高效口服脯氨酰内肽酶的合理化设计与发现及其机制研究

Enzymes drugs are an important class of biopharmaceutics. Now many natural enzymes with potential therapeutic value have low druggability, because of their low catalytic activity and poor stability. Prolyl endopeptidase form Sphingomonas capsulata (SC PEP) is a drug candidate for celiac sprue, which is one of the human genetic diseases with the highest incidence, but SC PEP has low catalytic activity and is easily biodegradable. Unfortunately, enzymes designed by using the conventional design methods have the problems of low catalytic activities and stability, and the enzyme therapy development is limited by the commonly recognized “stability-function tradeoff” theory. Therefore, the conventional design methods could not significantly increase catalytic activity and stability of SC PEP. There is no research revealing the enzymatic reaction pathway of SC PEP and the dynamic changes of enzyme structure. Therefore the catalytic mechanism of SC PEP is still not accurately clarified. Nevertheless, our recently developed unique computational enzyme modeling strategies and methods (e.g. the structure and transition-state based modeling) are promising in solving these challenges. Taking advantage of these recently developed computational enzyme modeling and design strategies and methods, in this project, we will reveal the catalytic mechanism of SC PEP, rationally redesign, discover, and develop prolyl endopeptidase (PEP), aiming to develop an engineered PEP with significantly improved catalytic activities against immunodominant gluten peptides and stability at low pH suitable for use as a novel oral enzyme therapy. To achieve the goal, we will first computationally uncover the detailed mechanism for PEP-catalyzed hydrolysis of immunodominant gluten peptides, followed by structure and mechanism based design of new mutants that are stable at low pH and potentially have improved catalytic activities against immunodominant gluten peptides. The computationally designed mutants will be evaluated in vitro for their actual catalytic activities, selectivity, and stability that in turn will be used to refine the computational design protocol and to make the next round of computational design more reliable. Thus, the iterative computational-experimental studies should eventually lead to reveal the catalytic mechanism of SC PEP, and to discovery of an PEP form with the desired catalytic activities, selectivity, and stability. Completion of this project will result in discovery of highly efficient prolyl endopeptidases metabolizing immunodominant gluten peptides suitable for further development as novel oral enzyme therapies to treat gluten-related diseases, and will demonstrate a generally-applicable protocol to rationally design and discover highly efficient therapeutic enzymes.

治疗酶是一类重要的生物药物，目前许多具有潜在治疗价值的天然酶分子因催化活性低、稳定性差，导致成药性低。脯氨酰内肽酶（SC PEP）是治疗发病率最高的人类遗传病之一的乳糜泻的候选药物，但其催化活性低、口服给药易降解，采用已有的酶分子设计方法仍未能显著提高SC PEP的催化活性与稳定性，难以成药，其原因在于SC PEP催化反应路径和酶分子结构的动态变化等关键机理至今尚未阐明。为此本项目深入模拟SC PEP催化反应过程，确定SC PEP催化反应路径，揭示SC PEP催化活性与关键氨基酸残基的关系，系统阐明其催化机理和稳定性的机理，根据催化机理设计与发现用于治疗乳糜泻的具有高催化活性和稳定性的口服SC PEP新药候选分子，为合理化设计与发现高效稳定的酶类新药提供科学依据和新策略新方法。

乳糜泻是一种系统性疾病，会导致慢性小肠吸收不良综合征，引发肿瘤的比例约11%；影响皮肤、中枢神经等多个组织或器官，造成肠外症状和神经疾病。乳糜泻是人类发病率最高的遗传性疾病之一，全球的发病率高达0.2%-1.0%。目前，乳糜泻仅依靠无谷蛋白饮食治疗，亟需开发治疗药物。口服酶治疗药物有望水解致病性谷蛋白多肽，从致病因子源头消除疾病。本研究针对野生型SCPEP在胃液中活性、稳定性不足的问题，采用基于酶结构的设计和基于机器学习的两种策略，设计筛选SCPEP突变体，并采用PEG修饰进一步提高SCPEP及其突变体在胃蛋白酶酸性溶液中的活性和稳定性。经过对SCPEP的结构模拟、SCPEP与底物复合物结构模拟，构建了SCPEP（V500W）等对13肽底物活性提高1倍的活性突变体。采用自主构建的二维氨基酸描述符，基于机器学习，设计筛选了18个在胃蛋白酸性溶液中活性与稳定性增强的SCPEP突变体，其中突变体B18的稳定性是野生型SCPEP的12.3倍。采用10 KDa的PEG修饰通过Lys侧链的NH2修饰野生型SCPEP及突变体B18，PEG修饰进一步将B18突变体的稳定性提高1倍。本项目经过4年的实施，发现了33个活性提高的SCPEP突变体或修饰物。相关成果发表在European Journal of Medicinal Chemistry（IF 6.514）等期刊上，并申请中国发明专利1项。本项目系统地构建了口服酶分子的设计、筛选与评价体系，为乳糜泻的治疗提供新的候选分子。