Ponzar Nathan, Chinnaraj Mathivanan, Pagotto Anna, De Filippis Vincenzo, Flaumenhaft Robert, Pozzi Nicola
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St Louis, Missouri, USA.
Department of Pharmaceutical and Pharmacological Sciences, School of Medicine, University of Padova, Padua, Italy.
J Thromb Haemost. 2025 Feb;23(2):577-587. doi: 10.1016/j.jtha.2024.09.036. Epub 2024 Oct 23.
Protein disulfide isomerase (PDI) is a promising target for combating thrombosis. Extensive research over the past decade has identified numerous PDI-targeting compounds. However, limited information exists regarding how these compounds control PDI activity, which complicates further development.
To define the mechanism of action of 2 allosteric antithrombotic compounds of therapeutic interest, quercetin-3-O-rutinoside and bepristat-2a.
A multipronged approach that integrates single-molecule spectroscopy, steady-state kinetics, single-turnover kinetics, and site-specific mutagenesis.
PDI is a thiol isomerase consisting of 2 catalytic a domains and 2 inactive b domains arranged in the order a-b-b'-a'. The active sites CGHC are located in the a and a' domains. The binding site of quercetin-3-O-rutinoside and bepristat-2a is in the b' domain. Using a library of 9 Förster resonance energy transfer sensors, we showed that quercetin-3-O-rutinoside and bepristat-2a globally alter PDI structure and dynamics, leading to ligand-specific modifications of its shape and reorientation of the active sites. Combined with enzyme kinetics and mutagenesis of the active sites, Förster resonance energy transfer data reveal that binding of quercetin-3-O-rutinoside results in a twisted enzyme with reduced affinity for the substrate. In contrast, bepristat-2a promotes a more compact conformation of PDI, in which a greater enzymatic activity is achieved by accelerating the nucleophilic step of the a domain, leading to faster formation of the covalent enzyme-substrate complex.
This work reveals the mechanistic basis underlying PDI regulation by antithrombotic compounds quercetin-3-O-rutinoside and bepristat-2a and points to novel strategies for furthering the development of PDI-targeting compounds into drugs.
蛋白质二硫键异构酶（PDI）是抗血栓治疗中一个很有前景的靶点。过去十年的广泛研究已鉴定出众多靶向PDI的化合物。然而，关于这些化合物如何控制PDI活性的信息有限，这使得进一步开发变得复杂。
确定两种具有治疗意义的变构抗血栓化合物槲皮素 - 3 - O - 芸香糖苷和贝普司他 - 2a的作用机制。
采用多管齐下的方法，整合单分子光谱、稳态动力学、单周转动力学和位点特异性诱变技术。
PDI是一种硫醇异构酶，由2个催化性a结构域和2个无活性的b结构域按a - b - b' - a'顺序排列组成。活性位点CGHC位于a和a'结构域中。槲皮素 - 3 - O - 芸香糖苷和贝普司他 - 2a的结合位点在b'结构域。使用9种Förster共振能量转移传感器库，我们表明槲皮素 - 3 - O - 芸香糖苷和贝普司他 - 2a全局改变了PDI的结构和动力学，导致其形状的配体特异性修饰以及活性位点的重新定向。结合酶动力学和活性位点诱变，Förster共振能量转移数据表明，槲皮素 - 3 - O - 芸香糖苷的结合导致酶发生扭曲，对底物的亲和力降低。相比之下，贝普司他 - 2a促进了PDI更紧凑的构象，其中通过加速a结构域的亲核步骤实现了更高的酶活性，导致共价酶 - 底物复合物形成更快。
这项工作揭示了抗血栓化合物槲皮素 - 3 - O - 芸香糖苷和贝普司他 - 2a对PDI调节的机制基础，并指出了将靶向PDI的化合物进一步开发成药物的新策略。
