Temperature effect in the inhibition of PLA₂ activity of <i>Bothrops brazili</i> venom by Rosmarinic and Chlorogenic acids, experimental and computational approaches

Myotoxicity caused by snakebite envenoming emerges as one of the main problems of ophidic accidents as it is not well neutralized by the current serum therapy. A promising alternative is to search for efficient small molecule inhibitors that can act against multiple venom components. Phospholipase A2 (PLA2) is frequently found in snake venom and is usually associated with myotoxicity. Thus it represents an excellent target for the search of new treatments. This work reports the effect of temperature in the inhibition of catalytic properties of PLA2 from Bothrops brazili venom by Rosmarinic (RSM) and Chlorogenic (CHL) acids through experimental and computational approaches. Three temperatures were evaluated (25, 37 and 50 °C). In the experimental section, enzymatic assays showed that RSM is a better inhibitor in all three temperatures. At 50 °C, the inhibition efficiency decayed significantly for both acids. Docking studies revealed that both ligands bind to the hydrophobic channel of the protein dimer where the phospholipid binds in the catalytic process, interacting with several functional residues. In this context, RSM presents better interaction energies due to stronger interactions with chain B of the dimer. Molecular dynamics simulations showed that RSM can establish selective interactions with ARG112B of PLA2, which is located next to residues of the putative Membrane Disruption Site in PLA2-like structures. The affinity of RSM and CHL acids towards PLA2 is mainly driven by electrostatic interactions, especially salt bridge interactions established with residues ARG33B (for CHL) and ARG112B (RSM) and hydrogen bonds with residue ASP89A. The inability of CHL to establish a stable interaction with ARG112B was identified as the reason for its lower inhibition efficiency compared to RSM at the three temperatures. Furthermore, extensive structural analysis was performed to explain the lower inhibition efficiency at 50 °C for both ligands. The analysis performed in this work provides important information for the future design of new inhibitors. Communicated by Ramaswamy H. Sarma蛇咬伤中毒引发的肌毒性是蛇伤事故的主要问题之一，当前血清疗法无法对其实现良好中和。颇具前景的替代方案是筛选可靶向多种毒液组分的高效小分子抑制剂。磷脂酶A₂（Phospholipase A2, PLA2）广泛存在于蛇毒中，且通常与肌毒性密切相关，因此是新型治疗手段研发的理想靶点。本研究通过实验与计算相结合的方法，探究了迷迭香酸（Rosmarinic, RSM）与绿原酸（Chlorogenic, CHL）对巴西矛头蝮（Bothrops brazili）毒液中PLA2催化活性的抑制作用受温度的影响。实验设置了25、37与50℃三个温度梯度。实验环节的酶活性测定结果显示，在三个温度下，RSM均展现出更优的抑制效果；而在50℃时，两种酸的抑制效率均出现显著下降。分子对接研究表明，两种配体均结合于PLA2二聚体的疏水通道——该通道为催化过程中磷脂结合的位点，并与多个功能残基产生相互作用。其中，RSM可与二聚体的B链形成更强相互作用，因此展现出更优的相互作用能。分子动力学模拟结果显示，RSM可与PLA2的ARG112B残基形成选择性相互作用，该残基紧邻PLA2类结构中推定的膜破坏位点残基。RSM与CHL对PLA2的亲和力主要由静电相互作用驱动，尤其是分别与ARG33B（CHL）、ARG112B（RSM）形成的盐桥相互作用，以及与ASP89A残基形成的氢键相互作用。研究发现，CHL无法与ARG112B形成稳定相互作用，这正是其在三个测试温度下的抑制效率均低于RSM的原因。此外，本研究还开展了全面的结构分析，以阐释两种配体在50℃时抑制效率下降的机制。本研究的分析结果可为未来新型抑制剂的设计提供重要参考。本文由Ramaswamy H. Sarma通讯。