Cunha Neto Rene Dos Santos, Vigerelli Hugo, Jared Carlos, Antoniazzi Marta Maria, Chaves Luciana Botelho, da Silva Andréa de Cássia Rodrigues, Melo Robson Lopes de, Sciani Juliana Mozer, Pimenta Daniel C
Butantan Institute, Laboratory of Biochemistry and Biophysics, Av. Vital Brazil, 1500, São Paulo, SP 05503-900 Brazil ; Pasteur Institute, Laboratory of Rabies Diagnostic, Serology, Avenida Paulista, 393, São Paulo, 01311-000 SP Brazil.
Butantan Institute, Laboratory of Biochemistry and Biophysics, Av. Vital Brazil, 1500, São Paulo, SP 05503-900 Brazil.
J Venom Anim Toxins Incl Trop Dis. 2015 Dec 2;21:50. doi: 10.1186/s40409-015-0048-1. eCollection 2015.
Rabies is an incurable neglected zoonosis with worldwide distribution characterized as a lethal progressive acute encephalitis caused by a lyssavirus. Animal venoms and secretions have long been studied as new bioactive molecular sources, presenting a wide spectrum of biological effects, including new antiviral agents. Bufotenine, for instance, is an alkaloid isolated from the skin secretion of the anuran Rhinella jimi that inhibits cellular penetration by the rabies virus. Antimicrobial peptides, such as ocellatin-P1 and ocellatin-F1, are present in the skin secretion of anurans from the genus Leptodactylus and provide chemical defense against predators and microorganisms.
Skin secretion from captive Leptodactylus labyrinthicus was collected by mechanical stimulation, analyzed by liquid chromatography and mass spectrometry, and assayed for antiviral and cytotoxic activities. Synthetic peptides were obtained using solid phase peptide synthesis, purified by liquid chromatography and structurally characterized by mass spectrometry, and assayed in the same models. Cytotoxicity assays based on changes in cellular morphology were performed using baby hamster kidney (BHK-21) cells. Fixed Rabies virus (Pasteur Virus - PV) strain was used for virological assays based on rapid fluorescent focus inhibition test.
Herein, we describe a synergic effect between ocellatin-F1 and bufotenine. This synergism was observed when screening the L. labyrinthicus skin secretion for antiviral activities. The active fraction major component was the antimicrobial peptide ocellatin-F1. Nevertheless, when the pure synthetic peptide was assayed, little antiviral activity was detectable. In-depth analyses of the active fraction revealed the presence of residual alkaloids together with ocellatin-F1. By adding sub-effective doses (e.g. < IC50) of pure bufotenine to synthetic ocellatin-F1, the antiviral effect was regained. Moreover, a tetrapetide derived from ocellatin-F1, based on alignment with the virus's glycoprotein region inferred as a possible cell ligand, was able to maintain the synergic antiviral activity displayed by the full peptide.
This novel antiviral synergic effect between a peptide and an alkaloid may present an innovative lead for the study of new antiviral drugs.
狂犬病是一种无法治愈的被忽视的人畜共患病，在全球范围内分布，其特征是由狂犬病病毒引起的致命性进行性急性脑炎。动物毒液和分泌物长期以来一直被作为新的生物活性分子来源进行研究，呈现出广泛的生物学效应，包括新型抗病毒药物。例如，蟾毒色胺是从无尾两栖动物奇异多指节蟾的皮肤分泌物中分离出的一种生物碱，它能抑制狂犬病病毒的细胞穿透。抗菌肽，如小眼蛙素-P1和小眼蛙素-F1，存在于细趾蟾属无尾两栖动物的皮肤分泌物中，为抵御捕食者和微生物提供化学防御。
通过机械刺激收集圈养的迷宫巧蛙的皮肤分泌物，采用液相色谱和质谱分析，并检测其抗病毒和细胞毒性活性。使用固相肽合成法获得合成肽，通过液相色谱纯化，用质谱进行结构表征，并在相同模型中进行检测。使用幼仓鼠肾（BHK-21）细胞进行基于细胞形态变化的细胞毒性检测。固定的狂犬病病毒（巴斯德病毒-PV）株用于基于快速荧光灶抑制试验的病毒学检测。
在此，我们描述了小眼蛙素-F1和蟾毒色胺之间的协同作用。在筛选迷宫巧蛙皮肤分泌物的抗病毒活性时观察到了这种协同作用。活性组分的主要成分是抗菌肽小眼蛙素-F1。然而，当检测纯合成肽时，几乎检测不到抗病毒活性。对活性组分的深入分析表明，除小眼蛙素-F1外还存在残留生物碱。通过向合成的小眼蛙素-F1中添加亚有效剂量（例如<IC50）的纯蟾毒色胺，抗病毒效果得以恢复。此外，基于与推测为可能的细胞配体的病毒糖蛋白区域比对，从小眼蛙素-F1衍生的四肽能够维持全长肽所显示的协同抗病毒活性。
这种肽与生物碱之间新型的抗病毒协同作用可能为新型抗病毒药物的研究提供创新性线索。
