Ncokazi Kanyile K, Egan Timothy J
Department of Chemistry, University of Cape Town, Private Bag, Rondebosch 7701, South Africa.
Anal Biochem. 2005 Mar 15;338(2):306-19. doi: 10.1016/j.ab.2004.11.022.
Antimalarial drugs such as chloroquine are believed to act by inhibiting hemozoin formation in the food vacuole of the malaria parasite. We have developed a new assay for measuring and detecting inhibition of synthetic hemozoin (beta-hematin) formation. Aqueous pyridine (5% v/v, pH 7.5) forms a low-spin complex with hematin but not with beta-hematin. Its absorbance obeys Beer's law, making it useful for quantitating hematin concentration in hematin/beta-hematin mixtures, allowing compounds to be investigated for inhibition of beta-hematin formation. The assay is rapid (60 min incubation) and requires no centrifugation. The beta-hematin inhibition data show good agreement with alternative assay methods reported by four laboratories. The assay was adapted for high-throughput colorimetric screening, allowing visual identification of beta-hematin inhibitors. In this mode, the assay successfully detected all 18 beta-hematin inhibitors in a set of 47 compounds tested, with no false positive results. The quantitative in vitro antimalarial activities of a set of 13 aminoquinolines and quinoline methanols were found to correlate significantly with beta-hematin inhibition values determined using the assay.
诸如氯喹之类的抗疟药物被认为是通过抑制疟原虫食物泡中的疟色素形成而起作用的。我们开发了一种新的测定方法，用于测量和检测合成疟色素（β-血红素）形成的抑制情况。吡啶水溶液（5% v/v，pH 7.5）与血红素形成低自旋复合物，但不与β-血红素形成复合物。其吸光度符合比尔定律，这使得它可用于定量血红素/β-血红素混合物中的血红素浓度，从而能够研究化合物对β-血红素形成的抑制作用。该测定方法快速（孵育60分钟），且无需离心。β-血红素抑制数据与四个实验室报告的其他测定方法显示出良好的一致性。该测定方法适用于高通量比色筛选，能够直观鉴定β-血红素抑制剂。在此模式下，该测定方法成功检测出了所测试的47种化合物中的所有18种β-血红素抑制剂，没有假阳性结果。发现一组13种氨基喹啉和喹啉甲醇的体外定量抗疟活性与使用该测定方法确定的β-血红素抑制值显著相关。
