肽基精氨酸脱亚胺酶4(PAD4)的失调涉及多种疾病,包括类风湿性关节炎(RA)和阿尔茨海默病(AD),它已经成为潜在和有希望的治疗靶点。然而,没有PAD4抑制剂可供临床使用。固定化酶筛选技术因其低成本而受到越来越多的关注,可重用性,易于从反应混合物中分离,以及对环境条件变化的抵抗力。在这项研究中,将PAD4固定在磁性纳米粒子(MNP)上,以延长其活性稳定性,建立了基于固定化PAD4的中药抑制剂的简便快速筛选策略。使用戊二醛(GA)作为交联剂,通过席夫碱反应将PAD4酶固定在磁性纳米颗粒(MNP)上。与游离PAD4相比,所得MNP@GA@PAD4表现出增强的温度耐受性和储存稳定性,回收10次后,其可重用性大大提高,初始酶活性为66%。使用两种已知的PAD4抑制剂GSK484和BB-Cl-脒评估固定化PAD4的抑制活性。GSK484和BB-Cl-脒对MNP@GA@PAD4的半最大抑制浓度(IC50)分别为1.00和0.97μM,分别,对于游离的PAD4分别为0.64和0.85μM,分别。最后,MNP@GA@PAD4用于从40种中药(TCM)中快速筛选天然PAD4抑制剂。在同样的条件下,用游离PAD4进行对照实验。TCMs抑制剂对MNP@GA@PAD4和游离PAD4的筛选结果相似,肉桂和石竹的醇提物对PAD4酶活性有显著的抑制作用。确定肉桂提取物对MNP@GA@PAD4和游离PAD4的IC50值为27和48μg/mL,分别。石竹提取物对MNP@GA@PAD4和游离PAD4的IC50值分别为48和32μg/mL,分别。第一次,本研究提出了一种将PAD4固定在磁性材料上的方法,并发展了一种快速的,从中药中筛选天然PAD4抑制剂的可重复使用和可行的策略。
Dysregulation of peptidyl arginine deiminase 4 (PAD4) is involved in a variety of diseases including rheumatoid arthritis (RA) and Alzheimer\'s disease (AD), and it has emerged as potential and promising therapeutic target. However, no PAD4 inhibitor is ready for clinical use. Immobilized enzyme screening technology has gained increasing attention due to its low cost, reusability, easy separation from the reaction mixture, and resistance to changes in environmental conditions. In this study, PAD4 was immobilized on the magnetic nanoparticles (MNP) to prolong its activity stability, and a simple and rapid screening strategy of traditional Chinese medicine inhibitors based on immobilized PAD4 was established. The PAD4 enzyme was immobilized on magnetic nanoparticles (MNP) via Schiff base reaction using glutaraldehyde (GA) as crosslinking agent. Compared with free PAD4, the resulting MNP@GA@PAD4 exhibited an enhanced tolerance to temperature and storage stability, and its reusability was greatly improved with 66 % of initial enzyme activity after being recycled 10 times. The inhibitory activity of the immobilized PAD4 was assessed using two known PAD4 inhibitors GSK484 and BB-Cl-amidine. The semi-maximum inhibitory concentrations (IC50) of GSK484 and BB-Cl-amidine for MNP@GA@PAD4 were 1.00 and 0.97 μM, respectively, for free PAD4 were 0.64 and 0.85 μM, respectively. Finally, the MNP@GA@PAD4 was employed to rapid screen of natural PAD4 inhibitors from forty traditional Chinese medicines (TCMs). Under the same conditions, the controlled experiment was conducted with free PAD4. The screening results of TCMs inhibitors on MNP@GA@PAD4 and free PAD4 were similar, the alcohol extracts of Cinnamomi Cortex and Caryophylli Flos had significant inhibitory effects on PAD4 enzyme activity. The IC50 values of Cinnamomi Cortex extract for MNP@GA@PAD4 and free PAD4 were determined as 27 and 48 μg/mL, respectively. The IC50 values of Caryophylli Flos extracts for MNP@GA@PAD4 and free PAD4 were determined as 48 and 32 μg/mL, respectively. For the first time, this study proposed a method to immobilize PAD4 on magnetic materials, and developed a rapid, reusable and feasible strategy to screening natural PAD4 inhibitors from TCMs.