Lead identification and pre-clinical studies on allosteric inhibitors of coagulation factor XIa

抗凝剂是治疗血栓性疾病的主要药物，如心脏病发作和中风， 也可能用于其他疾病，如癌症和新冠肺炎。尽管有几种抗凝剂 包括肝素、华法林和直接口服抗凝剂(DOAC)在临床上使用，每种药物都受到 减轻大出血和轻微出血的不良反应。到目前为止，一种安全的抗凝血剂可以在没有 出血风险尚未形成。在NHLBI的R01机制下，我们早先发现 人凝血因子Xia的肝素结合Exosite 2(HFXIa)与其他同源凝血系统有显著不同 蛋白水解酶(如hFIIa、hFXa等)。我们开发了一种新的设计策略，依赖于阴离子硫酸盐 和芳环对hFXIa的外显子2进行高选择性识别，从而抑制酶 活动。我们开发了一种很有前途的变构抑制剂，名为硫酸D-环肌醇(SCI)，它是 分四步合成，在Exosite 2中显示了100倍的hFXIa；结合血浆FXIa的选择性 亲和力为20-60 nM，即使当酶的活性部位被共价阻断时也是如此；并抑制体内动脉 大鼠静脉血栓形成，每只动物250微克(~1 mg/kg)，但不增加尾巴出血。SCI曾经是 耐受剂量高达每只大鼠25毫克，提示治疗窗口为~100。SCI是一个很有前途的 抗凝剂；但其药代动力学并不是最好的。根据肝素类药物的线索，我们假设 优化了D-chiro肌醇支架上硫酸盐基团的数目和位置，同时也进行了筛选 它的各种立体异构体将提高hFXIa亲和力、抑制效力、PK、药效学(PD)和 化学、制造和控制(CMC)属性，然后开始支持IND的研究。海流 提案的重点是研究一个由20个合理设计的SCI类似物组成的库，目标是至少识别出 一种用于晚期临床前开发的先导剂。具体目标是1)合成， 基于脊髓损伤结构的20种可能的XIA因子抑制剂的抗凝和出血作用；2)体内 抑制剂的抗血栓疗效、DMPK、体外和体内毒性；以及3)放大的非GMP合成， CMC、剂量递增效应和PD研究以确定一个或两个铅分子。脊髓损伤已在 一项美国专利(#9,758,459 B2，标题为‘作为抗凝剂的XIA因子的变构调节剂’)，其PI AS 发明者之一。关于合成、体外和体内疗效、体外和体内疗效的定量里程碑 体内毒性将被用来指导从R61到R33阶段的过渡。或者，最好的1(或2)类比 将被确定为最有希望的药物，从20%开始进行进一步的IND研究 设计了SCI类比。

Anticoagulants are the mainstay in the treatment of thrombotic diseases, such as heart attack and stroke, and may also be used in other diseases such as cancer and COVID-19. Although several anticoagulants including heparins, warfarin, and direct oral anticoagulants (DOACs) are used in the clinic, each agent suffers from major and minor bleeding adverse effects. As of now, a safe anticoagulant that inhibits coagulation without bleeding risk has not been developed as yet. Under the NHLBI's R01 mechanism, we earlier discovered that the heparin-binding exosite 2 of human factor XIa (hFXIa) is remarkably different from other homologous coagulation proteases (e.g., hFIIa, hFXa and others). We developed a novel design strategy that relies on anionic sulfates and aromatic rings to effect highly selective recognition of hFXIa's exosite 2 resulting in inhibition of enzyme activity. We developed a highly promising allosteric inhibitor, named sulfated D-chiro-inositol (SCI), which was synthesized in four steps, displayed >100-fold selectivity for hFXIa; bound plasma FXIa in exosite 2 with an affinity of 20–60 nM even when the enzyme's active site was covalently blocked; and inhibited in vivo arterial and venous thrombosis in the rat at 250 µg per animal (~1 mg/kg) without enhancing tail bleeding. SCI was tolerated at doses as high as 25 mg/rat suggesting a therapeutic window of ~100. SCI is a highly promising anticoagulant; yet its pharmacokinetics is not the best. Using cues from heparin-based drugs, we hypothesize that optimizing the number and position of sulfate groups on the D-chiro inositol scaffold, while also screening its various stereoisomers would improve hFXIa affinity, inhibition potency, PK, pharmacodynamics (PD), and chemistry, manufacturing and controls (CMC) properties before embarking on IND-enabling studies. The current proposal focuses on studying a library of 20 rationally designed SCI analogs with the goal of identifying at least one LEAD AGENT for advanced stage pre-clinical development. The specific aims are 1) synthesis, anticoagulation efficacy, and bleeding of 20 putative factor XIa inhibitors based on the SCI structure; 2) in vivo antithrombotic efficacy, DMPK, in vitro and in vivo toxicity of inhibitors; and 3) scaled-up non-GMP synthesis, CMC, dose escalation efficacy, and PD studies to identify one or two lead molecules. SCI has been claimed in a US patent (#9,758,459 B2 titled `Allosteric modulators of factor XIa as anticoagulant agents') with the PI as one of the inventors. Quantitative milestones with regard to synthesis, in vitro & in vivo efficacy, in vitro and in vivo toxicity will be used to guide the transition from the R61 to R33 Phase. Alternatively, the best 1 (or 2) analogs of SCI would be identified as the most promising agent for further IND-enabling studies starting from the 20 designed SCI analogs.