Dual Direct Inhibitors of Thrombin and Factor Xa

凝血酶和 Xa 因子双重直接抑制剂

• 批准号: 7879296
• 负责人: Umesh Ramanlal Desai
• 金额: $ 37.38万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7879296
• 关键词: A549; Activated Partial Thromboplastin Time measurement; Adverse reactions; Affinity; Anticoagulants; Anticoagulation; Antithrombins; Argatroban; Binding; Biochemical; Biological Assay; Biological Availability; Blood Clot; Blood coagulation; Cell Line; Coagulation Process; Competitive Binding; Complement; Coumarins; Coupling; Deep Vein Thrombosis; Diagnosis; Disease; Drug Interactions; Electrostatics; Enoxaparin; Enzymes; Factor IIa; Factor IXa; Factor VIIa; Factor XIa; Factor Xa; Food; Genetic Variation; Hemorrhage; Hemostatic function; Heparin; Hepatocyte; Hepatotoxicity; Hirudin; Hirudins; Hydrogen Bonding; In Vitro; Inhibitory Concentration 50; Inorganic Sulfates; Kininogenase; Left; Lepirudin; Lignin; Low-Molecular-Weight Heparin; Lung; Malignant Neoplasms; Measures; Mediating; Metabolism; Molecular; Molecular Mechanisms of Action; Monitor; Names; Nature; Needles; New Agents; Oral; Organic Synthesis; Patients; Plasma; Plasmin; Prevention; Property; Proteins; Prothrombin time assay; Pulmonary Embolism; Rattus; Reaction; Research; Risk; Screening procedure; Site; Specificity; Structure; Systems Analysis; Text; Therapeutic Index; Thrombelastography; Thrombin; Thrombus; Time; Toxic effect; Trypsin; Unspecified or Sulfate Ion Sulfates; Virtual Library; Warfarin; Whole Blood; Work; activated Protein C; alpha benzopyrone; base; bivalirudin; chemical synthesis; combinatorial; computer studies; cost; design; desirudin; dimer; fondaparinux; idraparinux; improved; in vitro activity; in vivo; inhibitor/antagonist; interest; monomer; novel; peptidomimetics; public health relevance; response; scaffold; small molecule; stoichiometry

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.

描述（由申请人提供）：血栓性疾病困扰着很多人。仅在美国，每年就诊断出近 576,000 例深静脉血栓和肺栓塞（两种最常见的血栓性疾病）新病例。更令人担忧的是，癌症患者发生血栓性疾病的可能性大约是癌症患者的三倍。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大量不良反应，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毒性。此外，患者之间的反应差异、治疗指数窄、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性疾病的治疗复杂化。我们推断，为了减少与当前抗凝治疗相关的问题，应该发现与所有现有药物（肝素、华法林、水蛭素和肽模拟物）完全不同的分子。我们发现化学酶法合成的木质素，以三种硫酸化脱氢聚合物（DHP）分子（CD、FD 和 SD）为代表，具有极其有趣的抗凝血特性和新颖的作用机制。 1) 硫酸化 DHP（CD、FD 和 SD）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，而在活化部分凝血活酶时间测定中，它们需要高 2-6 倍的浓度。 2) 使用血栓弹力图和止血分析系统进行的全血凝固研究表明，我们的新型抗凝剂抑制凝血的效力仅比依诺肝素弱 18-30 倍。 3) 从机制上讲，新分子抑制凝血酶、因子 Xa 和因子 XIa，IC50 值在 10-240 nM 范围内。 4) 相反，它们抑制因子 IXa 和因子 VIIa，IC50 值分别高出 60-170 倍和 >840 倍，表明对凝血酶和因子 Xa 具有高选择性。 5) 这种有效的抑制主要来自凝血酶和因子 Xa 的直接抑制，尽管抗凝血酶介导的间接抑制也可能起作用。 6) 直接抑制源于凝血酶催化装置的变构破坏（kCAT 的减少）。 7) 竞争性结合研究表明，CD 与凝血酶的外部位点 II 相互作用，该位点通常与抑制无关。 8) 化学合成的基于 CD 的单体抑制凝血酶和 Xa 因子，IC50 约为 30 <M。 9) 使用 A549 肺和 HepG2 肝细胞系的研究表明，CD、FD 和 2-5MSC 在浓度高达 50 mg/L 时不会诱导毒性。因此，总体而言，CD、FD 和 SD 具有强效抗凝活性（体外、血浆和全血）的现象，辅以新颖的分子作用机制和与现有抗凝剂截然不同的结构支架，为发现新型抗凝剂提供了强大的可能性。我们建议1）在分子水平上研究硫酸化DHP和基于DHP的合成分子与蛋白质（因子IIa、Xa、XIa、XIIa、XIIIa、IXa、VIIa、抗凝血酶、激肽释放酶、活化蛋白C纤溶酶、胰蛋白酶等）的相互作用； 2) 对与凝血酶、因子 Xa 和抗凝血酶结合的 CD、FD 和 SD 序列进行组合虚拟库筛选，以识别“大海捞针”； 3）化学合成特定的均质分子，研究其细胞毒性，并测量其体外、离体和体内的抗凝潜力。公共卫生相关性：仅在美国，每年就诊断出近 576000 例深静脉血栓和肺栓塞（两种最常见的血栓性疾病）新病例。血栓性疾病在癌症患者中更为普遍。拟议的关于新型凝血酶和 Xa 因子双重直接抑制剂的研究旨在改善目前存在大量不良反应和局限性的抗凝治疗。