Potent and selective inhibition of the structurally homologous proteases of coagulation poses challenges for drug development. Hematophagous organisms frequently accomplish this by fashioning peptide inhibitors combining exosite and active site binding motifs. Inspired by this biological strategy, we create several EXACT inhibitors targeting thrombin and factor Xa de novo by linking EXosite-binding aptamers with small molecule ACTive site inhibitors. The aptamer component within the EXACT inhibitor (1) synergizes with and enhances the potency of small-molecule active site inhibitors by many hundred-fold (2) can redirect an active site inhibitor’s selectivity towards a different protease, and (3) enable efficient reversal of inhibition by an antidote that disrupts bivalent binding. One EXACT inhibitor, HD22-7A-DAB, demonstrates extraordinary anticoagulation activity, exhibiting great potential as a potent, rapid onset anticoagulant to support cardiovascular surgeries. Using this generalizable molecular engineering strategy, selective, potent, and rapidly reversible EXACT inhibitors can be created against many enzymes through simple oligonucleotide conjugation for numerous research and therapeutic applications.

结构同源的凝血蛋白酶的有效和选择性抑制对药物开发提出了挑战。食血生物经常通过形成结合外部位点和活性位点结合基序的肽抑制剂来实现这一点。受这种生物学策略的启发，我们通过将 EXosite 结合适体与小分子 ACTive 位点抑制剂连接，从头创建了几种针对凝血酶和 Xa 因子的 EXACT 抑制剂。 EXACT 抑制剂中的适体成分 (1) 与小分子活性位点抑制剂协同作用并将其效力增强数百倍 (2) 可以将活性位点抑制剂的选择性重新定向到不同的蛋白酶，(3) 实现有效逆转破坏二价结合的解毒剂的抑制作用。一种 EXACT 抑制剂 HD22-7A-DAB 表现出非凡的抗凝活性，作为有效、快速起效的抗凝剂，具有支持心血管手术的巨大潜力。使用这种通用的分子工程策略，可以通过简单的寡核苷酸缀合来针对多种酶创建选择性、有效且快速可逆的 EXACT 抑制剂，用于众多研究和治疗应用。