Dataset Information

Quantum dots: a new tool for anti-malarial drug assays.

ABSTRACT:

Background

Malaria infects over 300 million people every year and one of the major obstacles for the eradication of the disease is parasite's resistance to current chemotherapy, thus new drugs are urgently needed. Quantum dot (QD) is a fluorescent nanocrystal that has been in the spotlight as a robust tool for visualization of live cell processes in real time. Here, a simple and efficient method using QD to directly label Plasmodium falciparum-infected erythrocytes (iRBCs) was searched in order to use the QD as a probe in an anti-malarial drug-screening assay.

Methods

A range of QDs with different chemical coatings were tested for their ability to specifically bind iRBCs by immunofluorescence assay (IFA). One QD was selected and used to detect parasite growth and drug sensitivity by flow cytometry.

Results

PEGylated-cationic QD (PCQD) was found to specifically label infected erythrocytes preferentially with late stage parasites. The detection of QD-labelled infected erythrocytes by flow cytometry was sensitive enough to monitor chloroquine anti-malarial toxicity with a drug incubation period as short as 24 h (EC50 = 113nM). A comparison of our assay with another widely used anti-malarial drug screening assay, the pLDH assay, showed that PCQD-based assay had 50% improved sensitivity in detecting drug efficacy within a parasite life cycle. An excellent Z-factor of 0.8 shows that the QD assay is suitable for high-throughput screening.

Conclusions

This new assay can offer a rapid and robust platform to screen novel classes of anti-malarial drugs.

SUBMITTER: Ku MJ

PROVIDER: S-EPMC3112454 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Publications

Quantum dots: a new tool for anti-malarial drug assays.

Ku Min-Je MJ Dossin Fernando M FM Choi Youngseon Y Moraes Carolina B CB Ryu Jiyoung J Song Rita R Freitas-Junior Lucio H LH

Malaria journal 20110509

<h4>Background</h4>Malaria infects over 300 million people every year and one of the major obstacles for the eradication of the disease is parasite's resistance to current chemotherapy, thus new drugs are urgently needed. Quantum dot (QD) is a fluorescent nanocrystal that has been in the spotlight as a robust tool for visualization of live cell processes in real time. Here, a simple and efficient method using QD to directly label Plasmodium falciparum-infected erythrocytes (iRBCs) was searched i ...[more]

PMID: 21554712

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Project description:BackgroundMultidrug-resistant Plasmodium is of major concern today. Effective vaccines or successful applications of RNAi-based strategies for the treatment of malaria are currently unavailable. An unexplored area in the field of malaria research is the development of DNA-targeting drugs that can specifically interact with parasitic DNA and introduce deleterious changes, leading to loss of vital genome function and parasite death.Presentation of the hypothesisAdvances in the development of zinc finger nuclease (ZFN) with engineered DNA recognition domains allow us to design and develop nuclease of high target sequence specificity with a mega recognition site that typically occurs only once in the genome. Moreover, cell-penetrating peptides (CPP) can cross the cell plasma membrane and deliver conjugated protein, nucleic acid, or any other cargo to the cytoplasm, nucleus, or mitochondria. This article proposes that a drug from the combination of the CPP and ZFN systems can effectively enter the intracellular parasite, introduce deleterious changes in its genome, and eliminate the parasite from the infected cells.Testing the hypothesisAvailability of a DNA-binding motif for more than 45 triplets and its modular nature, with freedom to change number of fingers in a ZFN, makes development of customized ZFN against diverse target DNA sequence of any gene feasible. Since the Plasmodium genome is highly AT rich, there is considerable sequence site diversity even for the structurally and functionally conserved enzymes between Plasmodium and humans. CPP can be used to deliver ZFN to the intracellular nucleus of the parasite. Signal-peptide-based heterologous protein translocation to Plasmodium-infected RBCs (iRBCs) and different Plasmodium organelles have been achieved. With successful fusion of CPP with mitochondrial- and nuclear-targeting peptides, fusion of CPP with 1 more Plasmodium cell membrane translocation peptide seems achievable.Implications of the hypothesisTargeting of the Plasmodium genome using ZFN has great potential for the development of anti-malarial drugs. It allows the development of a single drug against all malarial infections, including multidrug-resistant strains. Availability of multiple ZFN target sites in a single gene will provide alternative drug target sites to combat the development of resistance in the future.

Project description:BackgroundRepositioning of existing drugs has been suggested as a fast track for developing new anti-malarial agents. The compound libraries of GlaxoSmithKline (GSK), Pfizer and AstraZeneca (AZ) comprising drugs that have undergone clinical studies in other therapeutic areas, but not achieved approval, and a set of US Food and Drug Administration (FDA)-approved drugs and other bio-actives were tested against Plasmodium falciparum blood stages.MethodsMolecules were tested initially against erythrocytic co-cultures of P. falciparum to measure proliferation inhibition using one of the following methods: SYBR®I dye DNA staining assay (3D7, K1 or NF54 strains); [(3)H] hypoxanthine radioisotope incorporation assay (3D7 and 3D7A strain); or 4',6-diamidino-2-phenylindole (DAPI) DNA imaging assay (3D7 and Dd2 strains). After review of the available clinical pharmacokinetic and safety data, selected compounds with low ?M activity and a suitable clinical profile were tested in vivo either in a Plasmodium berghei four-day test or in the P. falciparum Pf3D7(0087/N9) huSCID 'humanized' mouse model.ResultsOf the compounds included in the GSK and Pfizer sets, 3.8% (9/238) had relevant in vitro anti-malarial activity while 6/100 compounds from the AZ candidate drug library were active. In comparison, around 0.6% (24/3,800) of the FDA-approved drugs and other bio-actives were active. After evaluation of available clinical data, four investigational drugs, active in vitro were tested in the P. falciparum humanized mouse model: UK-112,214 (PAF-H1 inhibitor), CEP-701 (protein kinase inhibitor), CEP-1347 (protein kinase inhibitor), and PSC-833 (p-glycoprotein inhibitor). Only UK-112,214 showed significant efficacy against P. falciparum in vivo, although at high doses (ED90 131.3 mg/kg [95% CI 112.3, 156.7]), and parasitaemia was still present 96 hours after treatment commencement. Of the six actives from the AZ library, two compounds (AZ-1 and AZ-3) were marginally efficacious in vivo in a P. berghei model.ConclusionsRepositioning of existing therapeutics in malaria is an attractive proposal. Compounds active in vitro at ?M concentrations were identified. However, therapeutic concentrations may not be effectively achieved in mice or humans because of poor bio-availability and/or safety concerns. Stringent safety requirements for anti-malarial drugs, given their widespread use in children, make this a challenging area in which to reposition therapy.

Dataset Information

Quantum dots: a new tool for anti-malarial drug assays.

Background

Methods

Results

Conclusions

Publications

Quantum dots: a new tool for anti-malarial drug assays.

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