Deep Learning-Based Screening for MRTF-A Nuclear Translocation Agonists and Investigation of Its Neuroprotective Effects on Synapses Following Ischemic Stroke Reperfusion

Reperfusion injury following ischemic stroke treatment significantly affects patient prognosis. Myocardin-related transcription factor A (MRTF-A) has been shown to alleviate this injury, making it a promising therapeutic target. In this study, MRTF-A expression was modulated in rat middle cerebral artery occlusion/reperfusion (MCAO/R) models. MRTF-A mitigated reperfusion injury by enhancing the co-localization of PARD3 and Tiam1, thereby preserving synaptic structure and function. Since MRTF-A activation requires its dissociation from G-actin, an active molecular screening zone was defined based on the binding site of the G-actin/MRTF-A RPEL domain. A dataset was constructed using batch molecular docking to train an AttentiveFP deep learning regression model for predicting the binding affinity of small molecules to the G-actin domain. Through molecular scaffold analysis, ADME/T prediction, and molecular dynamics simulation, the potential agonist Pranlukast was identified. Pranlukast competes with the RPEL domain for G-actin binding, thereby promoting MRTF-A activation. Surface plasmon resonance (SPR) and immunofluorescence confirmed Pranlukast's strong binding affinity to G-actin and its ability to promote MRTF-A nuclear translocation. In vivo experiments demonstrated Pranlukast's efficacy in preventing ischemic stroke reperfusion injury and reversing synaptic structural and functional impairments caused by ischemic injury. This study, which integrates specific disease targets with deep learning-based intelligent screening, reveals for the first time that Pranlukast targets the G-actin/MRTF-A interaction via a non-classical pathway. By promoting MRTF-A nuclear translocation, it provides neurosynaptic protection post-ischemic reperfusion, offering a novel therapeutic strategy for ischemic stroke reperfusion injury.缺血性脑卒中治疗后引发的再灌注损伤，会显著影响患者预后。肌球蛋白相关转录因子A（Myocardin-related transcription factor A, MRTF-A）已被证实可缓解该类损伤，因此是极具潜力的治疗靶点。本研究在大鼠大脑中动脉闭塞/再灌注（middle cerebral artery occlusion/reperfusion, MCAO/R）模型中对MRTF-A的表达进行了调控。MRTF-A可通过增强PARD3与Tiam1的共定位，缓解再灌注损伤，进而维持突触结构与功能的完整性。由于MRTF-A的激活需要其脱离G-肌动蛋白（G-actin），本研究基于G-肌动蛋白/MRTF-A RPEL结构域的结合位点，确定了活性分子筛选区域。本研究借助批量分子对接技术构建数据集，用于训练AttentiveFP深度学习回归模型，以预测小分子与G-肌动蛋白结构域的结合亲和力。通过分子骨架分析、ADME/T预测以及分子动力学模拟，研究成功筛选得到潜在激动剂普仑司特（Pranlukast）。普仑司特可与RPEL结构域竞争结合G-肌动蛋白，从而促进MRTF-A的激活。表面等离子体共振（Surface plasmon resonance, SPR）与免疫荧光实验证实，普仑司特与G-肌动蛋白具有较强结合亲和力，且可促进MRTF-A的核转位。体内实验结果表明，普仑司特可有效预防缺血性脑卒中再灌注损伤，并逆转缺血损伤引发的突触结构与功能缺陷。本研究将特定疾病靶点与基于深度学习的智能筛选相结合，首次揭示普仑司特通过非经典通路靶向作用于G-肌动蛋白与MRTF-A的相互作用。通过促进MRTF-A核转位，普仑司特可在缺血再灌注后为神经突触提供保护，为缺血性脑卒中再灌注损伤提供了全新的治疗策略。