Exploiting new approaches for selective inhibition of trypsins

开发选择性抑制胰蛋白酶的新方法

• 批准号: 10542402
• 负责人: Evette S Radisky
• 金额: $ 29.38万
• 依托单位: MAYO CLINIC JACKSONVILLE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10542402
• 关键词: Active Sites; Affinity; Allosteric Regulation; Allosteric Site; Binding; Binding Proteins; Biochemical; Biological; Biological Models; Biological Process; Calibration; Catalysis; Computer Analysis; Data; Development; Dimerization; Directed Molecular Evolution; Disease; Disease model; Engineering; Enzymes; Family; Future; Generations; Goals; Heterogeneity; Human; Individual; International; Libraries; Ligands; Machine Learning; Malignant Neoplasms; Maps; Mediating; Medical; Membrane Proteins; Methodology; Methods; Molecular; Molecular Conformation; Mutation; Nature; Pancreatitis; Pathologic; Pathology; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Play; Positioning Attribute; Pre-Clinical Model; Process; Productivity; Protease Inhibitor; Protein Isoforms; Protein-Protein Interaction Map; Proteins; Proteome; Publishing; Regulation; Role; Serine Protease; Shapes; Specificity; Structure; Surface; System; Therapeutic; Therapeutic Agents; Trypsin; Trypsin Inhibitors; Validation; X-Ray Crystallography; Yeasts; combinatorial; design; dimer; in silico; inhibitor; inhibitor therapy; innovation; insight; lead optimization; mesotrypsin; molecular dynamics; molecular modeling; molecular recognition; monomer; next generation sequencing; novel; novel strategies; novel therapeutics; pharmacologic; protein complex; protein protein interaction; screening; therapeutic target; tool; trypsin-like serine protease; virtual screening

PROJECT SUMMARY/ABSTRACT The human trypsin isoforms, trypsin 1, trypsin 2, and mesotrypsin, are proteases that have been implicated in disease processes in cancer and pancreatitis, and may offer viable therapeutic targets. Trypsins belong to a large family of trypsin-like enzymes with similar active site topology, and hence existing inhibitors lack selectivity. There is a need for selective trypsin inhibitors and isoform-selective trypsin inhibitors as pharmacological tools to better define the functions of these individual enzymes in disease, and to evaluate trypsin inhibition as a therapeutic strategy in preclinical models of disease. In this project, we will take a multipronged approach to develop new strategies for potent and selective inhibition of each of the human trypsin isoforms. (1) Our preliminary data reveal a previously unsuspected auto-inhibited conformation of mesotrypsin with a ligand-targetable allosteric site that may be exploited for inhibitory effect. We will use high- throughput virtual screening and structure-based hit-to-lead optimization to develop potent and selective allosteric inhibitors of mesotrypsin. We will also use structural and molecular dynamics analyses to evaluate whether similar strategies may hold potential for trypsins 1 and 2. (2) Our published studies have shown that Kunitz domains can be engineered to create more selective protein-based inhibitors of trypsin-like proteases by using a yeast surface display (YSD) platform for directed evolution. To enable further optimization of such inhibitors, we seek to generate comprehensive maps of the binding specificity landscapes that can, for any possible combination of mutations within an inhibitor, predict the consequences on inhibitor affinity and specificity toward a set of target proteases. We will accomplish this task by integrating YSD combinatorial library screening with next-generation sequencing (NGS), machine-learning (ML) approaches, and experimental calibration to enable quantitative prediction of the impact of multiple potentially interacting mutations of an inhibitor. These data will enable us to identify the most potent and selective Kunitz domains that can be achieved for targeting each of the human trypsins. (3) Our preliminary data demonstrate an enhancement of trypsin affinity by bivalent inhibitors capable of binding simultaneously to two molecules of mesotrypsin. Here, we will dissect the mechanisms responsible for these affinity enhancements and design strategies to exploit this information toward development of more potent and selective polyvalent trypsin inhibitors. In addition to developing three complementary strategies, each of which has high potential to produce the desired selective inhibitors of human trypsins, our project will provide broader insights that can aid future development of inhibitors for many other important trypsin-like proteases. Finally, the novel methodology developed here for mapping protein-protein interaction (PPI) affinity and specificity landscapes will be of broad utility for characterizing the sequence and structural constraints governing affinity and selectivity of functional protein interactions in many other diverse systems.

项目摘要/摘要 人胰蛋白酶同工型，胰蛋白酶1，胰蛋白酶2和间替肽是与蛋白酶有关的蛋白酶 癌症和胰腺炎的疾病过程，可能会提供可行的治疗靶点。胰蛋白酶属于 具有相似活性现场拓扑的大型胰蛋白酶样酶，因此现有的抑制剂缺乏 选择性。需要选择性胰蛋白酶抑制剂和同工型选择性胰蛋白酶抑制剂作为 更好地定义这些单个酶在疾病中的功能的药理工具，并评估 胰蛋白酶抑制作临床前疾病模型中的治疗策略。在这个项目中，我们将 多收益的方法来制定对每个人的有效和选择性抑制的新策略 胰蛋白酶同工型。 （1）我们的初步数据揭示了先前未经使用的自动抑制构象 间替肽具有可用于抑制作用的可依赖配体的变构位点。我们将使用高 吞吐量虚拟筛选和基于结构的命中率优化，以发展有效和选择性 间替肾蛋白酶的变构抑制剂。我们还将使用结构和分子动力学分析来评估 （2）我们发表的研究表明，类似的策略是否可能具有胰蛋白酶1和2的潜力。（2） 可以设计Kunitz结构域以创建更有选择性的基于胰蛋白酶样蛋白酶的抑制剂 通过使用酵母表面显示（YSD）平台进行定向演变。为了进一步优化这种 抑制剂，我们寻求生成具有约束力特异性景观的全面地图 可能在抑制剂中结合突变，预测抑制剂亲和力的后果 针对一组靶蛋白酶的特异性。我们将通过集成YSD组合来完成这项任务 使用下一代测序（NGS），机器学习（ML）方法和 实验校准以实现对多个潜在相互作用的影响的定量预测 抑制剂的突变。这些数据将使我们能够识别最有效，最有选择的kunitz域 这是针对每个人类胰蛋白酶的目标。 （3）我们的初步数据证明了 通过二价抑制剂增强胰蛋白酶亲和力，能够同时结合两个分子 美索蛋白酶。在这里，我们将剖析负责这些亲和力增强和设计的机制 利用这些信息来开发更有效和选择性的多价胰蛋白酶的策略 抑制剂。除了制定三种互补策略外，每种策略都具有很高的潜力 产生所需的人类胰蛋白酶的选择性抑制剂，我们的项目将提供更广泛的见解，可以帮助 许多其他重要胰蛋白酶样蛋白酶的抑制剂的未来开发。最后，新方法 这里开发用于映射蛋白质 - 蛋白质相互作用（PPI）亲和力和特异性景观的开发 用于表征序列和结构约束的实用程序 许多其他不同系统中的蛋白质相互作用。