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

抗凝剂是治疗血栓性疾病如心脏病发作和中风的主要药物， 也可用于其他疾病，如癌症和COVID-19。虽然几种抗凝剂 包括肝素、华法林和直接口服抗凝剂（DOAC）在临床中使用，每种药剂都遭受 严重和轻微的出血不良反应。到目前为止，一种安全的抗凝剂，抑制凝血， 出血风险尚未出现。根据NHLBI的R 01机制，我们早先发现， 人凝血因子XIa（hFXIa）的肝素结合外位点2与其他同源凝血明显不同 蛋白酶（例如，hFIIa、hFXa和其他）。我们开发了一种新的设计策略， 和芳环，以实现hFXIa的外位点2的高度选择性识别，从而抑制酶 活动我们开发了一种非常有前途的变构抑制剂，命名为硫酸化D-手性肌醇（SCI）， 在四个步骤中合成，对hFXIa显示>100倍的选择性;在外位点2中结合血浆FXIa， 20-60 nM的亲和力，即使当酶的活性位点被共价阻断;并抑制体内动脉 和静脉血栓形成，每只动物250 µg（约1 mg/kg），不增加尾部出血。SCI是 在高达25 mg/大鼠的剂量下耐受，表明治疗窗为~100。SCI是一个非常有前途的 抗凝血剂;但其药代动力学不是最好的。利用肝素类药物的线索，我们假设 优化D-手性肌醇支架上硫酸酯基团的数量和位置，同时筛选 其各种立体异构体将改善hFXIa亲和力、抑制效力、PK、药效学（PD）， 化学、制造和控制（CMC）特性，然后再开始IND使能研究。当前 一项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药物。具体目标是：1）综合， 基于SCI结构的20种推定因子XIa抑制剂的抗凝效力和出血; 2）体内 抑制剂的抗血栓形成功效、DMPK、体外和体内毒性;和3）按比例增加的非GMP合成， CMC、剂量递增疗效和PD研究，以确定一种或两种先导分子。SCI已在 美国专利（#9，758，459 B2，标题为“作为抗凝剂的因子XIa的变构调节剂”），PI为 发明者之一。关于合成、体外和体内功效、体外和体内 体内毒性将用于指导从R61阶段到R33阶段的过渡。或者，最好的1（或2）类似物 SCI将被确定为从20年开始进行进一步IND支持研究的最有前途的药物 设计SCI类似物。

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.