Characterization and optimization of the haemozoin-like crystal (HLC) assay to determine Hz inhibiting effects of anti-malarial compounds.

Carolina Tempera,Vânia André,Ricardo Franco,Carlos Caro,Peter Eaton,Thomas Hänscheid,Peter Burke

doi:10.1186/s12936-015-0913-y

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0913-y) contains supplementary material, which is available to authorized users.

Highlights

The haem-haemozoin biocrystallization pathway is an attractive target where several efficacious and safe anti-malarial drugs act
Growth of haemozoin‐like crystals From the originally described medium to grow Ileal Fluid Dependent Organism” (IFDO), only four ingredients are necessary for growth: Mycoplasma Broth Base, Tween 80, lysed blood and pancreatin (Table 1)
Lysed blood can be from diverse species, including horse or sheep, or it can be substituted with haemin

Summary

Introduction

The haem-haemozoin biocrystallization pathway is an attractive target where several efficacious and safe anti-malarial drugs act. The prime example is the parasite specific haem-haemozoin biocrystallization pathway where many of the anti-malarial drugs seem to act, including quinine, chloroquine, mefloquine, amodiaquine, lumefantrine, halofantrine, and piperaquine [8, 9]. This pathway appears immutable and resistance is caused by removal of the compound from its site of action, as in the case of chloroquine (CQ) [10]. Several common themes have emerged [13, 14] including: Hz biocrystallization is autocatalytic; involvement of lipids, growth at the lipid aqueous interface, orientated nucleation at the digestive vacuole (DV) membrane and a presumed role for a haemozoin detoxification protein (HDP) All these proposed mechanisms have to be reconciled with other aspects. Another aspect is the fact that Hz does not grow motionless in the DV, but apparently shows a rapid non-Brownian movement [21]

Methods

Results

Conclusion