Dual Direct Inhibitors of Thrombin and Factor Xa

描述(申请人提供)：血栓性疾病困扰着大量的人。仅在美国，每年就有近57.6万例新病例被诊断出深静脉血栓形成和肺栓塞，这两种最常见的血栓形成疾病。更令人担忧的是，血栓性疾病在癌症患者中的可能性要高出约3倍。抗凝剂，包括肝素和香豆素，是治疗和预防血栓栓塞性疾病的主要药物。然而，目前的抗凝治疗存在着大量的不良反应，包括出血风险增加、免疫反应、代谢遗传变异、食物或药物相互作用以及肝脏毒性。此外，患者对患者的反应变异性、治疗指数狭窄、作用持续时间不足、口服生物利用度差、需要频繁的凝血监测以及高成本效益比等问题进一步使血栓形成条件的治疗复杂化。我们认为，为了减少与当前抗凝治疗相关的问题，应该发现与目前所有药物(肝素、华法林、水飞蓟素和肽类药物)根本不同的分子。我们发现，以硫酸盐脱氢聚合物(DHP)分子CDS、FDS和SDS为代表的化学酶法合成的木质素具有非常有趣的抗凝性能和新的作用机理。1)硫酸化DHP(CDS、FDS和SDS)在比临床使用的LMWH依诺肝素低2-6倍的浓度下延长凝血酶原时间，而在活化的部分凝血活酶时间测定中它们需要高2-6倍的浓度。2)利用血栓弹性成像和止血分析系统进行的全血凝血研究表明，我们的新型抗凝剂抑制凝血的效力仅为依诺肝素的18-30倍。3)新分子在机制上对凝血酶、凝血因子Xa和凝血因子Xia具有抑制作用，其IC50值在10-240 nM之间。4)相反，它们抑制凝血因子IXa和凝血因子VIIa，其IC50值分别高60-170倍和840倍，这表明它们对凝血酶和凝血因子Xa具有很高的选择性。5)这种有效的抑制作用主要来自凝血酶和凝血因子Xa的直接抑制，但抗凝血酶介导的间接抑制也可能起作用。6)直接抑制来自于凝血酶催化装置的变构破坏(kCAT降低)。7)竞争性结合研究表明，CDS与凝血酶的外切酶II相互作用，这是一个典型的与抑制无关的位置。8)一种化学合成的以CDS为基础的单体抑制凝血酶和凝血因子Xa，IC50为~30；M)使用A549肺和HepG2细胞株的研究表明，在高达50 mg/L的浓度下，CDS、FDS和2-5MSC没有诱导毒性。因此，总的来说，CDS、FDS和SDS具有强大的抗凝血活性(体外、血浆和全血)的现象，再加上目前每种抗凝剂的新的分子作用机制和完全不同的结构支架，为发现新型抗凝剂提供了极大的可能性。我们建议1)研究DHP和基于DHP的合成分子与蛋白质(因子IIa、Xa、Xia、XIIa、IXa、VIIa、抗凝血酶、激肽释放酶、活化蛋白C、纤溶酶、胰酶等)的相互作用。在分子水平上，2)对与凝血酶、凝血因子Xa和抗凝血酶结合的CDS、FDS和十二烷基硫酸酯序列进行组合虚拟文库筛选，以鉴定大海捞针(S)；3)化学合成特定的均一分子，研究它们的细胞毒性，并测定它们的体内外抗凝能力。与公共卫生相关：仅在美国，每年就诊断出近576000例深静脉血栓形成和肺栓塞的新病例，这两种最常见的血栓形成疾病。血栓性疾病在癌症患者中更为普遍。新的凝血酶和凝血因子Xa双重直接抑制物的研究旨在改善目前的抗凝治疗，该治疗方法存在着大量的不良反应和局限性。

DESCRIPTION (provided by applicant): Thrombotic disorders afflict a large number of people. Nearly 576,000 new cases of deep vein thrombosis and pulmonary embolism, two of the most common thrombotic conditions, are diagnosed every year in the US alone. Even more alarming is that thrombotic disorders are ~3-fold more likely in people with cancer. Anticoagulants, including heparins and coumarins, are the mainstay of treatment and prevention of thromboembolic disorders. Yet, the current anticoagulation therapy is beset with a significant number of adverse reactions including enhanced bleeding risk, immunological reaction, genetic variation in metabolism, food or drug interactions and liver toxicity. In addition, problems such as patient-to-patient response variability, narrow therapeutic index, inadequate duration of action, poor oral bioavailability, the need for frequent coagulation monitoring, and high cost to benefit ratio further complicate the treatment of thrombotic conditions. We reasoned that to reduce the problems associated with the current anticoagulation therapy, molecules radically different from all the current agents (heparins, warfarins, hirudins, and peptidomimetics) should be discovered. We have discovered that chemo-enzymatically synthesized lignins, represented by three sulfated dehydropolymer (DHP) molecules, named CDs, FDs and SDs, possess extremely interesting anticoagulant properties and a novel mechanism of action. 1) Sulfated DHPs (CDs, FDs and SDs) prolong prothrombin time at concentrations 2-6-fold below that of the clinically used LMWH enoxaparin, while in the activated partial thromboplastin time assay they required 2-6-fold higher concentration. 2) Whole blood clotting studies using thromboelastography and hemostasis analysis system reveal that our novel anticoagulants inhibit clotting with potency only 18-30-fold weaker than enoxaparin. 3) Mechanistically, the new molecules inhibit thrombin, factor Xa and factor XIa with IC50 values in the range of 10-240 nM. 4) In contrast, they inhibit factor IXa and factor VIIa with IC50 values 60-170-fold and >840-fold higher, respectively suggesting high selectivity for thrombin and factor Xa. 5) This potent inhibition arises primarily from direct inhibition of thrombin and factor Xa, although indirect inhibition mediated by antithrombin may also contribute. 6) Direct inhibition arises from an allosteric disruption of thrombin's catalytic apparatus (reduction in kCAT). 7) Competitive binding studies suggest that CDs interacts with exosite II of thrombin, a site not typically associated with inhibition. 8) A chemically synthesized CDs-based monomer inhibits thrombin and factor Xa with an IC50 of ~30 <M. 9) Studies using A549 lung and HepG2 liver cell lines show no induction of toxicity by CDs, FDs, and 2-5MSC at concentrations as high as 50 mg/L. Thus overall, the phenomenon of potent anticoagulant activity (in vitro, plasma and whole blood) by CDs, FDs and SDs, complemented by a novel molecular mechanism of action and a radically different structural scaffold from each of the current anticoagulants, raises a strong possibilities for discovering novel anticoagulants. We propose to 1) investigate the interaction of sulfated DHPs and DHP-based synthetic molecules with proteins (factors IIa, Xa, XIa, XIIa, XIIIa, IXa, VIIa, antithrombin, kallikrein, activated protein C plasmin, trypsin, etc.) at a molecular level; 2) perform combinatorial virtual library screening of CDs, FDs and SDs sequences binding to thrombin, factor Xa and antithrombin to identify `needle(s) in the haystack'; and 3) chemically synthesize specific homogeneous molecules, study their cellular toxicity, and measure their anticoagulation potential in vitro, ex vivo and in vivo. PUBLIC HEALTH RELEVANCE: Nearly 576000 new cases of deep vein thrombosis and pulmonary embolism, two of the most common thrombotic conditions, are diagnosed every year in the US alone. Thrombotic disorders are even more prevalent in people with cancer. The proposed research on novel dual direct inhibitors of thrombin and factor Xa aims to improve current anticoagulation therapy, which is beset with significant number of adverse reactions and limitations.