Aliter Kholoud F, Al-Horani Rami A
Department of Biology and Chemistry, School of STEM, Dillard University, New Orleans, Louisiana, 70122, USA.
Division of Pharmaceutical Sciences, College of Pharmacy, Xavier University of Louisiana, New Orleans, Louisiana, 70125, USA.
Lett Drug Des Discov. 2024;21(19):4919-4927. doi: 10.2174/0115701808340492250115102419. Epub 2025 Mar 7.
Activated Protein C (APC) is a plasma serine protease with antithrombotic function. APC acts as an anticoagulant by promoting the degradation of factors Va and VIIIa, thus inhibiting the formation of thrombin. Specific inhibition of APC has been proposed to benefit hemophilia therapy.
We used chromogenic tripeptide substrate hydrolysis assay to screen a series of arginine and arginine-like containing small molecules to identify inhibitors of APC. Similar hydrolysis assays were used to determine selectivity against other serine proteases and blood-clotting enzymes. Molecular modeling was exploited to illustrate the binding of the most potent and selective inhibitor onto the putative binding site.
We identified inhibitor as a potent inhibitor with an value of 1.1 μM. The molecule demonstrated >100-fold selectivity against thrombin, factor XIa, and neutrophil elastase, >50-fold selectivity against factor XIIIa, 10-fold selectivity against factor Xa, and 8-fold selectivity against human plasmin. Molecular modeling reveals that inhibitor binds to the active site of APC with the best-docked structure, indicating that one protonated amidino group establishes a salt bridge to the side chain carboxylate of Asp189 residue. Another inhibitor was identified, yet it was not as selective to APC. Importantly, inhibitor demonstrates favorable physicochemical, pharmacokinetic, and drug-likeness properties.
Inhibitor is a selective and potent inhibitor of APC that serves as a powerful lead for the development of hemophilia therapy.
活化蛋白C（APC）是一种具有抗血栓形成功能的血浆丝氨酸蛋白酶。APC通过促进因子Va和VIIIa的降解发挥抗凝作用，从而抑制凝血酶的形成。有人提出特异性抑制APC对血友病治疗有益。
我们使用发色三肽底物水解试验筛选了一系列含精氨酸和类精氨酸的小分子，以鉴定APC的抑制剂。采用类似的水解试验来确定对其他丝氨酸蛋白酶和凝血酶的选择性。利用分子建模来说明最有效和最具选择性的抑制剂与假定结合位点的结合情况。
我们鉴定出抑制剂 为一种强效抑制剂，其 值为1.1 μM。该分子对凝血酶、因子XIa和中性粒细胞弹性蛋白酶的选择性大于100倍，对因子XIIIa的选择性大于50倍，对因子Xa的选择性为10倍，对人纤溶酶的选择性为8倍。分子建模显示抑制剂 以最佳对接结构与APC的活性位点结合，表明一个质子化的脒基与Asp189残基的侧链羧酸盐形成盐桥。还鉴定出了另一种抑制剂，但它对APC的选择性不如前者。重要的是，抑制剂 具有良好的物理化学、药代动力学和类药性质。
抑制剂 是APC的一种选择性强效抑制剂，可作为血友病治疗药物开发的有力先导。
