Quantitative structure-activity relationship studies of acteylcholinesterase-inhibitory peptides

生物活性肽是存在于食物蛋白质线性链中的氨基酸短序列（<20 个残基），可以通过仔细的酶消化释放。释放后，这些肽成为潜在的强大药物，可以恢复正常的生理过程。科学文献已证实，氨基酸的线性排列是肽的类型和活性效力的重要决定因素。因此，该提案的重点是确定影响乙酰胆碱酯酶（AChE）抑制肽效力的氨基酸位置排列。人乙酰胆碱酯酶是一种水解乙酰胆碱的酯酶，乙酰胆碱是神经系统中的主要神经递质。乙酰胆碱酯酶 (AChE) 的过度活性与神经系统疾病有关，尤其是痴呆和阿尔茨海默病。然而，关于肽链上氨基酸类型和位置与抑制 AChE 活性的能力之间的关系的信息很少。我的 NSERC 资助的发现研究计划的初步工作证明了利用酶消化从大麻籽蛋白中释放乙酰胆碱酯酶 (AChE) 抑制肽的可行性。该提案旨在继续开展胃蛋白酶工作，将其他食物蛋白（豌豆、油菜、亚麻籽、大豆、鹰嘴豆）纳入其中，并最终发现新型乙酰胆碱酯酶抑制肽。因此，本提案的主要目标是分级、纯化和确定这些植物蛋白酶解产物中存在的 AChE 抑制肽的氨基酸序列。目标肽是那些含有 10 个或更少氨基酸的肽，因为它们的小尺寸增加了穿过血脑屏障 (BBB)、进入大脑、与 AChE 结合并减少乙酰胆碱过度分解的潜力。因此，水解产物将通过 1 kDa 超滤膜过滤，并收集流出物（渗透物）并用于肽纯化。纯化的肽将经过液相色谱/串联质谱分析来鉴定氨基酸序列，这将用于建立偏最小二乘（PLS）回归分析的数据库。 PLS 模型将用于进一步发现新的 AChE 抑制肽。使用 Wistar 大鼠模型，将评估具有最强抗 AChE 效力的肽穿过 BBB 并降低 AChE 大脑活动的能力。拟议的工作意义重大，因为结果将提供有关乙酰胆碱酯酶抑制肽的氨基酸结构要求的基本科学信息。这些结构信息将有助于开发基于肽的营养工具，使与神经系统相关的生理紊乱正常化。这个五年周期的拟议工作将培训 3 名博士后研究员、2 名博士生、3 名硕士生和 5 名本科生暑期研究生。

Bioactive peptides are short sequences (<20 residues) of amino acids present within the linear chain of food proteins and can be released through careful enzymatic digestion. Upon release, these peptides become potentially powerful agents that can restore normal physiological processes. It has been established in scientific literature that the linear arrangement of amino acids is an important determinant of the type and potency of activity of the peptide. Therefore, the focus of this proposal is to determine the amino acid positional arrangements that influence potency of acetylcholinesterase (AChE)-inhibitory peptides. Human AChE is an esterase that hydrolyzes acetylcholine, the main neurotransmitter in the nervous system. Excessive activities of AChE have been implicated in disorders of the nervous system, especially dementia and Alzheimer's disease. However, there is scant information on the relationships of amino acid type and position on the peptide chain with ability to inhibit AChE activity. Preliminary work from my NSERC-funded Discovery research program has demonstrated the feasibility of using enzyme digestion to release AChE-inhibitory peptides from hemp seed proteins. This proposal seeks to continue the pepsin work to include other other food proteins (peas, canola, flaxseed, soybean, chickpea) and ultimately lead to the discovery of novel AChE-inhibitory peptides. Therefore, the main objectives of this proposal are to fractionate, purify, and determine the amino sequence of AChE-inhibitory peptides present in the enzymatic hydrolysates of these plant proteins. Target peptides are those with 10 or less amino acids because their small sizes increase the potential to cross the blood-brain barrier (BBB), enter the brain, bind to AChE and reduce excessive breakdown of acetylcholine. Thus, the hydrolysates will be filtered through a 1 kDa ultrafiltration membrane and the flow-through (permeate) collected and used for peptide purification. Purified peptides will be subjected to liquid chromatography/tandem mass spectrometry to identify amino acid sequences, which will be used to build databases for partial least square (PLS) regression analysis. PLS models will be used for additional discovery of new AChE-inhibitory peptides. Using the Wistar rat model, peptides with strongest potency against AChE will be evaluated for ability to cross the BBB and reduce brain activity of AChE. The proposed work is significant because the results will provide fundamental scientific information on the amino acid structural requirements for AChE-inhibitory peptides. Such structural information will enable development of peptide-based nutritional tools that can normalize physiological disorders associated with the nervous system. The proposed work for this 5-yr cycle will train 3 postdoctoral fellows, 2 PhD students, 3 MSc students and 5 undergraduate summer research students.