Peter Burke

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. 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. 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. 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.

Hemozoin crystals are normally formed in vivo by Plasmodium parasites to detoxify free heme released after hemoglobin digestion during its intraerythrocytic stage. Inhibition of hemozoin formation by various drugs results in free heme concentration toxic for the parasites. As a consequence, in vitro assays have been developed to screen and select candidate antimalarial drugs based on their capacity to inhibit hemozoin formation. In this report we describe new ways to form hemozoin-like crystals that were incidentally discovered during research in the field of prion inactivation. We investigated the use of a new assay based on naturally occurring “self-replicating” particles and previously described as presenting resistance to decontamination comparable to prions. The nature of these particles was determined using electron microscopy, Maldi-Tof analysis and X-ray diffraction. They were compared to synthetic hemozoin and to hemozoin obtained from Plasmodium falciparum. We then used the assay to evaluate the capacity of various antimalarial and anti-prion compounds to inhibit “self-replication” (crystallisation) of these particles. We identified these particles as being similar to ferriprotoporphyrin IX crystal and confirmed the ability of these particles to serve as nuclei for growth of new hemozoin-like crystals (HLC). HLC are morphologically similar to natural and synthetic hemozoin. Growth of HLC in a simple assay format confirmed inhibition by quinolines antimalarials at potencies described in the literature. Interestingly, artemisinins and tetracyclines also seemed to inhibit HLC growth. The described HLC assay is simple and easy to perform and may have the potential to be used as an additional tool to screen antimalarial drugs for their hemozoin inhibiting activity. As already described by others, drugs that inhibit hemozoin crystal formation have also the potential to inhibit misfolded proteins assemblies formation.