Dehydrobufotenin extracted from the Amazonian toad Rhinella marina (Anura: Bufonidae) as a prototype molecule for the development of antiplasmodial drugs

Abstract Background: The resistance against antimalarial drugs represents a global challenge in the fight and control of malaria. The Brazilian biodiversity can be an important tool for research and development of new medicinal products. In this context, toxinology is a multidisciplinary approach on the development of new drugs, including the isolation, purification, and evaluation of the pharmacological activities of natural toxins. The present study aimed to evaluate the cytotoxicity, as well as the antimalarial activity in silico and in vitro of four compounds isolated from Rhinella marina venom as potential oral drug prototypes. Methods: Four compounds were challenged against 35 target proteins from P. falciparum and screened to evaluate their physicochemical properties using docking assay in Brazilian Malaria Molecular Targets (BraMMT) software and in silico assay in OCTOPUS® software. The in vitro antimalarial activity of the compounds against the 3D7 Plasmodium falciparum clones were assessed using the SYBR Green I based assay (IC50). For the cytotoxic tests, the LD50 was determined in human pulmonary fibroblast cell line using the [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay. Results: All compounds presented a ligand-receptor interaction with ten Plasmodium falciparum-related protein targets, as well as antimalarial activity against chloroquine resistant strain (IC50 = 3.44 μM to 19.11 μM). Three of them (dehydrobufotenine, marinobufagin, and bufalin) showed adequate conditions for oral drug prototypes, with satisfactory prediction of absorption, permeability, and absence of toxicity. In the cell viability assay, only dehydrobufotenin was selective for the parasite. Conclusions: Dehydrobufotenin revealed to be a potential oral drug prototype presenting adequate antimalarial activity and absence of cytotoxicity, therefore should be subjected to further studies.

摘要 背景：抗疟药物耐药性是疟疾防控工作面临的全球性挑战。巴西生物多样性可为新型药物的研发提供重要支撑。在此背景下，毒素学（toxinology）作为研发新药的多学科研究手段，涵盖天然毒素的分离、纯化与药理学活性评价。本研究旨在评估4种分离自海蟾蜍（Rhinella marina）毒液的化合物作为口服药物原型的细胞毒性，以及其计算机模拟（in silico）与体外（in vitro）抗疟活性。

方法：以恶性疟原虫（Plasmodium falciparum）的35种靶蛋白为研究对象，对4种化合物进行筛选；采用巴西疟疾分子靶标（BraMMT）软件开展分子对接实验，借助OCTOPUS®软件进行计算机模拟实验，以评价其理化性质。采用基于SYBR Green I荧光染料的检测法，测定化合物对3D7恶性疟原虫克隆株的体外抗疟活性，计算半数抑制浓度（IC50）。细胞毒性实验中，采用[3(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐]（MTT）法，在人肺成纤维细胞系中测定半数致死剂量（LD50）。

结果：所有化合物均与10种恶性疟原虫相关蛋白靶标存在配体-受体相互作用，且对氯喹耐药株具有抗疟活性（IC50范围为3.44 μM至19.11 μM）。其中3种化合物（脱氢蟾毒它灵、海蟾蜍毒苷、蟾毒灵）具备口服药物原型的适宜条件，其吸收、渗透性预测结果良好，且无潜在毒性。细胞活力实验显示，仅脱氢蟾毒它灵对疟原虫具有选择性。

结论：脱氢蟾毒它灵展现出良好的抗疟活性且无细胞毒性，是极具潜力的口服药物原型，有待开展进一步研究。