Prolyl endopeptidases to treat Celiac Sprue

脯氨酰内肽酶治疗乳糜泻

• 批准号: 6932724
• 负责人: JEREMY MINSHULL
• 金额: $ 10万
• 依托单位: DNA TWOPOINTO, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-15 至 2006-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6932724
• 关键词: celiac disease; drug design /synthesis /production; endopeptidases; enzyme activity; enzyme structure; gastrointestinal agents; proline; protein engineering; proteolysis

DESCRIPTION (provided by applicant): The goal of this proposal is to engineer enzyme candidates suitable for the treatment of Celiac Sprue. Celiac Sprue is a widely prevalent immune disease of the small intestine induced by dietary gluten in genetically susceptible individuals. By damaging the intestinal villi, Celiac Sprue causes a range of symptoms, including diarrhea, iron and vitamin deficiencies, reduced bone density, stunted growth, fatigue, anemia and osteoporosis, increased risks of intestinal cancer and lymphomas. The only treatment for the disease currently available is complete avoidance of gluten, which is very difficult because gluten proteins are present in almost all grains. Proteolytically resistant proline-rich peptides from gluten have been implicated as the immunotoxic agent for Celiac patients. Preliminary results have indicated that bacterial prolyl endopeptidases can degrade these peptides and may thus be an effective treatment for celiac patients. However the naturally occurring endopeptidases all have shortcomings that prevent their therapeutic application. The aim of this project is to use a sequence-activity based protein engineering technology to optimize the activities of prolyl endopeptidases for treatment of Celiac Sprue. Enzymes will be obtained with increased activity towards a full spectrum of gluten-derived proteolytically-resistant peptides, and optimal pH profiles for oral administration. Briefly the experimental methods are as follows. (1) Use phylogenetic and structural modeling to identify amino acid substitutions likely to improve these desired enzyme properties. (2) Design and synthesize specific enzyme variants. (3) Use HPLC to accurately measure the ability of each variant to degrade gluten and its immunotoxic peptides under appropriate conditions. (4) Use the results from activity testing to derive sequence-activity relationships and thus design further improved variants.

描述（由申请人提供）：本提案的目标是设计适用于治疗乳糜泻的候选酶。口炎性腹泻是一种广泛流行的小肠免疫性疾病，由遗传易感个体的膳食麸质引起。通过破坏肠绒毛，乳糜泻会引起一系列症状，包括腹泻、铁和维生素缺乏、骨密度降低、生长迟缓、疲劳、贫血和骨质疏松症，增加患肠癌和淋巴瘤的风险。目前唯一可用的治疗方法是完全避免面筋，这是非常困难的，因为面筋蛋白存在于几乎所有的谷物中。 来自麸质的蛋白水解抗性富含脯氨酸的肽被认为是乳糜泻患者的免疫毒性剂。初步结果表明，细菌脯氨酰内肽酶可以降解这些肽，因此可能是一种有效的治疗乳糜泻患者。然而，天然存在的内肽酶都具有阻碍其治疗应用的缺点。 本项目的目的是使用基于序列活性的蛋白质工程技术来优化脯氨酰内肽酶的活性以用于治疗乳糜泻。将获得对全谱的谷蛋白衍生的蛋白水解抗性肽具有增加的活性的酶，以及用于口服给药的最佳pH曲线。简单地说，实验方法如下。(1)使用系统发育和结构建模来识别可能改善这些期望的酶特性的氨基酸取代。(2)设计和合成特定的酶变体。(3)使用高效液相色谱法准确测定每种变体在适当条件下降解面筋及其免疫毒性肽的能力。(4)使用活性测试的结果来推导序列-活性关系，从而设计进一步改进的变体。