Phosphatidylinositol 3,4,5-trisphosphate- (PIP3-) dependent Rac exchanger 1 (P-Rex1) functions as Rho guanine nucleotide exchange factor and is activated by synergistic activity of Gβγ and PIP3 of the heterotrimeric G protein. P-Rex1 activates Rac GTPases for regulating cell invasion and migration and promotes metastasis in several human cancers including breast, prostate, and skin cancer. The protein is a promising therapeutic target because of its multifunction roles in human cancers. Herein, the present study attempts to identify selective P-Rex1 natural inhibitors by targeting PIP3-binding pocket using large-size multiple natural molecule libraries. Each library was filtered subsequently in FAF-Drugs4 based on Lipinski’s rule of five (RO5), toxicity, and filter pan assay interference compounds (PAINS). The output hits were virtually screened at the PIP3-binding pocket through PyRx AutoDock Vina and cross-checked by GOLD. The best binders at the PIP3-binding pocket were prioritized using a comparative analysis of the docking scores. Top-ranked two compounds with high GOLD fitness score (>80) and lowest AutoDock binding energy (< -12.7 kcal/mol) were complexed and deciphered for molecular dynamics along with control-P-Rex1 complex to validate compound binding conformation and disclosed binding interaction pattern. Both the systems were seen in good equilibrium, and along the simulation time, the compounds are in strong contact with the P-Rex1 PIP3-binding site. Hydrogen bonding analysis towards simulation end identified the formation of 16 and 22 short- and long-distance hydrogen bonds with different percent of occupancy to the PIP3 residues for compound I and compound 2, respectively. Radial distribution function (RDF) analysis of the key hydrogen bonds between the compound and the PIP3 residues demonstrated a strong affinity of the compounds to the mentioned PIP3 pocket. Additionally, MMGB/PBSA energies were performed that confirmed the dominance of Van der Waals energy in complex formation along with favorable contribution from hydrogen bonding. These findings were also cross-validated by a more robust WaterSwap binding energy predictor, and the results are in good agreement with a strong binding affinity of the compounds for the protein. Lastly, the key contribution of residues in interaction with the compounds was understood by binding free energy decomposition and alanine scanning methods. In short, the results of this study suggest that P-Rex1 is a good druggable target to suppress cancer metastasis; therefore, the screened druglike molecules of this study need in vitro and in vivo anti-P-Rex1 validation and may serve as potent leads to fight cancer.

中文翻译：

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P-Rex1 与天然先导化合物复合的结构动力学使该蛋白质成为抑制癌症转移的有吸引力的治疗靶点

磷脂酰肌醇 3,4,5-三磷酸 (PIP3-) 依赖性 Rac 交换器 1 (P-Rex1) 充当 Rho 鸟嘌呤核苷酸交换因子，并由异源三聚体 G 蛋白的G βγ和 PIP3 的协同活性激活。P-Rex1 激活 Rac GTPases 来调节细胞侵袭和迁移，并促进多种人类癌症（包括乳腺癌、前列腺癌和皮肤癌）的转移。该蛋白质由于其在人类癌症中的多功能作用而成为一个有前途的治疗靶点。在此，本研究试图通过使用大型多个天然分子库靶向 PIP3 结合袋来鉴定选择性 P-Rex1 天然抑制剂。随后根据 Lipinski 五法则 (RO5)、毒性和滤盘测定干扰化合物 (PAINS) 在 FAF-Drugs4 中对每个文库进行过滤。通过 PyRx AutoDock Vina 在 PIP3 结合口袋处对输出命中进行虚拟筛选，并由 GOLD 进行交叉检查。通过对接分数的比较分析，优先考虑 PIP3 结合口袋处的最佳结合物。将具有高 GOLD 适应度得分 (>80) 和最低 AutoDock 结合能 (< -12.7 kcal/mol) 的排名靠前的两种化合物与对照-P-Rex1 复合物一起进行复合和破译，以进行分子动力学分析，以验证化合物结合构象并公开结合交互模式。两个系统都处于良好的平衡状态，并且在模拟过程中，化合物与 P-Rex1 PIP3 结合位点紧密接触。模拟结束时的氢键分析表明，化合物 I 和化合物 2 分别形成了 16 个和 22 个短距离和长距离氢键，其 PIP3 残基的占据百分比不同。对化合物与 PIP3 残基之间关键氢键的径向分布函数 (RDF) 分析表明，化合物与上述 PIP3 口袋具有很强的亲和力。此外，还进行了 MMGB/PBSA 能量分析，证实了范德华能量在复合物形成中的主导地位以及氢键的有利贡献。这些发现还通过更强大的 WaterSwap 结合能预测器进行了交叉验证，结果与化合物对蛋白质的强结合亲和力非常一致。最后，通过结合自由能分解和丙氨酸扫描方法了解残基与化合物相互作用的关键贡献。简而言之，这项研究的结果表明 P-Rex1 是抑制癌症转移的良好药物靶点；因此，本研究筛选出的药物样分子需要体外和体内抗 P-Rex1 验证，并可能作为抗癌的有效线索。