Abstract：
14-3-3ζ protein, the key target in the regulation and control of integrin β3 outside-in signaling, is an attractive new strategy to inhibit thrombosis without affecting hemostasis. In this study, 4\'-O-methylbavachalconeB (4-O-MB) in Psoraleae Fructus was identified as a 14-3-3ζ ligand with antithrombosis activity by target fishing combined with ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) analysis. The competitive inhibition analysis showed that 4-O-MB targeted 14-3-3ζ and blocked the 14-3-3ζ/integrin β3 interaction with inhibition constant (Ki) values of 9.98 ± 0.22 μM. Molecular docking and amino acid mutation experiments confirmed that 4-O-MB specifically bound to 14-3-3ζ through LSY9 and SER28 to regulate the 14-3-3ζ/integrin β3 interaction. Besides, 4-O-MB affected the integrin β3 early outside-in signal by inhibiting AKT and c-Src phosphorylation. Meanwhile, 4-O-MB could inhibit ADP-, collagen-, or thrombin-induced platelet aggregation function but had no effect on platelet adhesion to collagen-coated surfaces in vivo. Administration of 4-O-MB could significantly inhibit thrombosis formation without disturbing hemostasis in mice. These findings provide new prospects for the antithrombotic effects of Psoraleae Fructus and the potential application of 4-O-MB as lead compounds in the therapy of thrombosis by targeting 14-3-3ζ.
摘要：
14-3-3ζ蛋白，整合素β3外-内信号调节和控制的关键靶标，是抑制血栓形成而不影响止血的有吸引力的新策略。在这项研究中,补骨脂中的4'-O-甲基bavachalconeB（4-O-MB）通过靶向捕捞结合超高效液相色谱-四极杆飞行时间质谱（UHPLC-Q-TOF-MS）分析被鉴定为具有抗血栓形成活性的14-3-3ζ配体。竞争性抑制分析表明，4-O-MB靶向14-3-3ζ并阻断14-3-3ζ/整联蛋白β3相互作用，抑制常数（Ki）值为9.98±0.22μM。分子对接和氨基酸突变实验证实，4-O-MB通过LSY9和SER28与14-3-3ζ特异性结合，调节14-3-3ζ/整联蛋白β3相互作用。此外,4-O-MB通过抑制AKT和c-Src磷酸化影响整合素β3的早期外-内信号。同时,4-O-MB可以抑制ADP-，胶原蛋白-，或凝血酶诱导的血小板聚集功能，但对体内血小板与胶原蛋白涂层表面的粘附没有影响。给药4-O-MB可以显着抑制血栓形成，而不会干扰小鼠的止血。这些发现为补骨脂的抗血栓作用以及4-O-MB作为先导化合物在靶向14-3-3ζ治疗血栓形成的潜在应用提供了新的前景。
