Tempera Carolina, Franco Ricardo, Caro Carlos, André Vânia, Eaton Peter, Burke Peter, Hänscheid Thomas
Faculdade de Medicina de Lisboa, Instituto de Medicina Molecular, Av. Prof. Egas Moniz, 1649-028, Lisbon, Portugal.
Departamento de Química, Faculdade de Ciências e Tecnologia, UCIBIO, REQUIMTE, Universidade NOVA de Lisboa, 2829-516, Caparica, Portugal.
Malar J. 2015 Oct 12;14:403. doi: 10.1186/s12936-015-0913-y.
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.
血红素-疟原虫色素生物结晶途径是几个有效且安全的抗疟药物作用的一个有吸引力的靶点。因此，已经开发了体外疟原虫色素（Hz）抑制试验来鉴定新型化合物。然而，不同试验的结果可能存在差异，并且通常需要复杂的方法或精密的仪器设备。最近报道的类疟原虫色素晶体（HLC）的生长似乎是一种简单的替代方法，尽管其终产物在结构上与Hz不同。本研究旨在深入表征该试验，对其进行优化，并评估其性能。
采用先前描述的HLC试验，但检查了一系列不同的生长条件。使用扫描电子显微镜、粉末X射线衍射（PXRD）、傅里叶变换红外光谱（FTIR）和拉曼光谱（RS）对获得的HLC进行研究，并与合成疟原虫色素（sHz）和天然疟原虫色素（nHz）进行比较。使用RS分析HLC与喹啉的相互作用。确定了当前使用的抗疟药物在四种最终生长条件下的抑制作用。
HLC生长需要支原体肉汤基础培养基、吐温80、胰酶以及裂解的血液或血红素。HLC在溶解度、宏观和微观外观方面与nHz和sHz相似，尽管PXRD、FTIR和RS证实HLC的血红素聚集体在结构上有所不同。RS显示氯喹似乎以与Hz相似的方式与HLC相互作用。喹啉类药物的抑制作用范围为62.5μM（氯喹、阿莫地喹、哌喹）至甲氟喹的500μM。
HLC试验提供了关于化合物抑制特性的数据。即使终产物在结构上与Hz不完全相同，其抑制作用似乎与sHz试验获得的结果一致，喹啉类药物的结果就说明了这一点。该试验简单、廉价、稳健、可重复，并且可以在基本实验室条件下进行，通过简单的视觉阳性/阴性读数即可得出结果。
