Maslovski Franco, Pereañez Jaime Andrés, Hernández David, Alonso María, Echeverría Silvina, Gay Carolina, Leiva Laura, Angelina Emilio, Fusco Luciano
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Química Básica y Aplicada del Nordeste Argentino (IQUIBA-NEA), W3400, Corrientes, Argentina.
Facultad de Ciencias Exactas y Naturales y Agrimensura, Universidad Nacional del Nordeste, W3400, Corrientes, Argentina.
Arch Toxicol. 2025 Sep 11. doi: 10.1007/s00204-025-04188-9.
In the anti-venom production process, it is essential to use whole venom during immunization to generate specific antibodies capable of neutralizing all venom components. However, efforts are being made to improve the traditional immunization process by reducing the toxic effects of venom components on the immunized animals while ensuring an optimal immune response in accordance with good manufacturing practices. In this context, we aimed to evaluate the capacity of ASC16 to inhibit the enzymatic activity of the main components of B. alternatus venom. In vitro studies were conducted to evaluate the inhibitory effect of ASC16 on metalloproteinases (SVMP), serine proteinases (SVSP) and phospholipases A (PLA) using specific substrates. In vivo assays measured edema-forming activity, and histological analysis with hematoxylin and eosin staining were performed. Additionally, hemorrhage inhibition was tested using a murine model. In silico studies were also carried out using bothropasin as a model. The results of enzyme inhibition showed that ASC16 significantly inhibited SVMP (49.31% ± 0.62), SVSP (63.11 ± 3.86%) and PLA activity (36.52% ± 0.09). ASC16 did not reduce edema but it significantly inhibited hemorrhage (66.32%). In silico analysis suggested that ASC16's hydrophobic portion binds to critical residues involved in catalysis of the SVMP (Glu146 and His145), while the hydrophilic fraction also interacts with the protein (Pro109, Thr110, Gly112, and Tyr132). These findings position ASC16 as a promising candidate for use as an additive inhibitor in adjuvant formulations in antivenom immunization schemes for B. alternatus.
在抗蛇毒血清生产过程中，免疫时使用全毒液以产生能够中和所有毒液成分的特异性抗体至关重要。然而，人们正在努力改进传统免疫过程，在确保符合良好生产规范的最佳免疫反应的同时，降低毒液成分对免疫动物的毒性作用。在此背景下，我们旨在评估ASC16抑制松材线虫毒液主要成分酶活性的能力。进行了体外研究，使用特异性底物评估ASC16对金属蛋白酶（SVMP）、丝氨酸蛋白酶（SVSP）和磷脂酶A（PLA）的抑制作用。进行了体内试验以测量水肿形成活性，并进行苏木精和伊红染色的组织学分析。此外，使用小鼠模型测试了出血抑制情况。还使用矛头蝮蛋白酶作为模型进行了计算机模拟研究。酶抑制结果表明，ASC16显著抑制SVMP（49.31%±0.62）、SVSP（63.11±3.86%）和PLA活性（36.52%±0.09）。ASC16没有减轻水肿，但显著抑制了出血（66.32%）。计算机模拟分析表明，ASC16的疏水部分与参与SVMP催化的关键残基（Glu146和His145）结合，而亲水部分也与该蛋白相互作用（Pro109、Thr110、Gly112和Tyr132）。这些发现使ASC16成为在松材线虫抗蛇毒血清免疫方案的佐剂配方中用作添加剂抑制剂的有前景的候选物。
