The accurate resolution of the binding mechanism of a ligand to its molecular target is fundamental to develop a successful drug design campaign. Free-energy calculations, which provide the energy value of the ligand–protein binding complex, are essential for resolving the binding mode of the ligand. The accuracy of free-energy calculation methods is counteracted by their poor user-friendliness, which hampers their broad application. Here we present the Funnel-Metadynamics Advanced Protocol (FMAP), which is a flexible and user-friendly graphical user interface (GUI)-based protocol to perform funnel metadynamics, a binding free-energy method that employs a funnel-shape restraint potential to reveal the ligand binding mode and accurately calculate the absolute ligand–protein binding free energy. FMAP guides the user through all phases of the free-energy calculation process, from preparation of the input files, to production simulation, to analysis of the results. FMAP delivers the ligand binding mode and the absolute protein–ligand binding free energy as outputs. Alternative binding modes and the role of waters are also elucidated, providing a detailed description of the ligand binding mechanism. The entire protocol on the paradigmatic system benzamidine–trypsin, composed of ~105 k atoms, took ~2.8 d using the Cray XC50 piz Daint cluster at the Swiss National Supercomputing Centre.

准确解析配体与其分子靶标的结合机制是开展成功的药物设计活动的基础。自由能计算提供配体-蛋白质结合复合物的能量值，对于解析配体的结合模式至关重要。自由能计算方法的准确性因其用户友好性差而受到抵消，这阻碍了它们的广泛应用。在这里，我们介绍了漏斗元动力学高级协议 (FMAP)，它是一种灵活且用户友好的基于图形用户界面 (GUI) 的协议，用于执行漏斗元动力学，一种结合自由能的方法，采用漏斗形约束潜力来揭示配体结合模式并准确计算绝对配体-蛋白质结合自由能。FMAP 指导用户完成自由能计算过程的所有阶段，从准备输入文件到生产模拟，再到结果分析。FMAP 提供配体结合模式和绝对蛋白质-配体结合自由能作为输出。还阐明了替代结合模式和水的作用，提供了配体结合机制的详细描述。使用瑞士国家超级计算中心的 Cray XC50 piz Daint 集群，由约 105 k 原子组成的典型系统苯甲脒-胰蛋白酶的整个协议耗时约 2.8 天。还阐明了替代结合模式和水的作用，提供了配体结合机制的详细描述。使用瑞士国家超级计算中心的 Cray XC50 piz Daint 集群，由约 105 k 原子组成的典型系统苯甲脒-胰蛋白酶的整个协议耗时约 2.8 天。还阐明了替代结合模式和水的作用，提供了配体结合机制的详细描述。使用瑞士国家超级计算中心的 Cray XC50 piz Daint 集群，由约 105 k 原子组成的典型系统苯甲脒-胰蛋白酶的整个协议耗时约 2.8 天。