”

Section: Transgenic Parasites Expressing Reporter Proteinsmentioning

confidence: 99%

The use of transgenic parasites in malaria vaccine research

Othman

Marin-Mogollon

Salman

et al. 2017

Expert Review of Vaccines

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Introduction: Transgenic malaria parasites expressing foreign genes, for example fluorescent and luminescent proteins, are used extensively to interrogate parasite biology and host-parasite interactions associated with malaria pathology. Increasingly transgenic parasites are also exploited to advance malaria vaccine development. Areas covered: We review how transgenic malaria parasites are used, in vitro and in vivo, to determine protective efficacy of different antigens and vaccination strategies and to determine immunological correlates of protection. We describe how chimeric rodent parasites expressing P. falciparum or P. vivax antigens are being used to directly evaluate and rank order human malaria vaccines before their advancement to clinical testing. In addition, we describe how transgenic human and rodent parasites are used to develop and evaluate live (genetically) attenuated vaccines. Expert commentary: Transgenic rodent and human malaria parasites are being used to both identify vaccine candidate antigens and to evaluate both sub-unit and whole organism vaccines before they are advanced into clinical testing. Transgenic parasites combined with in vivo pre-clinical testing models (e.g. mice) are used to evaluate vaccine safety, potency and the durability of protection as well as to uncover critical protective immune responses and to refine vaccination strategies. ARTICLE HISTORY

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“…lacZ Escherichia coli lactose -galactosidase -galactosidase (Kopp et al, 2007;Shimada et al, 2012) (Adám et al, 2000;Verri et al, 2003;Zheng et al, 2006) (Arenal et al, 2004) . lacZ , , -galactosidase -galactosidase (Dube et al, 2009). lacZ .…”

Section: 서 론mentioning

confidence: 99%

Availability of the lacZ gene as a Reporter Gene for Production of Transgenic Artemia franciscana

정효선¹,

김동수

2013

Korean Journal of Fisheries and Aquatic Sciences

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We examined the availability of the lacZ gene ( -galactosidase gene) as a reporter of foreign gene transfer in the cysts of Artemia franciscana (A. franciscana) to conduct a risk assessment of living genetically modified organisms (LMOs) in the marine ecosystem. The LacZ gene was transferred to decapsulated cysts by particle bombardment, and its insertion and expression were assessed by means of polymerase chain reaction (PCR) and X-gal staining. X-gal staining indicated lacZ expression in all A. franciscana examined (including the control group), which exhibited not only negative but also positive PCR amplification. Endogenous -galactosidase is highly active in the whole body of A. franciscana during all stages of the life cycle. Thus, the lacZ gene is unsuitable as a reporter for foreign gene transfer in A. franciscana cysts, because it is difficult to discriminate between exogenous and endogenous -galactosidase activity.

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Reporter genes facilitating discovery of drugs targeting protozoan parasites

Cited by 72 publications

References 78 publications

A Replicative In Vitro Assay for Drug Discovery against Leishmania donovani

A Replicative In Vitro Assay for Drug Discovery against Leishmania donovani

The use of transgenic parasites in malaria vaccine research

Availability of the lacZ gene as a Reporter Gene for Production of Transgenic Artemia franciscana

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