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短序列氨基酸（<20个残基），可以通过仔细的酶消化释放出来。释放后，这些肽成为潜在的强大的药物，可以恢复正常的生理过程。科学文献已经证实，氨基酸的线性排列是决定肽活性类型和效力的重要因素。因此，本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酯酶，可水解乙酰胆碱，乙酰胆碱是神经系统中的主要神经递质。乙酰胆碱酯酶的过度活动与神经系统紊乱有关，特别是痴呆和阿尔茨海默病。然而，关于氨基酸类型和肽链上的位置与抑制乙酰胆碱酯酶活性的关系的信息很少。我的国家科学研究委员会资助的发现研究项目的初步工作已经证明了利用酶消化从大麻籽蛋白中释放乙酰胆酸抑制肽的可行性。该提案旨在继续胃蛋白酶的工作，包括其他食物蛋白质（豌豆，油菜籽，亚麻籽，大豆，鹰嘴豆），并最终导致发现新的疼痛抑制肽。因此，本提案的主要目的是分离、纯化和确定这些植物蛋白酶解物中存在的乙酰胆碱抑制肽的氨基酸序列。靶肽是指含有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、与乙酰胆碱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的超滤膜过滤，并收集流过的液体（渗透液）并用于肽纯化。纯化后的肽段将采用液相色谱/串联质谱法鉴定氨基酸序列，并用于建立偏最小二乘（PLS）回归分析的数据库。PLS模型将用于发现新的疼痛抑制肽。在Wistar大鼠模型中，研究人员将评估抗乙酰胆碱酯酶最强的肽穿过血脑屏障和降低乙酰胆碱酯酶脑活性的能力。这项工作具有重要意义，因为结果将为乙酰胆酸抑制肽的氨基酸结构需求提供基础科学信息。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障碍正常化。该项目为期5年，将培养3名博士后、2名博士、3名硕士和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.