Aqeel Yousuf, Siddiqui Ruqaiyyah, Anwar Ayaz, Shah Muhammad Raza, Khoja Shahrukh, Khan Naveed Ahmed
Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan.
International Center for Chemical and Biological Sciences, H. E. J. Research Institute of Chemistry, University of Karachi, Karachi, Pakistan.
Antimicrob Agents Chemother. 2015;59(6):3031-41. doi: 10.1128/AAC.05126-14. Epub 2015 Mar 9.
Acanthamoeba is a protist pathogen that can cause serious human infections, including blinding keratitis and a granulomatous amoebic encephalitis that almost always results in death. The current treatment for these infections includes a mixture of drugs, and even then, a recurrence can occur. Photochemotherapy has shown promise in the treatment of Acanthamoeba infections; however, the selective targeting of pathogenic Acanthamoeba has remained a major concern. The mannose-binding protein is an important adhesin expressed on the surface membranes of pathogenic Acanthamoeba organisms. To specifically target Acanthamoeba, the overall aim of this study was to synthesize a photosensitizing compound (porphyrin) conjugated with mannose and test its efficacy in vitro. The synthesis of mannose-conjugated porphyrin was achieved by mixing benzaldehyde and pyrrole, yielding tetraphenylporphyrin. Tetraphenylporphyrin was then converted into mono-nitrophenylporphyrin by selectively nitrating the para position of the phenyl rings, as confirmed by nuclear magnetic resonance (NMR) spectroscopy. The mono-nitrophenylporphyrin was reduced to mono-aminophenylporphyrin in the presence of tin dichloride and confirmed by a peak at m/z 629. Finally, mono-aminoporphyrin was conjugated with mannose, resulting in the formation of an imine bond. Mannose-conjugated porphyrin was confirmed through spectroscopic analysis and showed that it absorbed light of wavelengths ranging from 425 to 475 nm. To determine the antiacanthamoebic effects of the derived product, amoebae were incubated with mannose-conjugated porphyrin for 1 h and washed 3 times to remove extracellular compound. Next, the amoebae were exposed to light of the appropriate wavelength for 1 h. The results revealed that mannose-conjugated porphyrin produced potent trophicidal effects and blocked excystation. In contrast, Acanthamoeba castellanii incubated with mannose alone and porphyrin alone did not exhibit an antiamoebic effect. Consistently, pretreatment with mannose-conjugated porphyrin reduced the A. castellanii-mediated host cell cytotoxicity from 97% to 4.9%. In contrast, treatment with porphyrin, mannose, or solvent alone had no protective effects on the host cells. These data suggest that mannose-conjugated porphyrin has application for the targeted photodynamic therapy of Acanthamoeba infections and may serve as a model in the development of therapeutic interventions against other eukaryotic infections.
棘阿米巴是一种原生生物病原体，可导致严重的人类感染，包括致盲性角膜炎和几乎总会导致死亡的肉芽肿性阿米巴脑炎。目前针对这些感染的治疗方法包括多种药物混合使用，即便如此，仍可能复发。光化学疗法在棘阿米巴感染的治疗中显示出前景；然而，对致病性棘阿米巴的选择性靶向仍然是一个主要问题。甘露糖结合蛋白是致病性棘阿米巴生物体表面膜上表达的一种重要粘附素。为了特异性靶向棘阿米巴，本研究的总体目标是合成一种与甘露糖偶联的光敏化合物（卟啉）并在体外测试其疗效。甘露糖偶联卟啉的合成是通过将苯甲醛和吡咯混合实现的，生成四苯基卟啉。然后通过选择性硝化苯环的对位将四苯基卟啉转化为单硝基苯基卟啉，这通过核磁共振（NMR）光谱得到证实。在二氯化锡存在下，单硝基苯基卟啉被还原为单氨基苯基卟啉，并通过质荷比为629处的一个峰得到证实。最后，单氨基卟啉与甘露糖偶联，形成亚胺键。甘露糖偶联卟啉通过光谱分析得到证实，并表明它吸收波长范围为425至475nm的光。为了确定衍生产品的抗棘阿米巴作用，将阿米巴与甘露糖偶联卟啉孵育1小时，然后洗涤3次以去除细胞外化合物。接下来，将阿米巴暴露于适当波长的光下1小时。结果显示，甘露糖偶联卟啉产生了强大的杀滋养体作用并阻断了脱囊。相比之下，单独用甘露糖和单独用卟啉孵育的卡氏棘阿米巴没有表现出抗阿米巴作用。同样地，用甘露糖偶联卟啉预处理可将卡氏棘阿米巴介导的宿主细胞细胞毒性从97%降低至4.9%。相比之下，单独用卟啉、甘露糖或溶剂处理对宿主细胞没有保护作用。这些数据表明，甘露糖偶联卟啉可用于棘阿米巴感染的靶向光动力治疗，并可能作为开发针对其他真核生物感染的治疗干预措施的一个模型。
