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Characterization and optimization of the haemozoin-like crystal (HLC) assay to determine Hz inhibiting effects of anti-malarial compounds
BACKGROUND: The haem-haemozoin biocrystallization pathway is an attractive target where several efficacious and safe anti-malarial drugs act. Consequently, in vitro haemozoin (Hz) inhibition assays have been developed to identify novel compounds. However, results may differ between assays and often...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4603294/ https://www.ncbi.nlm.nih.gov/pubmed/26458401 http://dx.doi.org/10.1186/s12936-015-0913-y |
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author | Tempera, Carolina Franco, Ricardo Caro, Carlos André, Vânia Eaton, Peter Burke, Peter Hänscheid, Thomas |
author_facet | Tempera, Carolina Franco, Ricardo Caro, Carlos André, Vânia Eaton, Peter Burke, Peter Hänscheid, Thomas |
author_sort | Tempera, Carolina |
collection | PubMed |
description | BACKGROUND: The haem-haemozoin biocrystallization pathway is an attractive target where several efficacious and safe anti-malarial drugs act. Consequently, in vitro haemozoin (Hz) inhibition assays have been developed to identify novel compounds. However, results may differ between assays and often require complex methods or sophisticated infrastructure. The recently reported growth of haemozoin-like crystals (HLC) appears to be a simple alternative although the endproduct is structurally different to Hz. This study set out to characterize this assay in depth, optimize it, and assess its performance. METHODS: The HLC assay was used as previously described but a range of different growth conditions were examined. Obtained HLCs were investigated and compared to synthetic (sHz) and natural haemozoin (nHz) using scanning electron microscopy, powder X-ray diffraction (PXRD), Fourier Transform Infrared spectroscopy (FTIR) and Raman spectroscopy (RS). Interactions of HLC with quinolines was analysed using RS. Inhibitory effects of currently used anti-malarial drugs under four final growth conditions were established. RESULTS: HLC growth requires Mycoplasma Broth Base, Tween 80, pancreatin, and lysed blood or haemin. HLCs are similar to nHz and sHz in terms of solubility, macroscopic and microscopic appearance although PXRD, FTIR and RS confirm that the haem aggregates of HLCs are structurally different. RS reveals that CQ seems to interact with HLCs in similar ways as with Hz. Inhibition of quinoline drugs ranged from 62.5 µM (chloroquine, amodiaquine, piperaquine) to 500 µM in mefloquine. CONCLUSIONS: The HLC assay provides data on inhibiting properties of compounds. Even if the end-product is not structurally identical to Hz, the inhibitory effects appear consistent with those obtained with sHz assays, as illustrated by the results obtained for quinolines. The assay is simple, inexpensive, robust, reproducible and can be performed under basic laboratory conditions with a simple visual positive/negative read-out. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12936-015-0913-y) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-4603294 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-46032942015-10-14 Characterization and optimization of the haemozoin-like crystal (HLC) assay to determine Hz inhibiting effects of anti-malarial compounds Tempera, Carolina Franco, Ricardo Caro, Carlos André, Vânia Eaton, Peter Burke, Peter Hänscheid, Thomas Malar J Methodology BACKGROUND: The haem-haemozoin biocrystallization pathway is an attractive target where several efficacious and safe anti-malarial drugs act. Consequently, in vitro haemozoin (Hz) inhibition assays have been developed to identify novel compounds. However, results may differ between assays and often require complex methods or sophisticated infrastructure. The recently reported growth of haemozoin-like crystals (HLC) appears to be a simple alternative although the endproduct is structurally different to Hz. This study set out to characterize this assay in depth, optimize it, and assess its performance. METHODS: The HLC assay was used as previously described but a range of different growth conditions were examined. Obtained HLCs were investigated and compared to synthetic (sHz) and natural haemozoin (nHz) using scanning electron microscopy, powder X-ray diffraction (PXRD), Fourier Transform Infrared spectroscopy (FTIR) and Raman spectroscopy (RS). Interactions of HLC with quinolines was analysed using RS. Inhibitory effects of currently used anti-malarial drugs under four final growth conditions were established. RESULTS: HLC growth requires Mycoplasma Broth Base, Tween 80, pancreatin, and lysed blood or haemin. HLCs are similar to nHz and sHz in terms of solubility, macroscopic and microscopic appearance although PXRD, FTIR and RS confirm that the haem aggregates of HLCs are structurally different. RS reveals that CQ seems to interact with HLCs in similar ways as with Hz. Inhibition of quinoline drugs ranged from 62.5 µM (chloroquine, amodiaquine, piperaquine) to 500 µM in mefloquine. CONCLUSIONS: The HLC assay provides data on inhibiting properties of compounds. Even if the end-product is not structurally identical to Hz, the inhibitory effects appear consistent with those obtained with sHz assays, as illustrated by the results obtained for quinolines. The assay is simple, inexpensive, robust, reproducible and can be performed under basic laboratory conditions with a simple visual positive/negative read-out. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12936-015-0913-y) contains supplementary material, which is available to authorized users. BioMed Central 2015-10-12 /pmc/articles/PMC4603294/ /pubmed/26458401 http://dx.doi.org/10.1186/s12936-015-0913-y Text en © Tempera et al. 2015 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Tempera, Carolina Franco, Ricardo Caro, Carlos André, Vânia Eaton, Peter Burke, Peter Hänscheid, Thomas Characterization and optimization of the haemozoin-like crystal (HLC) assay to determine Hz inhibiting effects of anti-malarial compounds |
title | Characterization and optimization of the haemozoin-like crystal (HLC) assay to determine Hz inhibiting effects of anti-malarial compounds |
title_full | Characterization and optimization of the haemozoin-like crystal (HLC) assay to determine Hz inhibiting effects of anti-malarial compounds |
title_fullStr | Characterization and optimization of the haemozoin-like crystal (HLC) assay to determine Hz inhibiting effects of anti-malarial compounds |
title_full_unstemmed | Characterization and optimization of the haemozoin-like crystal (HLC) assay to determine Hz inhibiting effects of anti-malarial compounds |
title_short | Characterization and optimization of the haemozoin-like crystal (HLC) assay to determine Hz inhibiting effects of anti-malarial compounds |
title_sort | characterization and optimization of the haemozoin-like crystal (hlc) assay to determine hz inhibiting effects of anti-malarial compounds |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4603294/ https://www.ncbi.nlm.nih.gov/pubmed/26458401 http://dx.doi.org/10.1186/s12936-015-0913-y |
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