Department of Neurology, University of Michigan, Ann Arbor, MI, United States of America.
Department of Veterans Affairs, Neurology Service, VA Ann Arbor Healthcare System, Ann Arbor, MI, United States of America.
PLoS One. 2020 Sep 18;15(9):e0239464. doi: 10.1371/journal.pone.0239464. eCollection 2020.
Omeprazole is the most commonly used proton pump inhibitor (PPI), a class of medications whose therapeutic mechanism of action involves formation of a disulfide linkage to cysteine residues in the H+/K+ ATPase pump on gastric secretory cells. Covalent linkage between the sole sulfur group of omeprazole and selected cysteine residues of the pump protein results in inhibition of acid secretion in the stomach, an effect that ameliorates gastroesophageal reflux and peptic ulcer disease. PPIs, though useful for specific conditions when used transiently, are associated with diverse untoward effects when used long term. The mechanisms underlying these potential off-target effects remain unclear. PPIs may, in fact, interact with non-canonical target proteins (non-pump molecules) resulting in unexpected pathophysiological effects, but few studies describe off-target PPI binding. Here, we describe successful cloning of monoclonal antibodies against protein-bound omeprazole. We developed and used monoclonal antibodies to characterize the protein target range of omeprazole, stability of omeprazole-bound proteins, and the involvement of cysteines in binding of omeprazole to targets. We demonstrate that a wide range of diverse proteins are targeted by omeprazole. Protein complexes, detected by Western blotting, are resistant to heat, detergents, and reducing agents. Reaction of omeprazole occurs with cysteine-free proteins, is not fully inhibited by cysteine alkylation, occurs at neutral pH, and induces protein multimerization. At least two other clinically used PPIs, rabeprazole and tenatoprazole, are capable of binding to proteins in a similar fashion. We conclude that omeprazole binds to multiple proteins and is capable of forming highly stable complexes that are not dependent on disulfide linkages between the drug and protein targets. Further studies made possible by these antibodies may shed light on whether PPI-protein complexes underlie off-target untoward effects of chronic PPI use.
奥美拉唑是最常用的质子泵抑制剂（PPI），这类药物的治疗作用机制涉及与胃分泌细胞的 H+/K+ATP 酶泵中的半胱氨酸残基形成二硫键。奥美拉唑的唯一硫基团与泵蛋白的选定半胱氨酸残基之间的共价键合导致胃酸分泌抑制，从而改善胃食管反流和消化性溃疡病。PPI 在特定情况下短期使用时很有用，但长期使用时会引起多种不良影响。这些潜在的非靶点作用的机制尚不清楚。PPI 实际上可能与非典型靶蛋白（非泵分子）相互作用，导致意外的病理生理效应，但很少有研究描述非靶点 PPI 结合。在这里，我们成功克隆了针对蛋白结合型奥美拉唑的单克隆抗体。我们开发并使用单克隆抗体来描述奥美拉唑的蛋白靶标范围、奥美拉唑结合蛋白的稳定性以及半胱氨酸在奥美拉唑与靶标结合中的作用。我们证明了奥美拉唑靶向了广泛的不同蛋白。通过 Western 印迹检测到的蛋白复合物对热、去污剂和还原剂具有抗性。奥美拉唑的反应发生在不含半胱氨酸的蛋白上，半胱氨酸烷化不完全抑制，发生在中性 pH 值，并诱导蛋白多聚化。至少另外两种临床使用的 PPI，雷贝拉唑和泰妥拉唑，能够以类似的方式结合蛋白。我们得出结论，奥美拉唑结合多种蛋白并能够形成高度稳定的复合物，这些复合物不依赖于药物和蛋白靶标之间的二硫键。这些抗体进一步研究可能阐明慢性 PPI 使用引起的非靶点不良影响是否与 PPI-蛋白复合物有关。
