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

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

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Abstract

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.

Original languageEnglish
Article number403
JournalMalaria Journal
Volume14
Issue number1
DOIs
Publication statusPublished - 12 Oct 2015

Fingerprint

Antimalarials
Raman Spectrum Analysis
Quinolines
Fourier Transform Infrared Spectroscopy
Heme
X-Ray Diffraction
Powders
hemozoin
Growth
Pancreatin
Amodiaquine
Pharmaceutical Preparations
Mefloquine
Hemin
Polysorbates
Mycoplasma
Chloroquine
Crystallization
Electron Scanning Microscopy
Solubility

Keywords

  • Antimalarial drugs
  • Haemozoin inhibition
  • Malaria
  • Simple assay

Cite this

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. In: Malaria Journal. 2015 ; Vol. 14, No. 1.
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abstract = "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.",
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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.

In: Malaria Journal, Vol. 14, No. 1, 403, 12.10.2015.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Characterization and optimization of the haemozoin-like crystal (HLC) assay to determine Hz inhibiting effects of anti-malarial compounds

AU - Tempera, Carolina

AU - Franco, Ricardo

AU - Caro, Carlos

AU - André, Vânia

AU - Eaton, Peter

AU - Burke, Peter

AU - Hänscheid, Thomas

N1 - FCT-MEC Grants (PEst-C/EQB/LA0006/2011; PEst-C/EQB/LA0006/2013; UID/Multi/04378/2013; EXPL/CTM-NAN/0754/2013; RECI/QEQ-QIN/0189/2012; SFRH/BPD/78854/2011); Junta de Andalucia (P10 FQM 06615; P07 FQM 02595); STERIS Corporation

PY - 2015/10/12

Y1 - 2015/10/12

N2 - 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.

AB - 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.

KW - Antimalarial drugs

KW - Haemozoin inhibition

KW - Malaria

KW - Simple assay

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