Faculty of Pharma-Sciences, Teikyo University, Tokyo, Japan.
Faculty of Pharma-Sciences, Teikyo University, Tokyo, Japan.
Bioorg Med Chem. 2023 Jul 15;90:117362. doi: 10.1016/j.bmc.2023.117362. Epub 2023 Jun 2.
Proteins are vital constituents of all living organisms. As many therapeutic agents alter the activity of functional proteins, identifying functional target proteins of small bioactive molecules isessential for the rational design of stronger medicines. Flavonoids with antioxidant, anti-allergy, and anti-inflammatory effects are expected to have preventive effects for several diseases closely related to oxidation and inflammation, including heart disease, cancer, neurodegenerative disorders, and eye diseases. Therefore, identifying the proteins involved in the pharmacological actions of flavonoids, and designing a flavonoid structure-based medicine that strongly and specifically inhibits flavonoid target proteins, could aid the development of more effective medicines for treating heart disease, cancer, neurodegenerative disorders, and ocular diseases with few side effects. To isolate the flavonoid target protein, we conducted a novel affinity chromatography in a column wherein baicalin, a representative flavonoid, was attached to Affi-Gel 102. Through affinity chromatography and nano LC-MS/MS, we identified GAPDH as a flavonoid target protein. Then, we performed fluorescence quenching and an enzyme inhibition assay to experimentally confirmbaicalin's binding affinity for, and inhibition of, GAPDH. We also conducted in silico docking simulations to visualize the binding modes of baicalin and the newly identified flavonoid target protein, GAPDH. From the results of this study, it was considered that one of the reasons why baicalin exhibits the effects on cancer and neurodegenerative diseases is that it inhibits the activity of GAPDH. In summary, we showed that Affi-Gel102 could quickly and accurately isolate the target protein for bioactive small molecules, without the need for isotopic labeling or a fluorescent probe. By using the method presented here, it was possible to easily isolate the target protein of a medicine containing a carboxylic acid.
蛋白质是所有生物体的重要组成部分。由于许多治疗药物会改变功能蛋白的活性，因此鉴定小分子生物活性物质的功能靶蛋白对于合理设计更强效的药物至关重要。具有抗氧化、抗过敏和抗炎作用的黄酮类化合物有望对几种与氧化和炎症密切相关的疾病（包括心脏病、癌症、神经退行性疾病和眼部疾病）具有预防作用。因此，鉴定参与黄酮类化合物药理作用的蛋白质，并设计基于黄酮类化合物结构的药物，该药物可强烈且特异性地抑制黄酮类化合物靶蛋白，有助于开发更有效的治疗心脏病、癌症、神经退行性疾病和眼部疾病的药物，且副作用较少。为了分离黄酮类化合物的靶蛋白，我们在一个柱中进行了一种新颖的亲和层析，其中将黄芩苷（一种代表性的黄酮类化合物）连接到 Affi-Gel 102 上。通过亲和层析和纳升 LC-MS/MS，我们鉴定出 GAPDH 是一种黄酮类化合物靶蛋白。然后，我们进行荧光猝灭和酶抑制测定实验来验证黄芩苷与 GAPDH 的结合亲和力和抑制作用。我们还进行了计算机对接模拟以可视化黄芩苷和新鉴定的黄酮类化合物靶蛋白 GAPDH 的结合模式。从这项研究的结果来看，黄芩苷对癌症和神经退行性疾病表现出作用的原因之一是它抑制了 GAPDH 的活性。总之，我们表明 Affi-Gel102 可以快速准确地分离生物活性小分子的靶蛋白，而无需同位素标记或荧光探针。使用这里提出的方法，可以轻松分离含羧酸药物的靶蛋白。
