Characterization and optimization of the haemozoin-like crystal HLC assay to determine Hz inhibiting effects of anti-malarial compoundsReport as inadecuate




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Malaria Journal

, 14:403

First Online: 12 October 2015Received: 01 May 2015Accepted: 25 September 2015

Abstract

BackgroundThe 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.

MethodsThe 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.

ResultsHLC 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.

ConclusionsThe 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.

KeywordsMalaria Haemozoin inhibition Antimalarial drugs Simple assay Electronic supplementary materialThe online version of this article doi:10.1186-s12936-015-0913-y contains supplementary material, which is available to authorized users.

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Author: Carolina Tempera - Ricardo Franco - Carlos Caro - Vânia André - Peter Eaton - Peter Burke - Thomas Hänscheid

Source: https://link.springer.com/



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