凝血酶是最有效的血小板聚集剂。发现对治疗血小板聚集相关疾病有重要意义的选择性凝血酶抑制剂,进行了对接实验以对接(1R,3S)-2,3,4,9-四氢-β-咔啉-3-羧酸,[(1R,3S)-THCCA],和(1S,3S)-2,3,4,9-四氢-β-咔啉-3-羧酸,[(1S,3S)-THCCA],进入牛凝血酶的p口袋。理想的匹配支持(1R,3S)-THCCA可作为潜在的先导化合物。在这种情况下,理论上将20种天然氨基酸引入(1R,3S)-THCCA和20个衍生物,(1R,3S)-THCCA-氨基酸,将其停靠在牛凝血酶的p袋中进行虚拟筛选。筛查显示,与(1R,3S)-THCCA本身的16个衍生物的DockScore较高,和(1R,3S)-THCCA-Asn(4j)获得最高DockScore。因此,合成了16种衍生物进行实验研究。体外抗血小板聚集试验显示,在100μM浓度下,16种衍生物不能抑制由二磷酸腺苷和花生四烯酸诱导的血小板聚集。另一方面,然而,抑制血小板活化因子和凝血酶诱导的血小板聚集的16种衍生物的IC50值范围为9.44μM至194.64μM和0.07μM至9.56μM,分别。体外抗血小板聚集试验表明,16种衍生物选择性地抑制凝血酶诱导的血小板聚集。特别是,(1R,3S)-THCCA-Asn(4j)的值最低。在1nmol/kg剂量的大鼠模型上,16种衍生物可有效防止血栓形成。值得指出的是,即使在0.01nmol/kg的剂量下,4j仍然有效地防止血栓形成。4j对哺乳动物细胞增殖和大鼠尾部出血时间几乎没有影响。总之,虚拟筛选和生物分析的结合成功地导致4j作为一个有希望的候选凝血酶的选择性抑制剂的发现。
Thrombin is the most potent platelet aggregator. To discover the selective inhibitor of thrombin that is important to curing platelet aggregation-related diseases, docking experiments were performed to dock (1R,3S)-2,3,4,9-tetrahydro-β-carboline-3- carboxylic acid, [(1R,3S)-THCCA], and (1S,3S)-2,3,4,9-tetrahydro-β-carboline-3- carboxylic acid, [(1S,3S)-THCCA], into the p pocket of bovine thrombin. The ideal match supported that (1R,3S)-THCCA could be used as a potential lead compound. In this case 20 natural amino acids were theoretically introduced into the 3-carboxyl of (1R,3S)-THCCA and 20 derivatives, (1R,3S)-THCCA-amino acids, were docked into p pocket of bovine thrombin to perform virtual screening. The screening revealed that comparing to (1R,3S)-THCCA itself the DockScores of 16 derivatives were higher, and (1R,3S)-THCCA-Asn (4j) got the highest DockScore. Thus, 16 derivatives were synthesized for experimental study. The in vitro anti-platelet aggregation assay showed that at 100 μM of concentration the 16 derivatives failed to inhibit the platelet aggregation induced by both adenosine diphosphate and arachidonic acid. On the other hand, however, the IC50 value of the 16 derivatives inhibiting the platelet aggregation induced by platelet activating factor and thrombin ranged from 9.44 μM to 194.64 μM and from 0.07 μM to 9.56 μM, respectively. The in vitro anti-platelet aggregation assay suggested that the 16 derivatives selectively inhibited the platelet aggregation induced by thrombin. In particular, the IC50 of (1R,3S)-THCCA-Asn (4j) had the lowest value. On rat model at 1 nmol/kg of dosage the 16 derivatives effectively prevented thrombus formation. It is worth pointing out that even at 0.01 nmol/kg of dosage, 4j still effectively prevented thrombus formation. 4j hardly has effects on the proliferation of mammalian cells and rat tail bleeding time. In conclusion, the combination of virtual screening and biological assays successfully lead to the discovery of 4j as a promising candidate of selective inhibitor of thrombin.