title: Characterization and optimization of the haemozoin-like crystal (HLC) assay to determine Hz inhibiting effects of anti-malarial compounds.
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.
authors.affiliation: Faculdade de Medicina de Lisboa, Instituto de Medicina Molecular, Av. Prof. Egas Moniz, 1649-028, Lisbon, Portugal. ctempera@fm.ul.pt.
authors.forename: Carolina
authors.initials: C
authors.lastname: Tempera
authors.affiliation: Departamento de Química, Faculdade de Ciências e Tecnologia, UCIBIO, REQUIMTE, Universidade NOVA de Lisboa, 2829-516, Caparica, Portugal. ricardo.franco@fct.unl.pt.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Franco
authors.affiliation: Departamento de Química, Faculdade de Ciências e Tecnologia, UCIBIO, REQUIMTE, Universidade NOVA de Lisboa, 2829-516, Caparica, Portugal. cacarsal@gmail.com.
authors.forename: Carlos
authors.initials: C
authors.lastname: Caro
authors.affiliation: Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001, Lisbon, Portugal. vaniandre@tecnico.ulisboa.pt.
authors.forename: Vânia
authors.initials: V
authors.lastname: André
authors.affiliation: , Departamento de Química e Bioquímica, Faculdade de Ciências, REQUIMTE/UCIBIO, Universidade do Porto, 4169-007, Porto, Portugal. peter.eaton@fc.up.pt.
authors.forename: Peter
authors.initials: P
authors.lastname: Eaton
authors.affiliation: STERIS Corporation, 5960 Heisley Road, Mentor, OH, 44060, USA. Peter_Burke@steris.com.
authors.forename: Peter
authors.initials: P
authors.lastname: Burke
authors.affiliation: Faculdade de Medicina de Lisboa, Instituto de Medicina Molecular, Av. Prof. Egas Moniz, 1649-028, Lisbon, Portugal. t.hanscheid@fm.ul.pt.|Faculdade de Medicina, Instituto de Microbiologia, Lisbon, Portugal. t.hanscheid@fm.ul.pt.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hänscheid
mesh_terms: D000962:Antimalarials / Q000378:metabolism*; D006420:Hemeproteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D008855:Microscopy, Electron, Scanning; D011485:Protein Binding; D011804:Quinolines / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D014961:X-Ray Diffraction
chemical_list: D000962:Antimalarials; D006420:Hemeproteins; D011804:Quinolines; C020753:hemozoin